A dimethidazole derivative and its application

Abstract

This invention discloses a dimetronidazole derivative or its pharmaceutical salt, with one of the following general structural formulas: The compounds of this invention exhibit good antibacterial activity and specificity against *Fusobacterium nucleatum* in in vitro antibacterial activity tests; derivative B2 has excellent in vitro antibacterial activity and selectivity against *Fusobacterium nucleatum*. In vivo studies using a mouse xenograft model of colorectal cancer have shown that compound B2, when used alone, has a certain antitumor effect against xenografts caused by *Fusobacterium nucleatum*. Furthermore, when compound B2 is administered in combination with a PD-1 antibody, it exhibits excellent in vivo antitumor activity and can effectively reverse the resistance of *Fusobacterium nucleatum*-infected colorectal cancer to PD-1 antibodies. The compounds of this invention can be used to treat colorectal cancer caused by *Fusobacterium nucleatum*, as well as tumor metastasis and drug resistance that occur during treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically, it relates to a dimethidazole derivative that can treat colorectal cancer caused by Fusobacterium nucleatum and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide. Statistics show that approximately 900,000 people die from colorectal cancer globally each year. While it ranks third in incidence among all cancers, it ranks second in mortality, only behind lung cancer. In recent years, although some progress has been made in the prevention and treatment of colorectal cancer, existing treatments suffer from drawbacks such as significant side effects, easy drug resistance, and low response rates. Therefore, there is an urgent need to develop new, highly effective, and low-toxicity treatment strategies for colorectal cancer.

[0003] Fusobacterium nucleatum is a common anaerobic Gram-negative bacterium and an important component of the oral microbiota, as well as the gastrointestinal tract, upper respiratory tract, and genitals. Numerous studies have shown that Fusobacterium nucleatum can promote the occurrence and development of colorectal cancer; for example, it promotes chemotherapy resistance in colorectal cancer by regulating autophagy; and in Apc... Min / + In a mouse model of intestinal tumor development, *Fusobacterium nucleatum* can increase tumor diversity and selectively recruit tumor-infiltrating myeloid cells, thereby promoting tumor development. *Fusobacterium nucleatum* can also recruit tumor-infiltrating immune cells, creating a pro-inflammatory microenvironment that promotes colorectal cancer development. In colorectal cancer, *Fusobacterium nucleatum* aggregation can selectively promote the proliferation of immunosuppressed myeloid cells, generating an immunosuppressive tumor microenvironment (TME), further inhibiting T cell responses. Furthermore, *Fusobacterium nucleatum* within colorectal cancer tumors can bind to the inhibitory receptor TIGIT (T cell immune receptor with Ig and ITIM domains) on human natural killer cells (NK cells) and T cells, promoting immune evasion in colorectal cancer. In summary, *Fusobacterium nucleatum* affects the human immune system and tumor microenvironment, potentially influencing immunotherapy for colorectal cancer patients. Therefore, developing drugs against intestinal pathogens to improve the distribution of gut microbiota in colorectal cancer holds promise for overcoming the low efficacy and drug resistance of traditional anti-colorectal cancer drugs.

[0004] Literature review revealed that compounds containing nitroimidazole possess broad-spectrum antiparasitic, antibacterial, Gram-positive, and Gram-negative bacterial activity. Several marketed drugs are already used for antimicrobial purposes; for example, metronidazole and tinidazole are the only drugs approved in the United States for the treatment of trichomoniasis. In addition, metronidazole is also used to treat intestinal parasitic diseases caused by Giardia lamblia and anaerobic infections caused by Bacteroides fragilis, Clostridium difficile, and Fusobacterium nucleatum.

[0005] Traditional drug treatments often lead to drug resistance and reduced efficacy. Therefore, combination therapy is another effective treatment strategy. Literature review has found no cases of using metronidazole compounds to treat colorectal cancer caused by *Fusobacterium nucleatum*. Therefore, obtaining highly active antibacterial agents against *Fusobacterium nucleatum* is of great significance for improving the development and prognosis of colorectal cancer. By utilizing the additive or synergistic effects of combination drugs to improve the success rate of drug reuse, multiple targeting mechanisms can overcome drug resistance caused by *Fusobacterium nucleatum* or enhance the efficacy of immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to provide a dimethidazole derivative.

[0007] Another object of the present invention is to provide the use of the dimetronidazole derivative in the preparation of a drug for treating Clostridium nucleatum.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a dimetronidazole derivative or a pharmaceutical salt thereof, having one of the following general structural formulas:

[0010]

[0011] in,

[0012] R1 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl groups, C1-C10 alkoxy groups, etc.

[0013]

[0014] R2 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl groups, C1-C10 alkoxy groups, etc.

[0015]

[0016] R3 is selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), C1-C10 alkyl groups, C1-C10 alkoxy groups, etc.

[0017]

[0018] R7 is selected from hydrogen, C3-C8 cycloalkyl, C1-C5 alkyl, R8 is selected from hydrogen, C3-C8 cycloalkyl, C1-C5 alkyl, Alternatively, R7, R8, and the connected N, O, and C form a six-membered ring;

[0019] R9 is selected from hydrogen, C3-C8 cycloalkyl, C1-C5 alkyl, R 10 Selected from hydrogen, C3-C8 cycloalkyl, C1-C5 alkyl, R 11 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), and C1-C10 alkyl groups;

[0020] R 12 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), and C1-C10 alkyl groups;

[0021] R 13 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), and C1-C10 alkyl groups;

[0022] R 14 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), and C1-C10 alkyl groups;

[0023] R 15 Selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine), and C1-C10 alkyl groups;

[0024] R4 is selected from C1-C5 alkyl groups substituted with C3-C8 cycloalkyl groups, (CH3)2N(CH2)n-, n is selected from positive integers from 1 to 10 (preferably 1, 2, 3, 4, 5);

[0025] m is selected from positive integers from 1 to 10 (preferably 1, 2, 3, 4, 5);

[0026] R5 is selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, and halogens (fluorine, chlorine, bromine, iodine);

[0027] R6 is selected from hydrogen, C1-C10 alkyl, C1-C10 alkoxy, and halogens (fluorine, chlorine, bromine, iodine).

[0028] Preferably, in the dimetronidazole derivative,

[0029] R1 is selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, methoxy, ethoxy, tert-butoxy, fluorine, chlorine, bromine, etc.

[0030]

[0031]

[0032] R2 is selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, methoxy, ethoxy, tert-butoxy, fluorine, chlorine, bromine, etc.

[0033]

[0034] R3 is selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, methoxy, ethoxy, tert-butoxy, fluorine, chlorine, bromine, etc.

[0035]

[0036] R4 is selected from

[0037]

[0038] R5 is selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, methoxy, ethoxy, tert-butoxy, fluorine, chlorine, and bromine; R6 is selected from hydrogen, methyl, ethyl, n-propyl, tert-butyl, methoxy, ethoxy, tert-butoxy, fluorine, chlorine, and bromine. Most preferably, the structure of the dimetronidazole derivative is selected from one of the following structures:

[0039]

[0040]

[0041] The medicinal salt may be anhydrous or contain more than one molecule of water of crystallization, preferably 0.5-3 molecules of water of crystallization.

[0042] This invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned dimethicone derivative or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier. The carrier may be a commonly used carrier substance such as a flavoring agent, sweetener, liquid or solid filler, or diluent, and is prepared into a commonly used pharmaceutical formulation, such as tablets, capsules, powders, syrups, liquids, suspensions, or injections, using methods known in the art. The formulation typically contains 1-70% by weight of the active ingredient, preferably 5-50% by weight.

[0043] A second aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or a pharmaceutical salt thereof in the preparation of a medicament against Fusobacterium nucleatum.

[0044] A third aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or a pharmaceutical salt thereof in the preparation of a medicament for treating diseases caused by Fusobacterium nucleatum.

[0045] The diseases mentioned are selected from colon cancer, drug-resistant colon cancer, and metastatic colon cancer.

[0046] A fourth aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or a pharmaceutical salt thereof in the preparation of a medicament for treating colon cancer.

[0047] The fifth aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or its pharmaceutical salt in combination with metronidazole in the preparation of a drug for treating Fusobacterium nucleatum.

[0048] The sixth aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or its pharmaceutical salt in combination with metronidazole and PD-1 antibody in the preparation of a drug for treating Fusobacterium nucleatum.

[0049] A seventh aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or a pharmaceutical salt thereof in combination with metronidazole in the preparation of a medicament for treating colon cancer.

[0050] The eighth aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or its pharmaceutical salt in combination with metronidazole and PD-1 antibody in the preparation of a drug for treating colon cancer.

[0051] The ninth aspect of the present invention provides the use of the aforementioned dimetronidazole derivative or a pharmaceutical salt thereof in combination with metronidazole and a PD-1 antibody in the preparation of a medicament for reversing resistance to PD-1 antibody in colorectal cancer caused by Fusobacterium nucleatum infection.

[0052] This invention uses the half-dilution method for activity testing, with clindamycin as a positive control. Pharmacological experiments show that the compounds of this invention or their pharmaceutical salts exhibit in vitro antibacterial activity against *Fusobacterium nucleatum*. The results show that derivatives containing benzamide and ether bonds exhibit moderate to high antibacterial activity against *Fusobacterium nucleatum*, with some compounds showing increased activity compared to dimethylnitrazole, and comparable to the antibacterial activity of the positive control drug clindamycin.

[0053] The Class A compounds of this invention use dimethylnitroimidazole as a backbone, and various alkanes are linked at position 1. It was found that the activity of dimethylnitroimidazole was reduced or remained unchanged.

[0054] The B-type compounds of this invention have a benzamide active group linked at position 2 with dimethylnitroimidazole as the backbone. The derivatives with better activity are mainly B1-B10, with B2 being the most active derivative, which has an inhibitory activity of 0.25 μg / mL against Fusobacterium nucleatum, comparable to that of the positive control drug clindamycin.

[0055] The Class C compounds of this invention use dimethylnitroimidazole as a backbone, with a phenolic active group linked at position 2 via an amide bond. The activity of the derivatives is reduced or comparable to that of dimethylnitroimidazole.

[0056] The D and E class compounds of this invention use dimethylnitroimidazole as the backbone. Based on derivative B2, the amide bond of benzamide is modified, and the activity of the derivative disappears or is reduced.

[0057] In summary, in vitro antibacterial tests showed that the B-class compounds exhibited strong in vitro antibacterial activity, while showing no activity against the other five common intestinal bacteria, demonstrating strong specificity. They also exhibited low toxicity to normal human cells, indicating a certain degree of safety. This invention explored colorectal cancer caused by *Fusobacterium nucleatum*, laying the foundation for new treatment strategies for colorectal cancer. The compounds of this invention or their pharmaceutical salts possess a novel structural framework, making them innovative and practical for drug development against *Fusobacterium nucleatum*, and worthy of further research. Therefore, the compounds of this invention or their pharmaceutical salts can be used to prepare drugs for colorectal cancer caused by *Fusobacterium nucleatum*.

[0058] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0059] This invention discovered a dimethidazole derivative through phenotypic screening of a commercial compound library, which exhibits good antibacterial activity against Fusobacterium nucleatum.

[0060] This invention optimizes the structure of discovered dimetronidazole derivatives to identify highly active compounds. Then, it combines these highly active dimetronidazole derivatives with PD-1 (programmed cell death protein 1) antibodies, an immunotherapy drug for colorectal cancer, to investigate the therapeutic effect of this combination on colorectal cancer. The aim is to discover small molecule candidate drugs with therapeutic efficacy and provide a novel intervention strategy for the treatment of colorectal cancer.

[0061] This invention focuses on structure-activity relationship studies of dimetronidazole derivatives. Through a series of in vitro bioactivity evaluations, it was discovered that dimetronidazole derivative B2 exhibits excellent in vitro anti-Fusobacterium nucleatum activity and selectivity. At a concentration of 50 mg / kg, administered continuously for 15 days, mice showed intact organs without significant lesions, indicating that compound B2 is a highly effective, low-toxicity, and highly safe compound. In an in vivo model of colorectal cancer in mice, it was found that metronidazole and compound B2, when used alone, have certain anti-tumor effects, with compound B2 showing superior anti-tumor efficacy compared to metronidazole. The combined use of both compounds showed better anti-tumor efficacy than either alone. Furthermore, it was found that both compound B2 and metronidazole, when combined with PD-1 antibodies, exhibited excellent in vivo anti-tumor activity and effectively reversed resistance to PD-1 antibodies in colorectal cancer induced by Fusobacterium nucleatum infection. The combined use of compound B2, metronidazole, and PD-1 antibodies resulted in even better anti-tumor efficacy and the most significant reversal of resistance to PD-1 antibodies in Fusobacterium nucleatum-infected colorectal cancer. This indicates that compound B2 exhibits good antibacterial effects in vivo, potentially providing a precision treatment strategy for colorectal cancer patients with drug-resistant Fusobacterium nucleatum infection. It provides a new chemical entity for the eventual discovery of a highly active, highly selective, and drug-grade anti-Fusobacterium nucleatum agent, offering a novel strategy for the prevention and treatment of colorectal cancer, and laying the material and theoretical foundation for the development of original candidate drugs for the prevention and treatment of colorectal cancer.

[0062] The compounds of this invention exhibit good antibacterial activity and specificity against *Fusobacterium nucleatum* in in vitro antibacterial activity tests. They show no inhibitory effect on common intestinal bacteria such as *Escherichia coli* (ATCC 25922), *Salmonella paratyphi B* (ATCC CMCC 50094), *Staphylococcus aureus* (ATCC 6538), *Femtococcus* (ATCC 29212), and *Shigella flexneri* (ATCC 12022), but specifically inhibit *Fusobacterium nucleatum*. Derivative B2 exhibits excellent in vitro anti-*Fusobacterium nucleatum* activity and selectivity. In vivo studies using a mouse xenograft model of colorectal cancer have shown that compound B2, when used alone, has a certain anti-tumor effect on xenografts induced by *Fusobacterium nucleatum*. Furthermore, compound B2, when combined with a PD-1 antibody, exhibits excellent in vivo anti-tumor activity and can effectively reverse resistance to PD-1 antibodies in colorectal cancer infected with *Fusobacterium nucleatum*. The compounds of this invention can be used to treat colorectal cancer caused by *Fusobacterium nucleatum*, as well as tumor metastasis and drug resistance that occur during treatment. Attached Figure Description

[0063] Figure 1 This is a schematic diagram showing the changes in body weight of mice in the non-bacterial group during the drug administration period.

[0064] Figure 2 This is a schematic diagram showing the changes in body weight of mice in the bacterial enema group during the drug administration period.

[0065] Figure 3 This is a schematic diagram showing the results of PD-1 antibody treatment in mice in the inoculated group and the uninoculated group.

[0066] Figure 4 This is a schematic diagram showing the changes in tumor size after administration of the drug in the non-bacterial irrigation group.

[0067] Figure 5 This is a schematic diagram showing the changes in tumor size after administration of the bacteria to the infusion group.

[0068] Figure 6 This is a schematic diagram of the immunohistochemical and immunofluorescence results of mouse tumor tissue.

[0069] Figure 7 It is CD8 in mouse tumor tissue + Schematic diagram of T content results.

[0070] Figure 8 This is a schematic diagram showing the distribution of Fusobacterium nucleatum in mouse tumors and intestinal tissues. Detailed Implementation

[0071] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0072] Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0073] The chemical structural formulas, NMR and MS data of the compounds involved in the following examples are detailed in Table 1.

[0074] Table 1: NMR and MS data of the compounds of this invention

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Example 1: Preparation of 1-cyclopropylmethyl-2-methyl-5-nitro-1H-imidazolium (A1)

[0084]

[0085] In a 25 mL round-bottom flask, 1 mmol of compound 1 was dissolved in 5 mL of N,N-dimethylformamide, followed by the addition of 1.2 mmol of cesium carbonate and 1.2 mmol of chloromethylcyclopropane. The reaction was carried out at 85 °C for 6 h, monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate. The organic layers were combined and washed three times with saturated brine. After drying with anhydrous sodium sulfate, the anhydrous sodium sulfate was removed by filtration. The filtrate was collected and the ethyl acetate was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product A1 (0.7 g, yield 70.5%) was obtained by dry loading and silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:2).

[0086] Preparation method of compounds A2 to A6: Following the method of Example 1, the chloromethylcyclopropane in Example 1 was successively replaced with chloromethylcyclopentane, chloromethylcyclohexane, chloromethylcycloheptane, 3-chloro-1-(N,N-dimethyl)propylamine, and 4-methoxybenzyl chloride, with yields of 42.3%, 33.9%, 63.8%, 35.9%, and 35.3%, respectively.

[0087] Example 2: Preparation of 2-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)benzamide (B1)

[0088]

[0089] 30 mL of thionyl chloride was added to a 100 mL round-bottom flask, and 10 mmol of compound 3 was slowly added at 0 °C. After compound 3 was completely added, the reaction was carried out at room temperature for 2 h, and the reaction was monitored by TLC. After the reaction was completed, thionyl chloride was removed by vacuum distillation using a rotary evaporator to obtain compound 4.

[0090] In a 25 mL round-bottom flask, 1 mmol of compound 4 and 1.2 mmol of cesium carbonate were dissolved in 5 mL of N,N-dimethylformamide. After stirring at 85 °C for 30 min, o-hydroxybenzamide was slowly added, and the reaction was carried out at 85 °C for 6 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 30 mL of ice water, and a white solid precipitated out. The filtrate was filtered and the residue was collected. The residue was washed with ethyl acetate 3-5 times, and then the residue was collected and dried in a freeze dryer to obtain the target compound B1 (0.5 g, yield 31.8%).

[0091] Preparation method of compounds B2-B10: Following the method of Example 2, the o-hydroxybenzamide in Example 2 was successively replaced with 5-chlorosalicylic acid amide, 4-methylsalicylic acid amide, 5-bromo-2-hydroxybenzamide, 4-bromo-2-hydroxybenzamide, 2-hydroxy-5-methylbenzamide, 2-fluoro-5-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-bromo-3-hydroxybenzamide, and 4-hydroxybenzamide, with yields of 32.1%, 42.5%, 37.3%, 13.2%, 28.9%, 22.5%, 50.3%, 19.7%, and 36.9%, respectively.

[0092] Example 3: Preparation of 5-fluoro-2-hydroxy-N-((1-methyl-5-nitro-1H-imidazol-2-yl)methyl)benzamide (C1)

[0093]

[0094] Intermediate 4 (5 mmol) was placed in a 50 mL round-bottom flask, dissolved in 15 mL methanol (CH3OH), and then 15 mL ammonia (NH3·H2O) was added. The mixture was left to stand overnight at room temperature, and the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum distillation at room temperature. The oily substance in the round-bottom flask was redissolved with ethyl acetate and extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed once with saturated brine (300 mL). Finally, the mixture was dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The mixture was then loaded onto a silica gel column and eluted with a dichloromethane / methanol system (DCM:MeOH = 100:15) to obtain intermediate 7.

[0095] 1.2 mmol of intermediate 7 and 1.2 mmol of coupled 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) were dissolved in 5 mL of N,N-dimethylformamide (DMF). The mixture was reacted at room temperature for 30 min, followed by the addition of 4 mmol of N,N-diisopropylethylamine (DIPEA) and a reaction time of 5 min. Finally, 1 mmol of 5-fluoro-2-hydroxybenzoic acid was added and the mixture was reacted at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed three times with saturated brine (150 mL). The mixture was then dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The sample was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:2) to obtain the target compound C1 (0.4, yield 21.2%).

[0096] Preparation method of compounds C2 to C7: Following the method in Example 3, 5-fluoro-2-hydroxybenzoic acid in Example 3 was successively replaced with o-hydroxybenzoic acid, 2-hydroxy-5-methoxybenzoic acid, 5-chloro-2-hydroxybenzoic acid, 3-bromo-2-hydroxybenzoic acid, 2-hydroxy-5-methylbenzoic acid, and 3-chlorosalicylic acid, with yields of 37.8%, 39.9%, 15.7%, 18.3%, 23.6%, and 31.3%, respectively.

[0097] Example 4: Preparation of 5-chloro-N-cyclopentyl-2-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)benzamide (D1)

[0098]

[0099] 1.2 mmol of compound 10 and 1.2 mmol of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) were dissolved in 5 mL of DMF and reacted at room temperature for 30 min. Then, 4 mmol of N,N-diisopropylethylamine (DIPEA) was added and reacted for 5 min. Finally, compound 11 (cyclopentanamine, 1 mmol) was added and reacted at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed three times with saturated brine (150 mL). Finally, the solution was dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The solution was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:10) to obtain intermediate 12a.

[0100] Intermediate 12a (1.2 mmol) and cesium carbonate (1.2 mmol) were placed in a 25 mL round-bottom flask and dissolved in DMF (5 mL). The mixture was reacted at 85 °C for 20 min. Then, compound 4 (1.0 mmol) was added in portions and the mixture was reacted at 85 °C for 6 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed three times with saturated brine (150 mL). The mixture was then dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The sample was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:10) to obtain the target compound D1 (0.4, 25.3%).

[0101] Preparation method of compounds D2 to D8: Following the method in Example 4, the cyclopentylamine in Example 4 was successively replaced with isopropylamine, morpholine, aniline, p-toluidine, 4-bromoaniline, 4-chloroaniline, and 4-fluoroaniline, with yields of 11.7%, 12.2%, 17.8%, 11.8%, 14.3%, 12.7%, and 18.6%, respectively.

[0102] Example 5: Preparation of N-(5-chloro-2-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)phenyl)aniline (E1)

[0103]

[0104]

[0105] Compound 13 (1 mmol) was dissolved in anhydrous methanol (5 mL), and 1-2 drops of catalytic acetic acid were added. After stirring thoroughly, aniline (1 mmol) was added, and the reaction was carried out at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, intermediate 15a was obtained. Without any treatment, 2 mmol of sodium cyanoborohydride (NaBH3CN) was slowly added in portions at 0 °C. The mixture was then brought back to room temperature and stirred at room temperature for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated by vacuum distillation using a rotary evaporator to obtain an oily substance. The oil was extracted three times with ethyl acetate (20 mL), and the organic phases were combined. The organic phase was then washed once with saturated brine (150 mL). Finally, the mixture was dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The mixture was then loaded onto a silica gel column and eluted with an ethyl acetate / petroleum ether system (EA:PE = 1:6) to obtain the target intermediate 16a.

[0106] Intermediate 16a was dissolved in DCM (5 mL), triethylamine (1.2 mmol) was added, and the mixture was stirred thoroughly. Then, Boc anhydride (1.5 mmol) was added, and the mixture was reacted at room temperature for 3 h. The reaction was detected by TLC. After the reaction was complete, DCM was removed by vacuum distillation, and the sample was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:10) to obtain the target intermediate 17a.

[0107] Intermediate 17a and cesium carbonate (1.2 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at 85 °C for 30 min. Then, compound 4 was slowly added in portions and reacted at 85 °C for 6 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed three times with saturated brine (150 mL). Finally, the solution was dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The solution was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:2) to obtain the target intermediate 18a.

[0108] 1 mmol of intermediate 18a was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added at room temperature to make the volume ratio of dichloromethane to trifluoroacetic acid 4:1. The reaction was carried out at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate was added to quench the trifluoroacetic acid, and the mixture was extracted three times with dichloromethane (10 mL). The organic phases were combined and washed once with saturated brine (100 mL). The organic phase was then dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration, and DCM was removed by vacuum distillation. The target compound E1 (0.2, 10.6%) was obtained by silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:5).

[0109] Preparation of compounds E2-E5: Following the method in Example 5, the aniline in Example 5 was successively replaced with 4-fluoroaniline, 4-chloroaniline, 4-methylaniline, and 4-bromoaniline. The yields were 18.7%, 25.8%, 10.6%, and 13.7%, respectively.

[0110] Example 6: Preparation of (5-chloro-2-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)phenyl)methylamine (E6)

[0111]

[0112] Dissolve 1 mmol of compound 20 in 5 mL of dry tetrahydrofuran (THF). Under N2 protection and low temperature (0 °C), slowly add a tetrahydrofuran (THF) solution of lithium aluminum hydride (LiAlH4, 2.5 mmol) to the solution. After reacting at low temperature for 30 min, the temperature was restored to room temperature and the reaction was allowed to proceed for 4 h. The reaction was monitored by TLC. After the reaction was completed, ice water was slowly added until no more bubbles were generated to quench the LiAlH4. Then, the THF was evaporated to dryness, and potassium sodium tartrate was added and stirred overnight. After the colloidal substance in the solution becomes a sandy substance, it is filtered to remove potassium sodium tartrate. The filtrate is collected and extracted three times with ethyl acetate (20 mL). The organic phases are combined and washed once with saturated saline (150 mL). Finally, it is dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate is removed by filtration. The sample is loaded dry and subjected to silica gel column chromatography with a dichloromethane / methanol eluent system (DCM:MeOH = 100:10) to obtain the target intermediate 21.

[0113] Intermediate 21 (1 mmol) was dissolved in DCM (5 mL), triethylamine (1.2 mmol) was added, and the mixture was stirred thoroughly. Then, Boc anhydride (1.5 mmol) was added, and the mixture was reacted at room temperature for 3 h. The reaction was detected by TLC. After the reaction was complete, DCM was removed by vacuum distillation, and the sample was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:10) to obtain target intermediate 22.

[0114] Intermediate 22 (1 mmol) and cesium carbonate (1.2 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at 85 °C for 30 min. Then, compound 4 was slowly added in portions and reacted at 85 °C for 6 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into distilled water and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed three times with saturated brine (150 mL). Finally, the solution was dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration. The solution was loaded onto silica gel column chromatography with an ethyl acetate / petroleum ether eluent system (EA:PE = 1:2) to obtain the target intermediate 23.

[0115] 1 mmol of intermediate 23 was dissolved in 3 mL of dichloromethane, and 1 mL of trifluoroacetic acid (TFA) was added at room temperature to make the volume ratio of dichloromethane to trifluoroacetic acid 4:1. The reaction was carried out at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction was complete, saturated sodium bicarbonate was added to quench the trifluoroacetic acid, and the mixture was extracted three times with dichloromethane (10 mL). The organic phases were combined and washed once with saturated brine (100 mL). The organic phase was then dried with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was removed by filtration, and DCM was removed by vacuum distillation. The target compound E6 (0.2, 10.1%) was obtained by silica gel column chromatography with a dichloromethane / methanol eluent system (DCM:MeOH = 100:10).

[0116] Example 7: In vitro antibacterial activity test of the compounds of the present invention against Fusobacterium nucleatum

[0117] The in vitro antibacterial activity of the synthetic compounds of this invention was tested using a standardized broth dilution sensitivity test, following the methods of the Clinical and Laboratory Standards Institute (CLSI).

[0118] 1. Test strain: Fusobacterium nucleatum (ATCC 23726) was purchased from Huashan Hospital. The culture medium for Fusobacterium nucleatum was brain and heart broth (BHI) and Brucella broth.

[0119] 2. Instrument: Wellscan MK-2 fully automated microplate reader (manufacturer: Labsystems Dragon)

[0120] 3. Test method: Sample solution preparation: Dissolve the sample in DMSO (Merck) to prepare a 2 mg / mL solution. Prepare reference solutions of clindamycin and metronidazole under the same conditions.

[0121] Preparation of *Fusobacterium nucleatum* bacterial suspension: Shake a standard turbidimetric tube. After calibration, place the glass test tube in a turbidimeter, add 4 mL of Brucella broth, scrape well-grown, appropriately sized colonies with a cotton swab, and rub them inside the glass test tube until the turbidity reaches 0.5 McFarland turbidity. Pour the Brucella broth containing the bacterial suspension into a test tube containing 40 mL of Brucella broth, add 4.4 mL of defibrinated horse blood (repeatedly frozen and thawed), and vortex to mix (bacterial suspension: broth: horse blood = 1:10:1.1).

[0122] Half-dilution: Add the above bacterial culture to a 96-well plate, with 200 μL in the first column and 100 μL in the other wells. The initial concentration of the compound in the first column is 64 μg / mL. After half-dilution, place all 96-well plates in an anaerobic chamber with an anaerobic bag and incubate at 37°C for 48 hours. Visually observe the wells; the minimum inhibitory concentration (MIC) of the compound is indicated by the appearance of turbidity.

[0123] Table 2 Antibacterial activity of dimethylnitroimidazole derivatives (MIC)50 (μg / mL)

[0124]

[0125]

[0126] The in vitro anti-nucleated Fusobacterium activity and structure-activity relationship of the compound of the present invention were studied according to the above-described in vitro anti-nucleated Fusobacterium activity test methods:

[0127] The in vitro antibacterial activity of the target compounds was tested using a standardized broth dilution sensitivity test, with clindamycin and metronidazole used as positive controls. Preliminary in vitro bioactivity screening showed that most Class B dimetronidazole derivatives effectively inhibited the proliferation of *Fusobacterium nucleatum*. Structural modifications at positions 1 and 2 of dimetronidazole revealed varying degrees of activity reduction in derivatives synthesized after modification of position 1 (Class A derivatives) (Table 2), indicating that the methyl group at position 1 is essential. When the linker at position 2 was an amide (Class C derivatives), the activity of the derivative decreased or was lost. Destroying the amide group of compound B2 (o-phenylamide) (Class D and E derivatives) further reduced or eliminated the activity of the derivatives, indicating that the amide group is essential for this series of compounds. Some compounds exhibited antibacterial activity against *Fusobacterium nucleatum*, with some showing significantly improved activity compared to metronidazole, comparable to the marketed positive control drug clindamycin.

[0128] Example 8: In vitro antibacterial activity test of the compounds of the present invention against common intestinal bacteria

[0129] 1. Test strain

[0130] Escherichia coli (ATCC 25922), Salmonella paratyphi B (ATCC CMCC 50094), Staphylococcus aureus (ATCC 6538), Fecal cocci (ATCC 29212), and Shigella flexneri (ATCC 12022) were all purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0131] The cultures of Escherichia coli and Shigella flexneri were prepared in LB broth; the cultures of fecal cocci and Staphylococcus aureus (ATCC6538) were prepared in MH broth; and the cultures of Salmonella paratyphi B were prepared in nutrient broth.

[0132] Sample solution preparation: Dissolve the sample in DMSO (Merck) to prepare a 2 mg / mL solution.

[0133] Prepare reference solutions of cefoxitin and metronidazole under the same conditions.

[0134] Preparation of bacterial suspension: After calibration, place the glass test tube in the turbidimeter and add 4 mL of Brucella broth. Use a cotton swab to scrape well-grown, appropriately sized colonies and rub them inside the glass test tube until the turbidity reaches 0.5 McFarland turbidity. Pour the Brucella broth containing the bacterial suspension into a test tube containing 40 mL of MH broth. The bacterial concentration is 1 × 10⁻⁶. 6 CFU / mL, vortex mix.

[0135] Half-dilution: Add the above bacterial culture to 96-well plates, with 200 μL in the first column and 100 μL in the other wells. The initial concentration of the compound in the first column is 64 μg / mL. After half-dilution, incubate all 96-well plates at 37°C for 48 h. Measure the OD value at 630 nm using an MK-2 automated microplate reader and calculate the MIC. 50 .

[0136] MIC 50 = (OD value of blank control well - OD value of drug administration well) / OD value of blank control well × 100%.

[0137] Table 3. Antibacterial activity of target compounds in categories A and B against common intestinal bacteria.

[0138]

[0139] The B-type compounds showed no antibacterial activity against any of the five bacteria mentioned above, indicating that the B-type compounds have good antibacterial selectivity against Fusobacterium nucleatum.

[0140] Example 9: In vitro antitumor activity test of the compounds of the present invention

[0141] The cell proliferation activity of the compounds synthesized in this invention was tested using the CCK8 assay.

[0142] 1. Experimental cell lines

[0143] HUVEC (human umbilical vein endothelial cells) and HCT116 (human colon cancer cells) were both purchased from Shanghai Institute of Pharmaceutical Industry.

[0144] HUVEC culture medium consisted of ECM + 15% FBS + antibiotics, while HCT116 culture medium consisted of DMEM + 10% FBS + antibiotics.

[0145] 2. Instruments

[0146] Wellscan MK-2 fully automated microplate reader (manufacturer: Labsystems Dragon).

[0147] 3. Test methods

[0148] Camptothecin (CPT), metronidazole, and the preferred compound synthesized in this invention were formulated to a concentration of 10 mmol / L.

[0149] Solution preparation: Camptothecin in the 96-well plate was diluted to an initial concentration of 10 μmol / L with cell culture medium. The initial concentrations of camptothecin (CPT) and the preferred compounds of the present invention and synthesis were 50 μmol / L. Then, the solution was diluted three times with culture medium to obtain nine concentration gradients.

[0150] The concentration added to each well of the 96-well plate is 5 × 10⁻⁶. 4 100 μL of cell suspension (5000 cells / well) was added to each well and incubated at 37°C in a 5% CO2 incubator. After 24 hours, 10 μL of sample solution and control solution were added to each well, and the mixture was incubated at 37°C for 72 hours. After discarding the culture medium, 100 μL of 10% CCK8 DMEM solution was added to each well and incubated for 30 minutes. The OD value at 570 nm was measured using an automated microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated. 50 .

[0151] IC% = (OD value of blank control well - OD value of drug administration well) / OD value of blank control well × 100%.

[0152] Based on the IC% values ​​at each concentration, linear regression was performed to calculate the drug concentration that inhibits cell growth by 50%, i.e., IC50. 50 .

[0153] Table 4. Cell proliferation activity of target compounds in categories A and B

[0154]

[0155]

[0156] The cytotoxicity of class B compounds was assessed using the CCK8 assay, and the results are shown in Table 4. Class B compounds showed no anti-proliferative activity against HUVECs, with an IC50 value of [missing value]. 50 The values ​​were all greater than 50 μM. This indicates that the B-class compounds have low toxicity and excellent safety.

[0157] Example 10: In vivo antitumor activity test of the compounds of the present invention

[0158] (a) Grouping: Mice were divided into 16 groups, of which 8 groups were not given bacterial infusion and the other 8 groups were given bacterial infusion.

[0159] 1. Inoculation group, divided into the following groups:

[0160] Table 5. Grouping of the in vivo bacterial enema group in mice

[0161] Group test substance Should PD-1 antibody be injected? Group 1 / yes Group 2 / / Group 3 B2 yes Group 4 B2 / Group 5 Metronidazole yes Group 6 Metronidazole / Group 7 B2+ Metronidazole yes Group 8 B2+ Metronidazole /

[0162] 2. The group without inoculum treatment was divided into the following subgroups:

[0163] Table 6. Grouping of the non-bacterial perfusion group in mouse in vivo experiments

[0164] Group test substance Should PD-1 antibody be injected? Group 9 / yes Group 10 / / Group 11 B2 yes Group 12 B2 / Group 13 Metronidazole yes Group 14 Metronidazole / Group 15 B2+ Metronidazole yes Group 16 B2+ Metronidazole /

[0165] (b) Intestinal preparation: Accurately weigh streptomycin and clindamycin and dissolve them in the drinking water of C57 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) to make concentrations of 5 g / L and 0.1 g / L, respectively. Feed them for 1 week to eliminate intestinal bacteria.

[0166] (c) Tumor cell inoculation: C57 mice were inoculated subcutaneously at a concentration of 4 × 10⁻⁶ cells / mL under the armpit. 5 MC-38 cells (mouse colorectal cancer cells) per mL.

[0167] (d) Oral administration of Fusobacterium nucleatum: The concentration administered orally was 1×10⁻⁶. 8 CFU / mL (BHI dilution), 3 days / time, 500μL / animal, for a total of 4 times (or adjust appropriately according to the size of the xenograft).

[0168] (e) Drug preparation: B2 was suspended in physiological saline to prepare a concentration of 4 mg / mL, which contained 10% beroxam 168; metronidazole was dissolved in physiological saline to a concentration of 4 mg / mL; metronidazole and B2 were prepared together in physiological saline to a concentration of 2 mg / mL; PD-1 antibody was diluted with PBS to a concentration of 5 mg / kg.

[0169] (f) Administration: The infusion group was given Fusobacterium nucleatum by gavage once every 3 days; PD-1 antibody was given by intraperitoneal injection at a concentration of 5 mg / kg, and the injection dose could be selected as 100 μL or 200 μL depending on the size of the mass; the drug administration group was given 500 μL of the corresponding concentration of drug by gavage daily.

[0170] (g) Record tumor volume and weight: Record mouse weight and tumor size every 2 days, calculate tumor volume, and calculate mouse weight loss and tumor inhibition rate based on mouse weight and tumor volume.

[0171] (h) Immunohistochemistry and immunofluorescence of tumor tissue

[0172] (i) Flow cytometry detection of CD8 in mouse tumor tissue + Clustering of T

[0173] (j) rt-qPCR detection of the distribution of F. nucleatum in mouse tumors and intestinal tissues

[0174] The test results are as follows Figure 1 and 2 As shown, Figure 1 This is a schematic diagram showing the changes in body weight of mice in the non-bacterial group during the drug administration period. Figure 2This is a schematic diagram showing the changes in body weight of mice in the bacterial enema group during the drug administration period. The body weight of all mice fluctuated and increased during the drug administration period, indicating that regardless of whether it was the bacterial enema group (… Figure 2 ) or no-inoculation group ( Figure 1 Compound B2 and metronidazole had relatively little effect on the physical condition of mice.

[0175] Because *Fusobacterium nucleatum* can affect the tumor's immune microenvironment and thus have a certain impact on immunotherapy for colorectal cancer, this study investigated the effect of *Fusobacterium nucleatum* on PD-1 antibody therapy for colorectal cancer. The therapeutic effects of PD-1 antibody were observed in the infusion group and the non-infusion group, with simultaneous injection of PD-1 antibody. Figure 3 As shown, Figure 3 This is a schematic diagram showing the drug resistance results of PD-1 antibody in mice after treatment with the bacteria-infused group and the non-bacterial-infused group. Tumors grew faster under the influence of *Fusobacterium nucleatum*, and the therapeutic effect of PD-1 antibody on tumors was worse than that in the control group (group 9). Without bacteria infusion, the tumor inhibition rate of PD-1 antibody was 81.14% (group 9), while after bacteria infusion, the tumor inhibition rate was 62.92% (Table 7, group 1). This indicates that *Fusobacterium nucleatum*-induced colorectal cancer affected the therapeutic effect of PD-1 antibody, and the tumor developed resistance to PD-1 antibody, weakening its efficacy.

[0176] Figure 4 This is a schematic diagram showing the changes in tumor size after administration of the drug in the non-bacterial irrigation group. Figure 5 This is a schematic diagram showing the changes in tumor size after drug administration in the bacterial irrigation group. The changes in tumor size after individual drug administration show that, in the non-bacterial irrigation group, the compounds had no significant effect on tumor growth, nor on the efficacy of the PD-1 antibody. Figure 4 In the inoculation group () Figure 5In group 3, tumor growth was slower after oral administration of compound B2 alone compared to group 4, with a tumor inhibition rate of 25.39%. The tumor inhibition rate of metronidazole alone (group 6) was 15.77%. This suggests that compound B2 may control the growth of tumor cells and the tumor as a whole by inhibiting the growth of Fusobacterium nucleatum in the intestine. The results for metronidazole were consistent with those mentioned above. However, when compound B2 was combined with metronidazole (group 8), the tumor growth rate was slower than that of compound B2 and metronidazole alone, with a tumor inhibition rate of 36.67%. This suggests that the combined use of compound B2 and metronidazole has a better tumor-suppressing effect. When compound B2 was used in combination with PD-1 antibody (group 3), the tumor inhibition rate reached 85.20% due to the inhibition of Fusobacterium nucleatum growth by compound B2, which was similar to the tumor inhibition rate of the untreated group (TGI = 80.80%). When metronidazole was used in combination with PD-1 (group 5), the tumor inhibition rate was 67.95%, which was slightly better than the efficacy of PD-1 antibody alone (TGI = 62.92%), but the efficacy was worse than that of compound B2. When compound B2, metronidazole and PD-1 antibody were used in combination (group 7), the tumor inhibition rate reached 90.89%, and the efficacy was optimal (Tables 7 and 8).

[0177] In summary, compound B2 can effectively inhibit the growth of Fusobacterium nucleatum, thereby reversing the resistance of colorectal cancer to PD-1 antibodies caused by Fusobacterium nucleatum, and its tumor-suppressing effect is better than that of metronidazole. When compound B2 and metronidazole are used in combination, the effect of reversing drug resistance is better than that of single administration.

[0178] Table 7. Tumor inhibition rate (TGI) in mice in the bacterial irrigation group

[0179] Group test substance Should PD-1 antibody be injected? TGI (%) Group 1 / yes 62.92 Group 2 / / / Group 3 B2 yes 85.20 Group 4 B2 / 25.39 Group 5 Metronidazole yes 67.95 Group 6 Metronidazole / 15.77 Group 7 B2+ Metronidazole yes 90.89 Group 8 B2+ Metronidazole / 36.67

[0180] Table 8. Tumor inhibition rate (TGI) in mice without bacterial irrigation

[0181] Group test substance Should PD-1 antibody be injected? TGI (%) Group 9 / yes Group 10 / / 81.14 Group 11 B2 yes 80.80 Group 12 B2 / / Group 13 Metronidazole yes 80.99 Group 14 Metronidazole / / Group 15 B2+ Metronidazole yes 83.42 Group 16 B2+ Metronidazole / /

[0182] To further investigate the resistance of colorectal cancer guided by *Fusobacterium nucleatum* to PD-1 antibodies and the reversal of resistance to compounds B2 and metronidazole, CD8+ in tumor tissue was analyzed. + The T antibody was subjected to immunohistochemistry and immunofluorescence experiments. For example... Figure 6 As shown, Figure 6 This is a schematic diagram of the immunohistochemical and immunofluorescence results of mouse tumor tissue. Fusobacterium nucleatum reduces CD8 levels in mouse tumor tissue. + T antibodies, thereby leading to resistance to PD-1 antibodies in mouse colorectal cancer. When metronidazole or compound B2 is administered alone, CD8... + T antibodies were slightly higher in the control group (group 1), but lower than in the un-bacterial-filled group (CD8). +The T antibody (group 9) was similar; after metronidazole and compound B2 were used in combination with PD-1 antibody, CD8... + The significant increase in T antibodies indicates that metronidazole and compound B2 can inhibit the growth of Fusobacterium nucleatum, thereby increasing CD8 levels in tumor tissue. + The number of T cells reversed the drug resistance phenomenon in colorectal cancer caused by Fusobacterium nucleatum. The combined use of compound B2, metronidazole, and PD-1 antibody (group 7) reduced CD8+ levels in tumor tissue. + When T antibody reaches its highest level, it indicates the best outcome in reversing drug resistance, which is similar to the tumor volume change phenomenon mentioned above.

[0183] CD8 immune cells in mouse tumors + The T content was tested, and the results were as follows: Figure 7 As shown, Figure 7 It is CD8 in mouse tumor tissue + Schematic diagram of T content results. After administration of PD-1 antibody, regardless of whether the group was in the infusion group or the non-infusion group, the CD8+ levels in the tumor... + T was increased, while CD8 in the inoculation group was increased. + The T content was 7.8%, significantly lower than that of the un-inoculated group in terms of CD8 content. + The content of T (22.68%), after administration of metronidazole and compound B2 alone, the intratumoral CD8 content + The T content was similar to that of the un-drenched group, while the content of CD8 was slightly increased (3.05%) after combined administration of metronidazole and compound B2 compared to the single administration. In the drenched group, the content of CD8 was increased slightly (3.05%) after combined administration of compound B2 (22.35%) and metronidazole (20.3%) with PD-1 antibody. + The content of T was similar to that of the control group (19.65%) in the untreated group, and lower than that of the PD-1 antibody-treated group in the treated group. + High T content, including CD8 from compound B2 group + The T content was slightly higher in the group with metronidazole than in the group with PD-1 antibody, indicating that compound B2 and metronidazole can effectively inhibit *Fusobacterium nucleatum* and reverse the resistance of colorectal cancer to PD-1 antibody caused by *Fusobacterium nucleatum*. The antibacterial effect of compound B2 was slightly better than that of metronidazole. When compound B2, metronidazole and PD-1 antibody were used in combination, the intratumoral CD8... + The T content reached 26.66%, indicating that the combination of the three drugs had the best effect in reversing drug resistance, which corresponds to the results of immunohistochemistry and immunofluorescence.

[0184] The distribution of *Fusobacterium nucleatum* in the mouse intestine and tumor tissue was investigated, as well as the inhibitory effects of compound B2 and metronidazole on *Fusobacterium nucleatum* in vivo after drug administration. rt-qPCR experiments were performed for this purpose, and the results are as follows: Figure 8 As shown, Figure 8This is a schematic diagram showing the distribution of Fusobacterium nucleatum in mouse tumors and intestinal tissue. From... Figure 8 As can be seen, in the inoculation group, compound B2 was more effective than metronidazole in inhibiting the growth of *Fusobacterium nucleatum* in the intestine. Its effect on inhibiting *Fusobacterium nucleatum* in tumor tissue was comparable to metronidazole, but it failed to completely inhibit the growth of *Fusobacterium nucleatum* in both the tumor and the intestine. The optimal antibacterial effect was achieved when metronidazole was used in combination with compound B2 in both the intestine and the tumor. This indicates that compound B2 can effectively inhibit the growth of *Fusobacterium nucleatum* in vivo.

[0185] The above experimental results indicate that compound B2 can exert an effective antibacterial effect in vivo, inhibiting the growth of *Fusobacterium nucleatum* in the mouse intestine and tumor tissue. Immunohistochemical and immunofluorescence results show that compound B2 inhibits the growth of *Fusobacterium nucleatum*, thereby reducing the CD8+ oxidase activity in tumors. + The increase in T antibodies reversed the resistance to PD-1 antibodies in colorectal cancer induced by *Fusobacterium nucleatum*. Furthermore, the combination with metronidazole, leveraging metronidazole's ability to enter the tumor via the bloodstream and exert its bactericidal effect, along with the intestinal antibacterial properties of compound B2, effectively inhibited the growth of *Fusobacterium nucleatum*, reducing the adverse therapeutic effects of *Fusobacterium nucleatum* on colorectal cancer. This result also provides a new treatment option for the clinical treatment of colorectal cancer caused by *Fusobacterium nucleatum*.

[0186] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A dimetronidazole derivative or its pharmaceutical salt, characterized in that, The structure of the dimetronidazole derivative is selected from one of the following structures:

2. The use of a dimethidazole derivative or its pharmaceutical salt as described in claim 1 in the preparation of a drug for treating Fusobacterium nucleatum.

3. The use of a dimethidazole derivative of claim 1 or a pharmaceutical salt thereof in the preparation of a medicament for treating diseases caused by Fusobacterium nucleatum.

4. The use of a dimethidazole derivative or a pharmaceutical salt thereof as described in claim 1 in the preparation of a medicament for treating colon cancer.

5. The use of a dimetronidazole derivative or its pharmaceutical salt as described in claim 1 in combination with metronidazole in the preparation of a drug for treating Clostridium nucleatum.

6. The use of a dimetronidazole derivative or its pharmaceutical salt as described in claim 1 in combination with metronidazole and PD-1 antibody in the preparation of a drug against Fusobacterium nucleatum.

7. The use of a dimetronidazole derivative or its pharmaceutical salt as described in claim 1 in combination with metronidazole in the preparation of a drug for treating colon cancer.

8. The use of the dimetronidazole derivative or its pharmaceutical salt as described in claim 1 in combination with metronidazole and PD-1 antibody in the preparation of a drug for treating colorectal cancer, or the use of the dimetronidazole derivative or its pharmaceutical salt as described in claim 1 in combination with metronidazole and PD-1 antibody in the preparation of a drug for reversing resistance to PD-1 antibody in colorectal cancer caused by Fusobacterium nucleatum infection.

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