Application of fluorinated quinoline derivative in inhibition of colon cancer pathogenic bacteria

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a fluorinated quinoline derivative in inhibition of colon cancer pathogenic bacteria. The invention firstly provides a fluorinated quinoline derivative or a pharmaceutical salt thereof; the fluorinated quinoline derivative can inhibit fusobacterium nucleatum, has no inhibition effect on common intestinal bacteria such as bifidobacterium animalis subsp. Lactis, mucophilic protein-ackermania, bacteroides multiforme, clostridium sporogenes, lactobacillus reuteri, parabacteroides dielsii and the like, and has the characteristics of good fusobacterium nucleatum resistance activity and specificity; the fusobacterium nucleatum can be used for treating diseases caused by fusobacterium nucleatum infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a fluorinated quinoline derivative in inhibiting pathogenic bacteria of colon cancer. Background Art

[0002] Colorectal cancer (CRC) is the third most common malignancy globally and the second leading cause of cancer-related deaths. According to the World Health Organization (WHO) data, in 2020, there were approximately 1.93 million newly diagnosed cases of colorectal cancer globally, accounting for 9.7% of all newly diagnosed cancer cases, resulting in approximately 930,000 deaths, accounting for 9.4% of cancer-related deaths. In China, the incidence and mortality of colorectal cancer have been increasing year by year. In 2020, the number of newly diagnosed cases exceeded 550,000, and the number of deaths was approximately 280,000. Despite the continuous progress of treatment methods, approximately 86% of end-stage patients die due to disease progression within 5 years after initial diagnosis, indicating that the disease still has a high fatality rate in the advanced stage.

[0003] Fusobacterium nucleatum (Fn) is a Gram-negative anaerobic bacterium commonly found in the oral cavity and intestine. As an opportunistic pathogen, it can cause various infections. Studies have found that Fn is closely related to the occurrence and progression of colorectal cancer (CRC), especially highly expressed in colorectal cancer tissues. Fn enters the gastrointestinal tract through the oral cavity and colonizes in the colorectum, promoting cancer metastasis, recurrence, and drug resistance. In a mouse model, Fn induces colitis and accelerates the formation of intestinal tumors. Fn binds to E-cadherin on the surface of colorectal cancer cells, activates the inflammatory response, and promotes tumor development. Therefore, the important role of Fn in CRC makes it a potential diagnostic and therapeutic target.

[0004] Currently, there is no specific drug for Fusobacterium nucleatum. Summary of the Invention

[0005] In view of the above technical problems, the object of the present invention is to provide the application of a fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Fusobacterium nucleatum. Specifically, it includes the following content:

[0006] In a first aspect, the present invention provides the application of a fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting pathogenic bacteria of colon cancer; the structural formula of the fluorinated quinoline derivative is shown as the following formula (Ⅰ):

[0007]

[0008] Wherein, R1 is selected from:

[0009] Preferably, the structural formula of the fluorinated quinoline derivative is shown as the following formula (Ⅱ) or (Ⅲ):

[0010]

[0011] Preferably, the colon cancer pathogen is Fusobacterium nucleatum.

[0012] Preferably, the fluoroquinoline derivative or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or excipient to form any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions.

[0014] In a second aspect, the present invention provides an application of a fluoroquinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease caused by Fusobacterium nucleatum infection; the structural formula of the fluoroquinoline derivative is shown as the following formula (I):

[0015]

[0016] Wherein, R1 is selected from:

[0017] Preferably, the structural formula of the fluoroquinoline derivative is shown as the following formula (II) or (III):

[0018]

[0019] Preferably, the diseases caused by Fusobacterium nucleatum infection include colon cancer, colon cancer drug resistance caused by Fusobacterium nucleatum, and colon cancer metastasis caused by Fusobacterium nucleatum.

[0020] Preferably, the fluoroquinoline derivative or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or excipient to form any pharmaceutically acceptable dosage form.

[0021] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions.

[0022] In a third aspect, the present invention provides an application of a fluoroquinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colon cancer; the structural formula of the fluoroquinoline derivative is shown as the following formula (I):

[0023]

[0024] Wherein, R1 is selected from:

[0025] Preferably, the structural formula of the fluoroquinoline derivative is shown as the following formula (II) or (III):

[0026]

[0027] Preferably, the fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or excipient to form any pharmaceutically acceptable dosage form.

[0028] Preferably, the dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, suspensions.

[0029] The beneficial effects of the present invention are as follows: The present invention provides a fluorinated quinoline derivative for inhibiting colon cancer pathogenic bacteria; the fluorinated quinoline derivative exhibits good antibacterial activity against Fusobacterium nucleatum, and has low toxicity to common intestinal bacteria such as Bifidobacterium animalis subsp. lactis, Akkermansia muciniphila, Bacteroides thetaiotaomicron, Clostridium sporogenes, Lactobacillus reuteri, and Parabacteroides distasonis, etc., and has a specific inhibitory effect on Fusobacterium nucleatum; the fluorinated quinoline derivative of the present invention can be used to treat diseases caused by Fusobacterium nucleatum (such as colon cancer, colon cancer drug resistance caused by Fusobacterium nucleatum, and colon cancer metastasis caused by Fusobacterium nucleatum). Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0031] Figure 1 Results graph of the inhibition rate of the fluorinated quinoline derivative against Fusobacterium nucleatum detected by the CCK-8 method;

[0032] Figure 2 Results graph of the effect of the fluorinated quinoline derivative on the membrane permeability of Fusobacterium nucleatum; in the figure, A, D, E, G are 20× fields of view after fixation with paraformaldehyde and staining after treatment with EG medium, compound 166, compound 175, and penicillin-streptomycin double antibody, respectively; B, E, H, K are 20× fields of view of direct staining; C, F, I, L are 40× fields of view of direct staining;

[0033] Figure 3 Results graph of the fluorinated quinoline derivative destroying the cell wall structure of Fusobacterium nucleatum. Detailed Embodiments

[0034] In order to more clearly illustrate the present invention, the following further describes the present invention in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific descriptions are illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0035] In the following examples, the experimental methods without specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Reagents or instruments without indicating the manufacturer are all conventional products that can be obtained by commercial purchase.

[0036] The Fusobacterium nucleatum used in the following examples was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the number 1.2526. Its original strain was from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 and was preserved in this laboratory.

[0037] Bifidobacterium animalis subsp. Lactis, China General Microbiological Culture Collection Center, CGMCC 1.15623; Akkermansia muciniphila (A. muciniphila), China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1346; Bacteroides thetaiotaomicron, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1104; Clostridium sporogenes, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1481; Limosilactobacillus reuteri, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.614; Parabacteroides distasonis, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1564).

[0038] MTT thiazolyl blue (Beijing Solarbio Science & Technology Co., Ltd.); Propidium Iodide PI (Beijing Solarbio Science & Technology Co., Ltd.); Penicillin-Streptomycin Solution (100×) (Beijing Solarbio Science & Technology Co., Ltd.); Gluta Fixative (for electron microscopy, 2.5%) (Beijing Solarbio Science & Technology Co., Ltd.); 4% Tissue Cell Fixative (Beijing Solarbio Science & Technology Co., Ltd.); Soluble Starch (Beijing Solarbio Science & Technology Co., Ltd.); Yeast Extract (Beijing Solarbio Science & Technology Co., Ltd.); Agarose (Beijing Solarbio Science & Technology Co., Ltd.); Defibrinated Horse Blood (sterile) (Beijing Solarbio Science & Technology Co., Ltd.); L-Cysteine Hydrochloride (Beijing Solarbio Science & Technology Co., Ltd.); Dimethyl Sulfoxide (Beijing Solarbio Science & Technology Co., Ltd.); Beef Extract Powder (Beijing Aoboxing Biotechnology Co., Ltd.); Peptone (Beijing Aoboxing Biotechnology Co., Ltd.); Anhydrous Glucose (Sinopharm Chemical Reagent Co., Ltd.); Sodium Sulfate Dodecahydrate (Sinopharm Chemical Reagent Co., Ltd.); Glycerol (Sinopharm Chemical Reagent Co., Ltd.); 2.5L Anaerobic Gas Generator Pouch (Mitsubishi Gas Chemical Company, Inc., Japan); Gram Staining Kit (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.); Other reagents are commercially available.

[0039] Model 371 CO₂ Incubator (ThermoFisher Scientific, USA); Low-Speed Refrigerated Centrifuge L530R (Xiangyi Centrifuge Instrument Co., Ltd.); Desktop High-Speed Refrigerated Centrifuge H1850R (Xiangyi Centrifuge Instrument Co., Ltd.); Model 680 Microplate Reader (Bio-Rad, USA); UV-Visible Spectrophotometer (Shanghai Jinghe Analytical Instrument Co., Ltd.); CKX41 Inverted Microscope (Olympus, USA); 2.5L Sealed Anaerobic Culture Jar (Mitsubishi Gas Chemical Company, Inc., Japan).

[0040] References for the synthesis methods of compounds 126, 128, 131, 134, 145, 147, 151, 154, 157, 166, 171, 173, 175 described in the following examples: Chen Tang. Design, Synthesis and Bioactivity Evaluation of New Chemical Entities Based on Fluoroquinoline Directed [D]. Lanzhou University, 2022. DOI: 10.27204 / d.cnki.glzhu.2022.003156.

[0041] References for the synthesis methods of Compounds 31, 32, and 33: Yang, Y.D., He, Y.H., Ma, K.Y., Li, H., Zhang, Z.J., Sun, Y.,... Liu, Y.Q. (2021). Design and Discovery of Novel Antifungal Quinoline Derivatives with Acylhydrazide as a Promising Pharmacophore. J Agric Food Chem, 69(30), 8347-8357.

[0042] References for the synthesis methods of Compounds 346, 349, 362, 363, 364, and 371: Chen, Y.J., Ma, K.Y., Du, S.S., Zhang, Z.J., Wu, T.L., Sun, Y.,... Tang, C. (2021). Antifungal Exploration of Quinoline Derivatives against Phytopathogenic Fungi Inspired by Quinine Alkaloids. J Agric Food Chem, 69(41), 12156-12170.

[0043] The structural formulas of the compounds described in the following examples are as follows:

[0044]

[0045]

[0046] Example 1 Determination of the Activity of Fluorinated Quinoline Derivatives against Fusobacterium nucleatum

[0047] 1. Determination of Inhibition Rate

[0048] Counting by ultraviolet spectrophotometer method: Take Fusobacterium nucleatum in the logarithmic growth phase, centrifuge at 4000 rpm for 5 min, resuspend with the corresponding culture medium, measure the absorbance at a wavelength of 600 nm, and adjust the OD 600nm value to about 1.0 for standby. Dilute it in gradients with the culture medium.

[0049] Prepare a 50 μM solution of the drug with the liquid culture medium. Take a 96-well plate, add the bacterial solution into the wells, with 3 replicates, 50 μL per well. Then add 50 μL of the drug solution to each well. The liquid culture medium plus the bacterial solution is used as the negative control, the liquid culture medium is used as the blank control, and 0.2% DMSO is used as the solvent control. Place it in an anaerobic box (anaerobic bacteria) and culture it in an incubator at 37 °C for 24 h.

[0050] After 24 h of incubation, the absorbance was measured at a wavelength of 600 nm using a microplate reader. The results were expressed as the mean ± standard deviation.

[0051] The inhibition rate was calculated according to the following formula:

[0052] Inhibition rate (%) = (OD 阴性对照 - OD 实验组 ) / (OD 阴性对照 - OD 空白对照 ) × 100%.

[0053] Through screening, the inhibition rates of the above-mentioned fluorinated quinoline derivatives against *Fusobacterium nucleatum* are shown in Table 1. Among them, compounds 134, 166, 171, 173, and 175 had good inhibition rates against *Fusobacterium nucleatum* at 50 μM.

[0054] Table 1 Inhibition rates of fluorinated quinoline derivatives against *Fusobacterium nucleatum*

[0055] Compound Number Inhibition Rate (%) Compound Number Inhibition Rate (%) 126 72.69±14.81 173 94.96±9.78 128 7.12±1.86 175 99.94±1.97 131 -10.15±6.2 31 -1.43±5.61 134 99.85±1.06 32 29.12±12.05 145 -28.56±6.39 33 14.71±4.4 147 -26.82±2.19 346 20.07±3.58 151 0.39±2.6 349 -12.66±4.4 154 13.15±1.7 362 -5.02±7.11 157 30.08±4.88 363 -40.7±2.34 166 95.15±1.77 364 -11.84±4.96 171 99.61±2.99 371 -5.78±2.66

[0056] 2. MIC determination

[0057] Compounds 134, 166, 171, 173, and 175 were selected to detect the MIC against *Fusobacterium nucleatum*. The specific method was as follows:

[0058] (1) Take the *Fusobacterium nucleatum* bacterial solution in the logarithmic growth phase, centrifuge it at 8000 r / min for 5 minutes, discard the supernatant, and adjust the OD 600 value to about 1.0. Then dilute it with EG liquid medium to 2 × 10 6 CFU / mL for standby.

[0059] (2) Take the stock solutions of compounds 134, 166, 171, 173, and 175, and prepare solutions of 40 μM, 20 μM, 10 μM, and 5 μM with EG liquid medium (without horse blood) respectively.

[0060] (3) Take a 96-well plate, add 50 μL of *Fusobacterium nucleatum* bacterial solution to each well, and then add 50 μL of solutions of 134, 166, 171, 173, and 175 at different concentrations respectively. Set the following control groups: EG liquid medium plus *Fusobacterium nucleatum* bacterial solution as the negative control, EG liquid medium as the blank control, EG liquid medium plus 0.4% DMSO as the solvent control, and EG liquid medium plus antibiotic solution as the positive control. Place the 96-well plate in an anaerobic box and incubate it in a 37 °C incubator for 48 hours.

[0061] (4) After 48 hours of incubation, take out the 96-well plate and observe. The lowest drug concentration in the wells with clear solutions was taken as the minimum inhibitory concentration (MIC).

[0062] The results showed that the minimum inhibitory concentrations of compounds 134, 171 and 173 against Fusobacterium nucleatum were greater than 40 μM; while the minimum inhibitory concentrations of compounds 166 and 175 described in this application against Fusobacterium nucleatum were 10 μM and 20 μM, respectively. It indicated that compounds 166 and 175 described in this application had significant inhibitory effects on Fusobacterium nucleatum.

[0063] 3. MBC determination

[0064] Compounds 166 and 175 were selected to detect the MBC against Fusobacterium nucleatum. The specific method was as follows:

[0065] (1) Take the Fusobacterium nucleatum bacterial solution in the logarithmic growth phase, centrifuge at 8000 r / min for 5 minutes, discard the supernatant, and adjust the OD 600nm value to about 1.0. Then dilute it with EG liquid medium to 2×10 6 CFU / mL for standby.

[0066] (2) Take the stock solutions of compounds 166 and 175, and prepare solutions of 166 at 40 μM, 20 μM, 10 μM, and 5 μM, and 175 at 50 μM, 40 μM, 20 μM, and 10 μM with EG liquid medium (without horse blood). Take the 100× antibiotic solution (10,000 U, penicillin-streptomycin double antibody), and prepare solutions at 40 U, 10 U, 5 U, 2.5 U, 1.25 U, 0.6 U, and 0.3 U with EG liquid medium.

[0067] (3) Take a 96-well plate, add 50 μL of Fusobacterium nucleatum bacterial solution to each well, and then add 50 μL of solutions of different concentrations of 166 and 175 respectively. Set the following control groups: EG liquid medium plus Fusobacterium nucleatum bacterial solution as the negative control, EG liquid medium as the blank control, EG liquid medium plus 0.4% DMSO as the solvent control, and EG liquid medium plus antibiotic solution as the positive control. Place the 96-well plate in an anaerobic box and culture it in a 37 °C incubator for 48 hours.

[0068] (4) After culturing for 48 hours, take 50 μL of solution from the clear wells of different drug concentrations, add it to the EG medium plate, and spread it evenly on three plates corresponding to three replicates. The blank medium plate is used as the negative control, and the plate coated with Fusobacterium nucleatum bacterial solution is used as the positive control. Place the plate in an anaerobic box and culture it in a 37 °C incubator for 48 hours.

[0069] (5) Take out the medium plate and observe the colony growth. There is no colony growth on the negative control plate, and there is colony growth on the positive plate. The plate with the lowest drug concentration without colony growth is used as the minimum bactericidal concentration (MBC). The experiment was repeated three times.

[0070] The results are shown in Table 2. Fluoroquinoline derivative 166 could inhibit the growth of Fusobacterium nucleatum at 10 μM, with its MIC value being 10 μM and MBC value being 10 μM; fluoroquinoline derivative 175 could inhibit the growth of Fusobacterium nucleatum at 20 μM, with its MIC value being 20 μM and MBC value being 20 μM; the MIC value of the positive control antibiotic was 0.6 U and the MBC value was 1.2 U; the results of the solvent control group showed that 0.4% DMSO had no inhibitory effect on Fusobacterium nucleatum. In summary, the above results indicate that the fluoroquinoline derivatives 166 and 175 described in this application have significant effects against Fusobacterium nucleatum.

[0071] Table 2 MIC and MBC of Compounds 166 and 175 against Fusobacterium nucleatum

[0072] Name Compound 166 Compound 175 Bispecific Antibody MIC 10 μM 20 μM 0.6U MBC 20 μM 20 μM 1.2U

[0073] Example 2 Detection of Inhibition Rate by CCK-8 Method

[0074] The CCK-8 method uses the WST-8 compound, which is reduced by dehydrogenases in the mitochondria of living cells under the action of an electron coupling reagent to generate an orange-yellow water-soluble Formazan product. The more cell proliferation, the more Formazan product is generated and the deeper the color; the greater the cytotoxicity, the less Formazan product is generated and the lighter the color. By measuring the absorbance at a wavelength of 450 - 490 nm, the number of living cells and cell viability can be indirectly reflected. Fusobacterium nucleatum is a prokaryote without mitochondria, but there is still succinate dehydrogenase in its cells.

[0075] 1) Prepare solutions of the target compounds (166, 175, penicillin-streptomycin double antibody) at corresponding concentrations (the concentrations of 166 are 0.5 μM, 2.0 μM, 4.0 μM, 8.0 μM, 10.0 μM, 15.0 μM, 20.0 μM respectively, and the concentrations of 175 are 1.0 μM, 2.5 μM, 5.0 μM, 10.0 μM, 20.0 μM, 30.0 μM, 40.0 μM respectively) with EG liquid medium. Take the 100× penicillin-streptomycin solution (10,000 U) and prepare solutions of 20.0, 10.0, 5.0, 2.5, 1.2, 0.6, 0.3 U with EG liquid medium;

[0076] 2) Take a 96-well plate, add 50 μL of the bacterial solution into the wells, with 3 replicates, and then add 50 μL of the drug solution to each well. EG liquid medium is used as the blank control, EG liquid medium plus the bacterial solution is used as the negative control, and 0.25% DMSO is used as the solvent control. Place it in an anaerobic box and incubate at 37 °C for 24 h;

[0077] 3) After the incubation, add 10 μL of CCK-8 to each well and detect the OD after 2 h in the dark.600 Absorbance

[0078] 4) According to the following formula:

[0079] Inhibition rate (%) = (OD 阴性对照 - OD 实验组 ) / (OD 阴性对照 - OD 空白对照 ) × 100%.

[0080] The experiment was independently repeated three times.

[0081] The results are as Figure 1 shown. The inhibition rate of compound 166 was 78% at 10 μM, the inhibition rate of compound 175 was 82% at 20 μM, and the inhibition rate of penicillin - streptomycin double - antibody was 78% at 5.0 U. The above results indicate that the fluorinated quinoline derivatives 166 and 175 described in this application have good antibacterial activity against Fusobacterium nucleatum and show concentration - dependence.

[0082] Example 3 Effect of fluorinated quinoline derivatives on bacterial membrane permeability

[0083] Propidium iodide (PI) is a nuclear staining reagent for DNA staining, which can embed into double - stranded DNA and release red fluorescence. Although PI cannot penetrate intact cell membranes, it can penetrate the damaged cell membranes of late - apoptotic cells and dead cells. Therefore, in this example, the PI staining method was used to investigate the effect of compounds 166 and 175 on the membrane permeability of Fusobacterium nucleatum.

[0084] (1) Take the Fusobacterium nucleatum bacterial solution in the logarithmic growth phase, adjust its OD value to about 0.4 for standby. Take the stock solutions of compounds 166 and 175 respectively, and prepare solutions with MIC concentrations using EG liquid medium (without horse blood). Take the 100× concentration antibiotic solution (10,000 U), and also prepare a solution with MIC concentration using EG liquid medium (without horse blood). Take the PI solution, and prepare a 20 μg / mL solution with distilled water, wrap it with tin foil to avoid light, and use it immediately after preparation.

[0085] (2) Take 2 mL centrifuge tubes, add 0.5 mL of the bacterial solution and 0.5 mL of the drug solution. EG liquid medium (without horse blood) is used as the negative control, and the antibiotic solution is used as the positive control. Place the centrifuge tubes in an anaerobic box and culture them in a 37 °C incubator for 24 hours.

[0086] (3) After 24 hours of incubation, take a set of samples, centrifuge at 10,000 r / min for 5 minutes, discard the supernatant, and resuspend with 0.5 mL of 1×PBS. Subsequently, add 50 μL of PI solution and stain for 15 minutes; centrifuge again at 10,000 r / min for 5 minutes, discard the supernatant, wash twice with 1×PBS, and finally resuspend with 1 mL of 1×PBS. For another set of samples, first fix with 4% paraformaldehyde for 20 minutes, then centrifuge at 10,000 r / min for 5 minutes, discard the supernatant, resuspend with 0.5 mL of 1×PBS, and then add 50 μL of PI solution to stain for 15 minutes; repeat centrifugation and washing twice, and finally resuspend with 1 mL of 1×PBS.

[0087] (4) Take 10 μL of the bacterial solution and drop it onto a glass slide, cover it with a coverslip, and observe the staining situation under a fluorescence microscope.

[0088] The experimental results are as Figure 2 shown. After fixation with paraformaldehyde, the total number of bacteria in the solution can be determined. Compared with the group without drug treatment, the red staining under the fluorescence microscope increases after drug treatment, indicating that the compounds 166 and 175 described in this application can significantly reduce Fusobacterium nucleatum at the MIC concentration, increase the cell membrane permeability of Fusobacterium nucleatum, make the bacterial DNA stained by PI, increase the cell membrane permeability of bacteria, resulting in the outflow of contents, and promote bacterial death.

[0089] Example 4 Effect of Fluorinated Quinoline Derivatives on Bacterial Wall

[0090] In this example, a scanning electron microscope was used for intuitive observation:

[0091] (1) Take the Fusobacterium nucleatum bacterial solution in the logarithmic growth phase, centrifuge at 4000 rpm / min for 5 min, and adjust the OD 600 value to about 0.4 with EG medium (without horse blood) for standby.

[0092] (2) Prepare 20 μM of the 166 stock solution and 20 μM of the 175 stock solution;

[0093] (3) Take 2 mL centrifuge tubes, add 0.5 mL of bacterial solution and 0.5 mL of drug solution to each centrifuge tube, mix well, and incubate at 37 °C for 3 h.

[0094] (4) After incubation, centrifuge at 8000 rpm / min for 8 minutes, add 1 mL of Gluta fixative (for electron microscopy, 2.5%) solution to the bacterial pellet, mix well, and fix at 4 °C for 4 h.

[0095] (5) After fixation, centrifuge at 5000 rpm / min for 5 minutes and wash twice with PBS.

[0096] (6) Dehydrate with 20%, 50%, 80%, 100%, 100%, and 100% ethanol for 15 min in sequence. Finally, resuspend with absolute ethanol to an appropriate concentration.

[0097] (7) Air-dry, sputter with gold, and observe under a scanning electron microscope.

[0098] The experimental results are as Figure 3 shown. The surface of Fusobacterium nucleatum in the negative control group is smooth and the structure is complete. After compounds 166 and 175 act on Fusobacterium nucleatum respectively, the surface of the bacteria is no longer smooth (drug particles are adsorbed on the outer surface of the bacteria). At the same time, the bacterial structure shows swelling, breakage, elongation, shrinkage, and leakage of contents, etc. It shows that after the drug acts on Fusobacterium nucleatum, the integrity of the cell wall / cell membrane of Fusobacterium nucleatum is damaged, resulting in the death of the bacteria.

[0099] Example 5 Toxicity Test of Fluorinated Quinoline Derivatives on Common Intestinal Bacteria

[0100] The common intestinal pathogenic bacteria used in this example are selected from: Bifidobacterium animalis subsp. Lactis, China General Microbiological Culture Collection Center, CGMCC 1.15623; Akkermansia muciniphila (A. muciniphila), China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1346; Bacteroides thetaiotaomicron, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1104; Clostridium sporogenes, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1481; Limosilactobacillus reuteri, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.614; Parabacteroides distasonis, China Center for Industrial Culture Collection of Microorganisms, GDMCC 1.1564).

[0101] (1) Count by ultraviolet spectrophotometry: Take bacteria in the logarithmic growth phase, centrifuge at 4000 rpm for 8 minutes, and resuspend with GAM medium.

[0102] (2) Measure the absorbance at OD 600 and adjust the OD value to about 1.0 for standby, and dilute it stepwise with GAM medium.

[0103] (3) Prepare a solution of the target compound with a corresponding concentration using GAM liquid medium.

[0104] (4) Take a 96-well plate, add 50 μL of the bacterial solution into the wells, with 3 replicates, and then add 50 μL of the medicinal solution to each well. GAM liquid medium is used as the blank control, GAM liquid medium with the bacterial solution is used as the negative control, and 0.25% DMSO is used as the solvent control. Place it in an anaerobic box and incubate at 37 °C for 48 h;

[0105] (5) After culturing for 48 h, take out the 96-well plate and observe. The lowest drug concentration in the wells with clear solution is used as the minimum inhibitory concentration (MIC).

[0106] The experimental results are shown in Table 3 below. The experimental results indicate that Compounds 166 and 175 have relatively low toxicity to other normal intestinal flora.

[0107] Table 3 Toxicity of Compounds to Common Intestinal Bacteria (MIC)

[0108] Intestinal Bacteria Compound 166 Compound 175 Akkermansia muciniphila > 100 μM > 20 μM Bifidobacterium animalis subsp. lactis > 100 μM > 20 μM Bacteroides thetaiotaomicron > 100 μM > 50 μM Clostridium sporogenes > 100 μM > 100 μM Lactobacillus reuteri > 100 μM > 20 μM Parabacteroides distasonis > 100 μM > 20 μM

[0109] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. Use of a fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting colon cancer pathogenic bacteria; the structural formula of the fluorinated quinoline derivative is shown in the following formula (I): in, R1 is selected from:

2. The use according to claim 1, characterized in that The structural formula of the fluorinated quinoline derivative is shown in the following formula (II) or (III):

3. The use according to claim 1 or 2, characterized in that: The colon cancer pathogenic bacteria is Fusobacterium nucleatum.

4. Use of a fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease caused by Fusobacterium nucleatum infection; the structural formula of the fluorinated quinoline derivative is shown in the following formula (I): in, R1 is selected from:

5. The use according to claim 4, characterized in that The structural formula of the fluorinated quinoline derivative is shown in the following formula (II) or (III):

6. The use according to claim 4 or 5, characterized in that The diseases caused by Fusobacterium nucleatum infection include colon cancer, drug resistance of colon cancer caused by Fusobacterium nucleatum, and metastasis of colon cancer caused by Fusobacterium nucleatum.

7. Use of a fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating colon cancer; the structural formula of the fluorinated quinoline derivative is shown in the following formula (I): in, R1 is selected from:

8. The use according to claim 7, characterized in that The structural formula of the fluorinated quinoline derivative is shown in the following formula (II) or (III):

9. The use according to any one of claims 1 to 8, characterized in that: The fluorinated quinoline derivative or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or auxiliary material to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

Images