A class of compounds with anti-Fusobacterium nucleatum properties and their application

Abstract

The present invention discloses a class of compounds or pharmaceutically acceptable salts thereof having anti-Fusobacterium nucleatum activity, wherein the general structural formula is selected from one of the following structures: The compounds of the present invention were tested for anti-Fusobacterium nucleatum activity in vitro and found that the compounds of the present invention had good inhibitory activity against ATCC23726 strain, and compounds 19h and 19p showed better inhibitory activity against Fusobacterium nucleatum than nitisinone.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a class of compounds having anti-Fusobacterium nucleatum activity and applications thereof. Background Art

[0002] In the past few decades, colorectal cancer (CRC) has become the third most common malignancy and the fourth leading cause of cancer-related death worldwide. CRC is generally considered to be a multifactorial disease caused by multiple factors, including high-fat diet, obesity, smoking, alcohol consumption, inflammatory processes, environmental factors, and genetic changes. Recently, many studies have confirmed that CRC is significantly associated with gut microbiota (GMs). More than one thousand microorganisms inhabit the human gastrointestinal tract, with high heterogeneity, and the gut flora is causally related to the occurrence of colorectal cancer. Among them, Fusobacterium nucleatum (F. nucleatum) is an opportunistic commensal Gram-negative anaerobic bacterium that is ubiquitous in the human oral cavity and is involved in the occurrence of various periodontitis. Increasing evidence shows that F. nucleatum is specifically enriched in CRC compared with normal colon tissue and is associated with poor patient prognosis and CRC recurrence after treatment. In addition, F. nucleatum can promote the proliferation of intestinal tumor cells, inhibit immune cytotoxicity by interacting with T cells and NK cells, and support the immune escape of cancer cells. In summary, these studies confirm that Fusobacterium nucleatum is one of the pathogenic factors of CRC and may be a potential target for the prevention and treatment of CRC.

[0003] A previous preclinical study showed that antibiotic treatment effectively inhibited tumor growth in mice implanted with patient-derived Fusobacterium nucleatum xenografts, suggesting that inhibition of Fusobacterium nucleatum may be a promising strategy to combat CRC. Most antibiotics act primarily by inhibiting cell wall synthesis, protein synthesis, DNA synthesis, DNA-directed RNA polymerase, and essential metabolic enzymes. However, the widespread use of broad-spectrum antibiotics against Fusobacterium nucleatum and CRC poses the risk of developing severe drug resistance and disrupting the intestinal ecosystem, which is not conducive to practical application. Therefore, the identification of more potent and selective Fusobacterium nucleatum inhibitors is highly necessary.

[0004] The discovery of drug lead compounds is an important part of new drug research and development. Drug repurposing has begun to become an effective strategy for the discovery of lead compounds and an effective method for identifying new indications for existing drugs. Because of its complete toxicology, pharmacokinetics and pharmacodynamics profile, it is considered an effective and economical method in drug development. Therefore, a method for testing the minimum inhibitory concentration of compounds to inhibit Fusobacterium nucleatum was established, and compounds with inhibitory activity against Fusobacterium nucleatum were searched by screening the FDA-approved drug library. Among them, nitisinone was found to be a good lead compound, and the MIC 50The value was 8 μg / mL. Therefore, the structure of nitisinone was further optimized in order to find more active derivatives. Summary of the invention

[0005] The first object of the present invention is to provide a class of compounds having anti-Fusobacterium nucleatum activity.

[0006] The second object of the present invention is to provide a use of the compound having anti-Fusobacterium nucleatum activity in the preparation of an anti-Fusobacterium nucleatum drug or an anti-colon cancer drug.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a class of compounds or pharmaceutically acceptable salts thereof having anti-Fusobacterium nucleatum activity, wherein the general structural formula is selected from one of the following structures:

[0009]

[0010] in,

[0011] R1 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0012] R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0013] R3 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0014] R4 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0015] R5 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0016] R6 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0017] R7 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2, CF3;

[0018] R8 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2,

[0019] R9 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2,

[0020] R 10 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2,

[0021] R 11 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2,

[0022] R 12 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2,

[0023] R 13 Selected from hydrogen, NO2, CF3;

[0024] R 14 Selected from hydrogen, NO2, CF3;

[0025] R 15 Selected from hydrogen, NO2, CF3;

[0026] X is selected from C and N;

[0027] R 16 Selected from hydrogen, NO2, CF3;

[0028] R 17 Selected from hydrogen, NO2, CF3;

[0029] R 18 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl),

[0030] R 19 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl),

[0031] R 20 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl),

[0032] R 21 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl),

[0033] R 22 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl),

[0034] R 23 Selected from NO2, CF3;

[0035] R 24 Selected from NO2, CF3;

[0036] R 25 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl);

[0037] R 26 Selected from hydrogen, C1-C10 alkyl (such as methyl, ethyl, tert-butyl);

[0038] R 27 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0039] R 28 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0040] R 29 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0041] R 30 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0042] R 31 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0043] R 32 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0044] R 33 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0045] R 34 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0046] R 35 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0047] R 36 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0048] R 37 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0049] R 38 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0050] R 39 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0051] R 40 Selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), NO2;

[0052] R 41 Selected from hydrogen, NO2, CF3.

[0053] The pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt thereof; the inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid; the organic acid is acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, tannic acid, citric acid, trifluoroacetic acid, malic acid or benzenesulfonate.

[0054] Most preferably, the compound having anti-Fusobacterium nucleatum activity is selected from one of the following structures:

[0055]

[0056] The second aspect of the present invention provides a use of the compound having anti-Fusobacterium nucleatum activity or a pharmaceutically acceptable salt thereof in the preparation of an anti-Fusobacterium nucleatum medicament or an anti-colon cancer medicament.

[0057] The third aspect of the present invention provides a use of the compound having anti-Fusobacterium nucleatum activity or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases caused by Fusobacterium nucleatum.

[0058] The disease caused by nuclease-bearing bacteria is selected from colon cancer.

[0059] The compound with anti-Fusobacterium nucleatum activity of the present invention has excellent antibacterial activity in in vitro and in vivo antibacterial activity tests, has low cytotoxicity, and effectively inhibits the effect of Fusobacterium nucleatum on promoting tumor cell migration.

[0060] The Fusobacterium nucleatum is selected from ATCC23726 strain.

[0061] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0062] The compounds of the present invention were tested for their in vitro anti-Fusobacterium nucleatum activity and found that the compounds of the present invention had good inhibitory activity against ATCC23726 strain, and compounds 19h and 19p showed better inhibitory activity against Fusobacterium nucleatum than nitisinone (MIC 50 =4 μg / mL).

[0063] In the in vitro cytotoxicity test of the compounds of the present invention, it was found that the preferred compounds 19h and 19p had low toxicity to normal human HUCVEC cells and L02 cells, and are excellent anti-Fusobacterium nucleatum drugs.

[0064] In an in vitro cell migration experiment, it was found that the preferred compounds 19h and 19p of the present invention had a significant inhibitory effect on the effect of Fusobacterium nucleatum on promoting tumor cell migration.

[0065] The invention opens up a new way and provides a new strategy for in-depth research and development of new structural type anti-Fusobacterium nucleatum drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Schematic diagram of the growth curve of Fusobacterium avium with different concentrations over time.

[0067] Figure 2 Schematic diagram of the effects of different concentrations of compounds on tumor cell (MC-38) migration.

[0068] Figure 3 This is a schematic diagram showing the effects of different compounds on the nitroreductase gene.

[0069] Figure 4 Schematic diagram of the effects of different compounds on the tryptophanase gene. DETAILED DESCRIPTION

[0070] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0071] The reaction scheme of the compound prepared in the embodiment of the present invention is as follows:

[0072]

[0073]

[0074] Reagents and conditions: (a) SOCl2, 2h, 50℃; (b) DMF, K2CO3, 8h, 80℃

[0075] Example 1

[0076] Synthesis of 2-nitro-4-(trifluoromethyl)benzoyl chloride:

[0077] Under strict anhydrous and nitrogen protection conditions, thionyl chloride (5 mL) was added to 2-nitro-4-(trifluoromethyl)benzoic acid (16a, 200 mg, 0.85 mmol). The mixture was then stirred at 50°C for 3-4 hours. Afterwards, the remaining thionyl chloride was concentrated under reduced pressure to obtain 198 mg of the compound with a yield of 92.4%, which can be directly used in the next step as an oily liquid.

[0078] Synthesis of N-(3-chloro-5-nitrophenyl)-2-nitro-4-(trifluoromethyl)benzamide (19a):

[0079] Chloro-5-nitroaniline (18a, 100 mg, 0.58 mmoL), 2-nitro-4-(trifluoromethyl)benzoyl chloride (38.47 mg, 0.15 mmoL) and K2CO3 (61.27 mg, 0.44 mmoL) were added to anhydrous DMF (10 mL). Then, the mixture was stirred overnight at room temperature. The mixture was extracted three times with ethyl acetate (50 mL × 3), and the organic phase was collected and concentrated. The organic phase was purified by column chromatography (ethyl acetate / n-hexane = 1:6) to obtain the target compound N-(3-chloro-5-nitrophenyl)-2-nitro-4-(trifluoromethyl)benzamide as a white solid (22.1 mg) with a yield of 37.8%. 1 H NMR (600MHz, DMSO-d6) δ11.44 (s, 1H), 8.58 (d, J = 1.7Hz, 1H), 8.40 ~ 8.35 (m, 1H), 8.21 (d, J=8.9Hz, 1H), 8.13 (d, J=7.9Hz, 1H), 8.09 (d, J=2.2Hz, 1H), 7.75 (dd, J=8.9, 2.2Hz, 1H). 13 C NMR (151MHz, DMSO-d6) δ164.26, 146.95, 143.61, 142.84, 135.33, 132.06, 131.64, 131.35, 128.19, 127.32, 124.01, 122.35, 121.53, 118.94. ESI-HRMS:calcd for C 14 H7ClF3N3O5:m / z:[MH] - =387.9954; found [MH] - =387.9955.

[0080] Example 2

[0081] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by (2-(trifluoromethyl)phenyl)methanamine, and the rest was the same as in Example 1 to obtain compound 19b.

[0082] Example 3

[0083] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by pyridin-4-ylmethylamine, and the rest was the same as in Example 1 to obtain compound 19c.

[0084] Example 4

[0085] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by 4-aminobenzamide, and the rest was the same as in Example 1 to obtain compound 19d.

[0086] Example 5

[0087] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by 4-aminobenzyl methyl carbonate, and the rest was the same as in Example 1 to obtain compound 19e.

[0088] Example 6

[0089] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by 2-amino-5-methylbenzamide, and the rest was the same as in Example 1 to obtain compound 19f.

[0090] Example 7

[0091] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by 3-methylisoxazol-5-amine, and the rest was the same as in Example 1 to obtain compound 19g.

[0092] Example 8

[0093] The chloro-5-nitroaniline in step (2) of Example 1 was replaced by (5-nitrofuran-2-yl)methylamine, and the rest was the same as in Example 1 to obtain compound 19h.

[0094] Example 9

[0095] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 4-(trifluoromethyl)benzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by 4-aminobenzamide, and the rest was the same as in Example 1, to obtain compound 19i.

[0096] Example 10

[0097] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 2-fluoro-4-nitrobenzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by 3-chloro-4-nitroaniline. The other steps were the same as in Example 1 to obtain compound 19j.

[0098] Embodiment 11

[0099] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 2-(7-chloro-4-oxoquinazolin-1(4H)-yl)acetyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by 2-chloro-4-nitroaniline, and the rest was the same as in Example 1, to obtain compound 19k.

[0100] Example 12

[0101] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 3-bromo-4-nitrobenzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by isoxazol-3-amine, and the rest was the same as in Example 1, to obtain compound 191.

[0102] Embodiment 13

[0103] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 2-iodo-4-nitrobenzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by isoxazol-3-amine, and the rest was the same as in Example 1, to obtain compound 19m.

[0104] Embodiment 14

[0105] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 3-(4-nitrophenyl)propionyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by isoxazol-3-amine, and the rest was the same as in Example 1, to obtain compound 19n.

[0106] Embodiment 15

[0107] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 2-bromo-4-nitrobenzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by 4-chloro-3-nitroaniline. The other steps were the same as in Example 1 to obtain compound 19o.

[0108] Example 16

[0109] The 2-nitro-4-(trifluoromethyl)benzoyl chloride in step (1) of Example 1 was replaced by 3-bromo-4-nitrobenzoyl chloride, and the chloro-5-nitroaniline in step (2) was replaced by (5-nitrofuran-2-yl)methylamine, and the rest was the same as in Example 1, to obtain compound 19p.

[0110] The structural formula and NMR data of the compounds prepared in the examples of the present invention are shown in Table 1:

[0111] Table 1. Structural formula and NMR mass spectrometry data of the compounds of the present invention

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Embodiment 17

[0118] The target compound has inhibitory activity against Fusobacterium nucleatum

[0119] 1 Experimental materials: 15 mL centrifuge tubes, anaerobic bags, anaerobic jars, 96-well plates (Corning), cotton swabs, BHI medium (brain heart infusion broth), 50% horse blood.

[0120] 2Cell strain: Fusobacterium nucleatum (strain ATCC23726).

[0121] 3 Experimental methods

[0122] (1) Activation of the strain to be tested: Take out the strain to be tested from the -80℃ refrigerator, take out 1 mL of the original bacterial solution and add it to 8 mL of BHI medium, and culture it in an incubator at 37℃ under anaerobic conditions for 24 hours. After 24 hours, take out 1 mL of the BHI bacterial solution and add it to 8 mL of new BHI medium. When it is cultured in an incubator at 37℃ under anaerobic conditions for another 24 hours, the Fusobacterium nucleatum is in the late exponential growth stage.

[0123] (2) Preparation of bacterial solution: Centrifuge the activated strain to be tested (4000r, 10min), discard the supernatant, add 2mL of fortified Brucella broth and mix well, calibrate with standard turbidimetric tube, put the glass test tube into the turbidimeter, add 4mL of fortified Brucella broth, scrape a single colony of good growth and appropriate size with a cotton swab, and calibrate to 0.5 McFarland turbidity (1-2×10 8 CFU / mL). The prepared mixed bacterial solution was prepared into a suspension with Brucella broth: 50% horse blood: bacterial solution = 10:1.1:1.

[0124] (3) MIC determination: Add the prepared bacterial suspension to a 96-well plate (columns 1-11, rows BG), 200 μL for the first column and 100 μL for columns 2-11. Pipette 6.4 μL of the prepared drug solution into the first column and make three replicate wells to make the drug concentration 64 μg / mL. From the first column to the tenth column, dilute in half, that is, the concentration gradient is 0.125-64 μg / mL. No drug is added to the 11th column as a negative control group. Add blank broth culture solution to the outermost periphery as a blank control. Incubate in an incubator under anaerobic conditions at 37°C for 48 hours, and the lowest drug concentration that inhibits 50% bacterial growth is the minimum inhibitory concentration (MIC). 50 ) and observation was defined as disappearance of turbidity in the broth.

[0125] The experimental results are shown in Table 2. Most of the synthesized compounds had no significant antibacterial activity, but the synthesized compounds 19c and 19k showed slight antibacterial activity (MIC 50=64μg / mL), successfully found that the lead compound Nitisinone (MIC 50 =8 μg / mL) with better antibacterial activity for compounds 19h and 19p, the minimum antibacterial activity of which could reach 4 μg / mL.

[0126] Table 2. In vitro inhibitory activity of target compounds against Fusobacterium nucleatum (μg / mL)

[0127]

[0128] Embodiment 18

[0129] In vitro antitumor activity and cytotoxicity tests

[0130] 1. Sample preparation

[0131] The test compounds 19h and 19p were dissolved in DMSO (Merck) to a concentration of 10 mM.

[0132] 2 Cell lines

[0133] Mouse colon cancer cells (MC38), human colon cancer cells (HCT116), human umbilical vein epithelial cells (HUVEC), normal human hepatocytes (L02).

[0134] 3 Culture medium

[0135] DMEM medium, ECM medium.

[0136] 4 Test methods

[0137] This experiment used mouse colon cancer cells (MC-38). The cells were suspended in DMEM and seeded into 96-well transparent plates, 7000 cells / well. Incubate at 37°C and 5% CO2 for 24 hours. Then add the compound to the 96-well transparent plate to make the final drug concentration of 0.015-100μM, and make three-fold serial dilutions. After incubation at 37°C and 5% CO2 for 48h, add 10μL CCK8 to each well and incubate at 37°C for 30-50min. Use an enzyme reader to measure the optical density at 450nm (OD450). Calculate the cell growth inhibition rate of the compound at each concentration, and use GraphPad Prism nonlinear analysis to calculate the IC 50 .

[0138] In this experiment, human umbilical vein epithelial cells (HUVEC) were cultured in ECM medium with a cell concentration of 4000 cells / well. The rest of the test methods were based on the above anti-tumor activity test method.

[0139] The experimental results are shown in Table 3. Compounds 19h and 19p have higher inhibitory activity against mouse colon cancer cells and human colon cancer cells than the lead compound nitisinone. Compound 19p has lower hepatotoxicity to normal humans than nitisinone. Although compound 19h has higher cytotoxicity to normal humans than nitisinone, it is still in the low toxicity range. Therefore, in view of the low cytotoxicity of compounds 19h and 19p, compounds 19h and 19p are preferred for further biological experiments and preliminary exploration of mechanisms.

[0140] Table 3. In vitro antitumor activity and cytotoxicity (IC 50 , μM)

[0141]

[0142] Embodiment 19

[0143] Time-growth curve assay of compounds 19h and 19p

[0144] After the cells of Clostridium nucleatum ATCC 23726 have grown to the logarithmic growth phase, centrifuge at 8000 rpm for 10 min, discard the supernatant, and add fresh BHI medium. 8 CFU / mL. Then, different doses of compounds 19h and 19p, and nitisinone were added, respectively, so that the concentrations of nitisinone were 32, 64, and 128 μg / mL, and the concentrations of 19h and 19p were 8, 16, and 32 μg / mL. Then, the bacterial solution was cultured under anaerobic conditions at 37°C, and 100 μL of samples were taken at specific time points (0, 6, 12, 24, 36, and 48 h) in a 96-well plate, and OD595 Biotek Synergy H2 (Lab systems) was used for detection. All experiments were repeated at least 3 times. The effect of drugs on the growth of Fusobacterium nucleatum was investigated (the bacterial concentration is inversely proportional to the absorbance). The change in OD595 absorbance was plotted against time, as shown in Figure 1 As shown, Figure 1 Schematic diagram of the growth time curve of Fusobacterium nucleatum under the action of different concentrations of compounds. Figure 1 In the figure, A is a schematic diagram of the antibacterial growth curves of nitisinone, compound 19h, and compound 19p at the same concentration, B is a schematic diagram of the growth curves of nitisinone at different concentrations, C is a schematic diagram of the antibacterial growth curves of compound 19h at different concentrations, and D is a schematic diagram of the antibacterial growth curves of 19p at different concentrations.

[0145] The experimental results in Figure AD show that at the same drug concentration (32μg / mL), the inhibitory effects of compound 19h and compound 19p on Fusobacterium nucleatum are higher than those of nitisinone, and the changes in bacterial concentration and time of 19h and 19p at different concentrations show that the higher the drug concentration, the stronger the inhibitory effect on the bacteria. Figure 1 It can be seen that nitisinone starts to exert an antibacterial effect after incubation with Fusobacterium nucleatum for 8 hours, while the compound synthesized by the present invention exerts a strong antibacterial effect at the beginning.

[0146] Embodiment 20

[0147] Effects of Nitisinone and compounds 19h and 19p on the migration of MC-38 tumor cells

[0148] Studies have shown that the presence of Fusobacterium nucleatum promotes the migration of tumor cells and promotes the occurrence and development of tumor cells. Therefore, Fusobacterium nucleatum was co-incubated with tumor cells and compounds in vitro to observe the effect of Fusobacterium nucleatum on tumor cell migration, and at the same time, the effect of the compound on Fusobacterium nucleatum on tumor cell migration was investigated.

[0149] The cells grown to the logarithmic growth phase were suspended in DMEM medium containing 5% serum and inoculated into the upper chamber of a 24-well transwell plate (8 μm, Corning) at a density of 4×10 4 The serum-free DMEM medium in the lower chamber was prepared to 2-4×10 7 CFU / mL concentration of Fusobacterium nucleatum solution, Fusobacterium nucleatum as a chemoattractant. Fixed concentrations of different drugs were prepared in the lower chambers of different wells. After incubation for 48 hours at 37°C and 5% CO2, the upper chamber of the transwell was removed, the culture medium in the lower chamber was discarded, and the cells were rinsed with PBS. The cells were then fixed with paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 20 minutes. The non-migrated cells in the upper chamber were wiped off and placed under a fluorescence microscope for observation and counting. The results were expressed as a migration index, which was defined as: the average number of cells per well of the test compound / the average number of cells per well of the culture medium control. The experiment was repeated at least three times.

[0150] Figure 2 Schematic diagram of the effects of different concentrations of compounds on tumor cell (MC-38) migration.

[0151] Depend on Figure 2 It can be seen that the Fusobacterium nucleatum group ( Figure 2 E) Compared with the blank group cells ( Figure 2 A) The migration amount is too large, indicating that the presence of Fusobacterium nucleatum promotes the migration of tumor cells. The migration amount of cells in the drug-added group with the presence of Fusobacterium nucleatum is lower than that in the simple drug-added group (tumor cell migration amount Figure 2 Medium F< Figure 2 Middle B, Figure 2 Medium G< Figure 2 Middle C, Figure 2 Medium H< Figure 2D), indicating that the presence of the compound inhibits the migration-promoting effect of Fusobacterium nucleatum through its antibacterial effect, and the compound itself has a certain inhibitory effect on cells. The addition of the compound synthesized by the present invention has a greater effect on cell migration than Nitisinone, among which the addition of compound 19h to the bacterial solution leads to the lowest tumor cell migration (inhibition of tumor cell migration effect Figure 2 Medium F< Figure 2 Medium H< Figure 2 The above results show that both nitisinone and the compound of the present invention can inhibit the migration-promoting effect of Fusobacterium nucleatum by exerting their antibacterial effect, and the compound synthesized by the present invention is better than the lead compound nitisinone.

[0152] Embodiment 21

[0153] Regulation of nitroreductase gene and tryptophanase gene by lead compound and preferred compound 19h, 19p

[0154] Tryptophan has been shown to play a core role in physiology and physiological pathology, and nitroreductase is an important enzyme for the interaction between nitroaromatic compounds and bacteria. Tryptophanase genes and nitroreductase genes have been reported in a variety of microorganisms, including Fusobacterium nucleatum. Therefore, in order to preliminarily explore the mechanism of the antibacterial effect of the compound, the present invention uses RT-qPCR to quantitatively analyze the effect of the compound on the expression of the nitroreductase gene and tryptophanase gene of Fusobacterium nucleatum. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Fusobacterium nucleatum was resuspended in BHI medium (8 mL) to a concentration of 1 to 2 × 10 8 CFU / mL. The Fusobacterium nucleatum cells were treated with compounds nitisinone (32 μg / mL), 19p (8 μg / mL) and 19h (8 μg / mL), and the samples were incubated at 37°C for 48 h under anaerobic conditions. The Fusobacterium nucleatum cells were collected by centrifugation and washed with PBS (3×5 mL). The Fusobacterium nucleatum cells were collected and total RNA was extracted using the bacterial RNAout kit (Jianshi Biotechnology) according to the operating instructions. Reverse transcription was performed according to the instructions of the kit (PrimeScript TM RT Master Mix kit, TaKaRa), at 37°C for 15 min, 85°C for 5 s, and then cooled to 4°C. cDNA amplification was performed according to the following program: 1: 95°C, 15 s; 2: 60°C, 60 s, 40 cycles; 3: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s. The threshold cycle was measured using SYBR Green to monitor fluorescence during the cycles. The glucose-6-phosphate isomerase gene fn2054 was used as the internal reference gene, and 2 -ΔΔCtThe fold changes of gene expression were analyzed by relative quantitative analysis using GraphPad Prism 8 software. The data are presented as mean ± standard deviation of three experiments. Figure 3 As shown: First, the present invention studies the effects of different concentrations of nitisinone, compounds 19h and 19p on NTR gene expression. Figure 3 As shown in A, nitisinone and 19h up-regulated the expression of NTR gene in a concentration-dependent manner, especially high concentration 19h (32 μg / mL), while compound 19p down-regulated the expression of NTR gene in a dose-dependent manner. Secondly, in order to further verify whether the gene expression level is related to the drug incubation period, real-time quantitative PCR was performed on RNA extracted from the incubation period of Nitisinone, compound 19h and 19p (concentrations were 32 μg / mL, 8 μg / mL and 8 μg / mL, respectively) with Fusobacterium nucleatum at different incubation periods.

[0155] Figure 3 This is a schematic diagram showing the effects of different compounds on the nitroreductase gene. Figure 3 In the figure, A is a schematic diagram of the expression changes of the nitroreductase gene under different concentrations of different compounds and different incubation times at the same concentration. Figure 3 In Figure 2 , B is a schematic diagram of the expression changes of the nitroreductase gene at different concentrations of different compounds and different incubation times at the same concentration. Real-time fluorescence quantitative PCR was used to analyze the NTR cDNA expression of each sample, and the housekeeping gene fn2054 standard was used for normalization and expressed as a relative value, showing the average of three experiments.

[0156] Figure 4 Schematic diagram of the effects of different compounds on the tryptophanase gene. Figure 4 In the figure, A is a schematic diagram of the expression changes of tryptophanase gene at different concentrations of different compounds and different incubation times at the same concentration; Figure 4 In the figure, B is a schematic diagram of the expression changes of tryptophanase genes at different concentrations of different compounds and different incubation times at the same concentration, that is, the expression levels of tnaA at 4h, 8h, 12h, 24h, and 48h of the growth period when 32μg / mL, 8μg / mL, and 8μg / mL of nitisinone, compound 19h, and 19p were co-incubated with Fusobacterium nucleatum. The above data are expressed as the mean standard deviation of three experiments.

[0157] like Figure 3As shown in B, the NTR gene expression level is highly correlated with the incubation time. As the action time of the three compounds on Fusobacterium nucleatum increases, the expression of the NTR gene shows a trend of first increasing and then decreasing, indicating that nitisinone, compounds 19h and 19p may inhibit the growth of Fusobacterium nucleatum by regulating the expression of NTR. Subsequently, the present invention used different concentrations of nitisinone, compounds 19h and 19p (concentrations were 32 μg / mL, 8 μg / mL and 8 μg / mL, respectively) to co-incubate with Fusobacterium nucleatum under anaerobic conditions for 48 hours, and detected the expression level of the tnaA gene; as shown in FIG. Figure 4 As shown in Figure A, all three compounds down-regulated the expression of tnaA, especially low concentration compounds 19h and 19p. Secondly, the Fusobacterium nucleatum cells were treated with nitisinone, compounds 19h and 19p for 4h, 8h, 12h and 48h, respectively, to evaluate the expression of tnaA during the growth period of Fusobacterium nucleatum ( Figure 4 (B). After 8h and 12h of treatment with nitisinone and compound 19p, the transcription of tnaA was significantly higher than that at 4h (early log phase). When the incubation time was extended to 24h and 48h, the expression of tnaA decreased significantly. Compound 19h upregulated the expression of tnaA at the early log phase (4h) and downregulated it with the extension of incubation time. The above results show that nitisinone, compound 19h and 19p have different effects on the expression of tnaA in Fusobacterium nucleatum.

[0158] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof having anti-Fusobacterium nucleatum activity, characterized in that: The compound having resistance to Fusobacterium nucleatum is selected from one of the following structures:

2. Use of the compound having anti-Fusobacterium nucleatum activity according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-Fusobacterium nucleatum medicament or an anti-colon cancer medicament.

3. Use of the compound having anti-Fusobacterium nucleatum activity according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating diseases caused by Fusobacterium nucleatum.

4. The use according to claim 3, characterized in that: The disease caused by nuclease-bearing bacteria is selected from colon cancer.

Images