A kind of noraconitine derivative and its preparation method and application

Abstract

The invention discloses a noraconitine derivative or a pharmaceutical salt thereof. The structure of the noraconitine derivative is one of the following structures: the noraconitine derivative of the invention shows good anti-Fn activity and specificity in an in vitro antibacterial activity test; has no inhibitory effect on some common intestinal bacteria such as Escherichia coli, Salmonella paratyphi B, Staphylococcus aureus, Coprococcus, Shigella flexneri, etc., and has a specific inhibitory effect on Fn. The noraconitine derivative of the invention can be used to treat diseases caused by Fusobacterium nucleatum.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine preparation, and in particular relates to a noraconitine derivative and a preparation method and application thereof. Background Art

[0002] Colorectal cancer is the third most common cancer and the fourth most common cause of death worldwide, with approximately 1.2 million new cases and 600,000 deaths each year. Despite great advances in cancer treatment, nearly 86% of terminal cancer patients battle the disease and die within 5 years of initial diagnosis.

[0003] Fusobacterium nucleatum Fn is a Gram-negative obligate anaerobic bacillus that exists in many parts of the human body, including the oral cavity, intestines, and breasts, and is mainly colonized in the human oral cavity. For a long time, Fusobacterium nucleatum has been considered an opportunistic pathogen that can cause a variety of infectious diseases in the oropharynx and outside the oral cavity, including pericarditis, brain abscess, appendicitis, osteomyelitis, and pelvic inflammatory disease. Microbial infection is an important factor in the occurrence and development of tumors. Fusobacterium nucleatum can enter the gastrointestinal tract from the oral cavity and localize in colorectal cells.

[0004] There is increasing evidence that intestinal microorganisms play an important role in the development of colorectal cancer, and Fusobacterium nucleatum (Fn) is closely associated with colorectal cancer (CRC) and inflammatory bowel disease. With the deepening of research, the overexpression of Fn in CRC tissues has been clearly confirmed. Not only that, the enrichment of Fn is closely related to the stage and progression of cancer, and is related to the occurrence, metastasis, drug resistance and recurrence of colorectal cancer. High abundance of Fn affects the prognosis of CRC, triggers cancer recurrence, and promotes drug resistance in CRC patients. In a mouse colitis model, oral administration of Fusobacterium nucleatum can cause inflammation and the occurrence of small and large intestinal tumors. Fusobacterium nucleatum seems to easily attach to host CRC cells expressing endothelial cadherin, and then stimulates proinflammatory responses and cell proliferation.

[0005] Currently, there is no specific drug that can achieve specific protection against Fusobacterium nucleatum. Summary of the invention

[0006] The first object of the present invention is to provide a demethylaconitine derivative that is resistant to Fusobacterium nucleatum.

[0007] The second object of the present invention is to provide a method for preparing the noraconitine derivative.

[0008] The third object of the present invention is to provide a use of the demethylaconitine derivative in the preparation of drugs against nuclease-bearing Bacillus.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a noraconitine derivative or a pharmaceutically acceptable salt thereof, wherein the structure of the noraconitine derivative is one of the following structures:

[0011]

[0012] in,

[0013] R1 is selected from

[0014] R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, C1-C10 alkoxy, -CN, and -NO2;

[0015] R2 is selected from hydrogen;

[0016] R3 is selected from

[0017] More preferably, among the noraconitine derivatives,

[0018] R4, R5, R6, R7, and R8 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CN, and -NO2.

[0019] The pharmaceutically acceptable salt is selected from inorganic acid salts or organic acid salts.

[0020] The organic acid is selected from 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.

[0021] The inorganic acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid.

[0022] Most preferably, the structure of the noraconitine derivative is selected from one of the following structures:

[0023]

[0024]

[0025] The second aspect of the present invention provides a pharmaceutical composition, which contains the noraconitine derivative or a pharmaceutically acceptable salt thereof as a main active ingredient and a pharmaceutically acceptable carrier.

[0026] The pharmaceutically acceptable carrier is selected from common carrier materials such as flavors, sweeteners, liquid or solid fillers or diluents, and is prepared into common pharmaceutical preparations such as tablets, capsules, powders, syrups, liquids, suspensions or injections by methods known in the art. The preparations usually contain 1-70% by weight of active ingredients, and the preferred weight content is 5-50%.

[0027] The third aspect of the present invention provides a method for preparing the noraconitine derivative, comprising the following steps:

[0028] Dissolve noraconitine and a halogenated compound in a molar ratio of 1:(0.8-1.5) in N,N-dimethylformamide, add triethylamine, the molar ratio of triethylamine to noraconitine is (1.2-2):1, react at room temperature for 1-12 hours, and obtain the noraconitine derivative A;

[0029]

[0030] The halide is selected from 2-(bromomethyl)-5-nitrofuran, 3-fluorobenzyl bromide, 1-(bromomethyl)-4-methylbenzene, 4-cyanobenzyl bromide, 1-(chloromethyl)-4-methoxybenzene, 3-methoxybenzyl chloride, 4-nitrobenzyl bromide, 3-nitrobenzyl bromide, 4-fluorobenzyl bromide, 3-chlorobenzyl bromide, 2-chlorobenzyl bromide, 2-fluorobenzyl bromide, 1-(bromomethyl)-3-methylbenzene, benzyl bromide, p-bromobenzyl bromide, and m-bromobenzyl bromide.

[0031]

[0032] Dissolve noraconitine (compound 1) and di-tert-butyl dicarbonate in N,N-dimethylformamide at a molar ratio of 1:(1.01-2), add triethylamine, the molar ratio of triethylamine to noraconitine is (1.01-2):1, and react at room temperature for 1-12 hours to obtain compound 2;

[0033] Compound 2 and cesium carbonate in a molar ratio of 1:(1.05-2) are added to N,N-dimethylformamide, and reacted with diiodomethane at 80-90°C for 1-12h, wherein the molar ratio of diiodomethane to compound 2 is (0.5-1):1, to obtain compound 3;

[0034] Compound 3 and a halogenated heterocyclic compound in a molar ratio of 1:(1.05-2) are dissolved in N,N-dimethylformamide, cesium carbonate is added, and the molar ratio of cesium carbonate to compound 3 is (1.05-2):1, and the reaction is carried out at 80-90°C for 1-12h to obtain compound 4;

[0035] The halogenated heterocyclic compound is selected from

[0036] Compound 4 was dissolved in dichloromethane, and an excess of trifluoroacetic acid was added, followed by stirring at room temperature for 1 to 12 hours to obtain compound B.

[0037] The fourth aspect of the present invention provides a use of the noraconitine derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug against nuclease-bearing Bacillus.

[0038] The fifth aspect of the present invention provides a use of the noraconitine derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for a disease caused by nuclease-bearing bacteria.

[0039] The disease caused by Fusobacterium nucleatum is selected from colon cancer, drug resistance of colon cancer caused by Fusobacterium nucleatum, and metastasis of colon cancer caused by Fusobacterium nucleatum.

[0040] The present invention uses a standardized broth dilution sensitivity test for activity testing, and clindamycin and noraconitine are used as positive control drugs. Pharmacological experiments show that the compounds of the present invention or their pharmaceutically acceptable salts have in vitro antibacterial activity against Fusobacterium nucleatum. The results show that the derivatives (A1, B1) containing nitroheterocycles have above-moderate antibacterial activity against Fusobacterium nucleatum, but are weaker than the positive drug clindamycin.

[0041] The present invention uses a standardized broth dilution sensitivity test for activity testing, and cefoxitin and noraconitine are used as positive control drugs. Pharmacological experiments show that the compounds of the present invention or their pharmaceutically acceptable salts have in vitro antibacterial activity against Escherichia coli (ATCC25922), Salmonella paratyphi B (ATCCCMCC50094), Staphylococcus aureus (ATCC6538), Coccus (ATCC29212), and Shigella flexneri (ATCC12022). The results show that most of the compounds exhibit poor antibacterial activity against the four common intestinal bacteria, and the antibacterial activity is much lower than that of the positive drug cefoxitin.

[0042] Compound A1 of the present invention has strong antibacterial activity against Fusobacterium nucleatum, and MIC 50 It was 16 μg / mL, and the antibacterial activity of compound B1 against Fusobacterium nucleatum was 2 μg / mL.

[0043] The in vitro antibacterial activity test of the demethylaconitine derivatives of the present invention shows good anti-Fn activity and specificity; it has no inhibitory effect on some common intestinal bacteria such as Escherichia coli (ATCC25922), Salmonella paratyphi B (ATCCCMCC50094), Staphylococcus aureus (ATCC6538), Coccoccus faecalis (ATCC29212), Shigella flexneri (ATCC12022), etc., and has a specific inhibitory effect on Fn. The demethylaconitine derivatives of the present invention can be used to treat diseases caused by Fusobacterium nucleatum (such as colon cancer, colon cancer resistance caused by Fusobacterium nucleatum, and colon cancer metastasis caused by Fusobacterium nucleatum).

[0044] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0045] The present invention screened out demethylaconitine in a compound library through high-throughput screening, and found that demethylaconitine has a certain anti-Fusobacterium nucleatum effect (64 μg / mL). The systematic structural optimization and structure-activity relationship study of demethylaconitine were carried out to significantly improve its anti-Fusobacterium nucleatum activity. Furthermore, the present invention tested the anti-proliferation activity of these derivatives on human umbilical vein endothelial cells (HUVEC) and found that its IC 50 The minimum inhibitory activity (MIC) of the 50 μM flavonoids was greater than 50 μM. The 50 μM flavonoids ... 50 ) were greater than 64 μg / mL, and it was found that compounds A1 and B1 exhibited excellent anti-Fn activity and selectivity.

[0046] The antibacterial activity test of the present invention shows that the activities of compounds A1 and B1 against Fusobacterium nucleatum reach 16 μg / mL and 2 μg / mL, respectively, while the antibacterial activities against common intestinal bacteria are greater than 64 μg / mL, and the toxicity is relatively low. Therefore, the synthesized noraconitine derivatives of the present invention exhibit good antibacterial activity and specificity.

[0047] The present invention explores the design, synthesis, and antibacterial activity of noraconitine derivatives, and the activity of the optimal compound B1 reaches 2 μg / mL, while no obvious antibacterial activity is observed against other common intestinal bacteria, thus laying the foundation for subsequent research on anti-Fusobacterium nucleatum drugs. The noraconitine derivatives or their pharmaceutical salts of the present invention have a completely new structural skeleton and can be used as drugs for the development of anti-Fusobacterium nucleatum and anti-drug resistance and metastasis-related drugs for colorectal cancer, and are worth further research. DETAILED DESCRIPTION

[0048] 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.

[0049] The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0050] The chemical structural formula of the compounds involved in the following examples, 1The H-NMR and MS data are shown in Table 1. The numbers in Tables 1, 2, and 3 are consistent with those in the specification.

[0051] Table 1: Compounds of the present invention 1 H-NMR and MS data

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Example 1

[0060] Preparation of 8-(5-nitro-2-methylenefuran)noraconitine (A1)

[0061]

[0062] Dissolve noraconitine (0.20 g, 0.74 mmol) and 2-(bromomethyl)-5-nitrofuran (0.15 g, 0.74 mmol) in N,N-dimethylformamide (5 mL) and react at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). Monitor by TLC spot plate. After the reaction is completed, add 50 mL of water to dilute and extract three times with 50 mL of ethyl acetate respectively. Combine the organic layers, dry over anhydrous sodium sulfate, concentrate and column chromatograph (CH2Cl2:MeOH=100:5) to obtain compound A1 (0.05 g, yield 17.1%) as a yellow solid.

[0063] Example 2

[0064] Preparation of 8-(2-fluorobenzyl)noraconitine (A2)

[0065]

[0066] Dissolve noraconitine (0.20 g, 0.74 mmol) and 3-fluorobenzyl bromide (0.17 g, 0.88 mmol) in N,N-dimethylformamide (5 mL) and react at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). Monitor by TLC spot plate. After the reaction is completed, add 50 mL of water to dilute and extract three times with 50 mL of ethyl acetate respectively. Combine the organic layers, dry over anhydrous sodium sulfate, concentrate and column chromatograph (CH2Cl2:MeOH=100:5) to obtain compound A2 (0.13 g, yield 46.5%) as a yellow solid.

[0067] Example 3

[0068] Preparation of 8-(3-methylbenzyl)noraconitine A3

[0069]

[0070] Demethylaconitine (0.20 g, 0.74 mmol) and 1-(bromomethyl)-4-methylbenzene (0.16 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A3 (0.09 g, yield 32.5%) as a yellow solid.

[0071] Example 4

[0072] Preparation of 8-(3-cyanobenzyl)noraconitine A4

[0073]

[0074] Demethylaconitine (0.20 g, 0.74 mmol) and 4-cyanobenzyl bromide (0.17 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A4 (0.12 g, yield 42.1%) as a yellow solid.

[0075] Example 5

[0076] Preparation of 8-(3-methoxybenzyl)noraconitine A5

[0077]

[0078] Demethylaconitine (0.20 g, 0.74 mmol) and 1-(chloromethyl)-4-methoxybenzene (0.14 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A5 (0.11 g, yield 38.1%) as a yellow solid.

[0079] Example 6

[0080] Preparation of 8-(2-methoxybenzyl)noraconitine A6

[0081]

[0082] Demethylaconitine (0.20 g, 0.74 mmol) and 3-methoxybenzyl chloride (0.14 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A6 (0.12 g, yield 41.6%) as a yellow solid.

[0083] Example 7

[0084] Preparation of 8-(3-nitrobenzyl)noraconitine A7

[0085]

[0086] Demethylaconitine (0.20 g, 0.74 mmol) and 4-nitrobenzyl bromide (0.19 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2: MeOH = 100: 5) was performed to obtain compound A7 (0.15 g, yield 50.0%) as a yellow solid.

[0087] Example 8

[0088] Preparation of 8-(2-nitrobenzyl)noraconitine (A8)

[0089]

[0090] Demethylaconitine (0.20 g, 0.74 mmol) and 3-nitrobenzyl bromide (0.19 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A8 (0.13 g, yield 43.4%) as a yellow solid.

[0091] Example 9

[0092] Preparation of 8-(3-fluorobenzyl)noraconitine (A9)

[0093]

[0094] Demethylaconitine (0.20 g, 0.74 mmol) and 4-fluorobenzyl bromide (0.17 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A9 (0.10 g, yield 35.8%) as a yellow solid.

[0095] Example 10

[0096] Preparation of 8-(2-chlorobenzyl)noraconitine (A10)

[0097]

[0098] Demethylaconitine (0.20 g, 0.74 mmol) and 3-chlorobenzyl bromide (0.18 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A10 (0.15 g, yield 51.4%) as a yellow solid.

[0099] Embodiment 11

[0100] Preparation of 8-(1-chlorobenzyl)noraconitine (A11)

[0101]

[0102] Demethylaconitine (0.20 g, 0.74 mmol) and 2-chlorobenzyl bromide (0.18 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A11 (0.16 g, yield 54.8%) as a yellow solid.

[0103] Example 12

[0104] Preparation of 8-(1-fluorobenzyl)noraconitine (A12)

[0105]

[0106] Demethylaconitine (0.20 g, 0.74 mmol) and 2-fluorobenzyl bromide (0.17 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added to dilute, and 50 mL of ethyl acetate was used to extract three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2: MeOH = 100: 5) was performed to obtain compound A12 (0.14 g, yield 50.0%) as a yellow solid.

[0107] Example 13

[0108] Preparation of 8-(2-methylbenzyl)noraconitine (A13)

[0109]

[0110] Demethylaconitine (0.20 g, 0.74 mmol) and 1-(bromomethyl)-3-methylbenzene (0.16 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added to dilute, and 50 mL of ethyl acetate was used to extract three times respectively. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A13 (0.08 g, yield 28.9%) as a yellow solid.

[0111] Embodiment 14

[0112] Preparation of 8-Benzylnoraconitine (A14)

[0113]

[0114] Demethylaconitine (0.20 g, 0.74 mmol) and benzyl bromide (0.15 g, 0.88 mmol) were dissolved in N,N-dimethylformamide (5 mL) and reacted at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). TLC spot plate monitoring, after the reaction was completed, 50 mL of water was added for dilution, and 50 mL of ethyl acetate was used for extraction three times. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and column chromatography (CH2Cl2:MeOH=100:5) was performed to obtain compound A14 (0.11 g, yield 41.3%) as a yellow solid.

[0115] Embodiment 15

[0116] Preparation of 8-(3-bromobenzyl)noraconitine (A15)

[0117]

[0118] Dissolve noraconitine (0.20 g, 0.74 mmol) and p-bromobenzyl bromide (0.22 g, 0.88 mmol) in N,N-dimethylformamide (5 mL) and react at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). Monitor by TLC spot plate. After the reaction is completed, add 50 mL of water to dilute and extract three times with 50 mL of ethyl acetate respectively. Combine the organic layers, dry with anhydrous sodium sulfate, concentrate and column chromatography (CH2Cl2:MeOH=100:5) to obtain compound A15 (0.17 g, yield 52.4%) as a yellow solid.

[0119] Example 16

[0120] Preparation of 8-(2-bromobenzyl)noraconitine (A16)

[0121]

[0122] Dissolve noraconitine (0.20 g, 0.74 mmol) and m-bromobenzyl bromide (0.22 g, 0.88 mmol) in N,N-dimethylformamide (5 mL) and react at room temperature for 6 h under the action of triethylamine (0.13 g, 1.3 mmol). Monitor by TLC spot plate. After the reaction is completed, add 50 mL of water to dilute and extract three times with 50 mL of ethyl acetate respectively. Combine the organic layers, dry with anhydrous sodium sulfate, concentrate and column chromatography (CH2Cl2:MeOH=100:5) to obtain compound A16 (0.18 g, yield 55.6%) as a yellow solid.

[0123] Embodiment 17

[0124] Preparation of 1,3-dioxolane-14-(1-methyl-2-methylene-5-nitroimidazole)noraconitine (B1)

[0125]

[0126] Demethylaconitine, i.e., compound 1 (1.0 g, 3.7 mmol) and di-tert-butyl dicarbonate (1.0 g, 4.4 mmol) were dissolved in N,N-dimethylformamide (20 mL), triethylamine (0.4 g, 3.8 mmol) was added, and the mixture was reacted at room temperature for 6 h, and monitored by TLC. After the reaction was completed, 300 mL of water was added for dilution, and the mixture was extracted three times with 60 mL of ethyl acetate. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2:MeOH=100:6) to obtain pure compound 2 (0.54 g, yield 39.4%).

[0127] Compound 2 (0.54 g, 1.5 mmol) and cesium carbonate (0.95 g, 2.9 mmol) were added to N, N-dimethylformamide (10 mL), and reacted with diiodomethane (0.3 g, 1.2 mmol) at 85 ° C for 4 h, and monitored by TLC. After the reaction was completed, 200 mL of water was added for dilution, and 40 mL of ethyl acetate was used for extraction three times. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2: MeOH = 100: 2) to obtain pure compound 3 (0.3 g, yield 53.8%).

[0128] Compound 3 (0.3 g, 0.8 mmol) and 2-chloromethyl-1-methyl-5-nitroimidazole (0.17 g, 0.9 mmol) were dissolved in N,N-dimethylformamide (5 mL), cesium carbonate (0.5 g, 1.6 mmol) was added, and the mixture was reacted at 85 °C for 6 h, and monitored by TLC. After the reaction was completed, 100 mL of water was added for dilution, and 30 mL of ethyl acetate was used for extraction three times. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2: MeOH = 100: 1) to obtain pure compound 4-1 (0.2 g, yield 48.9%).

[0129] Compound 4-1 (0.2 g, 0.38 mmol) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 4 h and monitored by TLC. After the reaction was completed, 10 mL of saturated sodium bicarbonate solution was added to quench, 100 mL of water was added to dilute, and 30 mL of ethyl acetate was used to extract three times. The organic layers were combined, and the organic phase was washed once with 100 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2:MeOH=100:8) to obtain compound B1 (0.1 g, yield 61.8%).

[0130] Embodiment 18

[0131] 1,3-Dioxolane-14-(4-(3-chloropropyl)morpholino)noraconitine (B2)

[0132]

[0133] Demethylaconitine, i.e., compound 1 (1.0 g, 3.7 mmol) and di-tert-butyl dicarbonate (1.0 g, 4.4 mmol) were dissolved in N,N-dimethylformamide (20 mL), triethylamine (0.4 g, 3.8 mmol) was added, and the mixture was reacted at room temperature for 6 h, and monitored by TLC. After the reaction was completed, 300 mL of water was added for dilution, and the mixture was extracted three times with 60 mL of ethyl acetate. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2:MeOH=100:6) to obtain pure compound 2 (0.54 g, yield 39.4%).

[0134] Compound 2 (0.54 g, 1.5 mmol) and cesium carbonate (0.95 g, 2.9 mmol) were added to N, N-dimethylformamide (10 mL), and reacted with diiodomethane (0.3 g, 1.2 mmol) at 85 ° C for 4 h, and monitored by TLC. After the reaction was completed, 200 mL of water was added for dilution, and 40 mL of ethyl acetate was used for extraction three times. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2: MeOH = 100: 2) to obtain pure compound 3 (0.3 g, yield 53.8%).

[0135] Compound 3 (0.3 g, 0.8 mmol) and 4-(3-chloropropyl)morpholine (0.15 g, 0.9 mmol) were dissolved in N,N-dimethylformamide (5 mL), cesium carbonate (0.5 g, 1.6 mmol) was added, and the mixture was reacted at 85 °C for 6 h, and monitored by TLC. After the reaction was completed, 100 mL of water was added for dilution, and 30 mL of ethyl acetate was used for extraction three times. The organic layers were combined, and the organic phase was washed three times with 100 mL of saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2:MeOH=100:1) to obtain pure compound 4-2 (0.15 g, yield 37.6%).

[0136] Compound 4-2 (0.15 g, 0.3 mmol) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature for 4 h, and monitored by TLC. After the reaction was completed, 10 mL of saturated sodium bicarbonate solution was added to quench, 100 mL of water was added to dilute, and 30 mL of ethyl acetate was used to extract three times. The organic layers were combined, and the organic phase was washed once with 100 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and column chromatographed (CH2Cl2: MeOH = 100: 8) to obtain compound B2 (0.04 g, yield 33.2%).

[0137] Embodiment 19

[0138] In vitro anti-Fusobacterium nucleatum activity test of the compounds prepared in the examples of the present invention

[0139] The in vitro antimicrobial activity of the synthetic compounds of the present invention was tested using a standardized broth dilution susceptibility test according to the Clinical and Laboratory Standards Institute (CLSI) method.

[0140] 1. Test strain: Fusobacterium nucleatum (ATCC 23726) was provided by Huashan Hospital.

[0141] The culture medium for Fusobacterium nucleatum is brain heart infusion (BHI) broth and Brucella broth.

[0142] 2. Instruments: WellscanMK-2 fully automatic microplate reader (manufactured by Labsystems Dragon), Nephelometer turbidimeter (manufactured by Thermo Scientific).

[0143] 3. Test methods

[0144] Sample solution preparation: Dissolve in DMSO (Merck) and prepare a 2 mg / mL solution.

[0145] Clindamycin and noraconitine were prepared into reference solutions under the same conditions.

[0146] Fusobacterium nucleatum bacterial solution is equipped with: oscillate standard turbidimetric tube, after calibration, place the glass test tube into the turbidimeter, add 4mL of Brucella broth, scrape well-grown colonies of appropriate size with a cotton swab, and rub them in the glass test tube until the turbidity reaches 0.5. Pour the Brucella broth with bacterial solution into a test tube containing 40mL of Brucella broth, add 4.4mL of defibrinated horse blood (repeatedly frozen and thawed), and mix with a vortex oscillator (bacterial solution: broth: horse blood = 1:10:1.1).

[0147] Half-fold dilution: Add the above bacterial solution to a 96-well plate, 200 μL in the first column and 100 μL in other wells. The starting concentration of the compound in the first column is 64 μg / mL. After half-fold dilution, put all 96-well plates into an anaerobic box and add anaerobic bags, and culture them in a 37°C incubator for 48 hours. Observe with the naked eye, and the minimum inhibitory concentration of the compound is when turbid wells appear.

[0148] The data of minimum inhibitory concentration are shown in Table 2:

[0149] Table 2

[0150]

[0151]

[0152] According to the above in vitro anti-Fusobacterium nucleatum activity test method, the compounds of the present invention were subjected to in vitro anti-Fusobacterium nucleatum activity and structure-activity relationship studies:

[0153] The in vitro antibacterial activity of the target compounds was tested using a standardized broth dilution sensitivity test, with clindamycin and noraconitine used as positive control drugs. The activity results showed that some compounds A1 and B1 containing nitroheterocyclic rings had good activity, with activities of 16μg / mL and 2μg / mL, respectively. Among them, compound B1 containing nitroimidazole had the best activity and showed antibacterial activity against Fusobacterium nucleatum. Some compounds had improved activity compared with noraconitine, but were weaker than the listed positive drug clindamycin.

[0154] Embodiment 20

[0155] The in vitro activity test of the compounds prepared in the examples of the present invention was conducted on common intestinal bacteria: Escherichia coli (ATCC25922), Salmonella paratyphi B (ATCC CMCC 50094), Staphylococcus aureus (ATCC 6538), Coprococcus (ATCC 29212), and Shigella flexneri (ATCC 12022).

[0156] The in vitro antimicrobial activity of the synthetic compounds of the present invention was tested using a standardized broth dilution susceptibility test according to the Clinical and Laboratory Standards Institute (CLSI) method.

[0157] 1. Test strains: Escherichia coli (ATCC 25922), Salmonella paratyphi B (ATCC CMCC 50094), Staphylococcus aureus (ATCC 6538), Coccus faecalis (ATCC 29212), Shigella flexneri (ATCC 12022), all of which were purchased from Qingdao Haibo Biological Company

[0158] The culture medium for Escherichia coli and Shigella flexneri was LB broth, the culture medium for Coprococcus and Staphylococcus aureus (ATCC6538) was MH broth, and the culture medium for Salmonella paratyphi B was nutrient broth.

[0159] 2. Instruments: WellscanMK-2 fully automatic microplate reader (manufactured by Labsystems Dragon), Nephelometer turbidimeter (manufactured by Thermo Scientific).

[0160] 3. Test methods

[0161] Sample solution preparation: Dissolve in DMSO (Merck) and prepare a 2 mg / mL solution.

[0162] Cefoxitin and noraconitine were prepared into reference solutions under the same conditions.

[0163] Bacterial solution preparation: Oscillating standard turbidimetric tube, after calibration, put the glass test tube into the turbidimeter, add 4mL of Brucella broth, scrape well-grown and appropriately sized colonies with a cotton swab, and rub them in the glass test tube until the turbidity reaches 0.5. Pour the Brucella broth with bacterial solution into the test tube containing 40mL of MH broth, and the bacterial concentration is 1*10 6 CFU / mL, and mix by vortexing.

[0164] Half-fold dilution: Add the above bacterial solution to a 96-well plate, with 200 μL in the first column and 100 μL of bacterial solution in other wells. The starting concentration of the compound in the first column is 64 μg / mL. After half-fold dilution, place all 96-well plates in a 37°C incubator for 48 hours. Use an MK-2 fully automatic microplate reader to measure the 630nm OD value and calculate the MIC 50 .

[0165] MIC 50 =(OD value of blank control well-OD value of drug administration well) / OD value of blank control well×100%.

[0166] Table 3. Antibacterial activity of compounds against common intestinal bacteria (μg / mL)

[0167]

[0168] According to the above in vitro activity test method, the in vitro antibacterial activity and structure-activity relationship of the compounds of the present invention were studied:

[0169] The in vitro antibacterial activity of the target compound was tested using a standardized broth dilution sensitivity test, with cefoxitin and noraconitine as positive control drugs. The activity results showed that all noraconitine derivatives had no good activity against the above common intestinal bacteria, indicating that this type of noraconitine derivatives had strong selectivity.

[0170] Embodiment 21

[0171] In vitro antitumor activity test of the compounds of the present invention

[0172] The cell proliferation activity of the synthetic compound of the present invention was tested using the CCK8 method.

[0173] 1. Experimental cell lines

[0174] HUVEC (human umbilical vein endothelial cells) and HCT116 (human colorectal cancer cells) were purchased from Shanghai Institute of Pharmaceutical Industry.

[0175] The culture medium for HUVEC was ECM+15% FBS+double antibodies, and the culture medium for HCT116 was DMEM+10% FBS+double antibodies.

[0176] 2. Instrument: WellscanMK-2 fully automatic microplate reader (manufacturer: Labsystems Dragon).

[0177] 3. Test methods

[0178] Camptothecin (CPT), noraconitine and the compound synthesized by the present invention were prepared to 10 mmol / L.

[0179] Solution preparation: Camptothecin in a 96-well plate was diluted to an initial concentration of 10 μmol / L with cell culture medium, and the initial concentration of noraconitine and the compound synthesized by the present invention was 50 μmol / L, and then diluted three-fold with culture medium to 9 concentration gradients.

[0180] The concentration of 5 × 10 4 100 μL of cell suspension with a density of 5000 cells / mL, i.e. 5000 cells / well, was placed in a 37°C, 5% CO2 incubator. After 24 hours, 10 μL / well of sample solution and reference solution were added respectively, and the cells were incubated at 37°C for 72 hours. After discarding the culture medium, 100 μL of 10% CCK8 DMEM solution was added to each well for 30 minutes, and the 570 nm OD value was measured with an automatic microplate reader to calculate the half inhibitory concentration IC 50 .

[0181] IC%=(OD value of blank control well-OD value of drug administration well) / OD value of blank control well×100%.

[0182] According to the IC% value of each concentration, linear regression was performed to calculate the drug concentration that inhibited cell growth by 50%, i.e., IC 50 .

[0183] Table 4. Cell proliferation activity of target compounds of type A and B

[0184]

[0185] Based on the above in vitro cell proliferation activity results, in vitro cell proliferation activity and structure-activity relationship studies were conducted:

[0186] The in vitro cell proliferation activity of the target compound was tested by CCK8 method, and camptothecin CPT and noraconitine were used as positive control drugs. The activity results showed that the more active noraconitine derivatives A1 and B1 had no inhibitory activity on human umbilical vein endothelial cells (HUVEC) and human colorectal cancer cells (HCT116), indicating that the in vitro toxicity of this series of derivatives was very low.

[0187] In summary, the aconitine derivatives (A1, B1) in the present invention not only have excellent anti-Fusobacterium nucleatum activity, but also have no antibacterial activity against other intestinal bacteria, which shows that this type of compound has specificity. In addition, this type of compound has low toxicity and therefore has good research significance.

[0188] 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 noraconitine derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the noraconitine derivative is one of the following structures: in, R1 is selected from R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogen, C1-C10 alkyl, C1-C10 alkoxy, -CN, and -NO2; R2 is selected from hydrogen; R3 is selected from 2. The noraconitine derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the described norconitine derivatives, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, -CN, and -NO2.

3. The noraconitine derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The structure of the noraconitine derivative is selected from one of the following structures:

4. A pharmaceutical composition comprising the noraconitine derivative or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 as a main active ingredient and a pharmaceutically acceptable carrier.

5. A method for preparing a noraconitine derivative according to any one of claims 1 to 3, characterized in that: The following steps are involved: Dissolve noraconitine and a halogenated compound in a molar ratio of 1:(0.8-1.5) in N,N-dimethylformamide, add triethylamine, the molar ratio of triethylamine to noraconitine is (1.2-2):1, react at room temperature for 1-12 hours, and obtain the noraconitine derivative A; The halide is selected from 2-(bromomethyl)-5-nitrofuran, 3-fluorobenzyl bromide, 1-(bromomethyl)-4-methylbenzene, 4-cyanobenzyl bromide, 1-(chloromethyl)-4-methoxybenzene, 3-methoxybenzyl chloride, 4-nitrobenzyl bromide, 3-nitrobenzyl bromide, 4-fluorobenzyl bromide, 3-chlorobenzyl bromide, 2-chlorobenzyl bromide, 2-fluorobenzyl bromide, 1-(bromomethyl)-3-methylbenzene, benzyl bromide, p-bromobenzyl bromide, and m-bromobenzyl bromide; Dissolve noraconitine (compound 1) and di-tert-butyl dicarbonate in N,N-dimethylformamide at a molar ratio of 1:(1.01-2), add triethylamine, the molar ratio of triethylamine to noraconitine is (1.01-2):1, and react at room temperature for 1-12 hours to obtain compound 2; Compound 2 and cesium carbonate in a molar ratio of 1:(1.05-2) are added to N,N-dimethylformamide, and reacted with diiodomethane at 80-90°C for 1-12h, wherein the molar ratio of diiodomethane to compound 2 is (0.5-1):1, to obtain compound 3; Compound 3 and a halogenated heterocyclic compound in a molar ratio of 1:(1.05-2) are dissolved in N,N-dimethylformamide, cesium carbonate is added, and the molar ratio of cesium carbonate to compound 3 is (1.05-2):1, and the reaction is carried out at 80-90°C for 1-12h to obtain compound 4; The halogenated heterocyclic compound is selected from Compound 4 was dissolved in dichloromethane, and an excess of trifluoroacetic acid was added, followed by stirring at room temperature for 1 to 12 hours to obtain compound B.

6. Use of the noraconitine derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug against nuclease-bearing Bacillus.

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