CN109503510B - Antibacterial thiazole compound for preventing caries and preparation method thereof - Google Patents
Antibacterial thiazole compound for preventing caries and preparation method thereof Download PDFInfo
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Abstract
The invention provides an anti-caries antibacterial thiazole compound and a preparation method thereof, wherein the imidazole compound has a structure shown in a formula (I):
the compound has the characteristics of small molecular weight, relatively simple structure, strong inhibition, good killing effect and the like proved by bacteriostatic experiments, can obviously inhibit the formation of the variable chain bacteria biofilm, reduce the growth activity of the mature variable chain bacteria biofilm, inhibit the acid production of the variable chain bacteria, and is expected to be developed into a medicament for effectively preventing and treating the caries.
Description
Technical Field
The invention relates to the field of medicines, in particular to an anti-caries antibacterial thiazole compound and a preparation method thereof.
Background
Dental caries is a common chronic infectious disease occurring in hard tissues of a tooth body in an oral cavity, and finally generates the defect of the tooth body tissue through a series of processes of bacterial biofilm aggregation, metabolic acidogenesis, local demineralization of the tooth body tissue and the like. Streptococcus mutans (called as mutans bacteria for short) is a major cariogenic bacterium recognized nowadays, and generates an adhesion effect on the tooth surface through surface virulence factors (such as acid production and acid resistance, in-vitro adhesion and extracellular polysaccharide synthesis) on the basis of physical adsorption, and finally continuously aggregates to form a cariogenic biofilm. Because the inside of the biofilm tissue provides a relatively stable living environment for the growth of bacteria, the antibacterial drug can only penetrate through the biofilm to have an effect on cariogenic bacteria growing in the biofilm. If only antibacterial action is provided without inhibiting the biofilm, it is difficult to maintain long-lasting and effective antibacterial action, and thus bacterial resistance is very likely to develop. In view of this, the research on the inhibition effect of the biofilm is a hot research spot of anti-infection diseases, and the research on the effect of the streptococcus mutans cariogenic biofilm has particularly important significance and research value in the prevention of caries.
Dental plaque is a typical biofilm state, and from a planktonic growth state to the formation of a biofilm structure, bacteria undergo several stages, namely, an initial adhesion stage, a biofilm adhesion stage, a growth stage, a maturation stage, and a spreading stage. In the process of forming the biofilm, the biological characteristics of bacteria growing in the biofilm are changed, the toxicity is greatly increased, the resistance to the external environment and host immune defense is stronger, 10-1000 times of that of planktonic bacteria can be achieved, and the resistance to antibacterial drugs is easier to generate.
Based on these facts, the search for novel compounds capable of inhibiting oral pathogenic bacteria, inhibiting specific virulence factors associated with cariogenicity, would be of great importance in caries prevention.
Disclosure of Invention
In view of the shortcomings and needs of the prior art, the present invention aims to provide a novel thiazole compound which can be used for preventing and treating caries, and a preparation method and application thereof.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a compound of formula (I):
the chemical formula of the compound of formula (I) is C16H11ClN4O5S2The molecular weight of the compound is 438.86, wherein the name of the compound is 3- ((4-chlorphenyl) sulfonylamino) -N- (5-nitrothiazol-2-yl) benzamide, and the name of the compound is 3- ((4-chlorophenyl) sulfonamido) -N- (5-nitrothiazole-2-yl) benzamide. The compound of formula (I) is soluble in dimethyl sulfoxide and insoluble in water.
The invention includes compounds of formula (I) or a pharmaceutically acceptable salt or prodrug thereof. The invention also includes solvates of the compounds of formula (I). In addition, the invention also includes various crystal forms of the compound of formula (I).
The present invention provides pharmaceutically acceptable salts of the compounds of formula (I). Examples of the "pharmaceutically acceptable salt" according to the present invention include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and nitrate; organic acid salts such as acetate, propionate, oxalate, succinate, lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and ascorbate; inorganic base salts such as sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt and aluminum salt; and organic base salts such as arginine salt, benzathine salt, choline salt, diethylamine salt, dialcohol amine salt, glycinate salt, lysine salt, meglumine salt, ethanolamine salt, and tromethamine salt.
The "prodrug of the compound of formula (I)" of the present invention means that the prodrug reacts with an enzyme, gastric acid, etc. under physiological conditions in vivo, and is converted into the compound of formula (I); for example, by enzymatic oxidation, reduction, hydrolysis, etc., to a compound of formula (I); and to compounds of formula (I) by hydrolysis using gastric acid, and the like.
The compounds of formula (I) may exist in tautomeric forms. Accordingly, the invention also includes tautomers of the compounds of formula (I).
In a second aspect of the invention, there is provided a process for the preparation of a compound of formula (I), which process comprises: (1) preparation of tert-butyl (3- ((5-nitrothiazol-2-yl) carbamoyl) phenyl) carbamate (intermediate 1) using 2-amino-5-nitrothiazole and 3- (Boc-amino) benzoic acid as starting reactants; (2) the intermediate 1 is subjected to Boc removal reaction to obtain 3-amino-N- (5-nitrothiazole-2-yl) benzamide hydrochloride (intermediate 2); (3) and reacting the intermediate 2 with p-chlorobenzenesulfonyl chloride to generate the compound shown in the formula (I).
The process involves the following reaction scheme:
preferably, the reaction of step (1) is carried out in the presence of a solvent, a condensing agent and an active agent.
Preferably, in the step (1), the molar ratio of the 2-amino-5-nitrothiazole to the 3- (Boc-amino) benzoic acid to the condensing agent to the activating agent is 1: 1.2-1.7: 1.5-2: 1.5-3.0, preferably 1: 1.2: 2: 2.
preferably, the reaction temperature of step (1) is 0-60 ℃, preferably 30 ℃.
Preferably, the solvent is selected from one or more of anhydrous N, N-Dimethylformamide (DMF), tetrahydrofuran, acetonitrile, chloroform, dichloromethane, 1, 4-dioxane; anhydrous dichloromethane and DMF are preferred.
Preferably, the solvent is used in an amount of 10 to 15mL per millimole of 2-amino-5-nitrothiazole.
Preferably, the condensing agent is selected from Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), O- (7-azabenzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HBTU), O- (5-chlorobenzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HCTU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium tetrafluoroborate (TBTU), O- (N-succinimidyl) -bis (dimethylamino) carbonium tetrafluoroborate (TSTU), One or more of O- (N-endo-5-norbornene-2, 3-dicarboximide) -bis (dimethylamino) carbonium tetrafluoroborate (TNTU), benzotriazol-1-yloxy-tris (tetrahydropyrrolyl) phosphonium hexafluorophosphate (PyBOP); preferably EDCI and/or HATU.
Preferably, the activator is selected from one or more of 4-Dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), 4-pyrrolidinylpyridine (4-PPY), 1-hydroxy-7-azobenzotriazol (HOAT), N-hydroxysuccinimide (HOSu), N-hydroxyphthalimide (NHPI), N-hydroxyphthalimide (NHNI), pentafluorophenol (PFPOH); DMAP is preferred.
Preferably, the de-Boc reaction in step (2) is performed in an acidic solution at room temperature, wherein the acidic solution is selected from one or more of HCl/ethyl acetate solution, HCl/methanol solution and trifluoroacetic acid; preferably a HCl/ethyl acetate solution.
Preferably, the acidic solution is used in an amount of 15-20mL per millimole of intermediate 1.
Preferably, step (3) is carried out in the presence of an acid-binding agent, which is an inorganic base.
Preferably, the inorganic base is selected from one or more of potassium carbonate, sodium bicarbonate, preferably potassium carbonate.
Preferably, in the step (3), the intermediate 2 is dissolved in a solvent to participate in the reaction, wherein the solvent is a solvent/water system selected from one or more of dioxane/water solution, tetrahydrofuran/water solution and acetone/water solution; preferably a dioxane/water solution.
Preferably, in the solvent/water system, the volume ratio of the solvent to the water is 1: 1-3.
Preferably, the solvent/water system is used in an amount of 8-12mL per millimole of intermediate 2.
Preferably, in the step (3), the molar ratio of the intermediate 2, the acid-binding agent and the p-chlorobenzenesulfonyl chloride is 1: 2-4: 1-1.2, preferably 1: 3: 1.2.
in a third aspect of the invention, the invention provides a composition comprising a compound of formula (I) or an isomer, solvate, prodrug, and a pharmaceutically acceptable salt thereof. The inclusion is understood to mean that the compounds of formula (I) or isomers, solvates, prodrugs and pharmaceutically acceptable salts thereof, function as an adjunct to the ingredients in the compositions or as a primary active ingredient.
Preferably, the composition may comprise a pharmaceutically acceptable carrier, adjuvant and/or excipient.
In a fourth aspect of the invention, there is provided a pharmaceutical formulation, cosmetic or cleaning product comprising a compound of formula (I) or an isomer, solvate, prodrug thereof and a pharmaceutically acceptable salt thereof, and one or more pharmaceutically or cosmetically acceptable carriers, adjuvants and/or vehicles.
Preferably, the pharmaceutical preparation is a solid preparation, an external preparation, a spray or a liquid preparation.
Preferably, the cosmetic or cleaning product is a toothpaste, a mouthwash or a disinfectant.
The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form. The administration form may be a liquid form, a solid form, a preparation for external use, a spray, or the like. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, usually about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.
In addition, the solid preparation can also be made into toothpaste, solid collutory, chewing gum, buccal tablet, oral patch, etc.; the spray or liquid preparation for external use can also be made into collutory, lotion, etc.
In a fifth aspect of the present invention, the present invention also provides the use of the compound of formula (I) or its isomer, solvate, prodrug and pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same as an active ingredient in the preparation of a medicament, cosmetic or cleaning product for inhibiting and/or killing planktonic cells of oral bacteria, or in the preparation of a medicament, food, cosmetic or cleaning product for inhibiting and/or killing oral cariogenic bacteria.
The compounds of the present invention have broad-spectrum bacteriostatic and bactericidal activity against oral bacteria. Particularly, the minimum inhibitory concentration to actinomyces viscosus, mutans streptococci and streptococcus sanguis is as low as 0.25-2 mug/mL, and the minimum bactericidal concentration is as low as 0.5-4 mug/mL. Preferably, the oral bacteria are selected from the group consisting of actinomyces viscosus, mutans bacteria, and streptococcus sanguis.
In a sixth aspect of the present invention, the present invention also provides the use of a compound of formula (I), or an isomer, solvate, prodrug, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same as an active ingredient, in the preparation of a medicament, cosmetic, or cleaning product for inhibiting the formation of a biofilm caused by streptomyces, or in the preparation of a medicament, cosmetic, or cleaning product for removing a biofilm caused by streptomyces.
The MBIC value of the compound is 2 mu g/mL, and the inhibition rate of the compound on the mutans bacteria biofilm is 100%. Its MBEC50The value is 16 mu g/mL, and the reduction of the formed variable chain bacteria biofilm by at least 50 percent effectively reduces the growth activity of the mature variable chain bacteria biofilm.
In a seventh aspect of the present invention, the present invention also provides the use of a compound of formula (I) or its isomer, solvate, prodrug and pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same as an active ingredient for the preparation of an oral mutans bacteria biofilm inhibitor.
In an eighth aspect, the present invention provides the use of the above compound or its isomer, solvate, prodrug, pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same as an active ingredient in the preparation of a pharmaceutical preparation for combating caries.
The inhibitory effect of the compounds of formula (I) on oral cariogenic bacteria was determined. In the test, the compound shown in the formula (I) is dissolved in DMSO to prepare a mother solution with the final concentration of 1024mg/mL for storage.
Test results show that the oral cavity antibacterial agent has broad-spectrum antibacterial and bactericidal activity on oral cavity bacteria. Particularly, the minimum inhibitory concentration to actinomyces viscosus, mutans streptococci and streptococcus sanguis is as low as 0.25-2 mug/mL, and the minimum bactericidal concentration is as low as 0.5-4 mug/mL. The MBIC value of the compound is 2 mu g/mL, and the inhibition rate of the compound on the mutans bacteria biofilm is 100%. MBEC of the Compounds of the invention50The value is 16 mu g/mL, and the reduction of the formed variable chain bacteria biofilm by at least 50 percent effectively reduces the growth activity of the mature variable chain bacteria biofilm. The compound of the invention has obvious inhibition effect on acid production of the streptomyces variabilis under MIC (2 mug/mL) and 1/2MIC (1 mug/mL). In addition, the compound has no influence on normal oral bacteria, and can selectively act on cariogenic bacteria in a biofilm.
The invention has the following beneficial effects:
the compound of the invention has the characteristics of small molecular weight, relatively simple structure, strong inhibition, good killing effect and the like proved by bacteriostatic experiments, can obviously inhibit the formation of the variable chain bacteria biomembrane, reduce the growth activity of the mature biomembrane of the variable chain bacteria, inhibit the acid production of the variable chain bacteria, and is expected to be developed into a medicament for preventing and treating caries.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the effect of the compounds of the present invention on the acid-producing ability of a mutans bacterium.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
EXAMPLE 1 preparation of the Compound of formula (I)
The synthetic route is as follows:
reagents and reaction conditions used in the synthetic route: (a) EDCI, DMAP, anhydrous N, N-dimethylformamide, room temperature; (b) HCl/ethyl acetate saturated solution, room temperature, 15 min; (c) potassium carbonate, dioxane/water (V: V ═ 1:1), room temperature, 30 min.
The preparation process comprises the following steps:
(1) preparation of tert-butyl (3- ((5-nitrothiazol-2-yl) carbamoyl) phenyl) carbamate (intermediate 1)
2-amino-5-nitrothiazole (1mmol) and 3- (Boc-amino) benzoic acid (1.2mmol) were dissolved in 12mL of anhydrous N, N-dimethylformamide, EDCI (2mmol) and DMAP (2mmol) were added, respectively, and reacted at room temperature for 12 hours, 30mL of ethyl acetate was added to the reaction solution to dilute it, washed 2 times with 1mol/L aqueous HCl solution (2 × 30mL), washed 2 times with saturated sodium bicarbonate solution (1 × 30mL), washed 1 time with saturated brine (1 × 30mL), the organic phase was dried over anhydrous magnesium sulfate, filtered under reduced pressure, and evaporated and concentrated using a rotary evaporator to give a yellow solid 0.94g, with a yield of 94%.
(2) Preparation of 3-amino-N- (5-nitrothiazol-2-yl) benzamide hydrochloride (intermediate 2)
Slowly dropping acetyl chloride into absolute ethyl alcohol (V: V ═ 4:5), introducing generated HCl into ethyl acetate to prepare HCl/ethyl acetate saturated solution, dissolving the intermediate 1 in the previous step, stirring for 15 minutes at normal temperature, detecting the reaction by TLC completely, evaporating the solvent to obtain yellow solid, wherein the yield of the crude product is 100%, and directly carrying out the next step without treatment.
(3) Preparation of 3- ((4-chlorophenyl) sulfonylamino) -N- (5-nitrothiazol-2-yl) benzamide (compound of formula (I))
Dissolving the intermediate 2(1mmol) and potassium carbonate (3mmol) in 8mL dioxane aqueous solution at room temperature, adding p-chlorobenzenesulfonyl chloride (1.2mmol), stirring for 30min, monitoring the reaction by TLC until the raw material reaction is complete, evaporating dioxane, adding 25mL distilled water into the reaction solution, extracting the water phase with ethyl acetate for 3 times (3 × 25mL), combining the organic phases, washing with saturated sodium chloride solution for 1 time (1 × 25mL), drying with anhydrous magnesium sulfate, filtering under reduced pressure, and concentrating by vacuum evaporation to obtain a crude product, purifying and separating the crude product by a silica gel chromatographic column (200-300 meshes), wherein dichloromethane, methanol, 240: 1 is used as an eluent to obtain a yellow solid target product with the yield of 45%.1H NMR(400MHz,DMSO-d6)：δ13.65(s,1H),10.74(s,1H),8.73(s,1H),7.87(d,J＝7.8Hz,1H),7.80(m,3H),7.65(d,J＝8.4Hz,2H),7.47(t,J＝7.9Hz,1H),7.39(d,J＝8.1,2.1Hz,1H)ppm；ESI-MS:438.0[M-H]-.
Example 2 antimicrobial Activity assay of Compounds of formula (I)
1. Preparation of the Strain of mutans UA159 (S.mutans UA159) and the Compound of formula (I)
The mutant strain UA159 used in the present invention is a model strain, and the reference genome number in NCBI database (http:// www.ncbi.nlm.nih.gov /) is NC-004350. The mutans UA246 strain used in the present invention is a clinical strain isolated from the oral cavity of a patient suffering from dental caries. The most suitable medium is Brain Heart Infusion (Brain Heart Infusion) medium (Brain Infusion solids 12.5g/L, Beef heart Infusion solids 5.0g/L, protein peptone 10.0g/L, Glucose 2.0g/L, Sodium chloride 5.0g/L, Di-Sodium phosphate 2.5g/L, pH 7.4 + -0.2), and the medium is allowed to stand at 37 ℃ under the most suitable conditions for anaerobic culture.
(1) The culture medium for culturing the streptomyces variabilis is Brain Heart Infusion (Brain Heart Infusion) culture medium (brand OXOID, cat # CM1135), and the main components of the culture medium are Brain Infusion solids 12.5g/L, Beef Heart Infusion solids 5.0g/L, protein peptone 10.0g/L, Glucose 2.0g/L, Sodimchloride 5.0g/L, Di-sodium phosphate 2.5g/L and pH 7.4 +/-0.2. If the agar powder is prepared into solid, 15g/L of agar powder is needed to be added. Sterilizing at 115 deg.C for 30min, and cooling.
(2) The culture medium for culturing the mutan streptococci biofilm is a brain heart infusion-sucrose culture medium, namely, sucrose with the final concentration of 1% is added into the brain heart infusion culture medium. Sucrose was prepared as a 20% stock solution and sterilized by filtration through a 0.22 μm sterile filter.
(3) The strain UA159 of the mutant strain is streaked on a plate containing a brain heart infusion agar solid medium by using a sterile inoculating loop, and the plate is placed upside down in an anaerobic incubator at 37 ℃ for culture until a single colony is obvious.
(4) Scraping and taking out the mutans streptococci UA159 by using a sterile inoculating shovel, respectively transferring into test tubes filled with a brain-heart infusion liquid culture medium, standing in an anaerobic incubator at 37 ℃, and culturing until the liquid is turbid.
(5) Detecting absorbance value (OD) of mutans streptococci under 600nm by using ultraviolet-visible spectrophotometer600nm)。
(6) The compound of formula (I) was accurately weighed on an analytical balance, dissolved by adding DMSO, and then sterilized by filtration using a sterile filter having a pore size of 0.22 μm to prepare a stock solution having a final concentration of 1024mg/L, which was stored at-20 ℃ until use.
2. Experimental methods
(1) Culturing for preparing variable chain bacteria UA159 (OD) in logarithmic phase600nm0.8-1.0) was diluted to a final concentration of 5 × 10 with brain heart infusion medium5CFU/mL is ready for use.
(2) Respectively adding the compound solution of the formula (I) with different concentrations after dilution in multiple proportion into a sterile 96-well plate, wherein the 1 st to 11 th wells are experimental groups added with liquid medicine, the 12 th wells are growth control groups without adding medicine, and the final concentration of the bacterial liquid of the streptomyces in each well is 5 × 105CFU/mL, and the drug concentrations in the 1 st to 12 th wells are 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0mg/L respectively. The Minimum Inhibitory Concentration (MIC) was located at the lowest concentration that completely inhibited bacterial growth within the wells.
(3) After the bacterial liquid in the small holes is evenly coated on a brain-heart infusion agar solid culture medium, the culture is inverted in an anaerobic incubator at 37 ℃ for 24 hours, the Minimum Bactericidal Concentration (MBC) is positioned according to the minimum concentration without bacterial production, the experiment is effectively repeated for 3 times, and the experimental results are shown in Table 1.
Several species of s.sanguis ATCC 49295 (streptococcus sanguis), a.viscosus ATCC 27044 (actinomyces viscosus), s.sobrinus ATCC 33478 (streptococcus sobrinus), s.salivaria ATCC 7073 (streptococcus salivarius) and s.mitis ATCC 6249 (streptococcus mitis) were tested under the same conditions as described above, and the results of the experiments are listed in table 1.
TABLE 1 determination of the Activity of the Compounds of formula (I) on the planktonic cells of the bacterium mutans UA159 (unit. mu.g/mL)
MIC: a minimum inhibitory concentration; MBC: minimum germicidal concentration.
As can be seen from Table 1, the compound of formula (I) has excellent antibacterial activity and killing activity against oral cariogenic bacteria, comparable to the antibacterial activity of chlorhexidine, a positive control.
Example 3 Effect of Compounds of formula (I) on oral Normal bacteria
Experimental procedures As in example 2, compounds were tested for their Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) against normal oral bacteria L.delbrueckiiCICC 6032, V.dentriticiosi KQ-ETV-9, V.rogosae WZH3 n. The results are shown in Table 2.
TABLE 2 antibacterial, bactericidal Activity results (in. mu.g/mL) for compounds of formula (I) against normal oral bacteria
As can be seen from Table 2, the compound of formula (I) had no effect on normal oral bacteria, but less effect than the positive control, chlorhexidine.
Example 4 inhibition of the formation of a biofilm of mutans bacteria by Compounds of formula (I)
(1) Prepared as described in example 2The strain solution and the compound of formula (I) are diluted to a final concentration of 1 × 10 by brain heart infusion-sucrose medium7CFU/mL is ready for use.
(2) To a sterile 96-well plate, 100. mu.L of the bacterial suspension of formula (1) was added, and wells to which the compound of formula (I) was added were used as experimental groups, and wells to which the compound of formula (I) was not added were used as control groups. Placing the mixture in an anaerobic incubator at 37 ℃ for standing culture for 24 hours.
(3) The planktonic cells in each well were removed and the unadsorbed cells were rinsed with copious amounts of water.
(4) To each well was added 200. mu.L of a 0.1% crystal violet solution, and the staining was performed by standing at room temperature for 5min, after which the crystal violet solution was removed and unadsorbed crystal violet was removed by washing with a large amount of water.
(5) And adding 33% acetic acid solution 200uL into each hole to dissolve the adsorbed crystal violet, and then detecting the absorbance value at 590nm by using a microplate reader, wherein the above experiment is effectively repeated for 3 times, and the inhibition rate of the biofilm is calculated according to the formula that the inhibition rate is × 100% (1-experiment group/growth control group).
The results show that the MBIC value of the compound of the formula (I) is 2 mu g/mL, and the inhibition rate on the mutans streptococci biofilm is 100%.
Example 5 Effect of Compounds of formula (I) on the removal of mature biofilm from mutans
(1) The preparation of the inoculum and the compound of formula (I) was carried out as described in example 2, and the inoculum in log phase was diluted to a final concentration of 1 × 10 in brain heart infusion-sucrose medium7CFU/mL is ready for use.
(2) 100. mu.L of the bacterial solution of (1) was added to a sterile 96-well plate, and anaerobic culture was carried out at 37 ℃ for 24 hours to allow the Streptococcus mutans to form a biofilm.
(3) And after the biofilm is formed, discarding the supernatant, adding 150-200 mu L of 0.85% NaCl, standing for 2-3 min, discarding the waste liquid, and repeating for 3 times.
(4) After the waste liquid is discarded, 100uL of fresh BHI culture medium is sequentially added from the second hole to the last hole, 200 mu L (256 mu g/mL) of the compound shown in the formula (I) with the corresponding concentration is added into the first hole, after the first hole is uniformly mixed, 100 mu L of the compound is taken from the first hole and added into the second hole, the compound is sequentially added into the penultimate hole, the compound is not added into the last hole, and the compound is cultured in an anaerobic incubator at 37 ℃ for 24 hours.
(5) And (4) repeating the operation step (3).
(6) mu.L of fresh BHI culture medium was added to each well, 10. mu.L of MTT (concentration 5mg/mL) was added to each well, wrapped with tinfoil paper, and incubated at 37 ℃ for 2-4 h in the dark.
(7) Discarding the supernatant, washing with 0.85% NaCl for 3 times, adding 200 μ L DMSO, incubating at 37 deg.C for 10-15 min, adding 50 μ L DMSO, measuring absorbance at 490nm with microplate reader, and repeating the above experiment for 3 times.
The results show MBEC for the compound of formula (I)50The value is 16 mug/mL, which reduces the formed mutans bacteria biofilm by at least 50 percent, and obviously reduces the growth activity of the mature mutans bacteria biofilm.
Example 6 inhibition of acid-producing ability of the Compounds of formula (I)
(1) The preparation of the inoculum and the compound of formula (I) was carried out as described in example 2, and the inoculum in log phase was diluted to a final concentration of 5 × 10 in brain heart infusion-sucrose medium5CFU/mL is ready for use.
(2) According to the MIC determination of the compound of formula (I) for mutans, 2 concentrations (1 and 1/2MIC) below the MIC value were added, and the control group was medium without the compound of formula (I) in 3 replicates per group.
(3) Placing in an anaerobic incubator at 37 ℃ for standing culture for 0, 6, 12 and 24 hours, sucking supernatant, monitoring the decline by adopting a precise pH meter, and taking an average value of results, wherein the experimental result is shown in figure 1.
As can be seen from FIG. 1, the compound of formula (I) has a significant inhibitory effect on acid production by Streptomyces variabilis at MIC (2. mu.g/mL) and 1/2MIC (1. mu.g/mL) compared to the control without the compound of formula (I).
Claims (28)
1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:
2. a process for preparing a compound of formula (I), the process comprising: (1) preparing tert-butyl (3- ((5-nitrothiazol-2-yl) carbamoyl) phenyl) carbamate as an intermediate 1 from 2-amino-5-nitrothiazole and 3- (Boc-amino) benzoic acid serving as initial reactants; (2) the intermediate 1 is subjected to Boc removal reaction to obtain 3-amino-N- (5-nitrothiazole-2-yl) benzamide hydrochloride which is marked as an intermediate 2; (3) and reacting the intermediate 2 with p-chlorobenzenesulfonyl chloride to generate the compound shown in the formula (I).
3. The method according to claim 2, wherein the reaction of step (1) is carried out in the presence of a solvent, a condensing agent and an activating agent.
4. The method of claim 3, wherein in step (1), the molar ratio of 2-amino-5-nitrothiazole, 3- (Boc-amino) benzoic acid, condensing agent and activating agent is 1: 1.2-1.7: 1.5-2: 1.5-3.0.
5. The method of claim 3, wherein in step (1), the molar ratio of 2-amino-5-nitrothiazole, 3- (Boc-amino) benzoic acid, condensing agent and activating agent is 1: 1.2: 2: 2.
6. the process according to claim 2, wherein the reaction temperature in step (1) is 0 to 60 ℃.
7. The process according to claim 2, wherein the reaction temperature in step (1) is 30 ℃.
8. The method according to claim 3, wherein the solvent is selected from one or more of anhydrous N, N-dimethylformamide, tetrahydrofuran, acetonitrile, chloroform, dichloromethane, and 1, 4-dioxane.
9. The method according to claim 3, wherein the solvent is used in an amount of 10 to 15mL per mmol of 2-amino-5-nitrothiazole.
10. The method of claim 3, wherein the condensing agent is selected from one or more of DCC, DIC, EDCI, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, PyBOP.
11. The method of claim 3, wherein the activator is selected from one or more of DMAP, HOBt, 4-PPY, HOAT, HOSu, NHPI, NHNI, PFPOH.
12. The method of claim 2, wherein the de-Boc reaction in step (2) is performed at room temperature in an acidic solution selected from one or more of HCl/ethyl acetate solution, HCl/methanol solution, trifluoroacetic acid.
13. The method according to claim 12, wherein the amount of the acidic solution is 15-20mL per millimole of intermediate 1.
14. The process of claim 2, wherein step (3) is carried out in the presence of an acid scavenger which is an inorganic base.
15. The method according to claim 14, wherein the inorganic base is selected from one or more of potassium carbonate, sodium carbonate and sodium bicarbonate.
16. The process of claim 2, wherein in step (3), the intermediate 2 is dissolved in a solvent to react, wherein the solvent is a solvent/water system selected from one or more of dioxane/water solution, tetrahydrofuran/water solution and acetone/water solution.
17. The method of claim 16, wherein the solvent/water system has a solvent to water volume ratio of 1: 1-3.
18. The process according to claim 16, characterized in that the solvent/water system is used in an amount of 8-12mL per millimole of intermediate 2.
19. The process of claim 14, wherein in step (3), the molar ratio of intermediate 2, acid scavenger, and p-chlorobenzenesulfonyl chloride is 1: 2-4: 1-1.2.
20. The process of claim 14, wherein in step (3), the molar ratio of intermediate 2, acid scavenger, and p-chlorobenzenesulfonyl chloride is 1: 3: 1.2.
21. a composition comprising a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.
22. The composition according to claim 21, wherein the composition may comprise a pharmaceutically acceptable carrier, adjuvant and/or vehicle.
23. A pharmaceutical preparation, cosmetic or cleaning preparation comprising a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically or cosmetically acceptable carrier, adjuvant and/or vehicle.
24. The pharmaceutical preparation, cosmetic or cleaning product according to claim 23, wherein the pharmaceutical preparation is a solid preparation, an external preparation, a spray or a liquid preparation.
25. The pharmaceutical preparation, cosmetic or cleaning product according to claim 23, wherein the cosmetic or cleaning product is a toothpaste, mouthwash or disinfectant.
26. The application of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition taking the compound as an active ingredient in the preparation of medicines, cosmetics or cleaning products for inhibiting and/or killing planktonic cells of oral bacteria, wherein the structural formula of the compound of formula (I) is as follows:
27. the application of the compound of formula (I) or pharmaceutically acceptable salt thereof or a pharmaceutical composition taking the compound as an active ingredient in preparing medicines, cosmetics or cleaning products for inhibiting the formation of the biofilm caused by the streptomyces mutans or in preparing medicines, cosmetics or cleaning products for clearing the biofilm caused by the streptomyces mutans is disclosed, wherein the structural formula of the compound of formula (I) is as follows:
28. the application of the compound of formula (I) or pharmaceutically acceptable salt thereof or a pharmaceutical composition taking the compound as an active ingredient in preparing an oral mutans bacteria biofilm inhibitor or preparing a pharmaceutical preparation for resisting caries, wherein the structural formula of the compound of formula (I) is as follows:
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