CN109503510A - A kind of thiazole compound and preparation method thereof of preventing decayed tooth antibacterial - Google Patents
A kind of thiazole compound and preparation method thereof of preventing decayed tooth antibacterial Download PDFInfo
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- CN109503510A CN109503510A CN201910004830.4A CN201910004830A CN109503510A CN 109503510 A CN109503510 A CN 109503510A CN 201910004830 A CN201910004830 A CN 201910004830A CN 109503510 A CN109503510 A CN 109503510A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The present invention provides a kind of thiazole compound and preparation method thereof of preventing decayed tooth antibacterial, which has structure shown in formula (I):The compound molecular weight is small, structure is relatively easy, bacteriostatic experiment confirmation has the characteristics that strong inhibition capability, fragmentation effect are good, the formation of mutans streptococcus biomembrane can be significantly inhibited, reduce the growth vigor of mutans streptococcus biofilm, and the production acid for inhibiting mutans streptococcus, is expected to be developed into the drug for effectively preventing dental caries.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of thiazole compound and preparation method thereof of preventing decayed tooth antibacterial.
Background technique
Dental caries are chronic infectious disease of the common generation in oral cavity in dental hard tissue, pass through a series of bacterium living beings quilts
Film aggregation, metabolism produce acid, the processes such as tissue of tooth part demineralization, the final defect that tissue of tooth occurs.Streptococcus mutans are (referred to as
Mutans streptococcus) it is main cariogenic bacteria generally acknowledged now, it is (as resistance in produced acid by surface virulence factor on the basis of physical absorption
Acid sticks, synthetic cell exo polysaccharides in vitro) adhesive attraction is generated to dental surface, final constantly aggregation forms cariogenic biological quilt
Film.Since biofilm organization internal provides environment in a metastable existence for bacterial growth, antibacterials only have
It is perforated through biofilm, could be acted on cariogenic bacteria generation therein is grown on.If only antibacterial action and to biofilm
Inhibiting effect is not generated, then is difficult to maintain long-acting and effective antibacterial action, is also therefore very easy to generate bacterial resistance.In view of
This, the inhibiting effect research to biofilm is always the research hotspot of classes of anti-infective disease, and to the cariogenic life of Streptococcus mutans
The Effect study of object envelope has especially important meaning and researching value in prevention of caries.
Plaque is exactly a kind of typical biofilm state, from the growth conditions to swim to the shape of biofilm structure
At bacterium experienced several stages such as initial stage adherency, biofilm adherency phase, growth period, maturity period and the phase of sending out.In biological quilt
In film forming process, it is grown on biology of bacteria characteristic changing therein, virulence greatly increases, to external environment and host immune
Defence also has stronger resistance, can reach 10-1000 times of planktonic bacteria, is also more easier to generate antibacterials resistance to
Medicine.
Based on these facts, finds and be able to suppress oral cavity pathogen, inhibition specific virulence factor relevant to cariogenicity
New compound will be significant in prevention of caries.
Summary of the invention
In view of the deficiencies of the prior art with demand, it can be used for preventing and treating the new of dental caries the purpose of the present invention is to provide a kind of
Type thiazole compound and its preparation method and application.
Specifically, the present invention is achieved through the following technical solutions:
In the first aspect of the present invention, the present invention provides compounds shown in a kind of formula (I):
The chemical molecular formula of formula (I) compound is C16H11ClN4O5S2, Chinese is 3- ((4- chlorphenyl) sulphonyl ammonia
Base)-N- (5- nitrothiazole -2- base) benzamide, English name is 3- ((4-chlorophenyl) sulfonamido)-N-
(5-nitrothiazol-2-yl) benzamide, molecular weight 438.86.Formula (I) compound is dissolved in dimethyl sulfoxide, indissoluble
Yu Shui.
The present invention includes compound shown in formula (I) or its pharmaceutically acceptable salt or prodrug.The invention also includes formula (I)
The solvate of shown compound.In addition, the invention also includes the various crystal forms of formula (I) compound.
The present invention provides the pharmaceutically acceptable salts of formula (I) compound.It is of the present invention " pharmaceutically acceptable
The example of salt " includes inorganic acid salt, such as hydrochloride, hydrobromate, sulfate, phosphate and nitrate；Acylate, such as
Acetate, oxalates, succinate, lactate, malate, tartrate, citrate, maleate, prolongs recklessly propionate
Rope hydrochlorate, mesylate, benzene sulfonate, tosilate and ascorbate；Inorganic base salts, such as sodium salt, sylvite, calcium
Salt, zinc salt, magnesium salts and aluminium salt；And organic alkali salt, such as arginine salt, benzyl star salt, choline salt, diethylamine salt, glycol amine salt,
Glycinate, lysine salt, meglumine salt, ethanolamine salt and tromethamine salt.
" prodrug of the compound of formula (I) " of the present invention refers under physiological condition in vivo, can be with enzyme, gastric acid
Deng reaction, to convert the compound of an accepted way of doing sth (I)；For example, converting the change of an accepted way of doing sth (I) by the oxidation, reduction, hydrolysis etc. of enzyme
Close object；And the hydrolysis by using gastric acid, convert the compound etc. of an accepted way of doing sth (I).
The compound of formula (I) can exist in the form of tautomer.Therefore, the invention also includes the chemical combination of formula (I)
The tautomer of object.
In the second aspect of the present invention, the present invention provides a kind of method for preparing formula (I) compound, the method packets
Include: (1) 2- amino -5- nitrothiazole and 3- (Boc- amino) benzoic acid and 2- amino -5- nitrothiazole are initial reactant system
Standby (3- ((5- nitrothiazole -2- base) carbamoyl) phenyl) t-butyl carbamate (intermediate 1)；(2) intermediate 1 is through de-
3- amino-N- (5- nitrothiazole -2- base) benzamide hydrochloride salt (intermediate 2) is obtained after Boc reaction；(3) intermediate 2 with it is right
Chlorobenzene sulfonyl chloride reaction generates formula (I) compound.
The method is related to following reaction route:
Preferably, the reaction of step (1) need to carry out in the presence of solvent, condensing agent and activating agent.
Preferably, in step (1), 2- amino -5- nitrothiazole, 3- (Boc- amino) benzoic acid, condensing agent, activator
Molar ratio 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 DEG C, preferably 30 DEG C.
Preferably, the solvent be selected from anhydrous n,N-Dimethylformamide (DMF), tetrahydrofuran, acetonitrile, chloroform,
One of methylene chloride, 1,4- dioxane are a variety of；It is preferred that anhydrous methylene chloride and DMF.
Preferably, the dosage of the solvent is the 2- amino -5- nitrothiazole of every mM of 10~15mL.
Preferably, the condensing agent is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-
(3- dimethylamino-propyl) -3- ethyl carbodiimide (EDCI), O- (7- azepine benzotriazole -1- base)-two (dimethylamino)
Carbon hexafluorophosphate (HATU),-two (dimethylamino) carbon hexafluorophosphate (HBTU) of O- (benzotriazole -1- base), O-
(5- Chloro-Benzotriazole -1- base)-two (dimethylamino) carbon hexafluorophosphates (HCTU), O- (benzotriazole -1- base)-two
(dimethylamino) carbon tetrafluoroborate (TBTU) ,-two (dimethylamino) carbon tetrafluoroborate of O- (N- succimide base)
(TSTU), O- (two carbon imidodicarbonic diamide of N-endo-5- norcamphene -2,3-)-two (dimethylamino) carbon tetrafluoroborates (TNTU),
(one of nafoxidine base) Phosphonium hexafluorophosphate (PyBOP) is a variety of for benzotriazole -1- base oxygen-three；Preferably
EDCI and/or HATU.
Preferably, the activator is selected from 4-dimethylaminopyridine (DMAP), I-hydroxybenzotriazole (HOBt), 4- pyrroles
Alkyl pyridine (4-PPY), 1- hydroxyl -7- azo benzotriazole (HOAT), N- hydroxysuccinimide (HOSu), N- hydroxyl are adjacent
One of phthalimide (NHPI), n-Hydroxyphthalimide (NHNI), Pentafluorophenol (PFPOH) are a variety of；
Preferably DMAP.
Preferably, step (2) the de- Boc reaction carries out at room temperature in an acidic solution, and the acid solution is selected from
One of HCl/ ethyl acetate solution, HCl/ methanol solution, trifluoroacetic acid are a variety of；Preferably HCl/ ethyl acetate solution.
Preferably, the acid solution dosage is the intermediate 1 of every mM of 15-20mL.
Preferably, step (3) carries out in the presence of acid binding agent, and the acid binding agent is inorganic base.
Preferably, the inorganic base is selected from one of potassium carbonate, sodium carbonate, sodium bicarbonate or a variety of, preferably carbonic acid
Potassium.
Preferably, in step (3), intermediate 2, which is dissolved in solvent, participates in reaction, and the solvent is solvent/water system, should
System is selected from one of dioxane/water solution, tetrahydrofuran/aqueous solution, acetone/water solution or a variety of；Preferably dioxy
Six rings/aqueous solution.
Preferably, in the solvent/water system, the volume ratio of solvent and water is 1:1-3.
Preferably, the dosage of the solvent/water system is the intermediate 2 of every mM of 8-12mL.
Preferably, in step (3), the molar ratio of intermediate 2, acid binding agent and parachloroben-zenesulfonyl chloride is 1:2-4:1-1.2, excellent
Select 1:3:1.2.
In the third aspect of the present invention, the present invention provides a kind of compositions, and it includes formula (I) compound or its isomeries
Body, solvate, prodrug and its pharmaceutically acceptable salt.It is described comprising can be understood as formula (I) compound or its isomers,
Solvate, prodrug and its pharmaceutically acceptable salt help out as the ingredient in composition or as chief actives
Ingredient plays a major role.
Preferably, the composition may include pharmaceutically acceptable carrier, auxiliary material and/or excipient.
In the fourth aspect of the present invention, the present invention provides a kind of pharmaceutical preparation, cosmetics or cleaning supplies, and it includes formulas
(I) compound or its isomers, solvate, prodrug and its pharmaceutically acceptable salt and it is one or more pharmaceutically or makeup
Acceptable carrier, auxiliary material and/or excipient on product.
Preferably, the pharmaceutical preparation is solid pharmaceutical preparation, external preparation, spray or liquid preparation.
Preferably, the cosmetics or cleaning supplies are toothpaste, mouthwash or thimerosal.
The compounds of this invention can be administered in a unit containing its pharmaceutical composition.Form of administration can be liquid
Body dosage form, solid dosage forms, external preparation, spray etc..Liquid dosage form can be true solution class, colloidal type, particulate formulations, emulsion
Dosage form, mixed dosage form.Other dosage forms for example tablet, capsule, dripping pill, aerosol, pill, pulvis, solution, suspension, emulsion,
Granule, suppository, freeze drying powder injection, inclusion compound, landfill agent, patch, liniment etc..
Common carrier, pharmaceutical acceptable carrier packet described here can also be contained in pharmaceutical composition or pharmaceutical preparation of the invention
It includes but is not limited to: ion-exchanger, aluminium oxide, aluminum stearate, lecithin, haemocyanin such as human albumin, buffer substance
Such as phosphate, glycerol, sorb ester, potassium sorbate, the partial glyceride mixtures of saturated vegetable fatty acid, water, salt or electrolyte,
Such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, magnesium trisilicate, polyvinyl pyrrole
Alkanone, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, wool grease etc..Carrier is in medicine group
The content closed in object can be 1wt%-98wt%, generally about account for 80wt%.For convenience, local anesthetic, anti-corrosion
Agent, buffer etc. can be directly dissolved in carrier.
Oral tablet and capsule can contain excipient such as adhesive, such as syrup, Arabic gum, sorbierite, tragacanth, or
Polyvinylpyrrolidone, filler, such as lactose, sucrose, cornstarch, calcium phosphate, sorbierite, amion acetic acid, lubricant are such as hard
Fatty acid magnesium, talcum, polyethylene glycol, tripoli, disintegrating agent, such as potato starch or acceptable dibutyl phthalate, such as laurel sodium alkoxide sulfuric acid
Salt.Tablet can be coated with method well known in pharmaceutics.
The suspension of water and oil can be made in oral solution, and solution, emulsion, dry product can also be made in syrup, with preceding supplement
Water or other suitable mediums.This liquid preparation may include conventional additive, such as suspending agent, sorbierite, cellulose first
Ether, dextrose syrup, gel, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, the edible oil and fat of hydrogenation, emulsification
Agent, such as lecithin, sorbitan mono-oleate, gum arabic；Or nonaqueous carrier (may include edible oil), such as almond
Oil, grease such as glycerol, ethylene glycol or ethyl alcohol；Preservative, such as methyl p-hydroxybenzoate or propyl ester, sorbic acid.As needed can to add
Add flavoring agent or colorant.
In addition, such as toothpaste, solid mouthwash, chewing gum, lozenge, mouth paster can also be made in solid pharmaceutical preparation；External application
Such as mouthwash, lotion can also be made in spray or liquid preparation.
In the fifth aspect of the invention, the present invention also provides formula (I) compound or its isomers, solvate, prodrugs
And its pharmaceutically acceptable salt or using its as the pharmaceutical composition of active constituent preparation for inhibiting and/or killing oral cavity thin
Application in the drugs of bacterium planktonic cells, cosmetics or cleaning supplies, or it is used to prepare inhibition and/or the killing cariogenic bacterium in oral cavity
Drug, food, the application in cosmetics or cleaning supplies.
The compound of the present invention has the antibacterial and bactericidal activity of wide spectrum to oral bacteria.Especially to actinomyces viscosus,
The minimal inhibitory concentration of mutans streptococcus and Streptococcus sanguis is down to 0.25~2 μ g/mL, and minimum bactericidal concentration is down to 0.5~4 μ g/mL.It is excellent
Selection of land, the oral bacteria are selected from actinomyces viscosus, mutans streptococcus and Streptococcus sanguis.
In the sixth aspect of the present invention, the present invention also provides formula (I) compound or its isomers, solvate, prodrugs
And its pharmaceutically acceptable salt or using its as the pharmaceutical composition of active constituent in preparation for inhibiting caused by mutans streptococcus raw
Drug that object film is formed, the application in cosmetics or cleaning supplies prepare medicine for removing biomembrane caused by mutans streptococcus
Application in object, cosmetics or cleaning supplies.
The MBIC value of the compounds of this invention is 100% in 2 μ g/mL, the inhibiting rate to mutans streptococcus biofilm.Its
MBEC50Value reduces at least 50% in 16 μ g/mL, to established mutans streptococcus biofilm, and it is raw that mutans streptococcus maturation is effectively reduced
The growth vigor of object envelope.
In the seventh aspect of the present invention, the present invention also provides formula (I) compound or its isomers, solvate, prodrugs
And its pharmaceutically acceptable salt or using its be the pharmaceutical composition of active constituent preparation oral cavity mutans streptococcus bioflm inhibiting agents
In application.
And in the eighth aspect of the present invention, the present invention provides above compound or its isomers, solvate, preceding
Medicine and its pharmaceutically acceptable salt are resisted in dental caries pharmaceutical preparation using it as the pharmaceutical composition of active constituent in preparation
Using.
The present invention determines formula (I) compound to the inhibitory effect of cariogenic bacteria.With DMSO by formula (I) chemical combination when test
Object dissolution is made into the mother liquor storage of final concentration of 1024mg/mL.
Test result shows, the antibacterial and bactericidal activity to oral bacteria with wide spectrum of the invention.Especially to viscosity
The minimal inhibitory concentration of actinomyces, mutans streptococcus and Streptococcus sanguis is down to 0.25~2 μ g/mL, and minimum bactericidal concentration is down to 0.5~4 μ
g/mL.The MBIC value of the compounds of this invention is 100% in 2 μ g/mL, the inhibiting rate to mutans streptococcus biofilm.Of the present inventionization
Close the MBEC of object50Value reduces at least 50% in 16 μ g/mL, to established mutans streptococcus biofilm, and mutans streptococcus is effectively reduced
The growth vigor of mature biofilm.The compound of the present invention is at MIC (2 μ g/mL) and 1/2MIC (1 μ g/mL) to mutans streptococcus
Production acid have significant inhibiting effect.In addition, the compound is on oral cavity normal bacteria without influence, energy selectively acting and biological quilt
Cariogenic bacteria in film.
The invention has the following advantages:
The compound of the present invention molecular weight is small, structure is relatively easy, and bacteriostatic experiment confirms there is strong inhibition capability, fragmentation effect
The features such as good, can significantly inhibit the formation of mutans streptococcus biomembrane, reduce the growth vigor of mutans streptococcus biofilm, and press down
The production acid of mutans streptococcus processed is expected to be developed into the drug of prevention and treatment dental caries.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows influence of the compounds of this invention to mutans streptococcus acid producing ability.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part or according to the normal condition proposed by manufacturer.
Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art
Justice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
The preparation of 1 formula of embodiment (I) compound
Synthetic route are as follows:
Reagent used in synthetic route and reaction condition: (a) EDCI, DMAP, anhydrous n,N-Dimethylformamide, room
Temperature；(b) HCl/ ethyl acetate saturated solution, room temperature, 15min；(c) potassium carbonate, dioxane/water (V:V=1:1), room temperature,
30min。
Specific preparation process includes the following steps:
(1) preparation of (3- ((5- nitrothiazole -2- base) carbamoyl) phenyl) t-butyl carbamate (intermediate 1)
It is anhydrous that 2- amino -5- nitrothiazole (1mmol) and 3- (Boc- amino) benzoic acid (1.2mmol) are dissolved in 12mL
In n,N-Dimethylformamide, then it is separately added into EDCI (2mmol) and DMAP (2mmol), reacts 12h, Xiang Fanying at normal temperature
In liquid plus the ethyl acetate of 30mL dilutes, and is washed 2 times (2 × 30mL) with the HCL aqueous solution of 1mol/L, saturated sodium bicarbonate solution is washed
2 times (1 × 30mL), then 1 time (1 × 30mL) is washed with saturated common salt, organic phase is dry with anhydrous magnesium sulfate, it is filtered under diminished pressure,
Then it is concentrated by evaporation to obtain yellow solid 0.94g, yield 94% with Rotary Evaporators.
(2) preparation of 3- amino-N- (5- nitrothiazole -2- base) benzamide hydrochloride salt (intermediate 2)
Chloroacetic chloride is slowly instilled in dehydrated alcohol (V:V=4:5), the HCl of generation, which is passed through in ethyl acetate, is made HCl/
Ethyl acetate saturated solution, then previous step intermediate 1 is dissolved in wherein, it stirs 15 minutes, has been reacted with TLC detection at normal temperature
Entirely, solvent evaporated, obtains yellow solid, and crude product yield 100% is unprocessed directly to carry out in next step.
(3) 3- ((4- chlorphenyl) sulfonamido)-N- (5- nitrothiazole -2- base) benzamide (formula (I) compound)
Preparation
At room temperature, intermediate 2 (1mmol) and potassium carbonate (3mmol) are dissolved in 8mL dioxane aqueous solution
In, it adds parachloroben-zenesulfonyl chloride (1.2mmol), stirs 30min, with TLC monitoring reaction to raw material fully reacting.Steam dioxy
Six rings, into reaction solution plus the distilled water of 25mL, water phase is extracted with ethyl acetate 3 times (3 × 25mL), merges organic phase, with full
It is washed 1 time (1 × 25mL) with sodium chloride solution, anhydrous magnesium sulfate is dry, is filtered under diminished pressure, then obtains crude product through vacuum evaporation.
Crude product is through silica gel chromatographic column (200~300 mesh) purifies and separates, and methylene chloride: methanol=240:1 obtains yellow as eluant, eluent
Solid target compound, yield 45%.1H NMR(400MHz,DMSO-d6): δ 13.65 (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]-.
The antibacterial activity test of 2. formula of embodiment (I) compound
1, the preparation of mutans streptococcus UA159 bacterial strain (S.mutans UA159) and formula (I) compound
Mutans streptococcus UA159 bacterial strain used in the present invention be type strain, ncbi database (http: //
Www.ncbi.nlm.nih.gov/ the reference gene group # in) is NC_004350.Mutans streptococcus UA246 used in the present invention
Bacterial strain is the clinical strains being isolated from the oral cavity with saprodontia patient.Preferred brain heart infusion (the Brain of its most suitable culture medium
Heart Infusion) culture medium (Brain infusion solids 12.5g/L, Beef heart infusion
Solids 5.0g/L, Proteose peptone 10.0g/L, Glucose 2.0g/L, Sodium chloride 5.0g/L,
Di-sodium phosphate 2.5g/L, pH 7.4 ± 0.2), training is stood under 37 DEG C, the optimum of Anaerobic culturel
It supports.
(1) culture medium for cultivating mutans streptococcus is brain heart infusion (Brain Heart Infusion) culture medium (brand
OXOID, article No. CM1135), culture medium main component is Brain infusion solids 12.5g/L, Beef heart
Infusion solids 5.0g/L, Proteose peptone 10.0g/L, Glucose 2.0g/L, Sodium
Chloride 5.0g/L, Di-sodium phosphate 2.5g/L, pH 7.4 ± 0.2.If you need to be configured to solid, need to add
Add agar powder 15g/L.115 DEG C of sterilizing 30min, it is stand-by after cooling.
(2) culture medium for cultivating mutans streptococcus biofilm is brain heart infusion-sucrose culture medium, i.e., in brain-heart infusion medium
The sucrose of middle addition final concentration of 1%.Sucrose need to be made into 20% storage liquid and in advance with 0.22 μm of sterile filters filtration sterilization.
(3) use aseptic inoculation ring by mutans streptococcus type strain UA159 flat containing brain heart infusion agar solid medium
It crosses on plate, is inverted in culture in 37 DEG C of anaerobic culture box until there is apparent single colonie.
(4) with sterile inoculation shovel scraping mutans streptococcus UA159, it is transferred to the examination equipped with brain heart infusion fluid nutrient medium respectively
Guan Zhong is stood in 37 DEG C of anaerobic culture box, culture to liquid muddiness.
(5) absorbance value (OD with ultraviolet-uisible spectrophotometer detection mutans streptococcus at 600nm600nm)。
(6) assay balance accurate weighing formula (I) compound is used, DMSO is added and is dissolved, is then 0.22 μm with aperture
Sterile filters filtration sterilization, be made into the storage liquid of final concentration of 1024mg/L, deposit in -20 DEG C it is stand-by.
2, experimental method
(1) the mutans streptococcus UA159 (OD of logarithmic phase processed will be cultivated600nm=0.8~1.0) it is diluted to brain-heart infusion medium
Final concentration of 5 × 105CFU/mL is stand-by.
(2) using micro broth dilution method detection formula (I) compound to the minimal inhibitory concentration of mutans streptococcus UA159.It will
Formula (I) compound solution of various concentration is added separately in 96 sterile orifice plates after doubling dilution, the 1st to the 11st Kong Weijia medical fluid
Experimental group, the 12nd hole is that not dosing is used as growth control group, mutans streptococcus bacterium solution final concentration of 5 × 10 in each hole5CFU/mL,
At this point, the 1st hole to the 12nd hole drug concentration is respectively 256,128,64,32,16,8,4,2,1,0.5,0.25,0mg/L.With
The minimum concentration of complete inhibition bacterial growth positions minimal inhibitory concentration (MIC) in aperture.
(3) by after on the bacterium solution even spread to brain heart infusion agar solid medium in aperture, in 37 DEG C of Anaerobic culturels
It is inverted culture 24 hours in case, minimum bactericidal concentration (MBC) is positioned with the minimum concentration of no bacterium production, the above experiment is effective
It is repeated 3 times, experimental result is as shown in table 1.
It is (viscous to S.sanguis ATCC 49295 (Streptococcus sanguis), A.viscosus ATCC27044 according to the method described above
Property actinomyces), S.sobrinus ATCC 33478 (remote source streptococcus), S.salivarius ATCC7073 (streptococcus salivarius)
It is tested under the same conditions with S.mitis ATCC 6249 (streptococcus mitis) several strains, experimental result is listed in table 1
In.
Activity determination (unit μ g/mL) of 1. formula of table (I) compound to mutans streptococcus UA159 planktonic cells
MIC: minimal inhibitory concentration；MBC: minimum bactericidal concentration.
As it can be seen from table 1 formula (I) compound has good bacteriostatic activity and killing activity to cariogenic bacteria, with
The antibacterial activity of positive control drug chlorohexidene is suitable.
3. formula of embodiment (I) compound tests the effect of the normal bacterium in oral cavity
Experimental procedure with embodiment 2, test compound to normal oral bacterium L.delbrueckiiCICC6032,
The minimal inhibitory concentration (MIC) and minimum bactericidal concentration of V.denticariosi KQ-ETV-9, V.rogosae WZH3n
(MBC).The results are shown in Table 2.
Antibacterial, bactericidal activity result (unit μ g/mL) of 2. formula of table (I) compound to normal oral bacterium
From table 2 it can be seen that formula (I) compound does not influence oral cavity normal bacteria, than positive control drug chlorohexidene
Influence also wants small.
The Inhibition test that embodiment 4. formula (I) compound forms mutans streptococcus biofilm
(1) prepare mutans streptococcus bacterium solution and formula (I) compound according to method described in embodiment 2, with brain heart infusion-sucrose
Mutans streptococcus in logarithmic phase is diluted to final concentration of 1 × 10 by culture medium7CFU/mL is stand-by.
(2) the 100 μ L of bacterium solution in (1) is added into 96 sterile orifice plates, and the hole formula (I) compound is added is as in fact
Group is tested, the hole of formula (I) compound is not added as a control group.It is put in 37 DEG C of anaerobic culture boxes stationary culture for 24 hours.
(3) planktonic cells in each hole are removed, and rinses unadsorbed cell with a large amount of water.
(4) 0.1% 200 μ L of crystal violet solution is added into each hole, stands 5min at room temperature and is contaminated
Then color removes crystal violet solution, and is rinsed out with a large amount of water and remove unadsorbed crystal violet.
(5) crystal violet of 33% acetic acid solution 200uL dissolution absorption is added into each hole, is then examined using microplate reader
The absorbance value under 590nm is surveyed, the above experiment is effectively repeated 3 times.The inhibiting rate of biofilm is calculated, calculation formula is inhibiting rate
=(1- experimental group/growth control group) × 100%.
The MBIC value of formula (I) compound is 2 μ g/mL as the result is shown, and the inhibiting rate to mutans streptococcus biofilm is 100%.
5. formula of embodiment (I) compound is tested to the effect of mutans streptococcus maturation biofilm is removed
(1) prepare mutans streptococcus bacterium solution and formula (I) compound according to method described in embodiment 2, with brain heart infusion-sucrose
Mutans streptococcus in logarithmic phase is diluted to final concentration of 1 × 10 by culture medium7CFU/mL is stand-by.
(2) the 100 μ L of bacterium solution in (1) is added into 96 sterile orifice plates, 37 DEG C of Anaerobic culturels make mutans streptococcus form life for 24 hours
Object envelope.
(3) after forming biofilm, supernatant is abandoned, 2~3min is placed after 0.85% NaCl of 150~200 μ L is added, abandons
Waste liquid is repeated 3 times.
(4) after abandoning waste liquid, the second hole to a last hole sequentially adds the fresh BHI culture medium of 100uL, and phase is added in the first hole
After answering the 200 μ L of formula (I) compound (256 μ g/mL) of concentration, the first hole to mix, take 100 μ L that the second hole is added from the first hole, according to
Secondary to be added to hole second from the bottom, last hole is not added, and 37 DEG C of anaerobic culture box cultures are for 24 hours.
(5) repetitive operation step (3).
(6) the fresh BHI culture medium of 90 μ L is added in every hole, and every hole is added 10 μ L MTT (concentration 5mg/mL), uses masking foil
It wraps, 37 DEG C are protected from light 2~4h of incubation.
(7) abandon supernatant, after wash 3 times with 0.85%NaCl, 200 μ L DMSO be added, 37 DEG C of incubators incubations 10~
15min takes 50 μ L to be added in 150 μ L DMSO, measures its light absorption value at 490nm, the above experiment effectively weight with microplate reader
It is 3 times multiple.
The MBEC of formula (I) compound as the result is shown50Value is 16 μ g/mL, is reduced to established mutans streptococcus biofilm
At least 50%, significantly reduce the growth vigor of mutans streptococcus maturation biofilm.
Inhibition test of 6. formula of embodiment (I) compound to mutans streptococcus acid producing ability
(1) prepare mutans streptococcus bacterium solution and formula (I) compound according to method described in embodiment 2, with brain heart infusion-sucrose
Mutans streptococcus in logarithmic phase is diluted to final concentration of 5 × 10 by culture medium5CFU/mL is stand-by.
(2) MIC value 2 concentration (1 and 1/ below is added to the MIC measurement result of mutans streptococcus according to formula (I) compound
2MIC), control group is the culture medium without formula (I) compound, every group of 3 parts of formula.
(3) it is put in stationary culture 0 in 37 DEG C of anaerobic culture boxes, 6,12, for 24 hours, Aspirate supernatant is monitored using accurate pH meter
Decline, results are averaged, and experimental result is as shown in Figure 1.
It will be seen from figure 1 that compared with the control of the formula that is not added (I) compound, formula (I) compound in MIC (2 μ g/mL) and
There is significant inhibiting effect to the production acid of mutans streptococcus under 1/2MIC (1 μ g/mL).
Claims (10)
1. compound shown in formula (I) or its isomers, solvate, prodrug and its pharmaceutically acceptable salt:
2. a kind of method for preparing formula (I) compound, which comprises (1) 2- amino -5- nitrothiazole and 3- (Boc- ammonia
Base) benzoic acid and 2- amino -5- nitrothiazole be that initial reactant prepares (3- ((5- nitrothiazole -2- base) carbamoyl)
Phenyl) t-butyl carbamate (intermediate 1)；(2) intermediate 1 obtains 3- amino-N- (5- nitrothiazole-after de- Boc reaction
2- yl) benzamide hydrochloride salt (intermediate 2)；(3) intermediate 2 is reacted with parachloroben-zenesulfonyl chloride generates formula (I) compound.
3. according to the method described in claim 2, it is characterized in that, the reaction of step (1) need to be in solvent, condensing agent and activating agent
In the presence of carry out；
Preferably, in step (1), 2- amino -5- nitrothiazole, 3- (Boc- amino) benzoic acid, condensing agent, activator mole
Than for 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 DEG C, preferably 30 DEG C；
Preferably, the solvent be selected from anhydrous n,N-Dimethylformamide, tetrahydrofuran, acetonitrile, chloroform, methylene chloride,
One of 1,4- dioxane is a variety of；It is preferred that anhydrous methylene chloride and DMF；
Preferably, the dosage of the solvent is the 2- amino -5- nitrothiazole of every mM of 10~15mL；
Preferably, the condensing agent is in DCC, DIC, EDCI, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, PyBOP
It is one or more；Preferably EDCI and/or HATU；
Preferably, the activator be selected from one of DMAP, HOBt, 4-PPY, HOAT, HOSu, NHPI, NHNI, PFPOH or
It is a variety of；Preferably DMAP.
4. according to the method described in claim 2, it is characterized in that, step (2) the de- Boc reaction is in an acidic solution in room
Temperature is lower to be carried out, and the acid solution is selected from one of HCl/ ethyl acetate solution, HCl/ methanol solution, trifluoroacetic acid or more
Kind；Preferably HCl/ ethyl acetate solution；
Preferably, the acid solution dosage is the intermediate 1 of every mM of 15-20mL.
5. described to tie up acid according to the method described in claim 2, it is characterized in that, step (3) carries out in the presence of acid binding agent
Agent is inorganic base；
Preferably, the inorganic base is selected from one of potassium carbonate, sodium carbonate, sodium bicarbonate or a variety of, preferably potassium carbonate；
Preferably, in step (3), intermediate 2, which is dissolved in solvent, participates in reaction, and the solvent is solvent/water system, the system
Selected from one of dioxane/water solution, tetrahydrofuran/aqueous solution, acetone/water solution or a variety of；Preferably dioxane/
Aqueous solution；
Preferably, in the solvent/water system, the volume ratio of solvent and water is 1:1-3；
Preferably, the dosage of the solvent/water system is the intermediate 2 of every mM of 8-12mL；
Preferably, in step (3), the molar ratio of intermediate 2, acid binding agent and parachloroben-zenesulfonyl chloride is 1:2-4:1-1.2, preferably 1:
3:1.2.
6. composition, it includes formula described in claim 1 (I) compound or its isomers, solvate, prodrug and its pharmacy
Upper acceptable salt；
Preferably, the composition may include pharmaceutically acceptable carrier, auxiliary material and/or excipient.
7. pharmaceutical preparation, cosmetics or cleaning supplies, it includes formula described in claim 1 (I) compound or its isomers, molten
Agent compound, prodrug and its pharmaceutically acceptable salt and acceptable carrier, auxiliary material and/or figuration pharmaceutically or on cosmetics
Agent；
Preferably, the pharmaceutical preparation is solid pharmaceutical preparation, external preparation, spray or liquid preparation；
Preferably, the cosmetics or cleaning supplies are toothpaste, mouthwash or thimerosal.
8. formula (I) compound or its isomers, solvate, prodrug and its pharmaceutically acceptable salt or using it as active constituent
Pharmaceutical composition preparing drug, cosmetics or cleaning supplies for inhibiting and/or killing the planktonic cells of oral bacteria
In application.
9. formula (I) compound or its isomers, solvate, prodrug and its pharmaceutically acceptable salt or using it as active constituent
Pharmaceutical composition preparing answering in drug, cosmetics or cleaning supplies for inhibiting biofilm formation caused by mutans streptococcus
With or prepare application in drug, cosmetics or cleaning supplies for removing biomembrane caused by mutans streptococcus.
10. formula (I) compound or its isomers, solvate, prodrug and its pharmaceutically acceptable salt or with its for activity at
The pharmaceutical composition divided resists the application in dental caries pharmaceutical preparation in preparation oral cavity mutans streptococcus bioflm inhibiting agents or preparation.
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