CN103040826B - Preparation and medical application of substituted pyranone compound - Google Patents

Abstract

The invention relates to preparation and medical application of a substituted pyranone compound and particularly discloses a substituted pyranone compound 1-[6-(2-bromo-4,5-dimethoxyphenyl)-2-oxo-2H-pyran-3-yl-methyl]-piperidine-3-carboxylic acid amide or pharmaceutically acceptable salts thereof and a preparation method and medicinal application thereof. The in-vitro fungal growth inhibition activity of the compound is determined through a micro-dosage liquid-based dilution method; and shown by experimental results, the 80% inhibition concentration of the substituted pyranone compound to the growth inhibition of candida albicans is 250 micrograms/milliliter. Shown by pharmacodynamic results, the compound or pharmaceutically acceptable salts thereof can be expected to be applied to drugs for prevention and treatment of diseases caused by fungal infection.

Description

The preparation of a substituted pyrane ketonic compound and medicinal usage thereof

Technical field

The present invention relates to medical technical field, particularly, the present invention relates to substituted pyrane ketone compounds 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl with antifungal activity]-the purposes of piperidines-3-amine carboxylic acid in preparing anti-fungal infection medicine.

Background technology

Along with medical science applied development, various novel drugs and new technique are widely used as the medical procedure such as broad ectrum antibiotic, hormone, various intubation catheter technology, chemotherapy, transplanting, and HIV infects and tumor, the case increasing year by year that causes human immunity system to be destroyed gradually, thus cause the sickness rate of deep mycosis more and more high; And on the other hand, along with being widely used of mankind's abuse of antibiotics and clinical antifungal drug, cause traditional antibiotic can not effectively resist that some serious fungal infection and antibacterial infect, the drug resistance phenomenon of fungus is day by day serious.The antibiotic effect of tradition has met with remarkable bottleneck, and most of antibacterials, fungus have produced significant Drug resistance to common antibiotic; The concurrent fungal infections such as HIV sufferers, patient with severe symptoms, fire victim more make sufferer hang by a hair.Therefore, searching wide spectrum, antifungal new drug efficient, low toxicity have become the focus of drug research; Research to fastbacteria/intractable fungus is extremely urgent.

The disease that fungal infection causes can be divided into following four classes substantially: studies of invasive fungal infections and systemic mycosis (as aspergillosis and candidiasis etc.), mucosal pattern mycosis (as " thrush " etc.), shallow phenotype dermatomycosis (as " tinea pedis " or " tinea capitis ", " tinea unguium " etc.) and anaphylactic type mycosis (as asthma and chronic inflammatory disease etc.).In above four class mycosises, with the first kind, the mankind are endangered to maximum, then three class mycosises are corresponding light.According to the clinical statistics data from external, in dying from the crowd of infectious disease, have 4% to be to die from general aspergillosis mushroom fungal infection, about 2% people dies from general monilial infection.Clinical statistics data shows, once patient suffers from general aspergillosis, its mortality rate is up to 85%, as suffer from blood candidiasis its mortality rate can reach 40%.Utilize existing antifungal drug treatment systemic mycosis or the mycotic effect of blood infection type so far still cannot be satisfactory.Therefore, pharmacy circle is being found the newtype drug that can suppress systemic infection.

At present, the antifungal drug of having developed listing both at home and abroad mainly contains four large classes, i.e. echinocandin (echinocandins) class (as Caspofungin and meter Ka Min etc.) of polyenoid class (as amphotericin B), triazole type (as fluconazol, miconazole, econazole, Itraconazole and ketoconazole etc.), alkyl amine (as terbinafine) and listing newly developed.Front three major types antifungal agent is the old medicine of 20th century exploitation listing, and echinocandin class is the new drug of exploitation listing at the beginning of 21 century.But in said medicine, only have a few can be used for the treatment of systemic fungal infection disease.Antifungal drug in triazole class is the first generation or the second filial generation is all to there being the fungus-caused systemic infection unsatisfactory curative effect of silk no matter, and echinocandin class antifungal new drug is effective to this class disease.The greatest problem that echinocandin faces is at present: there is no peroral dosage form, be only limited to injection (and only limiting to hospital's use), therefore patient uses rather inconvenience.Secondly, although echinocandin is pretty good to the sick fungal infection curative effect that waits of Candida spp, it is expensive, so limited the sales volume in they markets beyond developed country.As it is reported, in China market, the retail price of every Caspofungin injection (dosage is 50 milligrams) is 3148 yuan, and the meter Ka Min price that Japan produces is slightly lower, but every (50 milligrams of dosage) also wants 650 yuan.China's hospital clinical at present the most frequently used general antifungal drug is still fluconazol and terbinafine.

The health azole of old kind is as the onset by lanosterol 14 demethylases in Antifungi ergosterol biosynthesis such as fluconazol, itraconazole, voriconazole, posaconazole, and this type of old brand triazole antifungal agent thing antimicrobial spectrum is compared with narrow and drug resistance grows with each passing day.Though new product antimicrobial spectrum expands, metabolisming property and physicochemical property are not good, and water solublity is low, bioavailability is poor; Needs of patients high fat diet is in order to drug absorption, or take the extraordinary dosage form of high price can onset; For increasing cyclodextrin that water solublity adds etc., also to renal insufficiency person, bring larger threat.

In sum, finding as early as possible new structure, differ from the antifungal lead compound of medicine in the past, is that many decades medicine workers need the promptly task of top priority of development from now on.In order to explore this field, the inventor is according to a large amount of literature surveies, structure activity relationship models coupling 3D-SAR evaluation work (Tripos database software) in conjunction with flood tide, design a class substituted pyrane ketonic compound, to finding the effectively lead compound of Antifungi growth, thereby even be developed further into the new medicinal products with energy Antifungi growth killing fungus.We synthesize the compound of preparing design thus, and have tested its growth inhibited effect to fungus.Found that formula (1) compound 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid has significant antifungal activity, completes thus the present invention.

Summary of the invention

The object of this invention is to provide a kind of structure substituted pyrane ketonic compound as the formula (1) for the preparation of the purposes of antifungal drug:

The present invention also provides the method for a kind of preparation formula (1) compound:

Wherein, OMe refers to methoxyl group; OEt refers to ethyoxyl.The name of formula (1) compound is called 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid.Synthesis condition and reagent: a) iodomethane, sodium metal, ethanol; B) Veratraldehyde, sodium hydride, n-butyl lithium, oxolane; C) sodium borohydride, methanol; D) Lithium hydrate (1 mol/L), ethanol; E) hydrochloric acid, water; F) dicyclohexylcarbodiimide, pyridine, dichloromethane; G) mesyl chloride, triethylamine, dichloromethane; H) 1,8-diazacyclo [5,4,0] 11 carbon-7-alkene (DBU), toluene; I) N-bromosuccinimide (NBS), azo-bis-isobutyl cyanide, carbon tetrachloride.

Another object of the present invention is to provide the substituted pyrane ketonic compound shown in formula (1) or the application of its officinal salt in the medicine of preparation prevention and treatment candida albicans infection disease.

Another object of the present invention is to provide the drug excipient or the carrier that contain the substituted pyrane ketonic compound shown in formula (1) and pharmaceutically useful salt and preparation permission and is prepared into pharmaceutical composition, and prepared pharmaceutical composition can also contain other antibacteriums and/or antifungal drug.Described medicine can be made multiple dosage form by means known in the art, comprises varnish, membrane, unguentum, injection, transdermal patch, aerosol, controlled release or slow releasing agent, and nanometer formulation.

Through the detailed Literature Consult of the inventor, up to the present, there is no relevant this compounds for the preparation of the report of antifungal drug.This substituted pyrane ketonic compound belongs to beyond thought discovery for the potent inhibition of conk, has definite originality, completes accordingly the present invention.

Usefulness of the present invention is: the substituted pyrane ketonic compound shown in discoverable type (1) has the patent medicine potentiality that Antifungi is grown, prepared anti-fungal infection aspect first, for exploitation becomes treatment fungal infection original new drug, provides new material base.There is potential huge Social benefit and economic benefit.

The specific embodiment

Below by embodiment, further illustrate the present invention.Embodiment has provided formula (1) compound 1-[6-, and (2-bromo-4,5-Dimethoxyphenyl)-2-oxygen-2H-pyrans-3-base-methyl] preparation method and the part physicochemical data of-piperidines-3-amine carboxylic acid, and the part pharmacologically active data of this substituted pyrane ketonic compound Antifungi growth.The following embodiment of mandatory declaration is for the present invention rather than limitation of the present invention are described.The simple modifications that essence according to the present invention is carried out the present invention all belongs to the scope of protection of present invention.

embodiment 1:1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl] preparation of-piperidines-3-amine carboxylic acid

1.1 instruments and reagent

Micro-meldometer for fusing point (production of Beijing Imtech) is measured, and temperature is not proofreaied and correct; Optically-active is produced on Polax-2L type automatic polarimeter and is measured in Japan; Infrared spectrum IR is by Bruker Vector-22 determination of infrared spectroscopy, through KBr tabletting; Ultraviolet spectra is measured with Shimadzu UV-240 ultraviolet spectrophotometer; Proton nmr spectra ¹h NMR, carbon-13 nmr spectra ¹³c NMR and 2D NMR measure (tetramethylsilane ether TMS is interior mark) by INOVA type NMR spectrometer with superconducting magnet (VARIAN INOVA-400MHz); Electrospray Mass Spectrometry ESI-MS is measured by Bruker Esquire 3000+ mass spectrograph, silica gel for column chromatography (100-200,200-300 and 300-400 order) and for thin layer chromatography silica GF254 (10-40 order) be Haiyang Chemical Plant, Qingdao's product; Agents useful for same is analytical pure, and wherein petroleum ether boiling range is 60-90 ° of C; The aluminium foil silica gel plate of Merck company for thin layer preparative chromatography (PTLC); Column chromatography adopts Sweden AmershamPharmacia Biotech AB company product with polydextran gel Sephadex LH-20; Thin plate (TLC) is surveyed the uviol lamp with 254nm and 365nm; Iodine vapor, 10% sulphuric acid-ethanol, phosphorus molybdenum acid solution for developer.

1.2 synthetic and purifications

1.2.1 key intermediate VIII's is synthetic: by ethyl acetoacetate (I), be starting material, through 9 step reactions (a-i), final purification obtains key intermediate compound VI II.

A) preparation of 2-methyl-acetoacetic ester (Compound I I):

In 300 milliliters of ethanol, add 13.8 grams of sodium metals (600 mMs), after dissolving completely, drip 80 milliliters of ethyl acetoacetates, heat up and reflux 1 hour, drip 50 milliliters of iodomethane, reflux two hours, be cooled to room temperature, rotary evaporation is removed ethanol, add extracted with diethyl ether for water (5 * 50 milliliters), merge organic facies, anhydrous sodium sulfate drying, sucking filtration, rotary evaporation is removed ether, distilling under reduced pressure, collect 78-80 ℃ of (13mmHg) fraction, can obtain the colourless transparent liquid of Compound I I.B) preparation of 2-methyl-3-oxygen-5-hydroxyl-5-(3,4-Dimethoxyphenyl)-ethyl valerate (compound III):

Cryosel is bathed under cooling condition, in the oxolane suspension (140 milliliters) of 2.3 grams of sodium hydrides, drip 5.78 grams of 2-methyl-acetoacetic esters, then drip 28 milliliters of the hexane solutions of 2.5M n-BuLi, after ten minutes, drip 3, the tetrahydrofuran solution that 4-dimethoxy benzaldehyde is 5.88 grams (10 milliliters), continue reaction 15 minutes, in reaction system, add saturated aqueous ammonium chloride (30 milliliters), with ether (3 * 50 milliliters), extract, merge organic facies, with saturated common salt water washing (3 * 20 milliliters), anhydrous sodium sulfate drying, sucking filtration, silica gel column chromatography (petroleum ether: ethyl acetate=2: 1) obtain yellow oily liquid, R _f(ethyl acetate: petroleum ether=1: 1)=0.59, is compound III.C) 2-methyl-3, the preparation of 5-dihydroxy-5-(3,4-Dimethoxyphenyl)-ethyl valerate (compound IV):

In ice-water bath, 3.06 grams of compound III are dissolved in oxolane (67.5 milliliters) and methanol (27 milliliters), slowly add 1.04 grams of sodium borohydrides, react 30 minutes.Add 30 milliliters of saturated aqueous ammonium chlorides, extracted with diethyl ether (3 * 40 milliliters), merge organic facies, saturated common salt water washing, anhydrous sodium sulfate drying, rotary evaporation is removed ether, obtains 3.2 grams of crude products, through silica gel column chromatography (petroleum ether: ethyl acetate=2: 1) obtain yellow oily liquid and be compound IV, R _f(ethyl acetate: petroleum ether=1: 1)=0.35.Proton nmr spectra ¹h NMR (400MHz, deuterochloroform): δ 1.22 (bimodal, J=7.2Hz, 3H, CHCH ₃), 1.31 (triplet, J=6.0Hz, 3H, CH ₂cH ₃), 1.65 – 1.93 (multiplet, 2H, H-4a, H-4b), 2.53 (multiplet, 1H, H-2), 3.82 (unimodal, 3H, 4 '-OMe), 3.88 (unimodal, 6H, 3 '-OMe), 4.17 (multiplet, 2H, CH ₂cH ₃), 4.27 (multiplet, 1H, H-3), 4.89 – 5.02 (multiplet, 1H, H-5), 6.81 – 6.93 (multiplet, 3H, H-2 ', H-5 ', H-6 '); ESI-MS:m/z 313[M+H] ⁺.

D/e) 2-dimethyl-3, the preparation of 5-dihydroxy-5-(3,4-Dimethoxyphenyl)-valeric acid (compound V):

3.1 grams of compound IV are dissolved in 72 milliliters of ethanol, add 10 milliliters of (1M) lithium hydroxide solutions to regulate the pH value of reaction system to 8-9 in batches.Ethanol is removed in decompression, adds 50 ml waters, and the mixture obtaining extracts by 30 milliliters of ethyl acetate.In water layer, add 15 milliliters of (1M) hydrochloric acid, be extracted with ethyl acetate (7 * 30 milliliters), the organic layer obtaining is through anhydrous sodium sulfate drying, and concentrating under reduced pressure obtains crude product, again through silica gel column chromatography (petroleum ether: ethyl acetate=1: 1) obtain yellow oily liquid, be compound V.Product structure process ¹h NMR, ESI-MS identifies.Proton nmr spectra ¹h NMR (400MHz, deuterochloroform): δ 1.22 (bimodal, J=7.2Hz, 3H, CHCH ₃), 1.68 – 1.87 (multiplet, 2H, H-4a, H-4b), 2.65 (multiplet, 1H, H-2), 3.82 is (unimodal, 3H, 4 '-OMe), 3.88 (unimodal, 6H, 3 '-OMe, 5 '-OMe), 4.30 (multiplet, 1H, H-3), 4.91 – 5.02 (multiplet, 1H, H-5), 6.57 (unimodal, 2H, H-2 ', H-6 '); ESI-MS:m/z 285[M+H] ⁺.

F) preparation of 3-methyl-4-hydroxyl-6-(3,4-Dimethoxyphenyl)-Pentamethylene oxide .-2-ketone (compound VI):

By 2.8, digest compound V and 1.9 grams of dicyclohexylcarbodiimides (DCC) are dissolved in 120 milliliters of dichloromethane, add 0.74 milliliter of pyridine, stirred overnight at room temperature, remove by filter the precipitation of generation, filtrate obtains crude product through concentrating under reduced pressure, again through silica gel column chromatography (petroleum ether: ethyl acetate=2: 1) obtain colourless jelly, be compound V, R _f(ethyl acetate: petroleum ether=1: 1)=0.34.Product structure process ¹h NMR, ESI-MS identifies.Proton nmr spectra ¹h NMR (400MHz, deuterochloroform): δ 1.41 (bimodal, 3H, J=7.2Hz, H-7), 2.11 (multiplet, 1H, H-3), 2.25 (multiplet, 2H, H-5a, H-5b), 3.88 (unimodal, 3H, 4 '-OMe), 3.89 (unimodal, 6H, 3 '-OMe), 4.28 (wide unimodal, 1H, H-4), 5.55 – 5.70 (multiplet, 1H, H-6), 6.81 – 6.93 (multiplets, 3H, H-2 ', H-5 ', H-6 '); ESI-MS:m/z 266[M] ⁺.

G/h) 3-methyl-6-(3,4-Dimethoxyphenyl)-5, the preparation of 6-dihydropyran-2-ketone (compound VI I):

837 milligrams of compound VI are dissolved in dichloromethane (43.5 milliliters), splash into 1.67 milliliters of triethylamines under ice-water bath condition, then slowly drip 0.872 milliliter of mesyl chloride.React 30 minutes.Add water washing (2 * 5 milliliters), saturated common salt water washing (2 * 5 milliliters), anhydrous sodium sulfate drying, concentrating under reduced pressure obtains crude product.Crude product is dissolved in to toluene (45 milliliters), under room temperature, stirs, slowly add 2.24 grams of DBU(14.7 mM), reaction is spent the night.Wash (2 * 10 milliliters) with water, saturated common salt water washing (2 * 5 milliliters), anhydrous sodium sulfate drying.Silica gel column chromatography (petroleum ether: ethyl acetate=2: 1) obtain the white needle-like crystals of compound VI I, R _f(ethyl acetate: petroleum ether=1: 1)=0.51.Fusing point m.p.115-116 ℃ (re-crystallizing in ethyl acetate).Product structure warp ¹h NMR and ESI-MS identify.Proton nmr spectra ¹h NMR (400MHz, deuterochloroform): δ 1.99 (unimodal, 3H, H-7), 2.49 – 2.71 (multiplet, 2H, H-5a, H-5b), 3.89 (unimodal, 3H, 4 '-OMe), 3.91 is (unimodal, 3H, 3 '-OMe), 5.36 (double doublets, J=12.4,3.6Hz, 1H, H-6), 6.67 (multiplet, 1H, H-4), 6.84 (bimodal, J=8.0Hz, 1H, H-5 '), 6.90 is (bimodal, J=8.0Hz, 1H, H-6 '), 6.97 (unimodal, 1H, H-2 '); ESI-MS:m/z 248[M] ⁺.

I) preparation of key intermediate 3-bromomethyl-6-(2-bromo-4,5-dimethoxy phenyl)-2H-pyran-2-one (compound VI II):

The mixture of 500 milligrams of compound VI I, 1.3 grams of NBS, 330 milligrams of azodiisobutyronitriles, 100 milliliters of carbon tetrachloride is stirred, temperature reaction was cooled to room temperature after 1.5 hours, add 30 ml waters, separatory takes off layer, and water layer is washed twice with 50 milliliters of chloroforms, merge organic facies with saturated common salt washing once, anhydrous sodium sulfate drying, sucking filtration, rotary evaporation is except desolventizing, silica gel column chromatography (chloroform: petroleum ether: 1: 1) obtains yellow solid, is compound VI II.R _f(ethyl acetate: petroleum ether=1: 3)=0.42,93～95 ℃ of fusing points (Gossypol recrystallized from chloroform).Product structure warp ¹h NMR and ESI-MS identify.Proton nmr spectra ¹h NMR (400MHz, deuterochloroform): δ 3.94 (unimodal, 3H, 4 '-OMe), 3.96 (unimodal, 3H, 5 '-OMe), 4.35 is (unimodal, 2H, H-7), 6.94 (bimodal, J=8.4Hz, 1H, H-5), 7.37 (unimodal, 1H, H-6 '), 7.54 (bimodal, J=8.4Hz, 1H, H-4), 7.66 (unimodal, 1H, H-3 '); ESI-MS:m/z 427[M+Na] ⁺.

1.2.2 by compound VI II, prepare 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid

20 milligrams of compound VI II are dissolved in 2 milliliters of acetonitrile liquid, and ice bath drips 3-formamido piperidines (19 milligrams) acetonitrile solution under stirring in this dry reaction bulb.Stir 120 minutes, concentrating under reduced pressure, adds 10 ml waters, with ethyl acetate extraction three times, and each 10 milliliters.The thick product of organic layer is again with silica gel column chromatography, petroleum ether-ethyl acetate (3:2) eluting, purification obtains 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid.Yellow powder, fusing point: 161-162 ℃ (chloroform); R _f(petroleum ether: ethyl acetate=1: 3)=0.25.

1.3 Structural Identification

IR (υ cm ^-1): 3370,3189,1715,1652,1511,1442; Proton nmr spectra ¹h NMR (400MHz, deuterochloroform, δ ppm): 1.73 (multiplet, 2H, H-4 "), 1.95 (multiplet, 2H, H-5 "), 2.20 (multiplet, 1H, H-6a "), 2.51 (multiplet; 1H, H-6b "), 2.56 (triplet, 1H, J=4.4Hz, H-3 "), 2.78 (multiplet, 1H; H-2a "), 2.98 (multiplet, 1H, H-2b "); 3.33 (bimodal, 1H, J=14.0Hz; H-7a), 3.47 (double doublet, 1H; J=14.0,0.8Hz, H-7b); 3.92 (unimodal, 3H, 4 '-OCH ₃), 3.94 (unimodal, 3H, 5 '-OCH ₃), 6.92 (bimodal, 1H, J=8.4Hz, H-5), 7.35 (bimodal, 1H, J=2.4Hz, H-6 '), 7.42 (unimodal, 1H, H-3 '), 7.52 (double doublet, 1H, J=8.4,2.4Hz, H-4); ESI-MS m/z:451[M] ⁺, 453[M+2] ⁺.Evaluation structure is as follows:

embodiment 2:the activity test of formula (1) compound Antifungi

The standardization antifungal sensitivity testing method proposing with reference to standard committee of American National clinical laboratory (NCCLS), the 1-[6-that test implementation example 1 prepares (2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl] extracorporeal antifungal activity of-piperidines-3-amine carboxylic acid.

2.1 fungus type strains:

Candida albicans ATCC10231: Chongqing Center for Disease Control (CDC) provides;

Candida albicans ATCC76615: attached Long March hospital of The 2nd Army Medical College provides.

2.2 reagent:

2.2.1 sabouraud's agar (sabouraud dextrose agar): Guangdong triumphant microorganism Science and Technology Ltd. product.

2.2.2 yeast extract (yeast extract): Hai Shenggong biotechnology Services Co., Ltd subpackage.

2.2.3 three morpholino nitrogen quinoline propane sulfonic acid (3-N-morpholinopropanesulfonicacid, MOPS).

2.2.4 testing drug: fluconazol (Fluconazole, FCZ), company of Yangzijiang Pharmaceutical Group; Formula (1) compound.The doubling dilution liquid that testing drug is configured to variable concentrations gradient is stand-by.

2.2.5 dimethyl sulfoxide (dimethylsulfoxide, DMSO) and dimethyl formamide (dimethylformamide, DMF): Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's subpackage product.

2.3 instruments:

2.3.1 electronic balance JA1203N(AB204-5, METTLER TOLEDO): Shanghai exact science instrument company product.

2.3.2SW-CJ-2FD the double one side clean work station of type: Purifying Equipment Co., Ltd., Suzhou's product.

2.3.3 water isolation type constant incubator (GSP-9160MBE): Shanghai Yiheng Scientific Instruments Co., Ltd's product.

2.3.4 electro-heating standing-temperature cultivator (HZQ-F160A): Shanghai Yiheng Scientific Instruments Co., Ltd's product.

2.3.5 U.S. Pedicellus et Pericarpium Trapae electric refrigerator (BCD-221CHC): Hefei Meiling Co. Ltd.'s product.

2.3.6 micropipettor (Proline Pipette, DragonMed Pipette): Finland Lei Bo company product.

2.3.7 cell counting count board (96well cell culture cluster): Shanghai refinement instrument company product.

2.3.8 ordinary optical microscope (Olympus): Japanese Olympus optical instrument Co., Ltd. product.

2.3.9 hot air oven (101A-2): Shanghai Chongming experimental apparatus company product.

2.3.10 vertical autoclave sterilizer (YXQ-LS-50S II): Shanghai Bo Xun Industrial Co., Ltd. product.

2.3.11 magnetic force heating stirrer (ARE): Italian VELP company product.

2.3.12 filter membrane, filter (0.22 μ m, Sartorius): Sartorius AG's product.

2.3.13 acidometer (PHSJ-4A): upper Nereid Ke Lei magnetic company product.

2.3.14 test tube oscillator (MS2): Guangzhou Yi Ke laboratory technique company product.

2.3.15 constant-temperature shaking incubator (THZ-18AB): Shanghai Yi Heng scientific instrument company product.

2.3.16 cryogenic refrigerator (20 ° of C, section dragon BCD-219WAK): Guangdong Ke Long electrical equipment Co., Ltd product.

2.3.1796 the flat microtest plate in hole: U.S. Corning Incorporated product.

2.4 experimental techniques:

2.4.1 experimental procedure:

2.4.1.1RPMI-1640 the preparation of liquid: get 10.4 grams of RPMI-1640 powder (containing L glutamine, not containing sodium bicarbonate, GIBCO) be dissolved in 900 ml sterile waters, adding 34.53 gram of three morpholino nitrogen quinoline propane sulfonic acid (MOPS) to its endpoint concentration is 0.165 mol/L, under room temperature, with magnetic stirring apparatus, mixes 2-3 hour, and it is fully dissolved, with sodium hydroxide (1 mol/L), regulate pH value to 7.0(25 ° of C), with aquesterilisa, be settled to 1 liter, Entkeimung, after subpackage ,-20 ° of C save backup.

2.4.1.2 the preparation of storing solution: storing solution concentration should be 10 times of application liquid, FCZ(fluconazol) be 1280 mcg/ml, with electronic balance, take FCZ10 milligram, with 1 milliliter of dimethyl formamide (DMF), dissolve, with RPMI-1640, be diluted to storage liquid concentration afterwards, after subpackage ,-70 ° of C save backup.

2.4.1.3 apply the preparation of liquid: with RPMI-1640 liquid, storing solution is done to after 1:10 dilutes, remake multiple proportions rare, FCZ is that 128 mcg/ml-0.25 mcg/ml are made 10 serial concentration, is 2 times of final concentrations.

2.4.1.4 the preparation of micro-susceptibility culture plate: use disposable 96 orifice plates, 1-10 row add respectively the application liquid of the testing drug of 10 Concentraton gradient, from high concentration to low concentration.The 11st row add RPMI-1640, every hole 100 microlitres, the 12nd row, as blank, are put into-20 ° of C refrigerators standby, during use through-4 ° of C, 4 ° of C and room temperature each 1 hour.

2.4.1.5 the activation of candidiasis and dilution: by bacterial strain to be measured at YPD Agar culture medium (1% yeast extract, 1% peptone, 2% glucose) upper activation after twice, cut-off footpath be greater than 1 millimeter bacterium colony a little in 3 milliliters of physiological saline solution, mix and make bacteria suspension, get a little, with the bacterial cell number in four large grids of hemocyte technology plate counting, the X that averages, now bacterial cell number is X * 10 ⁴cFU/ milliliter, with RPMI-1640 liquid, adjusting concentration is 3 * 10 ⁴cFU/ milliliter (twice final concentration).

2.4.1.6 application of sample: add candidiasis suspension on the above-mentioned culture plate that contains testing drug preparing, every hole 100 microlitre blanks do not add, and concussion is put into 35 ° of C of wet box (prevent evaporating of micro plate and affect the concentration of medicine) after mixing and hatched observed result after 24-48 hour.

2.4.1.7 naked eyes judged result: take growth control blank as foundation, (is MIC with 80% inhibition ₈₀) for observing terminal, growth obviously reduces, liquid is slightly muddy; Well-grown in positive growth simultaneously, blank is limpid, without bacterial growth.In the scope of Quality Control bacterial strain ZhiMICZhi U.S. Clinical microorganism laboratory standard CLSI M27-A2 scheme regulation, show that experimental result is effective.

2.5 experimental results: in Table 1.

Table 1 testing drug is to Candida albicans fungus inhibitory action (MIC ₈₀, unit: mcg/ml)

2.6 conclusions:

By adopting the extracorporeal antifungal activity of " Herbs By Broth Microdilution " Research-type (1) compound to two kinds of fungus reference cultures, experimental study shows: 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid has clear and definite antifungal activity to two kinds of candidiasis bacterial strains.Illustrate that it is potential Antifungi growth activity material, there is further exploitation and be worth.

Substituted pyrane ketonic compound shown in the formula preparing in the present invention (1) can be combined with pharmaceutically conventional adjuvant or carrier, prepares and has prevention and treat medicine and pharmaceutical composition or health product or the cosmetics of everyday use by fungus-caused infection such as Candida albicans.The dosage form that above-mentioned various kinds of drug compositions, health product or cosmetics of everyday use can adopt comprises varnish, membrane, unguentum, injection, transdermal patch, aerosol, controlled release or slow releasing agent, and nanometer formulation.

Substituted pyrane ketonic compound as the formula (1) of the present invention can also with medicine and the crude drug thereof of the treatment fungal infection associated conditions now having gone on the market, as: polyenoid class (as, amphotericin B), triazole type (as, fluconazol, miconazole, econazole, Itraconazole and ketoconazole etc.), alkyl amine (as, terbinafine) and the kind such as the echinocandin of listing newly developed (echinocandins) combine use, prepare compositions or the compound preparation with treatment fungal infection associated conditions effect activity, can expect and become treatment fungal infection disease medicine/health product or cosmetics of everyday use.Above-mentioned various kinds of drug compositions, medicine/health product or cosmetics of everyday use can adopt the dosage forms such as varnish, membrane, unguentum, injection, transdermal patch, aerosol, comprise the now conventional preparation of generally acknowledged pharmaceutics general knowledge of employing and various slow release, controlled release form or nanometer formulation.

When above-mentioned description elaboration is of the present invention, the object that embodiment is provided is simultaneously to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of within the scope of entering the claims in the present invention and its equivalent, practical application of the present invention comprises all general variations, cooperation, or improves.

Claims (1)

1. the substituted pyrane ketone compounds shown in a formula (1) is for the preparation of the medicinal usage of anti-fungal infection, the name of this compound is called: 1-[6-(2-bromo-4,5-dimethoxy phenyl)-2-oxygen-2H-pyrans-3-base-methyl]-piperidines-3-amine carboxylic acid;