CN100567299C - New antifungal compound, Preparation method and use - Google Patents
New antifungal compound, Preparation method and use Download PDFInfo
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- CN100567299C CN100567299C CNB2004100924434A CN200410092443A CN100567299C CN 100567299 C CN100567299 C CN 100567299C CN B2004100924434 A CNB2004100924434 A CN B2004100924434A CN 200410092443 A CN200410092443 A CN 200410092443A CN 100567299 C CN100567299 C CN 100567299C
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Abstract
The invention discloses the compound N CC00646 of (1) that has molecular formula and its pharmacologically acceptable salt, have anti-mycotic activity.The invention also discloses a kind of method for preparing compound N CC00646.The invention also discloses the composition that contains compound (1) and treat and/or prevent application in the fungi infestation medicine in preparation.
Description
Technical field
The invention relates to the new compound of anti-mycotic activity, Preparation method and use.Or rather, the invention discloses the compound of molecular formula (1), contain its medicinal compositions, utilize microbial fermentation to prepare the method for this compound, and the new microorganism of using and compound and medicinal compositions thereof treat and/or prevent purposes in the antifungal drug in preparation.
Background technology
After nineteen fifty, the research relevant with microbiotic is fast-developing and extensively universal, has developed the many medicines for various infectious diseases.On the other hand, occurred in recent years by candidiasis, aspergillus, the increase of the deep fungal that fungies such as cryptococcus cause and the phenomenon of seriousization, these phenomenons and leukemia, malignant lymphatic is swollen, and HIV infects the immunological incompetence that causes, the immunological competence that cancer therapy drug causes is low and a large amount of variation phenomenons of the bacterium that causes that use of microbiotic are in close relations.
Now, in the treatment of deep fungal, often use polyene macrolides, azole, flucytosine class medicine, a kind of amphotericin B of polyenoid class Macrocyclolactone lactone kind medicine for example, its anti-mycotic activity is strong, and good result of treatment is arranged.But because side effect such as its renal toxicity, pointed out to exist the problem (Gallis etc., Rev.Infect.Dis.Vol 12, p309～329,1990) of security, flucytosine class medicine also exists security low and have a problem of aspects such as resistant organism.
On the other hand, it is extensive that azole drug is considered to safety range, and side effect is low, but the problem of narrow antimicrobial spectrum is arranged, therefore low to the validity of serious illness, and be considered to resistance in increase.(Rex etc., Antimicrob.Agents Chemother Vol 39, p1～8,1995)
And cyclic peptide such as Micafungin and Caspofungin medicine (Denning, Lancet Vol362,9330 p1142～1151,2003) is developed in recent years, is used for the treatment of deep fungal infection.
Because above-mentioned present situation is arranged, the increase of the guaranteeing of variation, the security of the reply cause of disease, resistant organism, wishing can developing novel antifungal medicines.
Summary of the invention
Main purpose of the present invention provides a kind of new compound;
Another object of the present invention provides a kind of method for preparing this antifungal compound;
Further object of the present invention provides a kind of new pharmaceutical composition with anti-mycotic activity.
The present inventor, from the process of the new antifungal drug of screening of microbial metabolites, discovery belongs to the compound that has anti-mycotic activity in the fungal cultures that involves mould (Penicillum implicatum), then in order to determine active substance in the culture, carry out structure elucidation with the culture separation and purification and to compound, found that molecular formula (1) new compound has anti-mycotic activity, and finished the present invention on this basis.
The invention provides the new compound of molecular formula (1) with anti-mycotic activity, and the invention provides belonging to the culture of strains of Penicillium, from its meta-bolites, obtain the leaching process of the compound of formula (1) expression, the preparation method of formula (1) expression compound is provided.
The invention provides:
1. as the compound and the pharmaceutically useful salt thereof of molecular formula (1) expression;
2. the method for preparing above-claimed cpd, comprise cultivate a kind of can presentation (1) compound the Penicillium microorganism and from culture, extract the method for this compound of purifying;
3. produce the Penicillium microorganism of compound (1), involve mould (Penicillium implicatum) NCC00646 CGMCC 1079.
4. a pharmaceutical composition contains formula (1) compound as activeconstituents and pharmaceutical carrier.
5. aforementioned pharmaceutical compositions can be used for treating and/or preventing fungi infestation.
The compound of molecular formula of the present invention (1) expression has good anti-mycotic activity as fungi infestation treatment of diseases and/or prophylactic agent, very has application prospect.
The new compound (the following compound (1) that slightly is called) of above-mentioned molecular formula of the present invention (1) expression has the physical and chemical character of note down.
(1) color and outward appearance: colourless powder
(2) molecular formula: C
49H
79N
7O
16
(3) mass spectrum: (HRFAB-MS)
Observed value: m/z 1022.5658 [M+H]
+
Theoretical value: m/z 1022.5662[C
49H
80N
7O
16]
+
(4) relative specific rotation: [α]
D=-36.2 ° (c 0.8, MeOH, 25 ℃)
(5) uv-absorbing wave spectrum:
λ
max，nm(E
1％?
1cm)：223(sh，108)，276(15)，283(sh，13)/MeOH245(92)，291(16)/0.01N?KOH+MeOH
(6) infrared absorption wave spectrum: (KBr)
v
max，cm
-1：3360，2928，2855，1628，1520，1456，1232
(7)
1H NMR (Nuclear Magnetic Resonance) spectrum (CDCl
3, 400MHz)
δ(ppm)：0.85(3H，d)，0.87(3H，t)，1.04(3H，d)，1.20(6H，d)，1.25～1.35(19H，m)，1.58(2H，m)，1.90(1H，m)，2.05(1H，m)，2.08(1H，m)，2.21(2H，t)，2.41(1H，m)，2.51(1H，m)，3.37(1H，dd)，3.78(1H，dd)，3.85(1H，dd)，3.91(1H，m)，3.96(1H，dd)，4.14(1H，m)，4.17(1H，dd)，4.21(1H，dd)，4.29(1H，d)，4.30(1H，d)，4.32(1H，d)，4.38(1H，dd)，4.50(1H，dd)，4.54(1H，m)，4.57(1H，dd)，4.83(1H，d)，4.94(1H，d)，5.22(1H，d)，6.74(2H，dd)，7.13(2H，dd)
(8)
13C NMR (Nuclear Magnetic Resonance) spectrum (CDCl
3, 100MHz)
δ(ppm)：11.2，11.7，19.6(×2)，20.0，27.0，28.2，30.3，30.5，30.5，30.5，30.6，30.7，30.8，35.0，35.7，36.8，37.7，38.5，39.0，51.6，52.9，56.3，56.8，57.1，58.5，62.4，68.3，69.5，69.7，70.8，71.2，74.5，75.5，75.7，76.9，116.1(×2)，129.5(×2)，133.0，158.4，169.9，172.5(×2)，172.7，173.5，174.3，176.2
(9) solvability: be dissolved in methyl alcohol, chloroform, ethyl acetate and dimethyl sulfoxide (DMSO); Be insoluble to normal hexane and water.
And compound (1) similar compounds has sour jujube albomycin B (echinocandin B) (sandoz etc., Tetrahedron letters Vol 46, p4147～4150,1976) and Mulundocandin (Roy etc., J.Antibiotics Vol 40, P275-289,1987).But compound of the present invention (1) is different with the known compound physical and chemical character, can obviously distinguish.
The pharmacologically acceptable salt of compound (1) can be exemplified as sodium, potassium, alkaline earth salt such as basic metal such as lithium or calcium.
Above-claimed cpd (1) can use the Penicillium fungus culture that produces this compound, extracts this compound from culture.As the Penicillium fungi that produces compound (1), as long as can produce compound (1) and unrestricted.For example involve mould (Penicillium implicatum) fungi NCC00646.
The generation bacterium NCC00646 of compound (1) separates from the soil of Chinese yunnan Xishuangbanna (xi shuang ban na) and obtains.
The bacterial strain of NCC00646 is at Czapek ' s nutrient agar, in the potato dextrose nutrient agar, and 26 ℃.Bacterium colony is dark green to deep green, velvet-like, forms funiculus, the subiculum densification.Do not have solvable chromogenesis, the back side is brown.
Conidium has matrix to form, diameter 2～2.5 μ m, long 100 μ m.This mould is the multiple verticillate of single-wheel life or two metulae formation, and 8～12 of stigmas are intensive, 9～12 * 2～2.5 μ m, and conidium is spherical or subsphaeroidal, and 2.5～3 μ m are slightly coarse.
Involve mould (Penicilliumimplicatum) from what above mycology feature can think that NCC00646 belongs to fungi.
Bacterial strain NCC00646 was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on December 18th, 2003, preserving number: CGMCC No.1079.
The generation bacterium of compound among the present invention (1) can be used the mutant (both can spontaneous generation also can be mutagenesis) of NCC00646 origin, and plastid merges or the mutant strain of gene recombination.NCC00646 bacterial strain and mutant strain can also can be artificial variation's mutant strain by the mutant strain of nature existence.
Cultivate the generation bacterium of compound (1), can utilize the substratum of the nutrition source that contains common microorganism.
Nutrition source can utilize known fungi culture medium.For example, can be glucose, sucrose, molasses, dextrin, starch, glycerine, animal oil and vegetables oil as carbon source.As nitrogenous source usually is soyflour, wort, cotton seed, corn steep liquor, peptone, extractum carnis, casein food grade, yeast extract paste, urea, and ammonium salts such as ammonium sulfate and ammonium nitrate.It is also very effective to add some inorganic salt, as sodium, potassium, calcium, manganese, cobalt, mantoquita, and muriate, phosphoric acid salt and vitriol.In addition, add some organism that help fungal growth and inorganics and also help fungi growth.
Can be under aerobic conditions, adopt static culture method or oscillating culture to cultivate the generation bacterium of compound (1), leave standstill cultivation, shaking culture or jar cultivate all can, adopt wherein that to leave standstill cultivation more favourable.Culture temperature and time are unrestricted under the situation of compound (1) generation bacteria growing, can be according to the substratum different set.The suitable culture temperature is 20 ℃～30 ℃, and is best near 26 ℃.The culture cycle generally output in 2～21 days reaches maximum.After cultivating end, compound (1) stops when reaching maximum amount cultivating, and extracts compound (1).
The compound that extracts from fermented liquid (1) according to its proterties, can utilize common separation means.Separation means can be exemplified as solvent and extract, absorb-elute, the various resin chromatographies and the precipitator method etc.These means be used in combination better effects if.
Specifically, from the NCC00646 culture, extract target product, can be earlier with aqueous acetone solution extracting from the fermented liquid of bacterial strain object of 67%, the acetone in the crude extract is removed in evaporation, uses the ethyl acetate extracting then.The extract of ethyl acetate is purified with silica gel column chromatography and gel permeation chromatography, obtains compound (1) with TLC or HPLC purifying at last.
Compound (1) and its pharmacologically acceptable salt have anti-mycotic activity, wherein any pharmaceutical composition that contains effective constituent, and to comprising the people behind interior animals administer, effect is obvious.Specifically, to treating and/or preventing effectively of fungi infestation disease, can be used as antifungal drug.
As the preparation of oral administration administration, can make tablet, pill, capsule, powder, oral preparation, granule, pulvis, clouding agent, syrup, hypogloeeis agent etc.As para-oral preparation, can make injection, skin-absorbent agents, inhalation, suppository etc.Can add some acceptable medicated premixs in the prescription of pharmaceutical composition of the present invention, as tensio-active agent, vehicle, stablizer, wetting agent, disintegrating agent, solubilizing agent, isotonic agent, buffer reagent, tinting material, perfume compound etc.
Pharmaceutical carrier can use the carrier that allows use in the pharmaceutical industry, and its kind and composition can be decided according to route of administration and method.For example, liquid vehicle can be water, alcohol, and soybean oil, the sesame wet goods, solid carrier can be organic acid salts such as polysaccharide such as derivatived celluloses such as amino acid such as carbohydrates such as maltose, sucrose, Methionin, hydroxypropylcellulose, cyclodextrin, Magnesium Stearate.With the preparation of injection form, liquid vehicle is generally physiological saline, various damping fluids, saccharide solutions such as glucose etc.
Compound among the present invention (1) and its salt are during as the pharmaceutical composition administration, dosage is people's age and body weight due to illness, disease characteristic and seriousness, and route of administration and different, can be with reference to zooperal result and particular case, total dosage can not surpass certain limit.Specifically, be 1～1 to the oral dosage of being grown up, 000mg/kg/ days, intravenous dosage was 0.1～100mg/kg/ days.
Description of drawings
Accompanying drawing 1: the uv-spectrogram of compound N CC00646
Accompanying drawing 2: the infared spectrum of compound N CC00646
Accompanying drawing 3: the nuclear magnetic spectrum of compound N CC00646 (
1The H spectrum)
Accompanying drawing 4: the nuclear magnetic spectrum of compound N CC00646 (
13The C spectrum)
Embodiment
Being embodiments of the invention below, is not limitation of the invention, comprises the method and the modification means of the change that does not have demonstration here yet.
The preparation of embodiment 1 compound N CC00646
(1) cultivation of bacterial strain NCC00646
The monospore that will involve Penicillium notatum (Penicillium implicatum) was 24 ℃ of fresh malt extract agar (MEA) slant culture, 5 days.10 milliliters the spore suspension that contains 0.01% tween 80 is made on cultured inclined-plane.0.5 a milliliter spore suspension smoothens in the plate that contains malt extract agar (MEA), cultivates after 5 days, scrapes spore on the ware of making even with asepsis ring, is suspended in the sterile distilled water, is inoculated in the bio-reactor that contains 15 kilograms of wheat bran cultivation 7 days.Culture vessel (stainless steel) 200 * 80 * 17cm
(2) the extraction purifying of compound N CC00646
The part of above-mentioned culture (2kg) with the aqueous acetone solution extracting of 2L 67%, obtains crude extract earlier.Filtration also steams acetone, uses the extracting of 1.5L ethyl acetate then.Obtain the 6.8g crude extract after the evaporation of ethyl acetate extract.Crude extract 100ml dissolve with methanol, add 20g silica gel (WAKOGEL C-300, Wako Pure Chemical Industries) drying under reduced pressure, the silica gel that is adsorbed with crude extract is added on the chromatography column that 30g silica gel is housed, use chloroform then: methyl alcohol=99: 1 (100ml), chloroform: methyl alcohol=97: 3 (500ml), chloroform: the solvent sequentially eluting of methyl alcohol=90: 10 (300ml) etc.At methyl alcohol: chloroform=50: 50 (500ml) part, activeconstituents is gone out by wash-out, obtains the drying solid of 426mg behind the drying under reduced pressure.
Drying solid with dissolve with methanol after, carry out Sephadex LH-20 gel filtration chromatography (Pharmacia, 5cm i.d. * 90cm), and use methanol-eluted fractions.Elution amount is to obtain the 79mg solid after 600～650ml component drying partly.Then, these solid dissolve with methanol use HPLC chromatographic column (Cosmosil AR-II internal diameter 2cm * 25cm Nacalai Tesque) to be prepared, and moving phase is acetonitrile: distilled water=40: 30.The flow velocity of moving phase is 10ml/min, the composition that 13.5 minutes wash-outs go out, and concentrating under reduced pressure obtains compound (1) 6.2mg.
Embodiment 2: the anti-mycotic activity of compound N CC00646 is measured
The anti-mycotic activity of the compound that obtains among the embodiment 2 (1) adopts the minimal inhibitory concentration of strength of fluid dilution method mensuration to fungal bacterial strain.Fungi was cultivated 48 hours for 28 ℃ with YNBP culture medium culturing (contain the yeast nitrogen basis of buffer reagent, contain 0.5% glucose, adopt the DIFCO method to produce).Result such as table 1.
The anti-mycotic activity of table 1. compound (1)
| Test strain | Has the active Cmin (μ g/ml) of inhibition |
| Candida?albicans?TIMM1768 | 0.2 |
Table 1 shows that compound of the present invention (1) has good anti-mycotic activity.
Claims (4)
1, compound and the pharmaceutically useful salt of representing as molecular formula (1) thereof.
2, a kind of method for preparing claim 1 compound comprises the method that involves mould (Penicilliumimplicatum) NCC00646 CGMCC1079 and extract this compound of purifying from culture of cultivating.
3, a kind of pharmaceutical composition contains compound in the claim 1 as activeconstituents and pharmaceutical carrier.
4, the described pharmaceutical composition of claim 3 can be used for treating and/or preventing fungi infestation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100924434A CN100567299C (en) | 2004-12-27 | 2004-12-27 | New antifungal compound, Preparation method and use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100924434A CN100567299C (en) | 2004-12-27 | 2004-12-27 | New antifungal compound, Preparation method and use |
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| Publication Number | Publication Date |
|---|---|
| CN1796386A CN1796386A (en) | 2006-07-05 |
| CN100567299C true CN100567299C (en) | 2009-12-09 |
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ID=36817717
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100924434A Expired - Fee Related CN100567299C (en) | 2004-12-27 | 2004-12-27 | New antifungal compound, Preparation method and use |
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| Country | Link |
|---|---|
| CN (1) | CN100567299C (en) |
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2004
- 2004-12-27 CN CNB2004100924434A patent/CN100567299C/en not_active Expired - Fee Related
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|---|---|
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