CN103665071A - Gopalamicin derivatives and application of same in inhibition of infection by drug-resistant bacteria and drug-resistant mycobacterium tuberculosis - Google Patents
Gopalamicin derivatives and application of same in inhibition of infection by drug-resistant bacteria and drug-resistant mycobacterium tuberculosis Download PDFInfo
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Abstract
The invention relates to gopalamicin derivatives and application of the same in inhibition of infection by drug-resistant bacteria and drug-resistant mycobacterium tuberculosis. The derivatives are originated from fermentation products of marine actinomycete Streptomyces sp. 7-145. Results of experimental study show that discovered compounds with novel structures and known structures all have strong antibacterial activity on tested drug-resistant bacteria and drug-resistant mycobacterium tuberculosis and are expected to become clinically-useful novel drugs used for inhibition of infection by drug-resistant bacteria and drug-resistant mycobacterium tuberculosis.
Description
Technical field:
The present invention relates to obtain new compound from Marine Microorganisms; The present invention relates to the method for the described natural compounds of fermentation preparation; The invention still further relates to the application of described compound in anti-multiple drug-resistant bacteria and Drug-Resistant Mycobacterium tuberculosis infection.
Background technology:
At present, bacterial resistance is day by day serious, researches and develops new microbiotic extremely urgent.Elaiophylin (English name: Elaiophylin, Azalomycin B, Gopalamicin, or Salbomycin) be that a class has the large ring dilactone of dual rotational symmetric sixteen-ring microbiotic, there is antibacterial, parasiticide, immunosuppression, promotion cud animal and the biological activity ［ Yan Shuling etc. widely such as increase, microbiology circular 2002,29,103 – 107 ］.In recent years, a series of bioactive materials that have are developed in this laboratory from Marine Microorganisms, wherein find the new purposes of several novel compounds and known structure compound, especially drug-fast bacteria infection are had the compound of good anti-microbial activity.
The elaiophylin of hitherto reported produces bacterium and is streptomycete, comprise raw spore streptomycete (Streptomyces melanosporus) [Arcamone, F.M, et al.Giorn.Microbiol.1959, 7, 207 – 216], streptomyces hygroscopicus (Streptomyces hygroscopicus) [Arai, M.J.Antibiot.1960, 13, 46 – 50], Streptomyces violaceoniger (Streptomyces violaceoniger) [Fiedler, H.P., et al.J.Antibiot.1981, 34, 1107 – 1118], streptomyces albus (Streptomyces albus) [Klebarobva, E.I.et al.Farmatiya (Sofia), 1972, 22, 3.] etc.
The present invention derives from marine actinomycete streptomycete 7-145(Streptomyces sp.7-145 from a strain) fermented liquid activeconstituents, obtain two new elaiophylin derivatives, compound 1:11 ', 12 '-dehydration elaiophylin (11 ', 12 '-dehydroelaiophylin) and compound 2:11, 11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (11, 11 '-O-dimethyl-14 '-deethyl-14 '-methylelaiophylin), and four known structure elaiophylin class microbiotic, compound 3: elaiophylin (elaiophylin), compound 4:11-O-methyl elaiophylin (11-O-methylelaiophylin), compound 5:11, 11 '-O-dimethyl elaiophylin (11, 11 '-O-dimethylelaiophylin), compound 6:14 '-go ethyl-14 '-methyl elaiophylin (efomycin G).Antibacterial activity in vitro test result shows, these elaiophylin compounds are to comprising MRSA(methicillin-resistant staphylococcus aureus), MRSE(methicillin-resistant staphylococcus epidermidis), VRE(vancomycin resistance enterococcus faecalis, faecium), extensively the multiple drug-resistant bacteria of resistance (XDR) and multidrug resistant (MDR) mycobacterium tuberculosis has good anti-microbial activity.The anti-microbial activities of elaiophylin derivative to resistant organism and mycobacterium tuberculosis such as chemical structure, preparation method, antimicrobial agent and tuberculosis activity and compound 3～6 that relate to compound 1 and 2, up to now, there is not yet relevant report.
Summary of the invention:
One of the object of the invention is to provide a strain to produce the generation bacterium of elaiophylin derivative;
Two of the object of the invention is to provide elaiophylin derivative, especially new elaiophylin derivative;
Three of the object of the invention is that the preparation method of described elaiophylin derivative is provided;
Four of the object of the invention is that the application of described elaiophylin derivative in preparing antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infection medicine is provided.
The present invention has found the elaiophylin derivative 1 and 2 of two novel structures first from the tunning of marine actinomycete streptomycete (Streptomyces sp.) 7-145, and its structure is suc as formula shown in I, formula II:
Compound 1:11 ', 12 '-dehydration elaiophylin (11 ', 12 '-dehydroeliophylin)
Molecular formula C
54h
86o
17, molecular weight 1006.
Compound 2:11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (11,11 '-O-dimethyl-14 '-deethyl-14 '-methylelaiophylin)
Molecular formula C
55h
90o
18, molecular weight 1038.
The present invention, from the tunning of marine actinomycete streptomycete (Streptomyces sp.) 7-145, has also found that compound 3～6 has good antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis effect, its structure as shown in formula III 3～6.
Compound 3: elaiophylin;
Compound 4:11-O-methyl elaiophylin;
Compound 5:11,11 '-O-dimethyl elaiophylin;
Compound 6:14 '-go ethyl-14 '-methyl elaiophylin.
The preparation method of elaiophylin provided by the present invention and derivative thereof comprises the following steps:
Streptomycete (Streptomyces sp.) 7-145 fermented liquid obtains filtrate and mycelium after solid-liquid separation.Mycelium acetone supersound extraction; Filtrate is through the absorption of Amberlite XAD7HP macroporous adsorptive resins, successively water, 50% and 100% aqueous acetone solution wash-out; 50%, 100% acetone elutriant and mycelium acetone extract are merged, and concentrating under reduced pressure obtains the aqueous solution after removing acetone, makes to be extracted with ethyl acetate, and concentrating under reduced pressure obtains brown medicinal extract except after ethyl acetate; This medicinal extract utilizes forward silica gel column chromatography separated, uses successively petroleum ether-ethyl acetate (9:1,4:1,1:1), ethyl acetate, ethyl acetate-methyl alcohol (1:1) and methyl alcohol gradient elution; Wherein, ethyl acetate-methyl alcohol (1:1) wash-out part is separated through forward silica gel column chromatography again, uses successively methylene chloride-methanol (20:1,9:1,4:1,1:1 and 0:1) gradient elution, obtains 7 streams part (A～G); Stream part B is separated through anti-phase C18 silica gel flash column chromatography, and 30～100% methyl alcohol gradient elutions, obtain 1 main active constituent, then obtain compound 3(elaiophylin through chloroform-methanol solvent recrystallization); Stream part C and stream part D are respectively through the decolouring of Sephadex LH-20 column chromatography, and methylene chloride-methanol 1:1 wash-out, obtains active constituent; By flowing the mother liquor after part B crystallization and flowing part rear active constituent obtaining of C, D decolouring, through HPLC, prepare and partly prepare purifying respectively, obtain compound 1～6.
The streptomycete of using in described preparation method, be preferably streptomycete (Streptomyces sp.) 7-145, the separated marine bottom sediment from the about 40m of the DaLian, China Huanghai Sea black stone reef bay degree of depth of system, this bacterial strain is delivered the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms on July 12nd, 2013, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC No.7914.Streptomycete (Streptomyces sp.) 7-14516S rDNA gene order, as shown in SEQ ID NO.1, is submitted NCBI to, and GenBank accession number is JQ782979.
The present invention adopts agar dilution to test described elaiophylin and derivative thereof to comprising MRSA, VRE and multidrug resistant (MDR), the extensive minimal inhibitory concentration (MIC) of the multiple drug-resistant bacteria of resistance (XDR) mycobacterium tuberculosis.Experimental result shows that these compounds have good anti-microbial activity to tested Resistant strain.Show that described elaiophylin analog derivative can be used as preparing the medicine of antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infection; And using described derivative as activeconstituents, with pharmaceutically acceptable one or more carriers, vehicle or supplementary product compatibility, can be made into the pharmaceutical composition that antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infect.Described medicine and pharmaceutical composition can be used for the clinical treatment of drug-fast bacteria infection and Drug-Resistant Mycobacterium tuberculosis infection.Described compound also can form compound preparation for the treatment of drug-fast bacteria infection and Drug-Resistant Mycobacterium tuberculosis infection with known drug.
In the present invention, through the method for elaiophylin derivative 1 shown in microorganism fermentation preparation formula I and formula II and 2, applicable to other, can produce any microorganism of this compounds.
The present invention also comprises other substituted radical elaiophylin derivative shown in formula III, the application in preparing antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infection medicine, and described substituting group is because of as follows:
R
1, R
2=hydrogen or hydroxyl, include but not limited to C1～C5 alkoxy substituents such as methoxyl group, oxyethyl group, C2～the C6 such as vinyloxy group, propenyloxy group alkene oxy substituents, C1～C5 acyloxy such as C2～C6 alkynyloxy group, C3～C6 cycloalkyloxy, methanoyl, acetoxyl group; The C6 such as phenoxy group～C12 virtue epoxy group(ing)
C-11-C-12: can be singly-bound or two key
C-11 '-C-12 ': can be singly-bound or two key
R
3, R
4=hydrogen or methyl, include but not limited to C2～C5 saturated alkyl substituting groups such as ethyl, propyl group, C2～C6 alkenyl groups such as ethene, propylene, C2～C6 alkynyl, C3～C6 cycloalkyl
R
5=hydrogen, includes but not limited to C1～C5 saturated alkyl substituting groups such as methyl, ethyl, propyl group, C2～C6 alkenyl groups such as ethene, propylene, C1～C5 acyl groups such as C2～C6 alkynyl, C3～C6 cycloalkyl, formyl radical, ethanoyl; C6～C12 aromatic ring groups such as phenyl.
And using described derivative as activeconstituents, with pharmaceutically acceptable one or more carriers, vehicle or supplementary product compatibility, can be made into the pharmaceutical composition that antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infect.Described medicine and pharmaceutical composition can be used for the clinical treatment of drug-fast bacteria infection and Drug-Resistant Mycobacterium tuberculosis infection.Described compound also can form compound preparation for the treatment of drug-fast bacteria infection and Drug-Resistant Mycobacterium tuberculosis infection with known drug.
invention effect:
The present invention's separation from the tunning of strain marine actinomycete streptomycete (Streptomyces sp.) 7-145 prepare suc as formula novel elaiophylin derivative 1 shown in I, formula II and 2 and formula III shown in a compound 3～6 four known elaiophylin derivative; These compounds have good anti-microbial activity to comprising the multiple drug-resistant bacteria of MRSA, VRE, are expected to that exploitation becomes antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infect clinically newtype drug or for the control of agricultural pest.
Accompanying drawing explanation:
Fig. 1: the phylogenetic evolution tree of the 16S rRNA gene order of streptomycete (Streptomyces sp.) 7-145 is analyzed
Fig. 2: 11 ', the high resolution electrospray ionization mass spectrum of 12 '-dehydration elaiophylin (1)
Fig. 3: 11 ', 12 '-dehydration elaiophylin (1)
1h NMR spectrum
Fig. 4: 11 ', 12 '-dehydration elaiophylin (1)
13c NMR spectrum
Fig. 5: 11,11 '-O-dimethyl-14 '-remove the high resolution electrospray ionization mass spectrum of ethyl-14 '-methyl elaiophylin (2)
Fig. 6: 11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (2)
1h NMR spectrum
Fig. 7: 11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (2)
13c NMR spectrum
embodiment:
Embodiment of the present invention is applicable to from any microorganism, and is not limited to prepare elaiophylin and derivative thereof from streptomycete fermentation liquid.Listed embodiment is for helping those skilled in the art to understand better the present invention below, but does not limit the present invention in any way.
The evaluation of < embodiment 1> streptomycete (Streptomyces sp.) 7-145
A) bacterium source: streptomycete (Streptomyces sp.) 7-145 is by this laboratory separated acquisition from the oceanic sediment of DaLian, China Huanghai Sea black stone reef bay (38 ° of 49'N, 121 ° of 34'E) the about 40m of the degree of depth.
B) identification of strains: the conservative property according to 16S rRNA gene order in microorganism kind, 7-145 identifies to streptomycete (Streptomyces sp.).The genome that extracts streptomycete (Streptomyces sp.) 7-145, its 16S rRNA gene of pcr amplification also checks order, and being submitted to NCBI GenBank database acquisition accession number is JQ782979, and its sequence is as shown in SEQ ID NO.1.The 16S rRNA gene comparison result shows of streptomycete (Streptomyces sp.) 7-145, this bacterium and streptomyces bacterial strain Streptomyces samsunensis M1463
tsimilarity be 99.85%.By contiguous method, constructing system is grown evolutionary tree (Fig. 1).
SEQ?ID?NO.1
1?atgaaccggt?ttcggccggg?gattagtggc?gaacgggtga?gtaacacgtg?ggcaatctgc
61?cctgcactct?gggacaagcc?ctggaaacgg?ggtctaatac?cggatacgac?gcgttcccgc
121?atgggatacg?tgtggaaagc?tccggcggtg?caggatgagc?ccgcggccta?tcagcttgtt
181?ggtggggtga?tggcctacca?aggcgacgac?gggtagccgg?cctgagaggg?cgaccggcca
241?cactgggact?gagacacggc?ccagactcct?acgggaggca?gcagtgggga?atattgcaca
301?atgggcgcaa?gcctgatgca?gcgacgccgc?gtgagggatg?acggccttcg?ggttgtaaac
361?ctctttcagc?agggaagaag?cgtgagtgac?ggtacctgca?gaagaagcgc?cggctaacta
421?cgtgccagca?gccgcggtaa?tacgtagggc?gcaaagcgtt?gtccggaatt?attgggcgta
481?aagagctcgt?aggcggcttg?tcgcgtcgga?atgtgaaagc?ccggggctta?actccgggtc
541?tgcattcgat?acggggcagg?ctagagttcg?gtaggggaga?tcggaattcc?tggtgtagcg
601?gtgaaatgcg?cagatatcag?gaggaacacc?ggtggcgaag?gcggatctct?gggccgatac
661?tgacgctgag?gagcgaaagc?gtggggagcg?aacaggatta?gataccctgg?tagtccacgc
721?cgtaaacgtt?gggaactagg?tgtgggcgac?attccacgtt?gtccgtgccg?cagctaacgc
781?attaagttcc?ccgcctgggg?agtacggccg?caaggctaaa?actcaaagga?attgacgggg
841?gcccgcacaa?gcggcggagc?atgtggctta?attcgacgca?acgcgaagaa?ccttaccaag
901?gcttgacata?caccggaaaa?ctctggagac?agggtccccc?ttgtggtcgg?tgtacaggtg
961?gtgcatggct?gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca
1021?acccttgtcc?tgtgttgcca?gcgggttatg?ccggggactc?acaggagact?gccggggtca
1081?actcggagga?aggtggggac?gacgtcaagt?catcatgccc?cttatgtctt?gggctgcaca
1141?cgtgctacaa?tggccggtac?aatgagctgc?gaagccgtga?ggtggagcga?atctcaaaaa
1201?gccggtctca?gttcggattg?gggtctgcaa?ctcgacccca?tgaagtcgga?gtcgctagta
1261?atcgcagatc?agcattgctg?cggtgaatac?gttcccgggc?cttgtacaca?ccgcccgtca
1321?cgtcacgaaa?gtcggtaaca?cccgaagccg?gtggcccaac?ccttgtggag?ggag
Show, a kind of in Streptomycetaceae streptomyces of this Pseudomonas, accordingly by this bacterium called after streptomycete (Streptomyces sp.) 7-145, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 12nd, 2013, deposit number is: CGMCC No.7914.
The fermentation of < embodiment 2> streptomycete (Streptomyces sp.) 7-145
Streptomycete (Streptomyces sp.) 7-145 that gets activation is dull and stereotyped, the about 1cm of picking
2lawn be inoculated in 100mL fermention medium [formula is: starch 1.0g, glucose 2.5g, cottonseed meal 1.0g, peptone 0.3g, inorganic salt are as KH
2pO
40.01g, MgSO
40.01g, NaCl0.5g, CaCO
30.5g etc., deionized water 100mL], in 500mL shaking flask, 28 ℃, 200r/min shaking table is cultivated 120h.
The extraction of < embodiment 3> fermented liquid and the acquisition of medicinal extract
Streptomycete (Streptomyces sp.) 7-145 fermented liquid obtains filtrate and mycelium after solid-liquid separation; Mycelium is used acetone supersound extraction, obtains mycelium acetone extract; Filtrate is through the absorption of Amberlite XAD7HP macroporous adsorptive resins, successively water, 50%, 100% acetone wash-out; 50%, 100% acetone elutriant part merges with mycelium acetone extract, and concentrating under reduced pressure obtains the aqueous solution after removing acetone, through ethyl acetate, extracts; Ethyl acetate extraction part concentrating under reduced pressure, obtains fermented liquid medicinal extract.
Separation, preparation and the Structural Identification of < embodiment 4> compound 1～6
Get streptomycete (Streptomyces sp.) 7-145 fermented liquid medicinal extract (17g), separated through silica gel column chromatography, use successively petroleum ether-ethyl acetate (9:1,4:1,1:1), ethyl acetate, ethyl acetate-methyl alcohol (1:1) and methyl alcohol gradient elution; Ethyl acetate-methyl alcohol (1:1) wash-out stream part (6.5g), separated through forward silica gel column chromatography, use successively methylene chloride-methanol (20:1,9:1,4:1,1:1 and 0:1) gradient elution, obtain 7 streams part (A～G); Stream part B(2.7g) separated through anti-phase C18 silica gel flash column chromatography, 30～100% methyl alcohol gradient elutions obtain 1 main active constituent, through chloroform-methanol solvent recrystallization, obtain compound 3-elaiophylin (500mg); Flow a part C(0.9g) and a stream part D(0.2g) respectively through the decolouring of Sephadex LH-20 column chromatography, methylene chloride-methanol 1:1 wash-out, obtains active constituent; By the active constituent obtaining after the mother liquor after stream part B crystallization and stream part C, D decolouring, through HPLC preparation (Capcell-Pak C
18aQ5um, 20 * 250mm, 88% acetonitrile, 5mL/min) obtain 6 components, wherein 3 components are respectively the 3(of compound shown in formula III elaiophylin, 320mg), compound 4(11-O-methyl elaiophylin, and 520mg), compound 5(11,11 '-O-dimethyl elaiophylin, 650mg); All the other 3 components are again through HPLC half preparation (Capcell-Pak C
185um, 10 * 250mm, 68% or 87% acetonitrile, 2mL/min) purifying, obtains respectively structural compounds 1(11 ' shown in formula I, formula II and formula III, 12 '-dehydration elaiophylin, 12mg), compound 2 (11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin, 20mg) and compound 6(14 '-remove ethyl-14 '-methyl elaiophylin, 9mg).
A) compound 1:11 ' shown in formula I, the Structural Identification of 12 '-dehydration elaiophylin (1)
Compound 1 is white powder, is soluble in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, water insoluble, sherwood oil etc.High resolution electrospray ionization mass spectrum (HRESIMS) provides quasi-molecular ion peak m/z1029.5776[M+Na]
+(Fig. 2), syncaryon Magnetic Resonance Spectrum (NMR) data, determine that its molecular formula is C
54h
86o
17, than simultaneously the separated elaiophylin molecular formula obtaining is few from this fermented liquid 1 molecule H
2o.The hydrogen spectrum of compound 1 (
1h NMR) (Fig. 3) compose with carbon (
13c NMR) (Fig. 4) data with there is C
2the elaiophylin of-symmetrical structure (compound 3) ［ Kaiser, H., et al.Helv.Chim.Acta1981,64,407-424; Nair, M.G., et al.J.Agric.Food.Chem.1994,42,2308-2310.Lee, S.Y., et al.J.Microbiol.Biotechnol.1996,6,245-249 ］ bibliographical information data closely similar, but the signal of compound 1 is more complicated, prompting compound 1 is the unsymmetrical structure derivative of elaiophylin.In conjunction with
1h-
1relevant peaks in HCOSY spectrum, the hydrogen spectrum of compound 1 shows two 1, and 3-conjugated diolefine system and one is alkene hydrogen proton δ 4.74 (d, J=3.0Hz, H-12 ') independently, and prompting compound 1 may be the anhydro compounds of elaiophylin.Utilize two dimensional NMR wave spectrum (
1h-
1h COSY, HSQC, HMBC and ROESY spectrum) structure of compound 1 is confirmed.First, in the HMBC of compound 1 spectrum, demonstrate H-2, H-3, H-7 ' and C-1; H-2 ', H-3 ', H-7 and C-1 '; H-10, H-12eq, H-15, H
3-19, OH-11 and C-11; H-22 and C-13 and C-26; H-22 ' and C-13 ' and C-16 ' wait correlation peak signal, illustrate in the mode of connection of the structure fragment such as C-1～C-27, C-1 '～C-10 ', C-22 '～C-27 ' in compound 1 and elaiophylin in full accord; In addition, in HMBC spectrum, also demonstrate H-10 ', H-12 ', H-13 ', H-15 ', H
3-19 ' with C-11 ' (δ
c156.6) relevant peaks, chemical shift in conjunction with these protons and carbon, the two keys of explanation C-11 ' and C-12 ' quilt in compound 1 replace, at C-11 ' and C-12 ' position, dewater, prove that C-11 ' is connected with C-10 ' simultaneously, thereby locate to have formed 1 dihydropyrane ring structure unit at C-11 '～C-14 '; Finally, relevant according to H-13 ' in HMBC spectrum and C-22 ', confirms that other 1 2-deoxidation-L-fucose unit (C-22 '～C-27 ') is connected on C-13 '.Thereby determined the integral planar structure of compound 1.Except alkene hydrogen proton H-12 ' and double key carbon C-11 ', C-12 '; other proton coupling constant in compound 1 hydrogen spectrum; correlation peak signal in carbon spectrum data and ROESY spectrum; all similar to elaiophylin; show that these two compounds have identical relative configuration ［ Neupert-Laves, K., et al.Helv.Chim.Acta1982; 65,262-267; Paulus, E.F., et al.Acta Crystallogr.C1984,40,700-703 ］; Meanwhile, the CD of these two compounds spectrum highly overlaps, and due to they common source of students approach, illustrates that the absolute configuration of these two compounds is also identical.Therefore, the structure of compound 1 is defined as 11 ', 12 '-dehydration elaiophylin (11 ', 12 '-dehydroelaiophylin), this compound has no bibliographical information, is novel compound.
11 ', the spectral data of 12 '-dehydration elaiophylin (1): [α]
20 d-35.0 (c0.29, MeOH);
1h-NMR(Fig. 3) (DMSO-d
6, 500MHz) data are in Table 1;
13c-NMR(Fig. 4) (DMSO-d
6, 125MHz) data are in Table 2; HRESIMS m/z1029.5776[M+Na]
+(calculated value C
54h
86o
17na, 1029.5757), 1045.5514[M+K]
+(calculated value C
54h
86o
17k, 1045.5497).
B) compound 2:11 shown in formula II, 11 '-O-dimethyl-14 '-remove the Structural Identification of ethyl-14 '-methyl elaiophylin (2)
Compound 2 is white powder, is soluble in methyl alcohol, DMSO, is slightly soluble in chloroform, acetone, water insoluble, sherwood oil etc.High resolution electrospray ionization mass spectrum (HRESIMS) shows its quasi-molecular ion peak m/z1061.6019[M-H]
-(Fig. 5), in conjunction with NMR data, determine that its molecular formula is C
55h
90o
18.Compound 2 is closely similar with the data of elaiophylin, but different from elaiophylin, and the hydrogen spectrum of compound 2 has been lacked two tradable hydroxyl proton signals, but has had more two methoxyl group signal δ
h2.94 and 2.95; From the HMBC spectrum of compound 2, observing these two methoxyl group protons and C-11 and C-11 ' has coherent signal, shows that they are connected on C-11 and C-11 '; In addition, the NMR data of compound 2 than elaiophylin few 1 methylene signals; And demonstrate a bimodal methyl proton (H in its HMBC spectrum
3-20 ', δ
h0.85, d, J=6.0Hz) relevant to C-13 ', C-14 ', C-15 ' respectively, and
1h-
1in H COSY spectrum, also observe this methyl proton relevant to H-14 ', prompting methyl substituted in compound 2, at C-14 ', is different from C-14 ' in elaiophylin and is replaced by ethyl.The hydrogen spectrum proton coupling constant of compound 2, carbon spectrum data and ROESY spectrum are all similar to elaiophylin, illustrate that compound 2 is identical with elaiophylin configuration.Therefore, compound 2 structures be defined as 11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (11,11 '-O-dimethyl-14 '-deethyl-14 '-methylelaiophylin), be a novel compound.
11,11 '-O-dimethyl-14 '-remove the spectral data of ethyl-14 '-methyl elaiophylin (2): [α]
20 d+ 21.6 (c0.59, MeOH);
1h-NMR(Fig. 6) (DMSO-d
6, 500MHz) data are in Table 1;
13c-NMR(Fig. 7) (DMSO-d
6, 125MHZ) in Table 2; HRESIMS m/z1061.6019[M+Na]
+(calculated value C
55h
90o
18na, 1061.6019) and 1077.5760[M+K]
+(calculated value C
55h
90o
18k, 1077.5759).
C) compound shown in formula III 3: the Structural Identification of elaiophylin (3)
D) Structural Identification of the 4:11-O-of compound shown in formula III methyl elaiophylin (4)
Compound 4 is white powder, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, water insoluble, sherwood oil etc.[α]
20 d-26.8 (c0.64, MeOH);
1h-NMR(DMSO-d
6, 500MHz) data are in Table 4;
13c-NMR(DMSO-d
6, 125MHZ) in Table 2; ESIMS m/z1037[M-H]
-; 1061[M+Na]
+; 1077[M+K]
+.Above data and document ［ Ritzau, M.et al.J.Nat.Prod.1998,61,1337-1339; Lee, S.Y.et, al.J.Microbiol.Biotechnol.1997,7,272-277 ］ 11-O-methyl elaiophylin (11-O-methylelaiophylin) data consistent of report, so the structure of compound 4 is defined as 11-O-methyl elaiophylin.
E) 5:11 of compound shown in formula III, the Structural Identification of 11 '-O-dimethyl elaiophylin (5)
Compound 5 is white powder, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, water insoluble, sherwood oil etc.[α]
20 d+ 15.0 (c0.62, MeOH);
1h-NMR(DMSO-d
6, 500MHz) and
13c-NMR(DMSO-d
6, 125MHZ) in Table 3; ESIMS m/z1051[M-H]
-; 1075[M+Na]
+; 1091[M+K]
+.Above data and document ［ Ritzau, M.et al.J.Nat.Prod.1998,61,1337-1339 ］ report 11,11 '-O-dimethyl elaiophylin (11,11 '-O-dimethylelaiophylin) data consistent, so the structure of compound 5 is defined as 11,11 '-O-dimethyl elaiophylin.
F) 6:14 ' of compound shown in formula III-the go Structural Identification of ethyl-14 '-methyl elaiophylin (6)
Compound 6 is white powder, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, water insoluble, sherwood oil etc.[α]
20 d-47.4 (c0.24, MeOH);
1h-NMR(DMSO-d
6, 500MHz) data are in Table 4;
13c-NMR(DMSO-d
6, 125MHZ) data are in Table 2; ESIMS m/z1009[M-H]
-; 1033[M+Na]
+; 1049[M+Na]
+.Above data and document ［ Frobel, K., et al.U.S.Patent4927810,1990 ］ 14 of report '-remove ethyl-14 '-methyl elaiophylin (efomycin G) data consistent, so the structure of compound 6 be defined as 14 '-remove ethyl-14 '-methyl elaiophylin.
Table 1. compound 1 and 2
1h NMR data (500MHz, DMSO-d
6 a)
asignal ownership foundation
1h-
1h COSY, HSQC and the experiment of HMBC two dimensional NMR.
Table 2. compound 1,2,4 and 6
13c NMR data (125MHz, DMSO-d
6 a)
asignal ownership foundation
1h-
1h COSY, HSQC and the experiment of HMBC two dimensional NMR.
Table 3. compound 3 and 5 NMR data (DMSO-d
6 a)
a1h NMR data are measured at 500MHz;
13c NMR spectrum is measured at 125MHz; Signal ownership foundation
1h-
1h COSY, HSQC and the experiment of HMBC two dimensional NMR.
Table 4. compound 4 and 6
1h NMR data (δ
hmult.[J (Hz)], 500MHz, DMSO-d
6 a)
asignal ownership foundation
1h-
1h COSY, HSQC and the experiment of HMBC two dimensional NMR.
The anti-microbial activity test of < embodiment 5> elaiophylin derivative
1, experiment material
Assay strain: Staphylococcus aureus ATCC29213(MSSA); Staphylococcus aureus09-6(MSSA, clinical separation strain); Staphylococcus aureus ATCC33591(methicillin-resistant staphylococcus aureus); Staphylococcus aureus09-13(methicillin-resistant staphylococcus aureus, clinical separation strain); Staphylococcus aureus R6101(methicillin-resistant staphylococcus aureus, clinical separation strain in 2010); Staphylococcus aureus ATCC6538P(thiostrepton resistant Staphylococcus aureus, is derived from ATCC6538 mutagenesis); Staphylococcus epidermidis ATCC12228(methicillin-sensitivity staphylococcus epidermidis); Staphylococcus epidermidis09-9(methicillin-sensitivity staphylococcus epidermidis, clinical separation strain); Staphylococcus epidermidis09-3(methicillin resistance staphylococcus epidermidis, clinical separation strain); The responsive enterococcus faecalis of Enterococcus faecalis ATCC29212(vancomycin); The responsive enterococcus faecalis of Enterococcus faecalis09-8(vancomycin, clinical separation strain); Enterococcus faecalis ATCC51299(drug resistance of vancomycin enterococcus faecalis); Enterococcus faecalis W4138(drug resistance of vancomycin enterococcus faecalis, clinical separation strain in 2010); Enterococcus faecalis R6512(drug resistance of vancomycin enterococcus faecalis, clinical separation strain in 2010); The responsive faecium of Enterococcus faecium09-10(vancomycin); Enterococcus faecium ATCC700221(drug resistance of vancomycin faecium); Micrococcus luteus(apramycin resistance micrococcus luteus, is derived from ATCC10240 mutagenesis); Proteusbacillus vulgaris ATCC13315(Bacillus proteus); Bacillus subtilis CMCC63501(subtilis).Above bacterial strain is preserved by pharmacological room of Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences.
LB broth culture: yeast extract 0.5g, Tryptones 1.0g, NaCl0.5g, distilled water 100mL, pH7.4～7.6.
LB nutrient agar: yeast extract 0.5g, Tryptones 1.0g, NaCl0.5g, distilled water 100mL, agar 2.0g, pH7.4～7.6.
Mueller-Hinton broth culture: extractum carnis 0.2g, Zulkovsky starch 0.15g, acid hydrolyzed casein 1.75g, distilled water 100mL, pH value 7.4 ± 0.2.
Mueller-Hinton nutrient agar: extractum carnis 0.2g, Zulkovsky starch 0.15g, acid hydrolyzed casein 1.75g, distilled water 100mL, agar 2.0g, pH value 7.4 ± 0.2.
CM0984VRE BROTH BASE substratum (purchased from OXOID company).
CM0984VRE AGAR BASE substratum: add 2.0g agar in every 100mL CM0984VRE BROTH BASE.
Control sample: Azythromycin, erythromycin, Oxazacillin, vancomycin (purchased from Chinese food drug assay institute); Sulfuric acid Apramycin sulfate (Apramycin sulphate) (purchased from Shanghai Jiang Lai Bioisystech Co., Ltd).
2, experimental technique
Adopt the MIC value of agar dilution working sample to each strain test organism, specific as follows:
A) sample configuration: accurately take appropriate sulfuric acid Apramycin sulfate, be dissolved in pure water, be configured to certain final concentration solution, then become desired concn solution with pure water by doubling dilution, for active testing; Accurately take refining compound 1～6 sample of appropriate embodiment 4 and other control sample standard substance, be dissolved in dimethyl sulfoxide (DMSO) (DMSO), be configured to certain final concentration solution, then with DMSO, by doubling dilution, become desired concn solution, for active testing.
B) pastille agar plate preparation: the sample of the different concns of doubling dilution is added respectively to heating for dissolving, and in 45～50 ℃ of water-baths in the Mueller-Hinton nutrient agar (or LB nutrient agar, CM0984VRE AGAR BASE substratum) of balance, fully mix and topple over sterilizing plate, agar thickness 3～4mm; In 1: 9 ratio compounding pharmaceutical agar plate; The sample final concentration of each pastille agar plate is respectively 0.25,0.5,1.0,2.0,4.0,8.0,16,32,64,128,256 μ g/mL.
C) inoculum preparation and inoculation: get each test organism glycerine pipe by 1%(v/v) be inoculated in about 3mL liquid nutrient medium, wherein, Enterococcus faecalis R6512 and W4138 are used CM0984VRE BROTH BASE substratum, Micrococcus luteus, Proteusbacillus vulgaris ATCC13315, Bacillus subtilis CMCC63501, use LB broth culture, all the other test organisms are all inoculated in Mueller-Hinton broth culture; After inoculation in 37 ℃, 200rpm jolting overnight incubation; Bacterium liquid, according to the dense dilution of bacterium, prepares bacterium liquid (approximately 1～2 μ l) with multi-point inoculator absorption and is inoculated in agar plate surface, and each test organism is used the substratum same with preparation bacterium liquid phase to prepare agar plate.Every some bacterium number is about 10
4cFU(colony-forming unit), the bacterial plaque that formation diameter is 5～8mm, inoculates good rearmounted 35 ℃ and hatches 18h.
D) result judgement: flat board is placed on dead color, no-reflection body surface and judges test endpoint, and the minimum adding consistency of macroscopic bacteria growing inhibiting of take is minimum inhibitory concentration (MIC).
3, the Macrocyclic lactone compounds 1～6 of elaiophylin class shown in experimental result: formula I, II, III and control sample to the MIC value of 20 strain test organisms in Table 5.
Experimental result shows, 1～6 pair, elaiophylin and derivative thereof comprise in the 20 strain test organisms of MRSA and VRE, and the MIC values of the overwhelming majority, at 1～64 μ g/mL, demonstrate these compounds and have good anti-microbial activity.It is worthy of note; the anti-microbial activity of elaiophylin shown in formula I, II, III (3) and derivative thereof 1,2,4～6 couples of MRSA, MRSE and VRE is better than widely used first-selected macrolide antibiotics Azythromycin clinically; this prompting elaiophylin compounds may not be subject to impact [the Nakajima Y.J Infect Chemother of some macrolide resistance mechanism common in positive bacteria; 1999,5 (2): 61-74; Pechere J C.Int J Antimicrob Agents, 2001,18Suppl1:S25-28.].Elaiophylin and derivative thereof shown in formula I, II, III can be used as the medicine that antimicrobial agent infects, or take it as activeconstituents, with pharmaceutically acceptable one or more carriers, vehicle or supplementary product compatibility, make the pharmaceutical composition that antimicrobial agent infects.
Table 5. elaiophylin and derivative antibacterial minimal inhibitory concentration value thereof (MIC, the μ g/mL of unit)
Note:
a-do not detect;
bsulfuric acid Apramycin sulfate MIC value is 8 μ g/mL.
4, structure activity relationship
As can be seen from Table 5, when C-11 or one of them hydroxyl of C-11 ' are by methoxy substitution (as compound 4) or when dehydration occurring forming pair key at C-11～C-12 place (as compound 1), compare with elaiophylin (3), the anti-microbial activity of most of resistant organisms is reduced to approximately 2 times (MIC:1～2 μ g/mL is than 2～4 μ g/mL); And if two hydroxyls on C-11 and C-11 ' are during all by methoxy substitution (as compound 2 and 5), compare 8～32 times of activity decreaseds (MIC:8-64 μ g/mL is than 1-2 μ g/mL) for the derivative (as compound 6 and 3) that hydroxyl replaces with corresponding C-11 and C-11 '; These results show, the hemiacetal hydroxyl on C-11 and C-11 ' is most important to activity; In addition, when C-14 or C-14 ' are methyl substituted (as compound 2 and 6), than C-14 or C-14 ', be 2～4 times of the activity decreaseds of the corresponding derivative (as compound 5 and 3) that replaces of ethyl, show that the integrity of C-1～C-27 in substituted radical on elaiophylin C-14 or C-14 ' or its structure has material impact to anti-microbial activity.
< embodiment 6> elaiophylin derivative is tested mycobacterium tuberculosis bacteriostatic activity
1, experiment material
Assay strain: Mycobacterium tuberculosis H37Rv(Mycobacterium tuberculosis H37Rv reference culture, ATCC27294); The extensive persister XDR of M.tuberculosis FJ05436(, clinical separation strain), M.tuberculosis FJ05349(sensitive strain, clinical separation strain), M.tuberculosis FJ05060(sensitive strain, clinical separation strain), the extensive persister XDR of M.tuberculosis FJ05195(, clinical separation strain), M.tuberculosis FJ05120(multidrug resistant strain MDR, clinical separation strain).Above bacterial strain is preserved by CDC.
Prefabricated Middlebrook7H9 liquid nutrient medium: take 4.7g7H9 pulvis and add 900mL distilled water, add glycerine 2mL, 121 ℃ of autoclavings 15 minutes, after naturally cooling, put into 4 ℃ of refrigerators standby.
Prepare OADC (Oleic Acid Dextrose Catalase, includes OADC) nutrition enrichment liquid (can buy commercialization reagent).
The medicine storage liquid of preparation different concns: as 51.2mg/mL, 102.4mg/mL isoconcentration (concrete concentration can be prepared in conjunction with actual experiment demand), after 0.2 μ m filter membrane filter sterile filtration, puts-20 ℃ of refrigerator cold-storages.
purchased from U.S. company BD.
Other article: mill bacterium bottle (the interior aseptic screw-cap bottle with granulated glass sphere) several (each of each experimental strain), than turbid (examination), manage several (each of each experimental strain), several (each of each experimental strain of 15mL or 50mL centrifuge tube, press actual experiment demand and select specification), 10 μ L rifle heads, 200 μ L rifle heads, 1000 μ L rifle heads, transfering loop, physiological saline; 5%Tween-80 reagent.Above equipment all need, at 121 ℃, use after 15min sterilizing.
Separately need: each range pipettor, 1.0 1 of standard Maxwell opacity tube (MacFarland Mo.1) or than one, turbid instrument, cryopreservation tube, 96 orifice plates (band lid), sealed membrane, 37 ℃ of fixed temperature and humidity incubators, vortex vibrator, the mycobacterium of pure separation and Culture logarithmic phase (3～4 weeks) in L-J substratum, disposable calibrated pipet etc.
2, experimental technique
2.1 add OADC nutrition enrichment liquid according to 10% ratio in Middlebrook7H9 liquid nutrient medium
The preparation of 2.2 dilution bacterium liquid
2.2.1 in mill bacterium bottle (the interior aseptic screw-cap bottle with granulated glass sphere), add 1～2 5%Tween-80 reagent.And the mycobacterium of choosing pure separation and Culture logarithmic phase (3～4 weeks) in L-J substratum is cooked bacteria suspension.Bacterium with transfering loop from the L-J medium slant scraping semi-ring of this bacterium or a ring (5～10mg), is placed in the mill bacterium bottle about 1min that vibrates on vortex vibrator, until bacterium colony grind completely even after standing 15min stand-by;
2.2.2 carefully open mill bacterium bottle cap, add 2～3mL physiological saline, standingly make bulk matter precipitation in bacterium liquid;
2.2.3 with aseptic straw, get appropriate supernatant in opacity tube, and regulate concentration to a 1 Maxwell concentration (now bacteria concentration is about 1mg/mL) with physiological saline;
2.2.4 get again a 15mL or 50mL centrifuge tube (by actual experiment demand, selecting specification), in 1: the 20 Middlebrook7H9 liquid nutrient medium dilution bacterium liquid that adds OADC nutrition enrichment liquid for ratio.
2.3 build drug level gradient
2.3.1 in 96 orifice plates except each hole, edge, every hole adds 100 μ L Middlebrook7H9 liquid nutrient mediums, now except each hole, edge, the total 6*10 of every plate has added Middlebrook7H9 liquid nutrient medium in hole.
2.3.2 in the first row except each hole, edge (secondary series while starting to calculate from marginal pore), add again 98 μ L Middlebrook7H9 liquid nutrient mediums;
2.3.3 in the every hole of the first row except each hole, edge (secondary series while starting to calculate from marginal pore), add again the pre-configured medicine storage liquid of 2 μ l;
2.3.4 according to twice dilution principle, dilute successively, from adding the first row of medicine, every hole draw 100 μ L containing the Middlebrook7H9 liquid nutrient medium of medicine to secondary series, after mixing, from secondary series, draw 100 μ L containing Middlebrook7H9 liquid nutrient medium to the three row of medicine again, proceed to successively the tenth row, last row discard after drawing out 100 μ L.The medicine storage liquid concentration for example adding is: 51.2mg/mL, can obtain ten drug level gradients from 512～1 μ g/mL; When if experiment needs more concentration, can two 96 orifice plates of serial dilution, thus obtain more drug level gradient.
2.4 every strain bacterium inoculation a line, add 100 μ L dilution bacterium liquid to building every hole of drug level gradient.Still take the medicine storage liquid concentration that adds as 51.2mg/mL is example, and what now obtain is ten drug level gradients of 256～0.5 μ g/mL.
2.5 separately arrange one of blank plate (not containing medicine), and every hole adds 100 μ L Middlebrook7H9 liquid nutrient mediums and 100 μ L dilution bacterium liquid, and every strain bacterium at least arranges 8 holes; Control wells magnitude setting should be not very few, and more because of mycobacterium species kind, the speed of growth differs, and control wells quantity need to be suitably set, for experiment demand.
Each Kong Douying of the edge of 2.696 orifice plates drips physiological saline (or using sterilized water), and, with anti-drying, adding volume is 100 μ L～200 μ L
The sealed membrane of the about 1.5cm of 2.7 use width seals 96 orifice plates, dry to prevent experimental port liquid evaporation;
2.8 cultivate in 37 ℃ of fixed temperature and humidity incubators;
2.9 the interpretation of experimental result
2.9.1 from cultivate the 4th day, can in blank plate, add developer to observe strain growth situation.Each bacterial strain can be chosen a blank hole and add.Can be according to alamar Blue developer: 5%Tween-80 reagent=2: 5 ratio is carried out the premix dilution of developer, has diluted rear every hole and has added 70 these liquid of μ L, continues to put into 37 ℃ of fixed temperature and humidity incubators and cultivates, and observes every other day variable color situation.As this hole color becomes red-purple, represent this bacterial strain well-grown under its experiment condition, can in the experimental port of pastille, add developer to this bacterial strain; If do not have variable color or variable color degree more shallow, continue to add 70 μ L developer premixed liquids in another blank hole, cultivate and to observe to till variable color.
2.9.2 the second day after adding developer premixed liquid in experimental port, observes and result interpretation experimental port variable color situation.Do not have the maximum drug level having read in the minimum drug level reading in the experimental port of variable color and the experimental port that variable color has occurred to be the MIC value scope of this experimental strain to this medicine completely.
3, experimental result
Result demonstration, 1～6 pair of Mycobacterium tuberculosis H37Rv type strain of compound has stronger bacteriostatic activity, and MIC value is 0.5～4 μ g/mL; Compound 2 and 3 is except extensive persister FJ05436, other sensitive strain and persister are also had to stronger inhibition activity, MIC value is 0.0625～1 μ g/mL, and the inhibition activity of extensive persister FJ05195 is better than to the antitubercular agents such as Rimactazid, Tibutol and Streptomycin sulphate.
The active result of table 6 compound 1～6 Killing Mycobacterium Tuberculosis in vitro (MIC, unit: μ g/mL)
DS: medicaments insensitive strain; XDR: extensive persister; MDR: multidrug resistant strain
Sequence table
<110> Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
<120> elaiophylin derivative and the application in antimicrobial agent and resistance mycobacterial infections thereof
<160>?1
<210>?1
<211>?1374
<212>?DNA
<213> streptomycete (Streptomyces sp.) 7-145
<220>
<223>?16SrDNA
<400>
1?atgaaccggt?ttcggccggg?gattagtggc?gaacgggtga?gtaacacgtg?ggcaatctgc
61?cctgcactct?gggacaagcc?ctggaaacgg?ggtctaatac?cggatacgac?gcgttcccgc
121?atgggatacg?tgtggaaagc?tccggcggtg?caggatgagc?ccgcggccta?tcagcttgtt
181?ggtggggtga?tggcctacca?aggcgacgac?gggtagccgg?cctgagaggg?cgaccggcca
241?cactgggact?gagacacggc?ccagactcct?acgggaggca?gcagtgggga?atattgcaca
301?atgggcgcaa?gcctgatgca?gcgacgccgc?gtgagggatg?acggccttcg?ggttgtaaac
361?ctctttcagc?agggaagaag?cgtgagtgac?ggtacctgca?gaagaagcgc?cggctaacta
421?cgtgccagca?gccgcggtaa?tacgtagggc?gcaaagcgtt?gtccggaatt?attgggcgta
481?aagagctcgt?aggcggcttg?tcgcgtcgga?atgtgaaagc?ccggggctta?actccgggtc
541?tgcattcgat?acggggcagg?ctagagttcg?gtaggggaga?tcggaattcc?tggtgtagcg
601?gtgaaatgcg?cagatatcag?gaggaacacc?ggtggcgaag?gcggatctct?gggccgatac
661?tgacgctgag?gagcgaaagc?gtggggagcg?aacaggatta?gataccctgg?tagtccacgc
721?cgtaaacgtt?gggaactagg?tgtgggcgac?attccacgtt?gtccgtgccg?cagctaacgc
781?attaagttcc?ccgcctgggg?agtacggccg?caaggctaaa?actcaaagga?attgacgggg
841?gcccgcacaa?gcggcggagc?atgtggctta?attcgacgca?acgcgaagaa?ccttaccaag
901?gcttgacata?caccggaaaa?ctctggagac?agggtccccc?ttgtggtcgg?tgtacaggtg
961?gtgcatggct?gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca
1021?acccttgtcc?tgtgttgcca?gcgggttatg?ccggggactc?acaggagact?gccggggtca
1081?actcggagga?aggtggggac?gacgtcaagt?catcatgccc?cttatgtctt?gggctgcaca
1141?cgtgctacaa?tggccggtac?aatgagctgc?gaagccgtga?ggtggagcga?atctcaaaaa
1201?gccggtctca?gttcggattg?gggtctgcaa?ctcgacccca?tgaagtcgga?gtcgctagta
1261?atcgcagatc?agcattgctg?cggtgaatac?gttcccgggc?cttgtacaca?ccgcccgtca
1321?cgtcacgaaa?gtcggtaaca?cccgaagccg?gtggcccaac?ccttgtggag?ggag。
Claims (9)
1. an elaiophylin analog derivative, is characterized in that, described derivative derives from a strain deposit number and is
The marine actinomycete streptomycete 7-145 tunning of CGMCC No.7914, its structure is suc as formula shown in I:
11 ', 12 '-dehydration elaiophylin
Molecular formula C
54h
86o
17, molecular weight 1006.
2. an elaiophylin analog derivative, is characterized in that, described derivative derives from a strain deposit number and is
The marine actinomycete streptomycete 7-145 tunning of CGMCC No.7914, its structure is suc as formula shown in II:
11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin
Molecular formula C
55h
90o
18, molecular weight 1038.
3. the method for preparing derivative described in claim 1 or 2, is characterized in that, described method, applicable to any microorganism that can produce this compound especially tunning of streptomycete, mainly comprises the following steps:
1) by the microorganism of activation, preference chain mould 7-145 bacterial classification is inoculated in formula for the fermention medium of starch-glucose-cottonseed meal-peptone-inorganic salt, and in shaking flask, 28 ℃, shaking table is cultivated 120h;
2) fermented liquid is after solid-liquid separation, mycelium is partly used acetone supersound extraction, liquid portion adsorbs through macroporous adsorptive resins, water, 50%, 100% acetone wash-out successively, 50%, 100% acetone elutriant part merges with mycelium acetone extract, after concentrating under reduced pressure, be extracted with ethyl acetate, obtain fermented liquid medicinal extract;
3) gained medicinal extract utilizes forward silica gel column chromatography separated, use successively Shi You Mi ?ethyl acetate (9:1,4:1,1:1), ethyl acetate, Yi Suan Yi Zhi ?methyl alcohol (1:1) and methyl alcohol gradient elution; Wherein, Yi Suan Yi Zhi ?methyl alcohol (1:1) wash-out part separated through forward silica gel column chromatography again, use successively Er Lv Jia Wan ?methyl alcohol (20:1,9:1,4:1,1:1 and 0:1) gradient elution, obtain 7 streams part (an A～G); Stream part C and stream part D respectively through Sephadex LH ?20 column chromatographys decolourings, Er Lv Jia Wan ?methyl alcohol 1:1 wash-out, obtain active constituent; Active constituent after decolouring is prepared and partly prepares purifying through HPLC respectively, obtains compound 1 shown in formula I, formula II and compound 2.
4. the application of compound in preparing antimicrobial agent infection medicine described in claim 1 or 2.
5. described in claim 1 or 2, compound comprises the application in extensive resistance XDR, multidrug resistant MDR m tuberculosis infection medicine in preparation Killing Mycobacterium Tuberculosis.
6. the derivative described in claim 1 or 2 of take is effective constituent, the pharmaceutical composition forming with pharmaceutically acceptable one or more carriers.
7. the application of composition in preparing antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infection medicine described in claim 6.
8. one group of elaiophylin derivative application in preparing antimicrobial agent and Drug-Resistant Mycobacterium tuberculosis infection medicine, described derivative, deriving from a strain deposit number is the marine actinomycete streptomycete 7-145 tunning of CGMCC No.7914, is R shown in formula III
1=R
2=hydroxyl, R
3=R
4=methyl, R
5the compound 3 of=hydrogen: elaiophylin; R
1=methoxyl group, R
2=hydroxyl, R
3=R
4=methyl, R
5the compound 4:11-O-methyl elaiophylin of=hydrogen; R
1=R
2=methoxyl group, R
3=R
4=methyl, R
5the compound 5:11 of=hydrogen, 11 '-O-dimethyl elaiophylin; And R
1=R
2=hydroxyl, R
3=R
5=hydrogen, R
4the compound 6:14 ' of=methyl-go ethyl-14 '-methyl elaiophylin.
9. one group of elaiophylin derivative is in the application of preparing antimicrobial agent and m tuberculosis infection medicine, and described derivative contains following substituted radical shown in formula III:
R
1, R
2=hydrogen or hydroxyl, include but not limited to C1～C5 alkoxy substituents such as methoxyl group, oxyethyl group, C2～the C6 such as vinyloxy group, propenyloxy group alkene oxy substituents, C1～C5 acyloxy such as C2～C6 alkynyloxy group, C3～C6 cycloalkyloxy, methanoyl, acetoxyl group; The C6 such as phenoxy group～C12 virtue epoxy group(ing);
C-11-C-12: can be singly-bound or two key;
C-11 '-C-12 ': can be singly-bound or two key;
R
3, R
4=hydrogen or methyl, include but not limited to C2～C5 saturated alkyl substituting groups such as ethyl, propyl group, C2～C6 alkenyl groups such as ethene, propylene, C2～C6 alkynyl, C3～C6 cycloalkyl;
R
5=hydrogen, includes but not limited to C1～C5 saturated alkyl substituting groups such as methyl, ethyl, propyl group, C2～C6 alkenyl groups such as ethene, propylene, C1～C5 acyl groups such as C2～C6 alkynyl, C3～C6 cycloalkyl, formyl radical, ethanoyl; C6～C12 aromatic ring groups such as phenyl.
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