CN103665071B - Elaiophylin derivative and the application in antimicrobial agent and resistance m tuberculosis infection thereof - Google Patents

Abstract

The present invention relates to elaiophylin derivative and the application in antimicrobial agent and resistance m tuberculosis infection thereof, does described derivative system come from marine actinomycete streptomycete 7-145(Streptomyces? sp.7-145) tunning, experimental studies results proves, the new texture found and known structure compound, all tested resistant organism and resistance mycobacterium tuberculosis are had very strong anti-microbial activity, it is expected to become clinical useful antimicrobial agent and resistance m tuberculosis infection novel drugs.

Description

Elaiophylin derivative and the application in antimicrobial agent and resistance m tuberculosis infection thereof

Technical field:

The present invention relates to and obtain new compound from marine microorganism resource; The present invention relates to the method for fermentation for described natural compounds; The present invention also relates to the application of described compound in anti-multiple drug-resistant bacteria and resistance m tuberculosis infection.

Background technology:

At present, bacterial resistance is day by day serious, researches and develops new microbiotic extremely urgent. Elaiophylin (English name: Elaiophylin, AzalomycinB, Gopalamicin, or Salbomycin) it is the big ring dilactone microbiotic of sixteen-ring that a class has two-fold rotational symmetry, there are the biological activity [Yan Shuling etc. widely such as antibacterial, parasiticide, immunosuppression, promotion cud animal growth, microbiology circular 2002,29,103 107]. In recent years, a series of material with biological activity is developed in this laboratory from marine microorganism resource, wherein finds several novel compounds and the novelty teabag of known structure compound, drug-fast bacteria infection especially has the compound of good anti-microbial activity.

The elaiophylin producing strains of hitherto reported is streptomycete, comprise raw spore streptomycete (Streptomycesmelanosporus) [Arcamone, F.M, etal.Giorn.Microbiol.1959, 7, 207 216], streptomyces hygroscopicus (Streptomyceshygroscopicus) [Arai, M.J.Antibiot.1960, 13, 46 50], purple black streptomycete (Streptomycesviolaceoniger) [Fiedler, H.P., etal.J.Antibiot.1981, 34, 1107 1118], streptomyces albus (Streptomycesalbus) [Klebarobva, E.I.etal.Farmatiya (Sofia), 1972, 22, 3.] etc.

The present invention derives from the fermented liquid activeconstituents of marine actinomycete streptomycete 7-145 (Streptomycessp.7-145) from a strain, 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 '-remove ethyl-14 '-methyl elaiophylin (efomycinG). antibacterial activity in vitro test result shows, the multiple drug-resistant bacteria comprising MRSA (methicillin-resistant staphylococcus aureus), MRSE (Methicillin-resistant Staphylococcus epidermidis), VRE (vancomycin resistance enterococcus faecalis, faecium), extensive resistance (XDR) and multidrug resistant (MDR) mycobacterium tuberculosis is had good anti-microbial activity by these elaiophylin compounds. relate to that the chemical structure of compound 1 and 2, preparation method, antimicrobial agent and tuberculosis be active and the elaiophylin derivative such as compound 3��6 to the anti-microbial activity of resistant organism and mycobacterium tuberculosis, up to now, there is not yet relevant report.

Summary of the invention:

One of the object of the invention is, it is provided that a strain produces the producing strains of elaiophylin derivative;

The two of the object of the invention are, it is provided that elaiophylin derivative, especially new elaiophylin derivative;

The three of the object of the invention are, it is provided that the preparation method of described elaiophylin derivative;

The four of the object of the invention are, it is provided that described elaiophylin derivative is in the application prepared in antimicrobial agent and resistance m tuberculosis infection medicine.

The present invention has found the elaiophylin derivative 1 and 2 of two novel structures first from the tunning of marine actinomycete streptomycete (Streptomycessp.) 7-145, and its structure is such as formula shown in I, formula II:

Compound 1:11 ', 12 '-dehydration elaiophylin (11 ', 12 '-dehydroeliophylin)

Molecular formula C₅₄H₈₆O₁₇, 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 C₅₅H₉₀O₁₈, molecular weight 1038.

The present invention from, the tunning of marine actinomycete streptomycete (Streptomycessp.) 7-145, also having found that compound 3��6 has good antimicrobial agent and resistance 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 '-remove ethyl-14 '-methyl elaiophylin.

The preparation method of elaiophylin provided by the present invention and derivative thereof comprises the following steps:

Filtrate and mycelium is obtained after streptomycete (Streptomycessp.) 7-145 fermentation liquor solid-liquid separation. Mycelium acetone supersound extraction; Filtrate is adsorbed through AmberliteXAD7HP macroporous adsorptive resins, successively with water, 50% and 100% aqueous acetone solution wash-out; Being merged with mycelium acetone extract by 50%, 100% acetone elutriant, concentrating under reduced pressure obtains the aqueous solution after removing acetone, it may also be useful to extraction into ethyl acetate, and concentrating under reduced pressure obtains brown medicinal extract except after ethyl acetate; This medicinal extract utilizes just to silica gel column chromatography separation, successively with petroleum ether-ethyl acetate (9:1,4:1,1:1), ethyl acetate, acetate-methanol (1:1) and methyl alcohol gradient elution; Wherein, acetate-methanol (1:1) elution fraction, again through just to silica gel column chromatography separation, successively with 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 chief active group part, then obtain compound 3 (elaiophylin) through chloroform-methanol solvent recrystallization; Stream part C and stream part D is respectively through the decolouring of SephadexLH-20 column chromatography, and methylene chloride-methanol 1:1 wash-out, obtains active constituent; The active constituent obtained after mother liquor after stream part B crystallization and stream part C, D being decoloured, prepares through HPLC respectively and partly prepares purifying, obtain compound 1��6.

The streptomycete used in described preparation method, it is preferably streptomycete (Streptomycessp.) 7-145, system's separation is about the marine bottom sediment of 40m from the DaLian, China Huanghai Sea black Shi Jiao bay degree of depth, this bacterial strain delivers China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on July 12nd, 2013, deposit number: CGMCCNo.7914. Streptomycete (Streptomycessp.) 7-14516SrDNA gene order, as shown in SEQIDNO.1, submits to NCBI, GenBank accession number to be JQ782979.

The present invention adopts agar dilution to test described elaiophylin and derivative thereof to comprising MRSA, VRE and the minimal inhibitory concentration (MIC) of multidrug resistant (MDR), the extensively multiple drug-resistant bacteria of resistance (XDR) mycobacterium tuberculosis. Experimental result shows these compounds and tested Resistant strain is had good anti-microbial activity. Show that described elaiophylin analog derivative can be used as the medicine preparing antimicrobial agent and resistance m tuberculosis infection; And using described derivative as activeconstituents, with one or more carriers pharmaceutically acceptable, vehicle or supplementary product compatibility, can be made into antimicrobial agent and the pharmaceutical composition of resistance m tuberculosis infection. Described medicine and pharmaceutical composition can be used for drug-fast bacteria infection and the clinical treatment of resistance m tuberculosis infection. Described compound also can form compound preparation for the treatment of drug-fast bacteria infection and resistance m tuberculosis infection with known drug.

Through the method for elaiophylin derivative 1 and 2 shown in fermentable preparation formula I and formula II in the present invention, the applicable any microorganism that can produce this compounds in other.

The present invention also comprises other substituted radical elaiophylin derivative shown in formula III, and in the application prepared in antimicrobial agent and resistance m tuberculosis infection medicine, described substituting group is because of as follows:

R₁, R₂=hydrogen or hydroxyl, include but not limited to C1��C5 alkoxy substituent such as methoxyl group, oxyethyl group, C2��C6 alkene oxy substituents such as vinyloxy group, propylene oxygen base, C1��C5 acyloxy such as C2��C6 alkynyloxy group, C3��C6 ring alkoxyl group, methanoyl, acetoxyl group; C6��C12 virtue epoxy group(ing) such as phenoxy group

C-11-C-12: can be singly-bound or double bond

C-11 '-C-12 ': can be singly-bound or double bond

R₃, R₄=hydrogen or methyl, include but not limited to C2��C5 saturated alkyl substituting group such as ethyl, propyl group, C2��C6 alkenyl group such as ethene, propylene, C2��C6 alkynes base, C3��C6 cycloalkyl

R₅=hydrogen, includes but not limited to C1��C5 saturated alkyl substituting groups such as methyl, ethyl, propyl group, C2��C6 alkenyl group such as ethene, propylene, C1��C5 acyl groups such as C2��C6 alkynes base, C3��C6 cycloalkyl, formyl radical, ethanoyl; C6��C12 aromatic ring groups such as phenyl.

And using described derivative as activeconstituents, with one or more carriers pharmaceutically acceptable, vehicle or supplementary product compatibility, can be made into antimicrobial agent and the pharmaceutical composition of resistance m tuberculosis infection. Described medicine and pharmaceutical composition can be used for drug-fast bacteria infection and the clinical treatment of resistance m tuberculosis infection. Described compound also can form compound preparation for the treatment of drug-fast bacteria infection and resistance m tuberculosis infection with known drug.

Invention effect:

The present invention is separated from the tunning of strain marine actinomycete streptomycete (Streptomycessp.) 7-145 and prepares such as formula compound 3��6 four known elaiophylin derivatives shown in elaiophylin derivative 1 and 2 novel shown in I, formula II and formula III; The multiple drug-resistant bacteria comprising MRSA, VRE is had good anti-microbial activity by these compounds, is expected to develop the newtype drug becoming clinical upper antimicrobial agent and resistance m tuberculosis infection or the control for agricultural pest.

Accompanying drawing illustrates:

Fig. 1: the phylogenetic evolution tree of the 16SrRNA gene order of streptomycete (Streptomycessp.) 7-145 is analyzed

The high resolution electrospray ionization mass spectrum of Fig. 2: 11 ', 12 '-dehydration elaiophylin (1)

Fig. 3: 11 ', 12 '-dehydration elaiophylin (1)¹HNMR composes

Fig. 4: 11 ', 12 '-dehydration elaiophylin (1)¹³CNMR composes

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)¹HNMR composes

Fig. 7: 11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin (2)¹³CNMR composes

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 the present invention better below, but does not limit the present invention in any way.

The qualification of<embodiment 1>streptomycete (Streptomycessp.) 7-145

A) bacterium source: streptomycete (Streptomycessp.) 7-145 is about in the oceanic sediment of 40m by this laboratory from the black Shi Jiao bay of the DaLian, China Huanghai Sea (38 �� of 49'N, the 121 �� of 34'E) degree of depth to be separated obtain.

B) identification of strains: according to the conservative property of 16SrRNA gene order in microorganism kind, streptomycete (Streptomycessp.) 7-145 is identified. Extracting the genome of streptomycete (Streptomycessp.) 7-145, its 16SrRNA gene of pcr amplification also checks order, and being submitted to NCBIGenBank database acquisition accession number is JQ782979, and its sequence is as shown in SEQIDNO.1. The 16SrRNA gene comparison result display of streptomycete (Streptomycessp.) 7-145, this bacterium and streptomyces bacterial strain StreptomycessamsunensisM1463^TSimilarity be 99.85%. By contiguous method, constructing system grows evolutionary tree (Fig. 1).

SEQIDNO.1

Show, this bacterium belongs to the one in Streptomycetaceae streptomyces, accordingly by this bacterium called after streptomycete (Streptomycessp.) 7-145, and it being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 12nd, 2013, deposit number is: CGMCCNo.7914.

The fermentation of<embodiment 2>streptomycete (Streptomycessp.) 7-145

Streptomycete (Streptomycessp.) 7-145 getting activation is dull and stereotyped, chooses and gets about 1cm²Bacterium tongue is inoculated in 100mL fermention medium, and [formula is: starch 1.0g, glucose 2.5g, cottonseed meal 1.0g, and peptone 0.3g, inorganic salt are such as KH₂PO₄0.01g, MgSO₄0.01g, NaCl0.5g, CaCO₃0.5g etc., deionized water 100mL], in 500mL shaking flask, 28 DEG C, 120h cultivated by 200r/min shaking table.

<embodiment 3>extraction of fermented liquid and the acquisition of medicinal extract

Filtrate and mycelium is obtained after streptomycete (Streptomycessp.) 7-145 fermentation liquor solid-liquid separation; Mycelium uses acetone supersound extraction, obtains mycelium acetone extract; Filtrate through AmberliteXAD7HP macroporous adsorptive resins absorption, successively with 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 extraction into ethyl acetate; Ethyl acetate extraction part concentrating under reduced pressure, obtains fermented liquid medicinal extract.

The separation of<embodiment 4>compound 1��6, preparation and structure qualification

Get streptomycete (Streptomycessp.) 7-145 fermented liquid medicinal extract (17g), through silica gel column chromatography separation, successively with petroleum ether-ethyl acetate (9:1,4:1,1:1), ethyl acetate, acetate-methanol (1:1) and methyl alcohol gradient elution; Acetate-methanol (1:1) wash-out stream part (6.5g), through being just separated to silica gel column chromatography, successively with 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) is separated through anti-phase C18 silica gel flash column chromatography, and 30��100% methyl alcohol gradient elutions obtain 1 chief active group part, obtain compound 3 elaiophylin (500mg) through chloroform-methanol solvent recrystallization; Stream part C (0.9g) and stream part D (0.2g) are respectively through the decolouring of SephadexLH-20 column chromatography, and methylene chloride-methanol 1:1 wash-out, obtains active constituent; The active constituent obtained after mother liquor after stream part B crystallization and stream part C, D being decoloured, prepares (Capcell-PakC through HPLC₁₈AQ5um, 20 �� 250mm, 88% acetonitrile, 5mL/min) obtain 6 components, wherein 3 components are respectively compound 3 shown in formula III (elaiophylin, 320mg), compound 4 (11-O-methyl elaiophylin, 520mg), compound 5 (11,11 '-O-dimethyl elaiophylin, 650mg); All the other 3 components are again through HPLC half preparation (Capcell-PakC₁₈5um, 10 �� 250mm, 68% or 87% acetonitrile, 2mL/min) purifying, obtains (11 ', the 12 '-dehydration elaiophylin of structural compounds 1 shown in formula I, formula II and formula III respectively, 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 structure qualification of 12 '-dehydration elaiophylin (1)

Compound 1 is white powder, is soluble in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, is insoluble to water, 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, it is determined that its molecular formula is C₅₄H₈₆O₁₇, than being separated the few 1 molecule H of the elaiophylin molecular formula obtained from this fermented liquid simultaneously₂O. Compound 1 hydrogen spectrum (¹HNMR) (Fig. 3) and carbon spectrum (¹³CNMR) (Fig. 4) data with there is C₂Elaiophylin (compound 3) [Kaiser, H., the etal.Helv.Chim.Acta1981,64,407-424 of-symmetrical structure; Nair, M.G., etal.J.Agric.Food.Chem.1994,42,2308-2310.Lee, S.Y., etal.J.Microbiol.Biotechnol.1996,6,245-249] Reported data closely similar, but the signal of compound 1 is more complicated, prompting compound 1 is the unsymmetrical structure derivative of elaiophylin. In conjunction with¹H-¹Relevant peaks in HCOSY spectrum, hydrogen spectrum display two 1,3-conjugated diolefine systems of compound 1 and an independent alkene Hydrogen Proton �� 4.74 (d, J=3.0Hz, H-12 '), prompting compound 1 may be the anhydro compounds of elaiophylin. Utilize two dimensional NMR wave spectrum (¹H-¹HCOSY, HSQC, HMBC and ROESY compose) structure of compound 1 has been confirmed. First, the HMBC of compound 1 demonstrates H-2, H-3, H-7 ' and C-1 in composing; H-2 ', H-3 ', H-7 and C-1 '; H-10, H-12eq, H-15, H₃-19, 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 fragments such as C-1��C-27, C-1 '��C-10 ', C-22 '��C-27 ' in compound 1 and elaiophylin completely the same; In addition, HMBC also demonstrates H-10 ', H-12 ', H-13 ', H-15 ', H in composing₃-19 ' with C-11 ' (��_C156.6) relevant peaks, in conjunction with the chemical shift of these protons and carbon, illustrate that C-11 ' and C-12 ' is replaced by double bond in compound 1, namely dewater at C-11 ' and C-12 ' position, prove that C-11 ' is connected with C-10 ' simultaneously, thus define 1 dihydropyrane ring structure unit at C-11 '��C-14 ' place; Finally, in composing according to HMBC, H-13 ' is relevant to C-22 ', it was demonstrated that other 1 2-deoxidation-L-fucose unit (C-22 '��C-27 ') is connected on C-13 '. Thus the integral planar structure of compound 1 is determined. Except alkene Hydrogen Proton H-12 ' and double key carbon C-11 ', C-12 ', other proton coupling constant in compound 1 hydrogen spectrum, carbon modal data and ROESY compose in correlation peak signal, all similar to elaiophylin, show that these two compounds have identical relative configuration [Neupert-Laves, K., etal.Helv.Chim.Acta1982,65,262-267; Paulus, E.F., etal.ActaCrystallogr.C1984,40,700-703]; Meanwhile, the CD spectrum of these two compounds highly overlaps, and due to they common biological relations, 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), and this compound has no document report, is novel compound.

The spectral data of 11 ', 12 '-dehydration elaiophylin (1): [��]²⁰ _D-35.0 (c0.29, MeOH);¹H-NMR (Fig. 3) (DMSO-d₆, 500MHz) and data are in table 1;¹³C-NMR (Fig. 4) (DMSO-d₆, 125MHz) and data are in table 2; HRESIMSm/z1029.5776 [M+Na]⁺(calculated value C₅₄H₈₆O₁₇Na, 1029.5757), 1045.5514 [M+K]⁺(calculated value C₅₄H₈₆O₁₇K,1045.5497)��

B) 2:11,11 ' of compound shown in formula II-O-dimethyl-14 '-go the structure of ethyl-14 '-methyl elaiophylin (2) to identify

Compound 2 is white powder, is soluble in methyl alcohol, DMSO, is slightly soluble in chloroform, acetone, is insoluble to water, sherwood oil etc. High resolution electrospray ionization mass spectrum (HRESIMS) shows its quasi-molecular ion peak m/z1061.6019 [M-H]^-(Fig. 5), determine that its molecular formula is C in conjunction with NMR data₅₅H₉₀O₁₈. Compound 2 is closely similar with the data of elaiophylin, but with elaiophylin the difference is that, the hydrogen spectrum of compound 2 has lacked two tradable hydroxyl proton signals, but has gone out two methoxyl group signal �� more_H2.94 with 2.95; Observe these two methoxyl group protons composing from the HMBC of compound 2 and C-11 and C-11 ' has coherent signal, show that they are connected on C-11 and C-11 '; In addition, 1 methylene signals that the NMR data of compound 2 is fewer than elaiophylin; And demonstrate a bimodal methyl proton (H in its HMBC composes₃-20��,��_H0.85, d, J=6.0Hz) relevant to C-13 ', C-14 ', C-15 ' respectively, and¹H-¹HCOSY compose in also to observe this methyl proton relevant to H-14 ', point out in compound 2 methyl substituted at C-14 ', being different from elaiophylin C-14 ' is replaced by ethyl. The hydrogen spectrum proton coupling constant of compound 2, carbon modal data and ROESY spectrum are all similar to elaiophylin, illustrate that compound 2 is identical with elaiophylin configuration. Therefore, compound 2 structure 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): [��]²⁰ _D+ 21.6 (c0.59, MeOH);¹H-NMR (Fig. 6) (DMSO-d₆, 500MHz) and data are in table 1;¹³C-NMR (Fig. 7) (DMSO-d₆, 125MHZ) and in table 2; HRESIMSm/z1061.6019 [M+Na]⁺(calculated value C₅₅H₉₀O₁₈Na, 1061.6019) and 1077.5760 [M+K]⁺(calculated value C₅₅H₉₀O₁₈K,1077.5759)��

C) compound 3 shown in formula III: the structure qualification of elaiophylin (3)

Compound 3 is white column crystallization, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, is insoluble to water, sherwood oil etc. [��]²⁰ _D-56.4 (c0.50, MeOH);¹H-NMR(DMSO-d₆, 500MHz) and¹³C-NMR(DMSO-d₆, 125MHZ) and in table 3; ESIMSm/z1023 [M-H]^-; 1047 [M+Na]⁺; 1063 [M+K]⁺. Above data and document [Kaiser, H., etal.Helv.Chim.Acta1981,64,407-424; Nair, M.G., etal.J.Agric.Food.Chem.1994,42,2308-2310.Lee, S.Y., etal.J.Microbiol.Biotechnol.1996,6,245-249] elaiophylin (elaiophylin) data consistent reported, therefore the structure of compound 3 is defined as elaiophylin.

D) the structure qualification 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, is insoluble to water, sherwood oil etc. [��]²⁰ _D-26.8 (c0.64, MeOH);¹H-NMR(DMSO-d₆, 500MHz) and data are in table 4;¹³C-NMR(DMSO-d₆, 125MHZ) and in table 2; ESIMSm/z1037 [M-H]^-; 1061 [M+Na]⁺; 1077 [M+K]⁺. Above data and document [Ritzau, M.etal.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 reported, therefore the structure of compound 4 is defined as 11-O-methyl elaiophylin.

E) the structure qualification of the 5:11,11 ' of compound shown in formula III-O-dimethyl elaiophylin (5)

Compound 5 is white powder, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, is insoluble to water, sherwood oil etc. [��]²⁰ _D+ 15.0 (c0.62, MeOH);¹H-NMR(DMSO-d₆, 500MHz) and¹³C-NMR(DMSO-d₆, 125MHZ) and in table 3; ESIMSm/z1051 [M-H]^-; 1075 [M+Na]⁺; 1091 [M+K]⁺. Above data and document [Ritzau, M.etal.J.Nat.Prod.1998,61,1337-1339] report 11,11 '-O-dimethyl elaiophylin (11,11 '-O-dimethylelaiophylin) data consistent, therefore the structure of compound 5 is defined as 11,11 '-O-dimethyl elaiophylin.

F) 6:14 ' of compound shown in formula III-go the structure of ethyl-14 '-methyl elaiophylin (6) to identify

Compound 6 is white powder, is dissolved in methyl alcohol, DMSO, is slightly dissolved in chloroform, acetone, is insoluble to water, sherwood oil etc. [��]²⁰ _D-47.4 (c0.24, MeOH);¹H-NMR(DMSO-d₆, 500MHz) and data are in table 4;¹³C-NMR(DMSO-d₆, 125MHZ) and data are in table 2; ESIMSm/z1009 [M-H]^-; 1033 [M+Na]⁺; 1049 [M+Na]⁺. Above data and document [Frobel, K., etal.U.S.Patent4927810,1990] report 14 '-remove ethyl-14 '-methyl elaiophylin (efomycinG) data consistent, therefore the structure of compound 6 is defined as 14 '-removes ethyl-14 '-methyl elaiophylin.

Table 1. compound 1 and 2¹HNMR data (500MHz, DMSO-d₆ ^a)

^aSignals assignment foundation¹H-¹HCOSY, HSQC and HMBC two dimensional NMR is tested.

Table 2. compound 1,2,4 and 6¹³CNMR data (125MHz, DMSO-d₆ ^a)

^aSignals assignment foundation¹H-¹HCOSY, HSQC and HMBC two dimensional NMR is tested.

NMR data (the DMSO-d of table 3. compound 3 and 5₆ ^a)

^a1HNMR data measure at 500MHz;¹³CNMR spectrum measures at 125MHz; Signals assignment foundation¹H-¹HCOSY, HSQC and HMBC two dimensional NMR is tested.

Table 4. compound 4 and 6¹HNMR data (��_H[J (Hz)], mult. 500MHz, DMSO-d₆ ^a)

^aSignals assignment foundation¹H-¹HCOSY, HSQC and HMBC two dimensional NMR is tested.

The anti-microbial activity test of<embodiment 5>elaiophylin derivative

1, experiment material

Assay strain: StaphylococcusaureusATCC29213 (methicillin-sensitivity streptococcus aureus); Staphylococcusaureus09-6 (methicillin-sensitivity streptococcus aureus, clinical separation strain); StaphylococcusaureusATCC33591 (methicillin resistance streptococcus aureus); Staphylococcusaureus09-13 (methicillin resistance streptococcus aureus, clinical separation strain); StaphylococcusaureusR6101 (methicillin resistance streptococcus aureus, clinical separation strain in 2010); StaphylococcusaureusATCC6538P (thiostrepton resistant S staphylococcus, be derived from ATCC6538 mutagenesis); StaphylococcusepidermidisATCC12228 (methicillin-sensitivity staphylococcus epidermidis); Staphylococcusepidermidis09-9 (methicillin-sensitivity staphylococcus epidermidis, clinical separation strain); Staphylococcusepidermidis09-3 (methicillin resistance staphylococcus epidermidis, clinical separation strain); EnterococcusfaecalisATCC29212 (vancomycin sensitive enterococcus faecalis); Enterococcusfaecalis09-8 (vancomycin sensitive enterococcus faecalis, clinical separation strain); EnterococcusfaecalisATCC51299 (drug resistance of vancomycin enterococcus faecalis); EnterococcusfaecalisW4138 (drug resistance of vancomycin enterococcus faecalis, clinical separation strain in 2010); EnterococcusfaecalisR6512 (drug resistance of vancomycin enterococcus faecalis, clinical separation strain in 2010); Enterococcusfaecium09-10 (vancomycin sensitive faecium); EnterococcusfaeciumATCC700221 (drug resistance of vancomycin faecium); Micrococcusluteus (apramycin resistance micrococcus luteus, be derived from ATCC10240 mutagenesis); ProteusbacillusvulgarisATCC13315 (Bacillus proteus); BacillussubtilisCMCC63501 (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.

CM0984VREBROTHBASE substratum (purchased from OXOID company).

CM0984VREAGARBASE substratum: add 2.0g agar in every 100mLCM0984VREBROTHBASE.

Control sample: Azythromycin, erythromycin, Oxazacillin, vancomycin (examining and determine institute purchased from Chinese food medicine); Sulfuric acid Apramycin sulfate (Apramycin sulphate) (purchased from Shanghai Jiang Lai Bioisystech Co., Ltd).

2, experimental technique

Agar dilution is adopted to measure sample to the MIC value of each strain test organism, specific as follows:

A) sample configuration: accurately take moderate amount of 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 appropriate embodiment 4 refining compound 1��6 sample and other control sample standard substance, it is dissolved in dimethyl sulfoxide (DMSO) (DMSO), it is configured to certain final concentration solution, then becomes desired concn solution with DMSO by doubling dilution, for active testing.

B) prepare containing medicine agar plate: the sample of the different concns of doubling dilution is added heating for dissolving respectively, and in the Mueller-Hinton nutrient agar (or LB nutrient agar, CM0984VREAGARBASE substratum) balanced in 45��50 DEG C of water-baths, fully mixed even topple over sterilizing plate, agar thickness 3��4mm; In 1: 9 ratio compounding pharmaceutical agar plate; The sample final concentration respectively containing medicine agar plate is respectively 0.25,0.5,1.0,2.0,4.0,8.0,16,32,64,128,256 �� g/mL.

C) inoculum preparation and inoculation: get each test organism glycerine pipe and be inoculated in about 3mL liquid nutrient medium by 1% (v/v), wherein, EnterococcusfaecalisR6512 and W4138 uses CM0984VREBROTHBASE substratum, Micrococcusluteus, ProteusbacillusvulgarisATCC13315, BacillussubtilisCMCC63501, use LB broth culture, all the other test organisms are all inoculated in Mueller-Hinton broth culture; In 37 DEG C after inoculation, 200rpm jolting overnight incubation; Bacterium liquid, according to the dense dilution of bacterium, prepares bacterium liquid (about 1��2 �� l) with multi-point inoculator absorption and is inoculated in agar plate surface, and each test organism uses the medium preparing agar plate same with preparation bacterium liquid phase. Often some bacterium number is about 10⁴CFU (colony-forming unit), forming diameter is the bacterial plaque of 5��8mm, and 18h is hatched in good rearmounted 35 DEG C of inoculation.

D) result judges: is placed in by flat board on dead color, no-reflection body surface and judges test endpoint, taking the minimum adding consistency of macroscopic bacteria growing inhibiting as minimum inhibitory concentration (MIC).

3, the Macrocyclic lactone compounds of elaiophylin class shown in experimental result: formula I, II, III 1��6 and control sample to the MIC value of 20 strain test organisms in table 5.

Experimental result shows, elaiophylin and derivative 1��6 thereof are to, in the 20 strain test organisms comprising MRSA and VRE, the MIC value of the overwhelming majority is at 1��64 �� g/mL, it is shown that these compounds have good anti-microbial activity. It is worth being pointed out that, elaiophylin shown in formula I, II, III (3) and derivative 1,2,4��6 thereof the anti-microbial activity of MRSA, MRSE and VRE is better than clinical on the first-selected macrolide antibiotics Azythromycin that widely uses, this prompting elaiophylin compounds may not by the impact [NakajimaY.JInfectChemother of some macrolide resistance mechanism common in positive bacteria, 1999,5 (2): 61-74; PechereJC.IntJAntimicrobAgents, 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 taking it as activeconstituents, with one or more carriers pharmaceutically acceptable, vehicle or supplementary product compatibility, make the pharmaceutical composition that antimicrobial agent infects.

Table 5. elaiophylin and derivative antibacterial minimal inhibitory concentration value (MIC, unit �� g/mL) thereof

Note:^a-do not detect;^bSulfuric acid Apramycin sulfate MIC value is 8 �� g/mL.

4, structure activity relationship

As can be seen from Table 5, when one of them hydroxyl of C-11 or C-11 ' by methoxy substitution (such as compound 4) or occurs dehydration when C-11��C-12 place formation double bond (such as compound 1), compared with elaiophylin (3), the anti-microbial activity of most of resistant organism is reduced about 2 times (MIC:1��2 �� g/mL is than 2��4 �� g/mL); And if two hydroxyls on C-11 and C-11 ' are all by (such as compound 2 and 5) during methoxy substitution, the derivative (such as compound 6 and 3) replaced for hydroxyl with corresponding C-11 with C-11 ' is compared, active reduction by 8��32 times (MIC:8-64 �� g/mL is than 1-2 �� g/mL); 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 ' is (such as compound 2 and 6) during methyl substituted, the active reduction by 2��4 times of the corresponding derivative (such as compound 5 and 3) replaced for ethyl than C-14 or C-14 ', shows the substituted radical on elaiophylin C-14 or C-14 ' or in its structure, anti-microbial activity is had material impact by the integrity of C-1��C-27.

Mycobacterium tuberculosis bacteriostatic activity is tested by<embodiment 6>elaiophylin derivative

1, experiment material

Assay strain: MycobacteriumtuberculosisH37Rv (Mycobacterium tuberculosis H37Rv reference culture, ATCC27294); M.tuberculosisFJ05436 (extensive persister XDR, clinical separation strain), M.tuberculosisFJ05349 (responsive strain, clinical separation strain), M.tuberculosisFJ05060 (responsive strain, clinical separation strain), M.tuberculosisFJ05195 (extensive persister XDR, clinical separation strain), M.tuberculosisFJ05120 (multidrug resistant strain MDR, clinical separation strain). Above bacterial strain is preserved by Disease Control and Prevention in China center.

Prefabricated Middlebrook7H9 liquid nutrient medium: take 4.7g7H9 pulvis and add 900mL distilled water, add glycerine 2mL, 121 DEG C of autoclavings 15 minutes, after naturally cooling, put into 4 DEG C of refrigerators for subsequent use.

Prepare OADC (OleicAcidDextroseCatalase includes oleic acid, albumin, dextrose, catalase) nutrition enrichment liquid (commercial reagents can be bought).

The medicament storage liquid of preparation different concns: such as 51.2mg/mL, 102.4mg/mL isoconcentration (concrete concentration can be prepared in conjunction with actual experiment demand), after 0.2 ��m of filter membrane filter sterile filtration, put-20 DEG C of refrigerator cold-storages.

Assay: purchased from U.S. company BD.

Other article: several of mill bacterium bottle (the interior aseptic screw-cap bottle with granulated glass sphere) (each one of each experimental strain), than turbid (examination) pipe number only (each one of each experimental strain), 15mL or 50mL centrifuge tube number (each one of each experimental strain, specification is selected) by actual experiment demand, 10 �� L rifle heads, 200 �� L rifle heads, 1000 �� L rifle heads, transfering loop, physiological saline; 5%Tween-80 reagent. Above equipment all at 121 DEG C, need to use after 15min sterilizing.

Separately need: each range pipettor, 1.0 standard Maxwells are than turbid pipe (MacFarlandMo.1) 1 or than one, turbid instrument, cryopreservation tube, 96 orifice plates (band lid), sealing film, 37 DEG C of fixed temperature and humidity incubators, turbula shaker, 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 the ratio of 10% 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), 1��2 5%Tween-80 reagent is added. And the mycobacterium choosing pure separation and Culture logarithmic phase (3��4 weeks) in L-J substratum is cooked bacteria suspension. With transfering loop from the bacterium of the L-J medium slant of this bacterium scraping semi-ring or a ring (5��10mg), be placed in mill bacterium bottle and vibrate on turbula shaker about 1min, until bacterium colony grind completely even after to leave standstill 15min stand-by;

2.2.2 carefully open mill bacterium bottle cap, add 2��3mL physiological saline, leave standstill and make bulk matter precipitation in bacterium liquid;

2.2.3 get appropriate supernatant to than in turbid pipe with aseptic straw, and regulate concentration to 1 Maxwell concentration (now bacteria concentration is about 1mg/mL) with physiological saline;

2.2.4 a 15mL or 50mL centrifuge tube (selecting specification by actual experiment demand) is got again, in 1: the 20 ratio Middlebrook7H9 liquid nutrient medium dilution bacterium liquid adding OADC nutrition enrichment liquid.

2.3 structure drug concentration gradient

2.3.1 in 96 orifice plates except each hole, edge, every hole adds 100 �� LMiddlebrook7H9 liquid nutrient mediums, and now except each hole, edge, the total 6*10 hole of every plate with the addition of Middlebrook7H9 liquid nutrient medium.

2.3.2 98 �� LMiddlebrook7H9 liquid nutrient mediums are added again in the first row (the 2nd row when namely calculating from marginal pore) except each hole, edge;

2.3.3 the pre-configured medicament storage liquid of 2 �� l is added again in the first every hole of row (the 2nd row when namely calculating from marginal pore) except each hole, edge;

2.3.4 dilute principle according to twice to dilute successively, namely from the first row adding medicine, the Middlebrook7H9 liquid nutrient medium of 100 �� L drug containing is drawn to the 2nd row in every hole, again from the Middlebrook7H9 liquid nutrient medium of the 2nd row absorption 100 �� L drug containing to the 3rd row after mixed even, proceeding to the tenth row successively, last row are abandoned after drawing out 100 �� L. The medicament storage liquid concentration such as added is: 51.2mg/mL, then can obtain ten drug concentration gradient from 512��1 �� g/mL; If when experiment needs more concentration, can serial dilution two 96 orifice plates, thus obtain more drug concentration gradient.

2.4 every strain bacterium inoculate a line, namely the every hole building drug concentration gradient adds 100 �� L and dilutes bacterium liquid. Still for the medicament storage liquid concentration that adds as 51.2mg/mL, then what now obtain is ten drug concentration gradient of 256��0.5 �� g/mL.

2.5 separately arrange blank plate one piece (not drug containing), and every hole adds 100 �� LMiddlebrook7H9 liquid nutrient mediums and 100 �� L dilute bacterium liquid, and every strain bacterium at least arranges 8 holes; Control wells magnitude setting should be not very few, and because mycobacterium species kind is more, the speed of growth differs, it is necessary to suitably arrange control wells quantity, for experiment demand.

The each Kong Douying in the edge of 2.696 orifice plates drips and adds physiological saline (or use sterilized water), with anti-drying, adding volume is 100 �� L��200 �� L

2.7 are about sealing film phonograph seal 96 orifice plate of 1.5cm with width, to prevent the evaporation of experimental port liquid is dry;

2.8 cultivate in 37 DEG C of fixed temperature and humidity incubators;

The interpretation of 2.9 experimental results

2.9.1, from the 4th day cultivated, developer can be added in blank plate to observe strain growth situation. Each bacterial strain can be chosen a blank hole and add. Can according to alamarBlue developer: 5%Tween-80 reagent=2: the ratio of 5 carries out the premix dilution of developer, and after dilute, every hole adds 70 these liquid of �� L, continue to put into 37 DEG C of fixed temperature and humidity incubators and cultivate, observation variable color situations every other day. As this hole color becomes red-purple, then represent this bacterial strain well-grown under its experiment condition, then this bacterial strain can be added developer in containing the experimental port of medicine; If not having variable color or variable color degree more shallow, then continuing in another blank hole, add 70 �� L developer premixed liquids, cultivating and observing to variable color.

2.9.2 after adding developer premixed liquid in experimental port the 2nd day, observes and result interpretation experimental port variable color situation. The maximum drug level read in the minimum drug level completely not having to read in the experimental port of variable color and the experimental port that variable color has occurred is this experimental strain to the MIC value scope of this medicine.

3, experimental result

Result shows, and Mycobacterium tuberculosis H37Rv standard strain is had stronger bacteriostatic activity by compound 1��6, and MIC value is 0.5��4 �� g/mL; Compound 2 and 3 is except extensive persister FJ05436, other responsive strain and persister also had stronger inhibit activities, MIC value is 0.0625��1 �� g/mL, and the inhibit activities of extensive persister FJ05195 is better than the antitubercular agents such as Rifampin, vazadrine, Tibutol and Streptomycin sulphate.

The active result (MIC, unit: �� g/mL) of table 6 compound 1��6 Killing Mycobacterium Tuberculosis in vitro

DS: medicaments insensitive strain; XDR: extensively persister; MDR: multidrug resistant strain.

Claims (11)

1. an elaiophylin analog derivative, its structure is such as formula shown in II:

11,11 '-O-dimethyl-14 '-remove ethyl-14 '-methyl elaiophylin.

2. preparing the method for derivative described in claim 1, it is characterized in that, described method mainly comprises the following steps:

1) be the marine actinomycete streptomycete 7-145 strain inoculation of CGMCCNo.7914 by the deposit number of activation being the fermention medium of starch-glucose-cottonseed meal-peptone-inorganic salt in formula, in shaking flask, 28 DEG C, 120h cultivated by shaking table;

2) after fermentation liquor solid-liquid separation, mycelium part uses acetone supersound extraction, liquid portion adsorbs through macroporous adsorptive resins, successively with water, 50%, 100% acetone wash-out, 50%, 100% acetone elutriant part merges with mycelium acetone extract, it is extracted with ethyl acetate after concentrating under reduced pressure, obtains fermented liquid medicinal extract;

3) gained medicinal extract utilizes just to silica gel column chromatography separation, successively with the acetate-methanol of the petroleum ether-ethyl acetate of 9:1,4:1,1:1, ethyl acetate, 1:1 and methyl alcohol gradient elution; Wherein, acetate-methanol elution fraction, again through just to silica gel column chromatography separation, successively with the methylene chloride-methanol gradient elution of 20:1,9:1,4:1,1:1 and 0:1, obtains A��G7 stream part; Stream part C and stream part D is respectively through the decolouring of SephadexLH-20 column chromatography, and the methylene chloride-methanol wash-out of 1:1, obtains active constituent; Active constituent after decolouring is respectively through Capcell-PakC₁₈AQ5um, 20 �� 250mm, 88% acetonitrile, 5mL/minHPLC preparation and Capcell-PakC₁₈5um, 10 �� 250mm, 68% or 87% acetonitrile, 2mL/minHPLC half preparation carries out purifying, collects high resolution electrospray ionization mass spectrum (HRESIMS) quasi-molecular ion peak m/z1061.6019 part and obtains compound shown in formula II.

3. compound described in claim 1 is in the application prepared in methicillin-resistant resistant S staphylococcus (MRSA) infection medicine.

4. compound described in claim 1 is in the application prepared in anti-vancocin resistance enterococcus faecalis or anti-vancocin resistance Enterococcus faecium infections medicine.

5. compound described in claim 1 is in the application prepared in Killing Mycobacterium Tuberculosis infection medicine.

6. taking derivative described in claim 1 as effective constituent, with the pharmaceutical composition of one or more carriers pharmaceutically acceptable composition.

7. composition described in claim 6 is in the application prepared in methicillin-resistant resistant S staphylococcus (MRSA) infection medicine.

8. composition described in claim 6 is in the application prepared in anti-vancocin resistance enterococcus faecalis or anti-vancocin resistance Enterococcus faecium infections medicine.

9. composition described in claim 6 is in the application prepared in Killing Mycobacterium Tuberculosis infection medicine.

10. one group of elaiophylin derivative is in the application prepared in methicillin-resistant resistant S staphylococcus (MRSA), anti-vancocin resistance enterococcus faecalis, anti-vancocin resistance faecium or Killing Mycobacterium Tuberculosis infection medicine, described derivative is R shown in formula III₁=R₂=hydroxyl, R₃=R₄=methyl, R₅The compound 3 (elaiophylin) of=hydrogen; R₁=methoxyl group, R₂=hydroxyl, R₃=R₄=methyl, R₅The compound 4 (11-O-methyl elaiophylin) of=hydrogen; R₁=R₂=methoxyl group, R₃=R₄=methyl, R₅The compound 5 (11,11 '-O-dimethyl elaiophylin) of=hydrogen; And R₁=R₂=hydroxyl, R₃=R₅=hydrogen, R₄The compound 6 of=methyl (14 '-remove ethyl-14 '-methyl elaiophylin),

11. 1 kinds of elaiophylin analog derivatives are in the application prepared in methicillin-resistant resistant S grape ball (MRSA), anti-vancocin resistance enterococcus faecalis, anti-vancocin resistance faecium or Killing Mycobacterium Tuberculosis infection medicine, and the structure of described derivative is such as formula shown in I: