JP2019151565A - Compound, antibacterial agent, and secondary metabolite - Google Patents

Abstract

To provide an antibiotic effective against multidrug-resistant bacteria.SOLUTION: The invention provides a compound represented by formula (1) or a salt thereof.SELECTED DRAWING: None

Description

  The present invention particularly relates to novel compounds effective as antibiotics.

  The spread of methicillin-resistant Staphylococcus aureus (hereinafter abbreviated as “MRSA”) in the medical field has the potential to cause serious pathological conditions in patients with reduced infection-protection ability, and clinically. It has become a serious problem. In addition, vancomycin-resistant enterococcus (hereinafter abbreviated as “VRE”), which has acquired resistance to vancomycin effective for MRSA, has appeared, and many of the antibacterial drugs used for the treatment of infectious diseases are VRE. The current situation is invalid.

  For this reason, the search of the microorganisms which have the capability to produce a bioactive substance useful with respect to multi-drug resistant bacteria like MRSA and VRE is performed widely. For example, it has been reported that an antibiotic purified from a culture solution of a microorganism belonging to the genus Lysobacter exhibits antibacterial activity on both MRSA and VRE (see Patent Document 1).

JP 2012-5480 A

For screening of physiologically active substances effective against multidrug-resistant bacteria, it is common to use a pure culture of an isolated strain such as actinomycetes.
On the other hand, the present inventor activates the secondary metabolite-producing ability of actinomycetes by co-culturing (mixed culture) actinomycetes and mycolic acid-containing bacteria, and exhibits antibacterial properties not produced in pure culture. It has been found that metabolites can be obtained. By using this mixed culture technique to induce potential secondary metabolism of actinomycetes, it is expected to obtain novel antibiotics that cannot be produced by conventional pure culture.

  The object of the present invention is to obtain a compound effective against multidrug-resistant bacteria.

The present inventor has found that a novel physiologically active substance that cannot be obtained by pure culture is produced by mixing and culturing mycolic acid-containing bacteria and test bacteria, and has completed the present invention.
The present invention includes the following inventions.

  The present invention provides a novel compound having a chemical structure different from that of known antibiotics and exhibiting antibacterial action against multidrug-resistant bacteria such as MRSA and VRE. Here, the “antibacterial action” refers to an action that inhibits the growth or proliferation of bacteria, or an action that kills bacteria.

It is a chemical structure of a compound (1). It is NMR data of a compound (1). It is a figure which shows the COSY correlation and ROESY correlation of a compound (1). It is a figure which shows the HMBC correlation of a compound (1). It is a figure which shows the HMBC correlation of the partial structure of a compound (1).

で表される構造を有する。 Hereinafter, the present invention will be described in detail.
The compounds of the present invention

It has the structure represented by these.

  The compound (1) can be obtained as a secondary metabolite isolated from the culture solution obtained by combining (mixing) the mycolic acid-containing bacterium with the test bacterium and culturing. Hereinafter, the compound (1) is referred to as PTM904.

  A mycolic acid-containing bacterium is a microorganism that contains mycolic acid in the cell wall, and has the ability to induce secondary metabolite production of the test bacterium. The mycolic acid-containing bacterium used in the present invention is, for example, a microorganism belonging to the genus Tsukamurella.

The test bacteria are, for example, actinomycetes isolated from a soil sample in Japan (Furano City, Hokkaido) and named as HOK021 strain by the present inventor. The 16S rDNA base sequence of the HOK021 strain showed high homology (composition rate 99.6%) to that of the genus Streptomyces. Therefore, the HOK021 strain is a microorganism belonging to the genus Streptomyces.
The Streptomyces hygroscopicus HOK021 strain used as the test bacterium of Example 1 has been deposited as an accession number NITE P-02560 with the Patent Microorganism Depositary, National Institute of Technology and Evaluation (Accession date: October 17, 2017). Day).

  Mixed culture of mycolic acid-containing bacteria and test bacteria can be performed under culture conditions suitable for culturing the test bacteria alone, and is usually performed under aerobic conditions. The type of medium and culture conditions (medium nutrient source, culture time, temperature, pH, etc.) are the same as those usually used in the production of antibiotics by general microorganisms, and the properties of mycolic acid-containing bacteria and test bacteria Is determined as appropriate. Both the mycolic acid-containing bacterium and the test bacterium are mixed and cultured in the state of a living bacterium.

PTM904 is obtained from a culture obtained by mixing and culturing a mycolic acid-containing bacterium and a test bacterium, and isolated and purified.
As an isolation and purification method, a method usually used for obtaining secondary metabolites from microorganisms can be employed. For example, after culturing the culture, the cells and the supernatant are separated by a known method such as filtration or centrifugation, and the supernatant is extracted with an organic solvent or the like and then isolated and purified by various separation methods. PTM904 can be collected. Alternatively, PTM904 can be recovered by centrifuging the culture or filtering the culture, crushing the recovered cell pellets by an appropriate method, and extracting and purifying using an appropriate solvent or the like. As a separation method, reverse phase column chromatography such as an ODS column, fractionation by HPLC using reverse phase column chromatography, or the like can be performed. Furthermore, means such as crystallization, concentration under reduced pressure, and lyophilization can be used alone or in appropriate combination. Any isolation / purification method may be used as long as the target PTM904 is finally obtained.

  Streptomyces hygroscopicus strain HOK021 and mycolic acid-containing bacteria are mixed and cultured, and the secondary metabolite recovered from the culture is preferably an antibacterial substance, and is a known compound in addition to PTM904 Platensimycin, Enterobactin, ENT447 (N, N'-bis (2,3-dihydroxybenzoyl) -O- (α-aminoacryloyl) -O-serylserine), etc. Or in combination.

Of the secondary metabolites described above, platensymycin has been isolated in pure culture from Streptomyces platensis, a kind of actinomycetes present in South African soil.
Platensymycin exhibits an antibiotic action by inhibiting an enzyme (FabF) that synthesizes fatty acids in bacteria, is resistant to the emergence of resistant bacteria, and is effective for MRSA and VRE. Platensymycin has been reported to show a broad antibacterial spectrum against Gram-positive bacteria including MRSA and VRE. In view of the importance of platensymycin as an antibiotic, in the antibacterial action evaluation test described later, the MIC (minimum growth inhibitory concentration) of PTM904 and platensimimycin shall be measured.

  PTM904 may be synthesized using a known chemical synthesis method. However, the method for obtaining PTM904 by culturing microorganisms does not require a special metal catalyst or reagent, and has many advantages over chemical synthesis methods. PTM904 has an asymmetric carbon atom in the structure, and stereoisomerism occurs, but here, any stereoisomer is included, and it may be a racemate.

  PTM904 is not limited to a free form, and may exist as a salt. The salt of PTM904 is not particularly limited as long as it is a pharmacologically acceptable salt, and PTM904 can exist as a salt derived from an inorganic acid or an organic acid.

As the antibacterial agent of the present invention, PTM904 may be used as it is, but it is preferably processed into a preparation that is easy to use. For example, preparations for oral administration include tablets, capsules, fine granules, powders, granules, orally disintegrating tablets, solutions, syrups, etc., and preparations for parenteral administration include Injections, drops, suppositories, inhalants, transdermal absorbents, transmucosal absorbents, nasal drops, ear drops, ointments, creams and the like. These preparations can contain conventional adjuvants and carrier components depending on the type of each preparation. In addition, these preparations can be produced by conventional methods such as mixing, kneading, granulation, tableting, coating, sterilization, emulsification, etc., depending on the dosage form.
Moreover, the case where it adheres to medical gauze etc. and is used as a coating | covering material for preventing infectious diseases, such as a wound part, is also assumed.

<Example 1>
1. Production of PTM904 [Separation of HOK021]
Diluted phenol method and ISP4 medium (medium components: 1% Soluble starch, 0.1% K ₂ HPO ₄ , 0.1% MgSO) from the soil collected in the practice forest attached to the Graduate School of Agricultural and Life Sciences, University of Tokyo (Hokkaido Exercise Forest, Furano City, Hokkaido) Actinomycetes were isolated using ₄ , 0.1% NaCl, 0.2% (NH ₄ ) ₂ SO ₄ , 0.2% CaCO ₃ , 0.0001% FeSO ₄ , 0.0001% MnCl ₂ , 0.0001% ZnSO ₄ , 2.0% agar. When the partial 16S rDNA sequence of the isolated actinomycete HOK021 strain was amplified by PCR and the nucleotide sequence was determined by the Sanger method, Streptomyces hygroscopicus subsp. Glebosus (gene registration number AB184479) and 99.6% (1463-1469) ). The HOK021 strain was named Streptomyces hygroscopicus HOK021 strain.

[Culture of HOK021 and TP-B0596]
Inoculate Streptomyces hygroscopicus HOK021 strain spore solution glycerol stock (stored at -80 ° C) into ISP2 agar medium (medium components: 0.4% Yeast extract, 1% Malt extract, 0.4% glucose, 2.0% agar.) At 30 ° C Static culture was performed for 5 days. A cell glycerol stock (stored at -80 ° C) of Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium, was inoculated into an ISP2 agar medium, and statically cultured at 30 ° C for 3 days. In a K-1 flask, 100 ml of V-22 medium (medium components: 1% Soluble starch, 0.5% Glucose, 0.3% NZ-case, 0.2% Yeast extract, 0.1% Tryptone, 0.1% K ₂ HPO ₄ , 0.05% MgSO ₄ , 0.3% CaCO ₃ (pH 7)) was inoculated with HOK021 strain cells grown on an agar medium and precultured for 3 days (30 ° C., 200 rpm). TP-B0596 strain cells grown on an agar medium were inoculated into a medium containing 100 ml of V-22 medium in a K-1 type flask, and precultured for 2 days (30 ° C., 200 rpm). 3 ml of the preculture of the HOK021 strain and 1 ml of the preculture of the TP-B0596 strain were added to a 100 ml A-3M medium (medium components: 2% Soluble starch, 2% Glycerol, 1.5% Pharmamedia, 0.5 % Glucose, 0.3% Yeast extract, 1% Diaion (registered trademark) HP-20 (pH 7)) was inoculated at the same time, followed by main culture for 8 days (30 ° C., 200 rpm).

[Extraction of PTM904]
The main culture solution (3.6 L in total) is subjected to the first stirring extraction (about 1 hour) using an equal amount of ethyl acetate (3.6 L, Wako special grade reagent), and the ethyl acetate phase and the aqueous phase are separated by centrifugation. Separate and collect the ethyl acetate phase. The remaining aqueous phase is further subjected to a second stirring extraction (about 1 hour) with an equal amount of ethyl acetate (3.6 L), and the ethyl acetate phase and the aqueous phase are separated by centrifugation, and the ethyl acetate phase Was recovered. The collected first and second ethyl acetate phases were combined, and the ethyl acetate was distilled off under reduced pressure using an evaporator to obtain about 2.9 g of crude extract.

[PTM904 purification (1)]
First, the crude extract was roughly purified using an intermediate pressure ODS column using acetonitrile (Wako special grade reagent) and milli-Q (registered trademark) water as the mobile phase. About 0.6 g of the crude extract was dissolved in DMSO to a concentration of 0.1 g / ml and added to an ODS column (4id × 24 cm, about 300 ml) substituted with 20% acetonitrile solvent. Elution was performed using 300 ml each of 20% acetonitrile solvent, 40% acetonitrile solvent, 60% acetonitrile solvent, 80% acetonitrile solvent, and 100% acetonitrile solvent. A fraction containing the target PTM904 in the latter half 150 ml of the collected 60% acetonitrile solvent was obtained. Acetonitrile was distilled off using an evaporator, the remaining aqueous phase was frozen in a −80 ° C. deep freezer, and the remaining aqueous phase was removed by lyophilization to obtain 46.7 mg of a crude product.

[PTM904 purification (2)]
Next, HPLC purification was performed using a phenylethyl group column using acetonitrile (Wako special grade reagent) and 10 mM ammonium acetate buffer as a mobile phase. A COSMOSIL® 5PE-MS column (5 μm, 10 id × 250 mm, Nacalai) in which 46.7 mg of the crude product was dissolved in DMSO to a concentration of about 25 mg / ml and replaced with 30% acetonitrile solvent was applied to one column. 20 μL was injected at a time. Elution was performed with a flow rate of 4.2 ml / min and isocratic, and the peak eluted at a retention time of 8.5 minutes was collected to obtain a fraction containing the target PTM904. Acetonitrile was distilled off using an evaporator, the remaining buffer phase was frozen in a −80 ° C. deep freezer, and the remaining aqueous phase was lyophilized to obtain 4.2 mg of purified product.

[PTM904 purification (3)]
Next, HPLC purification of PTM904 was performed from the purified product using a _C18 column using acetonitrile (Wako special grade reagent) and 0.1% formic acid buffer as the mobile phase. 4.2 mg of the purified product was dissolved in about 0.5 ml of DMSO, and 20 μL was injected at a time onto a C ₁₈ -AR-II column (5 μm, 10 id × 250 mm, Nacalai) substituted with 55% acetonitrile solvent. . Elution was performed with a flow rate of 3.0 ml / min and isocratic, and a peak eluted at a retention time of 7.6 minutes was collected to obtain a fraction containing the target PTM904. Acetonitrile and the buffer phase were distilled off using an evaporator to obtain 3.4 mg of a purified product.

  A peak containing PTM904 was not observed in each single pure culture of HOK021 and TP-B0596. That is, PTM904 was confirmed for the first time by mixed culture of HOK021 and TP-B0596. In addition, in the main culture solution of HOK021 and TP-B0596, as the secondary metabolites other than PTM904, platensimycin, enterobactin, ENT447 (N, N′-bis (2,3 -dihydroxybenzoyl) -O- (α-aminoacryloyl) -O-serylserine).

2. Structural analysis of PTM904 The physicochemical properties of PTM904 isolated as described above are shown below.
(A) Appearance: pale yellow powder (B) Molecular weight: 903.9
(C) Molecular formula: C ₄₄ H ₄₅ N ₃ O ₁₆ S
(D) HR QTOF ESI MS (positive): measured value 904.2616 [M + H] ⁺
Calculated 904.2593 (for C ₄₄ H ₄₆ N ₃ O ₁₆ S ⁺ )
(E) HR QTOF ESI MS (negative): Measured value 902.2512 [MH] ^-

The NMR data of PTM904 isolated as described above is shown in FIG. The numbers in the “position” column in FIG. 2 indicate the position numbers of the carbons constituting the PTM 904 (see FIG. 1, not following the IUPAC nomenclature). Chemical shifts (δ _H , δ _C ) are expressed in ppm, ¹ H-NMR was analyzed at 500 MHz, ¹³ C-NMR was analyzed at 125 MHz, and pyridine-d5 was used as a deuterated solvent.

The molecular formula of this compound was determined to be C ₄₄ H ₄₅ N ₃ O ₁₆ S based on mass spectrometry data and NMR data. 44 carbon signals of the present compound were confirmed by ¹³ C-NMR spectrum. ^{The 13} C-NMR spectrum and ¹ H-NMR spectrum suggested the presence of aromatic rings, carboxyl groups, amide bonds, thioester bonds, and the like.

  By HH-COSY (Proton-Proton Correlation Spectroscopy), a connection between protons indicated by a thick line in FIG. 3 was recognized. By HMBC (Heteronuclear Multiple Bond Coherence), the proton-carbon correlation indicated by the arrow in FIG. 4 was recognized. In addition, a correlation between protons close to each other as shown by arrows in FIG. 3 was recognized by ROESY (Rotating Overhauser Enhancement and exchange Spectroscopy). Here, a correlation with C—H hydrogen adjacent to N—H hydrogen was observed.

The partial structure shown in FIG. 5 having a bridged ring structure and an enone structure of a tricyclo ring was determined for its binding mode and configuration by each spectrum analysis of HH-COSY, HMBC, and ROESY.
Based on the above analysis results, the structure of PTM904 was determined as shown in Formula (1).

3. Antibacterial action of PTM904 To measure the antibacterial action against methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) and vancomycin-resistant enterococci (hereinafter referred to as VRE), first, a drug susceptibility test (halo test) by a disk diffusion method was performed. It was.

  PTM904 was dissolved in DMSO, and 2 ml of the test solution was soaked in a paper disk having a diameter of 8 mm. Thus, the paper disk containing 10 μg and 1 μg of PTM904 was placed on the medium coated with the bacterial solution and cultured at 30 ° C. for 16 to 20 hours. Thereafter, the growth inhibition circle diameter (diameter (mm) passing through the center of the paper disk) was measured. The results are shown in Table 1. In the table, “-” means no growth inhibition circle.

  PTM904 showed antibacterial activity against multidrug resistant Gram-positive bacteria such as MRSA and VRE. In particular, it was revealed that PTM904 exhibits significant growth inhibition with respect to VRE in a concentration-dependent manner.

  Moreover, when the antibacterial action of PTM904 against other strains was tested by the same disk diffusion method, PTM904 was found to be Klebsiella pneumoniae DHA (+) and AAC (6 ′)-Ib (+), K. pneumoniae DHA (+) and No growth-inhibition circle formation was observed for gram-negative bacteria such as armA (+) and Acinetobacter faecalis vanB (+). For this reason, it was suggested that PTM904 has the effect | action which selectively suppresses or prevents the growth of specific multidrug-resistant bacteria, such as Staphylococcus genus and Enterococcus genus. Therefore, by using an antibacterial agent containing PTM904 as an active ingredient for treatment of infectious diseases, it is expected to exert a therapeutic effect without disturbing normal bacterial flora such as in the intestine.

Next, the MIC (minimum growth inhibitory concentration) of platensymycin (PTM) and PTM904 for various strains was measured by a micro liquid dilution method. The measurement conditions are shown below.
(A) Strain used: listed in Table 2
(C) Medium: CAMHB (Note 1) and ID-CAMHB (Note 2)
(D) Culture conditions: 37 ° C., 20 hours, dark place, aerobic conditions, standing (E) Number of bacteria at the start of culture: 5 (2-8) × 10 ⁵ CFU / ml
(F) Criteria: Visual observation of precipitates of 2 mm or more (CLSI standard)

(Note 1) CAMHB is Cation-adjusted Muller-Hinton broth [Mueller II Broth Cation-Adjusted 500g BBL 212322].
(Note 2) ID-CAMHB is Iron-depleted Cation-adjusted Mueller Hinton Broth. Specifically, ID-CAMHB is a sterilized medium in which 5 g of resin (Kirex (registered trademark) 100 resin # 14222842) is added per 100 ml of the above-mentioned CAMHB, and after stirring for 1 hour, the resin is removed by filtering.
The MIC measurement results are shown in Table 2.

As shown in Table 2, it was revealed that PTM904 has a smaller MIC against Gram-positive bacteria than MIC against Gram-negative bacteria, and particularly has a good antibacterial action against MRSA and VRE.
In addition, PTM904 is less effective in PTM904 than in P16, which is 4 to 16 times less effective in an iron-restricted medium (ID-CAMHB), which is an environment close to the body that lacks free iron. Furthermore, it was confirmed that MRSA was more effective than PTM.
For example, it is considered that a gauze or the like absorbs a drug containing PTM904 and covers the wound or catheter insertion site, thereby suppressing the growth of VRE and MRSA and preventing surgical site infection and catheter infection.

  The novel compounds of the present invention have industrial applicability that new antibiotics can be provided.

NITE P-02560

Claims (5)

A compound represented by formula (1) or a salt thereof.

  An antibacterial agent comprising the compound (1) according to claim 1 as an active ingredient.   A secondary metabolite recovered by culturing a mixture of Streptomyces hygroscopicus strain HOK021 with accession number NITE P-02560 and a microorganism containing mycolic acid in the cell wall, and recovering the culture.   The secondary metabolite according to claim 3, wherein the microorganism containing a mycolic acid in the cell wall belongs to the genus Tucumrela.   The secondary metabolite according to claim 3 or 4, which has an antibacterial action against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

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