U.S. patent application number 13/278269 was filed with the patent office on 2014-06-05 for compound amycolamicin, method for producing the same, and use of the same.
The applicant listed for this patent is Yoshiko Homma, Masayuki Igarashi, Ryuichi Sawa. Invention is credited to Yoshiko Homma, Masayuki Igarashi, Ryuichi Sawa.
Application Number | 20140155586 13/278269 |
Document ID | / |
Family ID | 43010804 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140155586 |
Kind Code |
A1 |
Igarashi; Masayuki ; et
al. |
June 5, 2014 |
Compound Amycolamicin, Method for Producing the Same, and Use of
the Same
Abstract
A compound having a structure expressed by the following
Structural Formula (1), tautomers thereof, or salts of the compound
or the tautomers. ##STR00001##
Inventors: |
Igarashi; Masayuki; (Tokyo,
JP) ; Sawa; Ryuichi; (Tokyo, JP) ; Homma;
Yoshiko; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Igarashi; Masayuki
Sawa; Ryuichi
Homma; Yoshiko |
Tokyo
Tokyo
Tokyo |
|
JP
JP
JP |
|
|
Family ID: |
43010804 |
Appl. No.: |
13/278269 |
Filed: |
October 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2009/058210 |
Apr 24, 2009 |
|
|
|
13278269 |
|
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|
Current U.S.
Class: |
536/16.8 ;
435/252.1; 435/74 |
Current CPC
Class: |
A61P 31/04 20180101;
C07H 19/044 20130101; C07D 405/14 20130101; C12P 19/44 20130101;
C12P 19/46 20130101; C12R 1/01 20130101; C12P 17/16 20130101 |
Class at
Publication: |
536/16.8 ;
435/74; 435/252.1 |
International
Class: |
C07H 19/044 20060101
C07H019/044; C12P 19/44 20060101 C12P019/44 |
Claims
1. A compound having a structure expressed by the following
Structural Formula (1), tautomers thereof, or salts of the compound
or the tautomers. ##STR00007##
2. A method for producing a compound having a structure expressed
by the following Structural Formula (1), tautomers thereof, or
salts of the compound or the tautomers, the method comprising:
culturing a microorganism belonging to the genus Amycolatopsis and
capable of producing the compound having a structure expressed by
the Structural Formula (1), the tautomers thereof, or the salts of
the compound or the tautomers, and isolating the compound having a
structure expressed by the Structural Formula (1), the tautomers
thereof, or the salts of the compound or the tautomers from a
culture obtained from the culturing. ##STR00008##
3. The method according to claim 2, wherein the microorganism is a
microorganism of Amycolatopsis sp. MK575-fF5 strain deposited under
accession number FERM P-21465.
4. A microorganism, wherein the microorganism belongs to the genus
Amycolatopsis and is capable of producing a compound having a
structure expressed by the following Structural Formula (1),
tautomers thereof, or salts of the compound or the tautomers.
##STR00009##
5. The microorganism according to claim 4, wherein the
microorganism is a microorganism of Amycolatopsis sp. MK575-fF5
strain deposited under accession number FERM P-21465.
6. An antibacterial agent comprising: a compound having a structure
expressed by the following Structural Formula (1), tautomers
thereof, or salts of the compound or the tautomers, serving as an
active ingredient. ##STR00010##
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation application of PCT/JP2009/058210,
filed on Apr. 24, 2009.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a new compound having an
excellent antibacterial activity against a wide variety of
pathogenic bacteria such as drug-resistant bacteria and bacteria
causing pneumonia in domestic livestock, a method for producing the
new compound, use of the new compound, and a new microorganism that
produces the new compound.
[0004] 2. Description of the Related Art
[0005] In general, infectious diseases caused by pathogenic
bacteria are treated with chemotherapy including administering
drugs such as antibiotics. However, conventionally, frequent use of
such drugs has allowed pathogenic bacteria to acquire an ability to
neutralize these drugs, leading to appearance of resistant bacteria
to which the drugs are ineffective. Actually, many drug-resistant
bacteria have been problematic mainly in medical facilities.
[0006] For example, as one clinically important problem,
Staphylococcus aureus, which is known as bacteria causing
suppurative diseases, pneumonia and food poisoning, acquires
multidrug resistances to methicillin or other antibiotics to be
methicilline-resistant Staphylococcus aureus (MRSA). At present,
vancomycin, teicoplanin, arbekacin, line zolid, etc. are used as
typical therapeutic drugs against MRSA. However, it is generally
difficult to completely eliminate MRSA. In particular, thorough
care should be taken about the use of vancomycin, since appearance
of vancomycin resistant Staphylococcus aureus (VRSA) has already
been reported (see Sievert D M, et al.: Staphylococcus aureus
Resistant to Vancomycin United States, 2002. MMWR Jul. 5, 2002; 51:
565-567.).
[0007] In order to overcome such problems relating to
drug-resistant bacteria, demand has arisen for the development of a
new compound that has a different chemical structural skeleton from
those of the conventional antibiotics and also has an excellent
antibacterial activity.
[0008] Furthermore, infectious diseases caused by pathogenic
bacteria can be greatly problematic in not only human but also
non-human animals. For example, pneumonia in domestic livestock
such as bovine and pig is expected to effectively prevent or treat
in the livestock industry. Therefore, at present, further demand
has arisen for the development for a new compound having an
excellent antibacterial activity against bacteria causing pneumonia
in domestic livestock.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention aims to solve the above existing
problems and achieve the following objects. That is, an object of
the present invention is to provide a new compound having an
excellent antibacterial activity against a wide variety of
pathogenic bacteria such as drug-resistant bacteria and bacteria
causing pneumonia in domestic livestock, tautomers thereof, or
salts of the new compound or the tautomers; a method for producing
them; a new microorganism that produces the new compound, the
tautomers thereof, or the salts of the new compound or the
tautomers; and an antibacterial agent containing the new compound,
the tautomers thereof, or the salts of the new compound or the
tautomers.
[0010] In order to solve the above existing problems, the present
inventors conducted extensive studies to succeed in isolating as a
new microorganism a bacterial strain belonging to the genus
Amycolatopsis, and have found that this bacterial strain produces
an antibiotic having a new structural skeleton. The present
inventors have found that this antibiotic has an excellent
antibacterial activity against a wide variety of pathogenic
bacteria such as drug-resistant bacteria and bacteria causing
pneumonia in domestic livestock, and have also confirmed that this
antibiotic is a new compound through analysis of its chemical
structure. The present invention has been accomplished on the basis
of these findings. Notably, the present inventors named this new
compound "amycolamicin."
[0011] The present invention is based on the above findings
obtained by the present inventors. Means for solving the above
existing problems are as follows.
[0012] <1> A compound having a structure represented by the
following Structural Formula (1), tautomers thereof, or salts of
the compound or the tautomers.
##STR00002##
[0013] <2> A method for producing the compound, the tautomers
or the salts according to <1>, the method including:
[0014] culturing a microorganism belonging to the genus
Amycolatopsis and capable of producing the compound, the tautomers
or the salts according to <1>, and isolating the compound,
the tautomers or the salts according to <1> from a culture
obtained from the culturing.
[0015] <3> The method according to <2>, wherein the
microorganism is a microorganism of Amycolatopsis sp. MK575-fF5
strain deposited under accession number FERM P-21465.
[0016] <4> A microorganism,
[0017] wherein the microorganism belongs to the genus Amycolatopsis
and is capable of producing the compound, the tautomers or the
salts according to <1>.
[0018] <5> The microorganism according to <4>, wherein
the microorganism is a microorganism of Amycolatopsis sp. MK575-fF5
strain deposited under accession number FERM P-21465.
[0019] <6> An antibacterial agent including:
[0020] the compound, the tautomers or the salts according to
<1> serving as an active ingredient.
[0021] The present invention can provide a new compound having an
excellent antibacterial activity against a wide variety of
pathogenic bacteria such as drug-resistant bacteria and bacteria
causing pneumonia in domestic livestock, tautomers thereof, or
salts of the compound or the tautomers; a method for producing the
new compound, the tautomers thereof, or the salts of the new
compound or the tautomers; a new microorganism that produces the
new compound, the tautomers thereof, or the salts of the new
compound or the tautomers; and an antibacterial agent containing
the new compound, the tautomers thereof, or the salts of the new
compound or the tautomers. These can solve the above existing
problems and achieve the above objects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is an infrared absorption spectrum chart of
amycolamicin measured by the KBr tablet method, where vertical
axis: transmittance (%) and horizontal axis: wavenumber
(cm.sup.-1).
[0023] FIG. 2 is a UV absorption spectrum chart of amycolamicin
measured in methanol, where vertical axis: absorbance (Abs) and
horizontal axis: wavelength (nm).
[0024] FIG. 3 is a .sup.1H nuclear magnetic resonance spectrum
chart of amycolamicin measured in deuterated methanol at 30.degree.
C. and 600 MHz, where the unit of the horizontal axis: ppm.
[0025] FIG. 4 is a .sup.13C-NMR spectrum chart of amycolamicin
measured in deuterated methanol at 30.degree. C. and 150 MHz, where
the unit of the horizontal axis: ppm.
DETAILED DESCRIPTION OF THE INVENTION
Compound, Tautomers Thereof, or Salts of the Compound or the
Tautomers
[0026] A compound of the present invention has a structure
expressed by the following Structural Formula (1). The compound
having the following Structural Formula (1) is a new compound
separated by the present inventors (hereinafter may be referred to
as "amycolamicin").
##STR00003##
[0027] Physico-chemical properties of the compound having the above
Structural Formula (1) (i.e., amycolamicin) are as follows.
(1) Appearance: white powder (2) Molecular formula:
C.sub.44H.sub.60Cl.sub.2N.sub.4O.sub.14 (3) High resolution mass
spectrometry (HRESIMS: negative ion mode)
[0028] Calcd: m/z 937.3386 (M-H).sup.-
[0029] Found: m/z 937.3405 (as
C.sub.44H.sub.59Cl.sub.2N.sub.4O.sub.14)
(4) Specific rotation [.alpha.].sub.D.sup.23=-21.6.degree. (c 0.5,
methanol) (5) Infrared absorption spectrum is as shown in FIG. 1.
(6) UV absorption spectrum is as shown in FIG. 2. (7) .sup.1H
nuclear magnetic resonance (NMR) spectrum measured in deuterated
methanol at 30.degree. C. and 600 MHz is as shown in FIG. 3. (8)
.sup.13C nuclear magnetic resonance (NMR) measured in deuterated
methanol at 30.degree. C. and 150 MHz is as shown in FIG. 4. (9)
Thin-layer chromatography using silica gel 60F.sub.254 (product of
Merck Co.) and a developing solvent of chloroform:methanol (9:1 by
volume), the Rf value is 0.31.
[0030] Whether a compound has a structure expressed by Structural
Formula (1) can be determined with appropriately selected various
analysis methods. This determination can be performed by measuring
the above .sup.1H nuclear magnetic resonance spectrum, .sup.13C
nuclear magnetic resonance spectrum, infrared absorption spectrum
or mass spectrum.
[0031] Notably, amycolamicin has tautomerism and thus encompasses
tautomers thereof. Non-limiting examples of the tautomers of
amycolamicin include those having the following four Structural
Formulas. Amycolamicin can have such several different structural
patterns, and is not considered that it exists at a certain fixed
state.
##STR00004## ##STR00005##
[0032] The spectrum charts of amycolamicin obtained through .sup.1H
nuclear magnetic resonance spectrum analysis, .sup.13C nuclear
magnetic resonance spectrum analysis, etc. may be somewhat
different from those shown in FIGS. 3 and 4. Here, those skilled in
the art can easily understand that the compound having Structural
Formula (1) can actually have several different structural patterns
and does not exist at a certain fixed state. Thus, even when a
compound shows a .sup.1H nuclear magnetic resonance spectrum chart
somewhat different from that of FIG. 3, a .sup.13C nuclear magnetic
resonance spectrum chart somewhat different from that of FIG. 4,
and other somewhat different spectrum charts, those skilled in the
art could easily identify the compound as amycolamicin.
[0033] Also, the amycolamicin may be in the form of a salt. The
salt is not particularly limited and may be appropriately selected
depending on the intended purpose. Examples thereof include salts
formed with alkali metals such as sodium and potassium; salts
formed with alkaline earth metals such as calcium and magnesium;
and salts formed with organic amines such as methylamine,
ethylamine and diethanolamine.
[0034] The amycolamicin, tautomers thereof, or salts of the
amycolamicin or the tautomers may be produced from the bacteria
that produce the amycolamicin or may be obtained through chemical
synthesis. Among them, the amycolamicin, the tautomers thereof, or
the salts of the amycolamicin or the tautomers are preferably
obtained by the below-described method of the present invention for
producing the compound, the tautomers thereof, or the salts of the
compound or the tautomers.
[0035] As shown in Test Examples 1 to 4 described below, the
amycolamicin, the tautomers thereof, or the salts of the
amycolamicin or the tautomers each have an excellent antibacterial
activity against a wide variety of Gram-positive and negative
bacteria such as drug-resistant bacteria and bacteria causing
pneumonia in domestic livestock. Thus, any of the amycolamicin,
tautomers thereof and salts of the amycolamicin and tautomers can
be suitably used as, for example, an active ingredient of the
below-described antibacterial agent of the present invention.
(Method for producing compound, tautomers thereof, or salts of the
compound or tautomers)
[0036] A method for producing the compound of the present
invention; i.e., amycolamicin, the tautomers thereof, or the salts
of the compound or the tautomers includes: culturing a
microorganism belonging to the genus Amycolatopsis and capable of
producing the amycolamicin, the tautomers thereof, or the salts of
the amycolamicin or the tautomers; and isotating the amycolamicin,
the tautomers thereof, or the salts of the amycolamicin or the
tautomers from a culture obtained from the culturing. Notably, the
amycolamicin obtained through culturing of the microorganism may be
referred to as "antibiotic amycolamicin" hereinafter.
[0037] The antibiotic amycolamicin is produced as follows.
Specifically, microorganisms that produce the antibiotic
amycolamicin (hereinafter may be referred to simply as
"amycolamicin-producing microorganisms") are inoculated into a
nutrient medium and cultured at a temperature suitable for the
production of the antibiotic amycolamicin, whereby a culture
containing the antibiotic amycolamicin is obtained.
[0038] The nutrient medium used for the above culturing is nutrient
media that can be used for culturing actinomycetes. Examples of the
nutrient sources added to the nutrient medium include nitrogen
sources such as commercially available soy flour, peptone, yeast
extract, meat extract, corn steep liquor and ammonium sulfate;
carbohydrates such as tomato paste, glycerin, starch, glucose,
galactose and dextrin; and carbon sources such as fats. In
addition, inorganic salts such as sodium chloride and calcium
carbonate may be added to the medium before use. If necessary, a
trace amount of a metal salt may be added to the medium before use.
Any known material for culture may be used so long as the material
can be utilized by the amycolamicin-producing microorganisms to
promote the production of the antibiotic amycolamicin.
[0039] The production of the antibiotic amycolamicin uses the
microorganism belonging to the genus Amycolatopsis and capable of
producing the antibiotic amycolamicin. Specifically, it has been
clarified that a microorganism of Amycolatopsis sp. MK575-fF5
strain isolated by the present inventors (FERM P-21465, details
will be described in the following section "Microorganism" of the
present invention) can produce the antibiotic amycolamicin. Also,
other strains that are capable of producing the antibiotic
amycolamicin can be isolated from the natural world by a routine
method for insolating bacteria that produce antibiotics. Notably,
through mutation treatments such as exposure to radiation, the
microorganism of Amycolatopsis sp. MK575-fF5 strain and other
microorganisms capable of producing the antibiotic amycolamicin can
be mutated so that they have increased production capability of the
antibiotic amycolamicin. Moreover, the antibiotic amycolamicin can
be produced through genetically engineering techniques.
[0040] The seed culture used for the production of the antibiotic
amycolamicin may be, for example, the growth culture obtained
through slant culturing of the amycolamicin-producing bacteria on
an agar medium. For the production of the antibiotic amycolamicin,
it is preferred that the amycolamicin-producing bacteria be
aerobically cultured in an appropriate medium. Also, for isolating
the target compound from the resultant culture, a routine method
can be used. The culturing temperature is not particularly limited
and may be determined depending on the type of the
amycolamicin-producing microorganisms, so long as the growth of the
amycolamicin-producing microorganisms is not substantially
inhibited and the amycolamicin-producing microorganisms can produce
the antibiotic amycolamicin. The culturing temperature is
preferably 25.degree. C. to 35.degree. C.
[0041] For example, the production of the antibiotic amycolamicin
by the Amycolatopsis sp. MK575-fF5 strain generally becomes maximum
for 3 days to 9 days. In general, it is preferred that the
production of the antibiotic amycolamicin be performed until the
medium exhibits sufficient antibacterial activity. A change over
time of the titer of the antibiotic amycolamicin in the culture can
be measured through, for example, HPLC or a cylinder plate method
using Staphylococcus aureus or other bacteria as tested
bacteria.
[0042] In the above production method, the antibiotic amycolamicin
is isolated from the obtained culture. The isolation method may
appropriately utilize a method used for recovering metabolites
produced by microoganisms. Examples of the isolation method for the
antibiotic amycolamicin include a method by extracting with a
water-immiscible solvent, a method utilizing differences in
adsorption affinity to various adsorbents, gel filtration,
chromatography utilizing countercurrent distribution and
combinations thereof. The separated microorganisms are treated with
an extracting method using an appropriate organic solvent or an
eluting method through disruption of microorganisms, whereby the
antibiotic amycolamicin can be recovered through extraction of the
microorganisms and isolation/purification as described above.
[0043] The production method can be performed as described above to
produce the antibiotic amycolamicin or the tautomers thereof.
[0044] Since the amycolamicin or tautomers thereof are each an
acidic compound, the salts of the amycolamicin or tautomers thereof
can generally be produced by a known method using, for example,
pharmaceutically acceptable various metals (e.g., alkali metals) or
organic bases (e.g., quaternary ammonium salts).
(Microorganism)
[0045] A microorganism of the present invention belongs to the
genus Amycolatopsis and is capable of producing the above-described
compound of the present invention; i.e., amycolamicin, the
tautomers thereof, or the salts of the amycolamicin or the
tautomers. The microorganism is not particularly limited and may be
appropriately selected depending on the intended purpose, so long
as it belongs to the genus Amycolatopsis and is capable of
producing the amycolamicin, the tautomers thereof, or the salts of
the amycolamicin or the tautomers, and thus can be used as the
amycolamicin-producing microorganisms in the above-described
production method for the compound of the present invention, the
tautomers thereof, or the salts of the compound or the
tautomers.
[0046] In particular, preferably used are actinomycetes isolated
from the soil of Sendai-shi, Miyagi and given accession number
MK575-fF5 strain in May, 1996 by the microbial chemistry research
center of Microbial Chemistry Research Foundation. The mycological
characteristics of the MK575-fF5 strain are as follows.
1. Morphology
[0047] Substrate hyphae are well branched in a zig-zag form and are
divided. Aerial hyphae are grown in some cases or not grown in
other cases. When aerial hyphae are grown, the aerial hyphae are
relatively long and linear or irregularly curved as well as divided
into cylindrical spores. The surface is smooth and the size is
about 0.4 .mu.m to 0.6 .mu.m.times.0.8 .mu.m to 2.2 .mu.m. Also,
the aerial hyphae may be tangled together to have a spherical
shape. Whorls, mycelial strands, sporangia and motile spores are
not observed.
2. Growth Conditions in Various Media
[0048] The standards in blankets relating to colors are based on
the color harmony manual of Container Corporation of America.
(1) Yeast-Malt Agar Medium (ISP-Medium 2, Culturing at 27.degree.
C.)
[0049] This strain is grown in pale yellow [2 gc, Bamboo] and
slightly forms white aerial hyphae in some cases or does not form
them in other cases. Soluble dyes are not produced.
(2) Oatmeal Agar Medium (ISP-Medium 3, Culturing at 27.degree.
C.)
[0050] This strain is grown in pale yellow [2 gc, Bamboo] and
slightly forms white aerial hyphae in some cases or does not form
them in other cases. Soluble dyes are not produced.
(3) Starch-Inorganic Salt Agar Medium (ISP-Medium 4, Culturing at
27.degree. C.)
[0051] This strain is grown in pale yellow [2 ea, Lt Wheat] to dull
yellow [3 nc, Amber] and slightly forms white aerial hyphae in some
cases. Soluble dyes are not produced.
(4) Glycerin-Asparagine Agar Medium (ISP-Medium 5, Culturing at
27.degree. C.)
[0052] This strain is grown in dull yellowish orange [3 lc, Amber]
and forms aerial hyphae of pale orange [4 ea, Light Apricot] in
some cases or does not form them in other cases. Soluble dyes are
not produced.
(5) Tyrosine Agar Medium (ISP-Medium 7, Culturing at 27.degree.
C.)
[0053] This strain is grown in pale yellow [3 ca, Pearl Pink] to
dull yellowish orange [3 lc, Amber] and forms aerial hyphae of pale
orange [4 ca, Fresh Pink] in some cases or does not form them in
other cases. Soluble dyes are not produced.
(6) Sucrose-Nitrate Agar Medium (Culturing at 27.degree. C.)
[0054] This strain is grown in colorless to pale yellowish orange
[3 ea, Lt Melon Yellow] and slightly forms white aerial hyphae in
some cases or does not form them in other cases. Soluble dyes are
not produced.
3. Physiological Properties
(1) Temperature Range of Growth
[0055] This strain was cultured on a glucose-aspartic acid agar
medium (glucose: 1.0%, L-aspartic acid: 0.05%, dipotassium
hydrogenphosphate: 0.05%, string agar: 3.0%, pH 7.0) at a
temperature of 10.degree. C., 20.degree. C., 24.degree. C.,
27.degree. C., 30.degree. C., 37.degree. C., 45.degree. C. or
50.degree. C. As a result, the strain was not grown at 10.degree.
C., 45.degree. C. or 50.degree. C. but was grown at 20.degree. C.
to 37.degree. C. The optimal growth temperature is about 30.degree.
C.
(2) Hydrolysis of Starch (Starch-Inorganic Salt Agar Medium,
ISP-Medium 4, Culturing at 27.degree. C.)
[0056] Negative on day 21 from the beginning of the culturing
(3) Production of Melanine-Like Dye (Tripton-Yeast-Broth,
ISP-Medium 1; Peptone-Yeast-Iron Agar Medium, ISP-Medium 6;
Tyrosine Agar Medium, ISP-Medium 7; Culturing at 27.degree. C. For
Each Medium)
[0057] Negative for each medium
(4) Availability of Carbon Source (Pridham-Godleave Agar Medium,
ISP-Medium 9; Culturing at 27.degree. C.)
[0058] This strain is grown by utilizing D-glucose, L-arabinose,
D-xylose, D-fructose, sucrose, inositol, rhamnose, raffinose and
D-mannitol.
(5) Reduction Reaction of Nitrate (0.1% Potassium
Nitrate-Containing Peptone Water, ISP-Medium 8, Culturing at
27.degree. C.)
[0059] Positive
4. Microbial Components
(1) Composition of Cell Wall
[0060] The cell wall contains meso-2,6-diaminopimelic acid.
(2) Reducing Sugar in Each Microorganism
[0061] Each microorganism contains arabinose and galactose (Type
A).
(3) Isoprenoid Quinone
[0062] MK-9 (H.sub.4) and a small amount of MK-10 (H.sub.4) are
contained as main menaquinone.
(4) Phospholipid
[0063] Type PII (phosphatidylethanolamine is contained but
phosphatidyl choline and unknown glucosamine-containing
phospholipids are not contained)
(5) Mycolic Acid
[0064] Not contained.
5. Analysis of 16S rRNA Gene
[0065] A partial nucleotide sequence (1,455 nt) of the 16S rRNA
gene was determined and searched for homology based on
international nucleotide sequence database (GenBank/DDBJ/EMBL). As
a result, the nucleotide sequence of the MK575-fF5 strain was found
to have high homology with those of the 16S rRNA genes of
actinomycetes belonging to the genus Amycolatopsis; i.e.,
Amycolatopsis kentuckyensis (99.1%), Amycolatopsis rifamycinica
(99.03%), Amycolatopsis mediterranei (98.9%), Amycolatopsis
balhimycetica (98.82%), etc. Note that the values in parentheses
are homology between the nucleotide sequences.
[0066] In summary, substrate hyphae of the MK575-fF5 strain are in
a zig-zag form and divided. Aerial hyphae are linear or irregularly
curved and divided into cylindrical spores. Whorls, mycelial
strands, sporangia and motile spores are not observed. The
MK575-fF5 strain is grown in colorless to yellowish orange in
various media. Aerial hyphae of white to pale orange are formed in
some cases or not formed in other cases. Soluble dyes are not
produced. The optimal growth temperature is about 30.degree. C.
This strain is negative in terms of the production of melanine-like
dye and the hydrolysis of starch, but is positive in terms of
reduction reaction of nitrate.
[0067] Regarding the microbial components of the MK575-fF5 strain,
the hydrolyzates of each microorganism contain
meso-2,6-diaminopimelic acid, arabinose and galactose, the cell
wall is of Type IV, and the reducing sugars of each microorganism
are of Type A. Also, mycolic acid is not contained and phospholipid
is of Type PII (phosphatidylethanolamine is contained but
phosphatidyl choline and unknown glucosamine-containing
phospholipids are not contained) as well as a main menaquinone is
MK-9(H.sub.4).
[0068] Through comparison of the partial nucleotide sequence of the
16S rRNA gene with corresponding data of known bacterial strains,
the nucleotide sequence was found to have high homology with those
of actinomycetes belonging to the genus Amycolatopsis.
[0069] In conclusion, the MK575-fF5 strain was thought to belong to
the genus Amycolatopsis (International Journal of Systematic
Bacteriology, Vol. 36, pp. 29-37, 1986). Then, the MK575-fF5 strain
was designated Amycolatopsis sp. MK575-fF5 strain. Notably, the
MK575-fF5 strain was requested for deposition to the National
Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary and was accepted as FERM
P-21465 on Dec. 12, 2007. (Antibacterial agent)
[0070] An antibacterial agent of the present invention contains, as
an active ingredient, the above-described compound of the present
invention; i.e., amycolamicin, the tautomers thereof, or the salts
of the amycolamicin or the tautomers; and, if necessary, further
contains other ingredients.
[0071] The amount of the amycolamicin, the tautomers thereof, or
the salts of the amycolamicin or the tautomers contained in the
antibacterial agent is not particularly limited and may be
appropriately selected depending on the intended purpose. The
antibacterial agent may be the amycolamicin, the tautomers thereof,
or the salts of the amycolamicin or the tautomers, itself or
themselves.
[0072] The other ingredients are not particularly limited and may
be appropriately selected depending on the intended purpose from,
for example, pharmacologically acceptable carriers. Examples of the
other ingredients include ethanol, water and starch. The amount of
the other ingredients contained in the antibacterial agent is not
particularly limited and may be appropriately selected depending on
the intended purpose so that the effects of the amycolamicin, the
tautomers thereof, or the salts of the amycolamicin or the
tautomers are not impaired.
[0073] The dosage form of the antibacterial agent is not
particularly limited and may be appropriately selected depending on
the intended purpose. Examples of the dosage form include powder,
capsules, tablets and liquids. The antibacterial agent can be
routinely formed into each of these dosage forms.
[0074] The administration method of the antibacterial agent is not
particularly limited and may be appropriately selected depending
on, for example, the dosage form of the antibacterial agent. The
antibacterial agent can be administered orally or parenterally.
[0075] The dose of the antibacterial agent is not particularly
limited and may be appropriately determined considering various
factors of target individuals such as their age, body weight,
constitution, symptoms and concomitant use of a drug containing
other active ingredients.
[0076] The animal species to which the antibacterial agent is to be
administered is not particularly limited and may be appropriately
selected depending on the intended purpose. Examples thereof
include humans, monkeys, pigs, bovines, sheep, goats, dogs, cats,
mice, rats and birds.
[0077] As shown in Test Examples 1 to 4 described below, since the
antibacterial agent contains as an active ingredient the
amycolamicin, the tautomers thereof, or the salts of the
amycolamicin or the tautomers, it has an excellent antibacterial
activity against a wide variety of Gram-positive and negative
bacteria such as drug-resistant bacteria and bacteria causing
pneumonia in domestic livestock. Thus, the antibacterial agent can
suitably used for the prevention or treatment of infectious
diseases caused by pathogenic bacteria such as drug-resistant
bacteria and bacteria causing pneumonia in domestic livestock as
shown in Test Examples 1 to 4 described below. The antibacterial
agent may be used alone or in combination with a drug containing
other active ingredients. Also, the antibacterial agent may be
incorporated before use into a drug containing other active
ingredients.
EXAMPLES
[0078] The present invention will next be described in detail by
way of Examples and Test Examples, which should not be construed as
limiting the present invention thereto. In Examples and Test
Examples, the unit "%" means "% by mass" unless otherwise
specified.
Example 1
Production of Amycolamicin
[0079] Cells of Amycolatopsis sp. MK575:fF5 strain (deposited as
FERM P-21465) were cultured in an agar slant medium. Separately, a
liquid medium containing galactose 2%, dextrin 2%, glycerin 1%,
Bacto Soytone (product of Difco Co., Ltd.) 1%, corn steep liquor
0.5%, ammonium sulfate 0.2% and calcium carbonate 0.2% (the pH of
the liquid medium being adjusted to 7.4) was dispensed in 500
mL-conical flasks so that each conical flask contained 110 mL of
the liquid medium, followed by routinely sterilizing at 120.degree.
C. for 20 min. The above-cultured cells were inoculated in the
liquid medium. Thereafter, the cells were shake-cultured through
rotation at 30.degree. C. for 4 days, to thereby obtain seed
culture liquids.
[0080] A medium (100 L) having the following composition: glycerin
0.5%, dextrin 0.5%, Bacto Soytone (product of Difco Co., Ltd.)
0.25%, yeast extract (product of NIHON PHARMACEUTICAL CO., LTD.)
0.075%, ammonium sulfate 0.05% and calcium carbonate 0.05% (the pH
of the medium being adjusted to 7.4) was prepared in a 200
L-culturing tank, followed by sterilizing, to thereby obtain a
production medium. Two percent by volume of each seed culture was
inoculated in the production medium, followed by culturing in the
tank for four days under the following conditions: 27.degree. C.,
200 rpm, air flow rate: 100 L/min.
[0081] The thus-obtained culture was centrifuged so as to be
separated into 80 L of the culture filtrate and 2.5 kg of the
microorganisms. Subsequently, 12 L of methanol was added to the
microorganisms, and the resultant mixture was thoroughly stirred.
Then, amycolamicin was extracted from the microorganisms with
methanol, followed by removal of methanol under reduce pressure, to
thereby obtain 2 L of a microorganism extract containing
amycolamicin. Then, 6 L of ethyl acetate was added to the
microorganism extract (2 L), and the resultant mixture was
thoroughly stirred so that amycolamicin was extracted with ethyl
acetate. The resultant mixture treated with 6 L of ethyl acetate
was dried with anhydrous sodium sulfate and then concentrated and
dried under reduced pressure, to thereby obtain 18 g of a crude
product containing amycolamicin. Hexane (200 mL) was added to 18 g
of the crude product containing amycolamicin and the hexane-soluble
ingredients were removed, to thereby obtain 3.3 g of a
hexane-insoluble fraction containing amycolamicin. The
hexane-insoluble fraction (3.3 g) containing amycolamicin was
dissolved in methanol, and the resultant solution was
chromatographically separated with a Sephadex LH-20 (inner
diameter: 28 mm.times.length: 450 mm, product of Pharmacia Biotech
Inc.) column. The solution was fractionated every 8 g (one
fraction) and, as a result, the active fractions were eluted as
fractions 12 to 15. The fractions were collected and concentrated
and dried under reduced pressure, to thereby obtain 1,584 mg of a
crude product containing amycolamicin. The thus-obtained crude
product (1,584 mg) was dissolved in a small amount of methanol, and
50 mL of kieselguhr (product of Merck Co.) was added to the
resultant solution. Then, the solvent was concentrated and dried
under reduce pressure. The kieselguhr was applied to a silica gel
column (inner diameter: 32 mm.times.length: 175 mm, product of
Merck Co.) to perform chromatography. The developing solvents
sequentially used were chloroform:methanol: water=100:0:0 (200 mL),
95:5:0 (450 mL), 1:95:5:0.25 (450 mL) and 1:90:10:0.5 (450 mL). The
chromatography was fractionated every 18 g (one fraction) and, as a
result, amycolamicin was eluted as fractions 76 to 103. The
fractions were collected and concentrated and dried under reduced
pressure, to thereby obtain 827 mg of pure amycolamicin.
[0082] Through analysis of the obtained amycolamicin for
physico-chemical properties, it was found to have the
physico-chemical properties as shown below. From the
physico-chemical properties, it was confirmed that the amycolamicin
was a new compound having a structure expressed by the following
Structural Formula (1). Also, this amycolamicin was found to have
tautomerism.
(1) Appearance: white powder (2) Molecular formula:
C.sub.44H.sub.60Cl.sub.2N.sub.4O.sub.14 (3) High resolution mass
spectrometry (HRESIMS: negative ion mode)
[0083] Calcd: m/z 937.3386 (M-H).sup.-
[0084] Found: m/z 937.3405 (as
C.sub.44H.sub.59Cl.sub.2N.sub.4O.sub.14)
(4) Specific rotation [.alpha.].sub.D.sup.23=-21.6.degree. (c0.5,
methanol) (5) Infrared absorption spectrum is as shown in FIG. 1.
(6) UV absorption spectrum is as shown in FIG. 2. (7) .sup.1H
nuclear magnetic resonance (NMR) spectrum measured in deuterated
methanol at 30.degree. C. and 600 MHz is as shown in FIG. 3. (8)
.sup.13C nuclear magnetic resonance (NMR) measured in deuterated
methanol at 30.degree. C. and 150 MHz is as shown in FIG. 4. (9)
Thin-layer chromatography using silica gel 60F.sub.254 (product of
Merck Co.) and a developing solvent of chloroform:methanol (9:1 by
volume), the Rf value is 0.31.
##STR00006##
[0085] Also, the obtained amycolamicin was measured for
antibacterial activity in the following Test Examples 1 to 4.
Test Example 1
[0086] According to the standard method of Japanese Society of
Chemotherapy, the amycolamicin was measured for antimicrobial
spectrum against various microorganisms including drug-resistant
bacteria (e.g., methicilline resistant bacteria and vancomycin
resistant bacteria) in the Muller Hinton agar medium by the
multiple dilution method. The minimum inhibitory concentrations
(MICs) measured are shown in Table 1.
Test Example 2
[0087] According to the standard method of Japanese Society of
Chemotherapy, the amycolamicin was measured for antimicrobial
spectrum against various microorganisms including drug-resistant
bacteria (e.g., penicillin resistant pneumococcus, macrolide
resistant pneumococcus and penicillin resistant influenza) in the
Muller Hinton agar medium supplemented with sheep blood (5% by
volume) by the multiple dilution method in an atmosphere of 5% by
volume CO.sub.2. The minimum inhibitory concentrations (MICs)
measured are shown in Table 2.
Test Example 3
[0088] According to the standard method of Japanese Society of
Chemotherapy, the amycolamicin was measured for antimicrobial
spectrum against bacteria causing pneumonia in domestic livestock
in the brain heart infusion agar medium by the multiple dilution
method. The minimum inhibitory concentrations (MICs) measured are
shown in Table 3.
Test Example 4
[0089] According to the standard method of Japanese Society of
Chemotherapy, the amycolamicin was measured for antimicrobial
spectrum against bacteria causing pneumonia in domestic livestock
other than those listed in Table 3 in the Muller Hinton agar medium
supplemented with Fields enrichment (5% by volume) by the multiple
dilution method in an atmosphere of 5% by volume CO.sub.2. The
minimum inhibitory concentrations (MICs) measured are shown in
Table 4.
TABLE-US-00001 TABLE 1 MIC (.mu.g/ml) Tested Microorganism Strains
MIC(.mu.g/ml) Staphylococcus aureus FDA209P 0.39 S. aureus Smith
0.39 S. aureus MS9610 0.39 S. aureus MRSA No. 5 0.39 S. aureus MRSA
No. 17 0.39 S. aureus MS16526(MRSA) 0.39 S. aureus TY-04282(MRSA)
0.39 Enterococcus faecalis JCM 5803 0.2 Ent. faecalis
NCTC12201(VRE) 0.1 Ent. faecalis NCTC12203(VRE) 0.2 Ent. faecium
JCM 5804 0.78 Ent. faecium NCTCI12202(VRE) 0.78 Ent. faecium
NCTCI12204(VRE) 0.78 Escherichia coli NIHJ 12.5 E. coli K-12 12.5
Klebsiella pneumoniae PCI602 6.25 MRSA: Methicilline resistant
Staphylococcus aureus VRE: Vancomycin resistant Enterococcus
TABLE-US-00002 TABLE 2 MIC(.mu.g/ml) Tested Microorganism MIC
Tested Microorganism MIC Strains (ug/ml) Strains (.mu.g/ml)
Streptococcus pneumoniae 0.39 Haemophilus infulenzae 0.78 S-223
IID938 S. pneumoniae CR-2 0.39 H. infulenzae IID985 0.78 (PRSP) S.
pneumoniae T037 0.39 H. infulenzae IID986 0.78 (PRSP) S. pneumoniae
H69 0.39 H. infulenzae IID987 0.78 (PRSP) S. pneumoniae KM99 0.39
H. infulenzae IID988 0.78 (PRSP) S. pneumoniae JPS326 0.39 H.
infulenzae T196 0.20 (ermB) S. pneumoniae JPS344 0.20 H. infulenzae
0.78 (ermB) ME570(BLNAR) S. pneumoniae JPS380 0.39 H. infulenzae
0.78 (ermB) ME587(BLNAR) S. pneumoniae JPS399 0.39 H. infulenzae
0.78 (ermB) ME870(BLNAR) S. pneumoniae JPS408 0.39 H. infulenzae
0.78 (ermB) MT066(BLNAR) S. pneumoniae JPS317 0.39 H. infulenzae
0.78 (mefA) ARD476(BLPAR) S. pneumoniae JPS390 0.39 (mefA) S.
pneumoniae JPS401 0.10 (mefA) S. pneumoniae JPS402 0.20 (mefA) S.
pneumoniae JPS403 0.20 (mefA) PRSP: Penicillin resistant
Streptococcus pneumiae ermB: Macrolide resistant Streptococcus
pneumiae mefA: Macrolide resistant Streptococcus pneumiae BLNAR:
Beta-lactamase-nonproducing penicillin resistant Haemophilus
influenza BLPAR: Beta-lactamase-producing penicillin resistant
Haemophilus influenza
TABLE-US-00003 TABLE 3 Tested Microorganisms Strains MIC (.mu.g/ml)
Mannheimia haemolytica N791 0.78 M. haemolytica N811 0.78
Pasteurella multocida No. 6 1.56 P. multocida Kobe 0.78 P.
multocida TS-8 0.78 P. multocida M-17 0.78
TABLE-US-00004 TABLE 4 Tested Microorganisms Strains MIC (.mu.g/ml)
Actinobacillus pleuropneumoniae NB001 1.56 A. pleuropneumoniae YN
0.78 A. pleuropneumoniae SHP-17 1.56 A. pleuropneumoniae CH-1 1.56
A. pleuropneumoniae CH-2(H4024) 1.56 A. pleuropneumoniae M-1 0.78
A. pleuropneumoniae O-223 1.56 A. pleuropneumoniae Shop1421 3.13 A.
pleuropneumoniae HPT-1 1.56 A. pleuropneumoniae Hi-1 3.13 A.
pleuropneumoniae 6H-8 1.56 A. pleuropneumoniae 7H-3 1.56
Histophilus somni 23N2359 0.20
[0090] From the results of Tables 1 to 4, the amycolamicin was
found to exhibit an excellent antibacterial activity against
Gram-positive and negative bacteria such as drug-resistant bacteria
and bacteria causing pneumonia in domestic livestock. Among others,
the amycolamicin was found to exhibit a particularly excellent
antibacterial activity against Staphylococcus aureus including
MRSA, Enterococcus faecalis/faecium including VRE, Streptococcus
pneumoniae including macrolide resistant bacteria and penicillin
resistant bacteria, Haemophilus infulenzae including penicillin
resistant bacteria, Mannheimia haemolytica, Pasteurella multocida
and Histophilus somni.
INDUSTRIAL APPLICABILITY
[0091] The new compound (amycolamicin) of the present invention,
the tautomers thereof or the salts of the compound (amycolamicin)
or the tautomers have an excellent antibacterial activity against a
wide variety of pathogenic bacteria such as drug-resistant bacteria
and bacteria causing pneumonia in domestic livestock and thus can
suitably used as an antibacterial agent.
Accession Number
[0092] FERM P-21465
* * * * *