CA1238594A - Antibiotics, tan-558, their production and use - Google Patents

Abstract

ANTIBIOTIC, THEIR PRODUCTION AND USE
Abstract of the Disclosure Antibiotic TAN-588 produced by a microorganism of the genus Empedobacter or genus Lysobacter, its p-nitrobenzyl or benzhydryl ester derivative, or their N-deacetylated derivatives or salts thereof possess antimicrobial activities against Gram-positive and Gram-negative bacteria, and can be used as a therapeutic agent for bacterial infections in mammals, fowls, etc.

Description

ANTIBIOTICS, THEIR PRODUCTION AND USE

The present invention relates to a novel antibiotic whlch is useful as an antimicrobial agent, etc., to a process for producing the same, and its use.
As the antibiotlc exhibiting inhibitory activity of cell wall synthesis which is produced by bacteria, heretofore, there have been known sulfazecin and isosulfazecin (Nature, 289, 590-591, 1981), and subsequently, there have been also discovered SQ 26180, 26700, 26823, 26875, 26970, 26~12, etc. (Nature, 291, 489-491, 1981). These are known as the antibiotic which exhibits principally antimicrobial activity against Gram-negative bacteria. In addition, the reports were recently presented on SQ 28332, 23502, 28503, etc. (J. Antibiotics, 36, 1245-1251: 1252~1257, 1983).
The present inventors, with a speciic view to the search for novel antibiotics, isolated a large number of microorganisms from soil, ànd performed screenings or identifying the antibiotics which the microorganisms produce. ~s a result, it was found that a certain micro-organism is able to produce a novel antibiotic, that the said microorganism is a novel species of the genus Empedobacter or the yenu~ L~sobacter, and that cultivation of the said microorganism in a suitable culture medium results in accumulation in the culture medium o~ the antibiotic which exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria, and this antibiotic was isolated; its physico-chemical and biological characteristics led to the :,, ~ .
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~2~ 24205-611 confirmation that the said antibiotic is a novel an-tibiotic, and it has been ~ecided that this is referred to as "Anti-biotic TAN-588".
The present inventors found that -the above An-tibiotic TAN-588 has N-acetyl and carboxyl groups in the molecule and that the said acetyl group can be removed.
Furthermore, the present inventors found that a micro-organism belonging to -the genus Empedobacter or the genus Lysobacter can produce a N-deace-tylated derivative of the Antibiotic TAN-588, and that when a microorganism belonging to the genus Acinetobacter and a microorganism belonging to the genus . _ _ _ . _ _ Empedobac-ter or the genus Lysobacter are cultivated in the culture medium, a N-deacetylated derivative of the Antibiotic TAN-588 is produced in a large amount than only the mieroorganism belonying to the genus ~mpedobacter or the genus Lysobacter is employed.

. .
These findings were followed by further research, whieh has eulminated into -the present invention.
The present invention generally provides novel compound Antibiotie TAN-588, N-deacetyl Antibiotie TAN-588, p-nitrobenzyl or benzhydryl ester derivative of Antibiotie TAN-588 or N-deaeetyl Antibiotie TAN-588 or a pharmaeeutieally acceptable salt of Anti-biotie TAN-588 or N-deaeetyl Antibiotie TAN-588, whieh is represented by the formula l~ N~l )_N r~~
~2 ~ (I) COOR

~ j .

.,, :

-2a-[wherein R is H or CH3CO- and R is H, -CH2 ~ NO2, -CH( ~ )2 or a pharmaceutically acceptable salt-forming cation].
The present invention also provides a process for producing such novel compound which comprises:
(A) for producing Antibiotic TAN-588 (Formula 1, R is CH3CO- and R is H) and/or N-deacetyl Antibiotic TAN-588 (Formula I, R is H and R is H), cultivating a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-S88 elaborated and accumulated in the culture medium, recovering the said antibiotic, (B) for producing N-deacetyl Antibiotic TAN-588, carrying out the mixed cultivation of a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or its N-deacetylated derivative and of a microorganism which belongs to the genus Acinetobacter and is capable of having said microorganism of the genus Empedobacter or Lysobacter produce N-deacetylated Antibiotic TAN-588 in a culture medium to have N-deacetylated Antibiotic TAN-S88 elaborated and accumulated in the culture broth, and recovering the said antibiotic, (C) for producing ~-de,acetyl Antibiotic TAN-588, deacetylating Antibiotic TAN-588 or its salt produced in variant (A), (D) for producing the p-nitrobenzyl or benzhydry]. ester derivative of Antibiotic TAN-S88 and/or N-deacetyl Antibiotic TAN-588, p-nitrobenzylating or benzhydrylating Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-S88, or 1~ ....

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-2b-(E) ~or producing the p-nitrobenzyl or benzhydryl ester derivative of N-deacetyl Antibiotic TAN-588, deacetylating p-nitrobenzyl or benzhydryl ester derivative of Antibiotic TAN-588 produced in variant (D), and if desired, converting Antibiotic TAN 588 or N-deacetyl Antibiotic TAN-588 produced by any o~ the above process variants into a pharmaceutically acceptable salt thereof.
The present invention can be broken down to:
(1) An-tibiotic TAN-588, its para-nitrobenzyl or benzhydryl derivativg, or their N-deacetylated derivatives or salts of these compounds;

(2) A method for producing Antibiotic TAN-588 and/or its N-deacetylated derivative or their salts, which comprises cultivat-ing a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or its N-deacetyla-ted derivative in a culture medium to have Antibiotic TAN-588 and/or its N-deacetylated derivative elaborated and accumulated in the culture broth and recovering the said antibiotic;
~3) A method Eor producing the N-deacetyl Antibiotic TAN-588 or its sa:lts, which compr.ises cleacetylating Anti.biotic TAN-588 or its salt;
(~) A process ~or producing the benzhydryl ester derivat:ive of the N-deacetyl Antibiotic TAN-588, which comprises reacting Antibiot.ic TAN-588 or its salts with a compound capable of allow-ing the introduction oE a benzhydryl group to convert into the benzhydryl ester derivative oE Antibio-tic TAN-588, and subjecting the benæhydryl ester derivative to deacetylation; and '~ ~

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(5) A method for producing N-deacetyl Antibiotic ~AN-588, which comprises carrying out the mixed cultivation of a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or its N-deacetylated derivative and of a microorganism which belongs to the genus Acinetobacter and is capable oE
hav.ing said microorganism of the genus Empedobacter or Lysobacter produce N-deacetyl Antibiotic TA~-588 in a culture medium to have N-deacetyl Antibiotic TAN-588 elaborated and accumulated in the culture broth and recovering the said antibiotic.
In this specification, Antibiotic TAN-588 is in some instances referred to merely as "TAN-588".
The microorganism capable of producing An-tibiotic TAN-588 and/or its N-deacetylated derivative, which is used in the present invention, may be any type of microorganisms, only when ~ey possess the capacity to produce Antibiotic TAN-588. The examples include, for example, Empedobacter lactamgenus which is a novel species of microorganism. As its speciic example, there may be mentioned mpedobacter lactamgenus YK-258 strain (here:inafter referred to, in some _ .
instances, brie1y as "YK-258 strain") which was isolated from a soil sample collected at Masuda city, Shimane Pre-fecture, Japan.
The microbiolo~ica? characteristics oE YK-25a strain are as described in the .~olJ.owin~.
(a) Morphological characteristics The observation a:~ter incubation of the strain on a nutrient agar slant at 2~C for 5 days indicates that the ... :

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~2~94 cells are in the form o~ elongated rod having a diameter of 0.4 to 0.6 ~m and a length of 2.0 to 3.0 ~m and are occasionally shaped like filament having a length of 12 to 30 ~m, but show no flagellum, with no cell motility ~ound;
and that the cells form no spore, are Gram-negative and not acid-fast.
(b) Growths on various culture media Cultivation was conducted at 24C, and observations were made over the period of 1 to 14 days.
(1) Nutrient agar plate culture:
The ~ormed colonies are translucent, pale yellowish, circular-shaped, with papillate surface and entire margin;
no diffusible pigment produced.
(~) Nutrient agar slant culture:
The colonies show good effuse growth and develop yellowish to amber color. -

(3) Nutrient broth culture:
The culture grows turbid and produces a precipitate, with pellicle formed.

(4) Nutrient gelatin stab culture:
Growth mainly on the upper part, with crater-form liquefaction. Liquefaction activity is relatively weak.

(5) Litmus milk:
No activity or reduction of Litmus, peptonization and coagulation observed.
(c) Physiological characteristics (1) Reduction o~ nitrates: -(2) Denitri~ication reaction: -(3) MR (Methyl red) test: -(4) VP (Voges-Proskauer) test: -(5) Production o~ indole: -

(6) Production of hydrogen sulfids (TSI agar and leadacetate paper): -

(7) ~ydrolysis o~ starch: -5 (8) Utilization o~ citrate (on Koser's, Christensen's andSimmons' culture media): ~

, . . . .

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(9) ~tilization of inorganic nitrogen sources:
i) Potassium nitrate: -ii) Ammonium sulfate:
(10) Production of pigments (on King A and B and mannit~
yeast extract agar culture media): No diffusible pigment production observed.
(11) Urease: -(12) Oxidase: +
(13) Catalase: -(14) Ranges for the growth:
i) pH: The optimal pH ranges from 5.8 to 6.6, though the microorganism grows at pH of 5.4 to 8.5.
Culture medium: 0.1% of glucose, 0.01% of yeast extract, 0.1~ of ammonium sulfate, 0.1% of sodium chloride~ 0.05~ of magnesium sulfate (heptahydrate) and 0.1 M of phosphate buffer (sterilized separately).
ii) Temperature: The cptimum temperature is 24 to 31C, though the microorganism grows at 20 to 32C.
Culture medium: Nutrient broth culture medium.
(15) Oxygen demand: Aerobic (16) O-F (Oxidative-Fermentative) test (Hugh-Leifson method):
Not reactive (17) Acid and gas production from sugars and their utilization:
Acid Gas Utilization (Aqueous (Aqueous (Davis culture _peptone)_ _ _eptone) medium) L-Arabinose - - ~
D-Xylose D-Glucose - - ~~
D-Mannose - - +
D-Fructose - - +
D-Galactose Maltose 5ucrose Lactose -Trehalose - ~ +
;

Acid Gas Utilization (~queous (~queous (Davis culture _ peptone ~ pe~tone medium) D Sorbitol - - -D Mannitol Inositol Glycerol Starch ~ ~ +-(18) The mol% G +C of the DNA: ~4.4 +1.5 (Tm method) 0 (19) Capacities to decompose polysaccharides:
Carboxymethylcellulose:
Colloidal chitin: +
Sodium alginate: -(20) Tolerance to actinomycin: Resistant The strain YK-258 having the above-described micro-biological characteristics was compared with the strains as described in "Bergey's Mannual of Determinative Bacteriology, 8th edition" and "International Journal of Systematic Bac~eriology, 30t 225-420 (1980) and 32, 146-149 (1982), and on the basis of the observations that the strain is a yellowish Gram-negative, with no cell motility, being aerobic, lacks the capacity to produce acid and gas rom sugars and shows a high GC content of DNA, it is considered appropriate that the strain belongs to the genus FlavobacteriumO
However, it has been pointed out from the standpoint of bacterial taxonomy that the Flavbbac-terium strains heretofore described are mingled with foreign, different species of microorganisms, and the definition of the genus Flavobacterium was recently corrected in "International Journal of Systematic Bacteriology", 29, 416~426 (1979). According to the said literature reference and "Annual Review of Microbiology", 37, 233-252 ~1983), it is regarded as more reasonable that the strain ~K-258 belongs to rather the genus Empedobacter than the genus Flavobacterium. Nevertheless, among these is not found the description of a species that exhibits the GC
content of the DNA in excess of 70~. The above finding , suggests that the strain YK-258 was identified as a strain belonging to the novel species of microorganism and that the said novel species of microorganism was named Empedobacter lactamgenus.
. . . _ .
The above strain Empedobacter lactamgenus YK-258 has been deposited at the Institute for Fermentation, Osaka (IFO: 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka, ~apan) as the accession number IFO 14322 as from the 20th February, 1984. The said microorganism has also been deposi-ted at the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (FRI: 1-3, Yatabe-cho higashi 1-chome, Tsukuba-gun, Ibaragi Prefecture,Japan) as the accession number FERM P-7558 as from the 26th March, 1984, and the deposit has been converbed t~ a deposit under the Budapest Treaty and has been stored at the FRI under the accession number of FERM BP-699.
Examples of the microorganism of the genus Lysobacter capable of producing Antibiotic TAN~588 and/or its N-deacetylated derivative, which is usable in the present invention,include, for example, Lysobacter albus, a new species of microorganism. As its speciic example, there may be mentioned the strain Lysobacter albus sp. nov. YK-422 (herelnafter referred to in some instances briefly as "YK-422"), which the present inventors isolated from the soil collected at Kanzaki-gun, Shiga Prefecture, ~apan.
The microbiological characteristics of the strain ~K~
422 are as described in the Eollowing:
(a) Morphological characteriskics The observation after incubation of the strain on a nutrient agar slant at 24C for 2 days indicates that the cells are in the form of elongated rod having a diameter of 0.4 to 0.7 ~m and a length of 2.0 to 4.4 ~m and are occasionally shaped like filament having a length of 20 to 30 ~m, but show no flagellum, with no cell motility found;
and that the cells form no spore nor microcyst, are negative 'I!' ~, ~æ3s~

to Gram-staining and not acid-fast.
(b) Growths on various culture media Cultivation was conducted at 24C, and observations were made over the periods of 1 to 14 days.
(1) Nutrient agar plate culture:
The formed colonies are translucent~ white, circular-shapedl with convex surface and entire margin, and show mucoidal surface growth; no diffusible pigment produced.
(2) Nutrient agar slant culture:
The colonies show good filamentous growth and develop white color.
(3) Nutrient broth culture:
The culture grows slightly turbid and produces a small amount of precipitate, with weak fairy ring formed.
(4) Nutrient gelatin stab culture:
Growth is observed on the upper part~ with stratiform liquefaction. Liquefaction activity is relatively strong.
(5) Litmus milk:
Reduction of Litmus and peptonization are observed, with no coagulation found.
(6) Dried yeast plate culture: Cleared zone is formed in the periphexies of colonies formed, with motility through gliding being shown.
(c) Physiological characteristics (1) Reduction of nitrates: -(2) Denitrification reaction: -(3) MR (Methyl red) test: -(~) VP (~oges-Proskauer) test: -(5) Production o~ indole: -(6) Production of hydrogen sulfide (TSI agar and lead acetate paper):
(7) Hydrolysis of starch: -

(8) Utilization of citrate (on Christensen's and Simmons culture media): + 5 (9) Utilization of inorganic nitrogen sources:i) Potassium nitrate: -

- 9 -ii) Ammonium sulfate: -

(10) Production of pigments (on King A and B and mannit-yeast extract agar culture media): No diffusible pigment production observed.

(11) Urease: -

(12) Oxidase: +

(13) Catalase: -

(14) Ranges for the growth:i) pH: The optimal pH ranges from 6.3 to 7.9, though the microorganism grows at a pH of 4.6 to 8.2.
Culture medium: 0.1% of glucose, 0.01~ o~ yeast extract, 0.1% o~ ammonium sulfate, 0.1% of sodium chloride and 0.05~ of magnesium sulfate (hepta-hydrate), with the pH adjusted with sodium hydroxide or sul~uric acid.
ii) Temperature: The optimum temperature is 21 to 28C, though the microorganism grows at 14 to 32C.
Culture medium: Nutrient broth medium.

(15) Oxygen demand: Aerobic 0 (16) O-F (Oxidative-Fermentative) test (Hugh-Leison method):
Not reactive.
(17) Acid and gas production from sugar~ and their utili-zation:
Acid Gas Utilization (Aqueous(Aqueous (Davis culture peptone)peptone) medium) L-Arabinose - -D-Xylose ~ ~
D-Glucose - ~ +
D-Mannose D-Fructose - _ +
D-Galactose ~ ~ +
Maltose ~ ~ +
Sucrose - ~ +
Lactose ~ ~ +
Trehalose ,, ~ .

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Acid Gas Utilization (Aqueous (Aqueous (Davis culture _pe~_ ne) peptone) medium) D-Sorbitol D-Mannitol - - +
Inos itol Glycerol - - +
Starch - - +
.
(18) The mol~ G +C of the DNA: 70.2 +1.5 (Tm method). 0 (19) Capacities to decompose polysaccharides:
Carboxymethylcellulose: +
Colloidal chitin: +
Sodium alginate: +
The strain YK-422 showing the above-described microbiological characteristics was compared with the strains as described in "Bergey's Mannual of Determinative Bacteriology, 8th edition" and !1 International Journal of Systematic Bacteriology", 30, 225-420 (1980) and 32, 146-149 (1982) as well as validation lists in the relevant literature, and on the basis of the observations that the microorganism is a ~ram-negative bacterium exhibiting mucoidal g~owth, being observed to occur partlally in the form of a ~ilamentous organism, shows motility through gliding~ possesses the ability to decompose colloidal chitin and drie,d yeast, forms no microcyst and shows a high GC content in DNA, it is considered appropriate that the strain belongs to the genus Lysobacter However, the strain differed from the heretofore .

described species of the genus Lysobacter ("International Journal of Systematic Bacteriology", 28r 367-393 (1978)~ ln that it is not reactive in O-F test, forms colonies showing no distinctive color tone and possesses no capability to assimilate and digest the inorganic nitrogen source. The above findings su~gest that the ~K-422 strain was identified as a strain belonging to the novel species of microorganism and that the said novel species of microorganism was named Lysobacter albus sp. nov.
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The above strain Lysobacter albus sp. nov. YK-422 has been deposited at the IFO as the accession number IFO
14384 as from the 5th October, 1~84. The said strain has also deposited at the FRI as the accession number FERM P-7938 as from 14th November, 1984, and the deposit has been converted t-o a deposit under the Budapest Treaty and has been stored at the FRI under the accession number of FERM
BP-698.
In the present mixed cultivation, a microorganism belonging to the genus Acinetobacter and capable of having TAN-588 and/or its N-deacetylated derivative-producing microorganism of the genus Empedobacter or Lysobacter produce N-deacetylated derivative of TAN-588 in a large amount.
Said large amount means that the amount of N-deacetyl Antibiotic TAN-588 in the mixed cultivation is much more than the amount of N-deacetyl Antibiotic TAN-588 in the cultivation employing only the microorganism of the genus Empedobacter or Lysobacter.
As the example of the microorganism of the genus Acinetobacter, there may be mentioned the strain Acinetobacter sp. YK-504 (hereinafter referred to in some instances briefly as "YK-504".), which the present inventors isolated from the sweet water collected at Yodogawa-ku, Osaka City, Osaka Prefecture, Japan.
The microbiological characteristics o the strain YK-504 are as described in the following:
~a) Morphological characteristics The observation after incubation of the strain on a nutrient agar slant at 24C for 5 days indicates that the cells are in the form of short rod or spherical having a diameter of 0.8 ~o 1.5 ~m and a length of 1.1 to 2.1 ~m and occures occasionally in pairs and filament, but show no flagellu~, with no cell motility found; and that the cells form no spore nor microcyst, are negative to Gram-staining and not acid-fast.
.

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(b) Growths on various culture media Cultivation was conducted at 24C, and observations were made over the periods of ~ to 14 days.
(1) Nutrient agar plate culture:
The ~ormed colonies are small, circular-shaped, with convex surface and-entire margin, no diffusible pigment produced.
(2) Nutrient agar slant culture:
The colonies show moderate filamentous growth and are colorless and glossy.
(3) Nutrient broth culture:
The culture grows turbid and produces a precipitate, with no ring formed.
(4) Nutrient gelatin stab culture:
Weak growth is observed on the upper part. Liquefaction activity is negatlve.
(5) Litmus milk:
Reduction of Litmus and peptonization and coagulation are not observed.
(c) Physiological characteristics (1) Reduction of nitrates: -(2) Denitrification reaction: -(3) MR (Methyl red) test: -(4) VP (Voges-Proskauer) test: -(5) Production of indole: -(6) Production of hydrogen sulide (TSI agar and lead acetate paper): -(7) Hydrolysis of starch: -t8) Utilization o citrate (on Koser's, Christensen's and Simmons' culture media): -(9) Utilization of inorganic nitrogen sources:
i) Potassium ni~rate: -ii) Ammonium sulfate: -(10) Production o pigments (on King A and B and mannit-yeast extract agar culture media): No diffusible pigment production observed.
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(11) Urease: -(12) Oxidase: -~13) Catalase: +
(14) Ranges for the growth:
i) pH: The optimal pH ranges from 6 to 7.5, though the microorganism grows at a pH of 5 to 8.
Culture medium: Nutrient broth with the pH adjusted with sodium hydroxide or sulfuric acid.
ii) Temperature: The optimum temperature is 15 to 22C, though the microorganism grows at 7.5 to 39C.
Culture medium: Nutrient broth medium.
tl5) Oxygen demand: Aerobic

(16) O-F (Oxidative-Fermentative) test (Hugh-Leison method):
Not reactive. 5 (17) Acid and gas production from sugars and their utilization:
Acid Gas Utilization (Aqueous (Aqueous (Davis culture pe~tone) peptone) medium) L-Arabinose - - -D-Xylose D-Glucose D-Mannose D-Fructose D-Galactose Maltose Sucrose Lactose - ~ ~
Trehalose D-Sorbitol D~Mannitol Inositol - ~ ~
Glycerol Starch .
_ .. _ _ . ,,,, ,,,,, , _ , _ , _ _ (18) The mol% G ~C of the DNA: 40.0 +1.5 (Tm method).

i~23~4 (19) Capacities to decompose polysaccharides:
Carboxymethylcellulose: -Colloidal chitin: -Sodium alginate: -The strain YK-504 showing the above-described micro-biological charac~erisLiCs was compared with the strains as described in "Bergey's Mannual of Determinative Bacteriology, 8th edltion" and "International Journal of Systematic Bacteriology", 30, 225-420 (1980) and 32, 146-149 (1982) as well as validation lists in the relevant literature, and on the basis of the observations that the microorganism is Gram-negative short rod or spherical and is not motile, aerobic, does not form acid and gas from sugars. Oxidase test is negative. Catalase test is positive. The mol %
G +C of the DNA is 40.0 +1.5, the strain YK-504 is determined to belong to the genus Acinetobacter, and then designated as Acinetobacter sp. YX-504.
The above strain Aeinetobaeter sp. YK-504 has been deposited at the IFO as the aceession number IFO 14420 as of January 31, 1985, and at the FRI as the accession number FERM BP-709 as of February 12, 1985.
The mieroorganism of the genus Empedobaeter or the genus Lysobaeter, whieh is usable in the present invention, generally shows p:roperties and eharaeteristies readily liable to undergo ehange or alteration, and is readily suseeptible to mutation through aritifieial mutation means using.for e~ample ultraviolet light r ~-rays and ehemieal agents ~e.g., nitrosoguanidine, ethylmethanesulonie aeid, ete.);
any o its mutants ean be used in the present invention, only if they are eapable of produeing TAN-588 and/or its N-deaeetylated derivative that the present invention for its objeet.
The ~.iaroorganism of the genus Aeinetobaeter, usable in the present invention r generally shows properties and eharaeteristies readily liable to undergo ehange or ...

alteration, and is readily susceptible to mutation through aritificial mutation means using for example ultraviolet light, X-rays and chemical agents (e.g., nitrosoguanidine, ethylmethanesulfonic acid, etc.); any of its mutants can be used in the present invention, only if they are capable of having Antibiotic TAN-588 and/or its N-deacetylated deriva-tive-producing strain produce N-deacetyl Antibiotic TAN-588 in a large amount by the mixed cultivation of the present invention.
In cultivating the TAN-588 and/or its N-deacetylated derivative-producer, as the carbon source, there may be suitably used substances which the microorganism can assimilate and digest, such as glucose, fructose, maltose, soluble strach, dextrin, oils and fats (e.g., soybean oil, olive oil, etc.) and organic acids (e.g., citric acid, succinic acid, gluconic acid, etc.). As the nitrogen source, there can be utilized organic nitrogen compounds/ such as soybean meal, cotton seed flour/ corn gluten meal, dried yeastt yeast extract, meat extract, peptone and urea. As the inorganic salt,there may be used, solely or suitable combinations, inorganic salts which are normally necessary for the cultivation of micro-organisms, such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate and disodium phosphate.
In addition, heavy metal salts, such as ferrous sulfate and copper sulfate, vitamins, such as vitamin B1, biotin, and the like are added, if necessary. Furthermore, anti-foams and surfactant, such as silicone oil or polyalkylene glycol ether, may be added to the culture medium. other organic and inorganic substances which help t.he micro-organism to grow and promote the production of TAN-588 and/or its N-deacetylated derivative may be suitably added.
With reference to the cultural method, cultivation may be conducted by a procedure similar to the process for producing general antibiotics, and either solid culture or liquid culture may be adoptable. In the case of liquid culture, stationary culture, shake culture, submerged culture, aerobic culture, etc. may all be carried out, though aerobic submer~ed culture is particularly preferred.
The incubation temperature is preferably in the range of about 15C to 32C, wher~y incuba~ion is carried out with a pH of the culture medium ranging from about 5 to 8 for 8 to 168 hours, preferably 24 to 144 hours.
The mixed cultivation of the present invention is carried out by the ~anner similar to the manner of the production of TAN-588 and/or its N-deacetylated derivative by the cultivation of the microorganism of the genus Empedobacter or Lysobacter.
The detection of the N-deacetyl Antibiotic TAN-588 is carried out by TLC-bioautography method employing Pseudomonas aeruginosa C-141.
The present mixed cuitivation employing a microorganism belonging to the genus Acinetobacter brings a production of N-deacetyl TAN-588 in a larger amount than the production employing only the microorganism belonging to the genus Empedobacter or the genus Lysobacter.
In order to harvest the objective AntibiOtic TAN-588 and/
or its N-deacetylated derivative from the resulting culture broth, there are suitably utilized separation means which are normally employed in recovering metabolites produced by a microorganism from its microbial culture. Since the antibiotic TAN-588 and its N-deacetylated derivative behaves like a water-soluble, and is contained mainly in the filtrate of the culture broth, or example, there is advantageously utilized a means which comprises firstly adding a filter aid to the culture broth to remove microbial cells by filtration or centrifugation, contacting the resulting filtrate of the culture broth with a suitable support to adsorb the active ingredient contained in the filtrate and desorbing the active substance with a suitable solvent to conduct fractionation and recovery. As the support, there are advantageously those utilizing the difference in ,

- 17 -adsorptivity of compounds, such as activated carbon, silica gel, crystalline cellulose and adsorptive resins, those utilizing ~he difference in functional groups of compounds, such as anion exchange resins and anion exchange cellulose, or those utilizing the difference in a molecular size, such as the medium for gel filtration. In order to elute the objective compound from these supports, for example, aqueous solutions of water-miscible solvents/ i.e., aqueous acetone and aqueous alcohols, or aqueous solutions containing acids, alkalies, buffers or inorganic or organic salts are used in suitable combinations, although such combinations varies depending upor. the type and characteristics of the support used. Also, the crude material of the present antibiotic obtained by these chromatographic procedures can be sub-jected to high-performance liquid chromatography (HPLC) for fractionation to perform ~urther purification. Alter-natively, the eluate desalted by activated carbon chromato-graphy is concentrated, and the present antibiotic can be recovered from the concentrate by the ion-paired extraction method, i.e. by use of an organic solvent containing quaternary alkylammonium halide.
Referring in more particular to the procedure, as TAN-588 is an acidic substance, when anion exchange resins, such as Dowex-l* (produced by Dow Chemical Co.l U.S.A.), Amberlite*
IRA-68, 400, 402 and 410 (produced by Rohm & Haaæ, U.S.A.) and Diaion*SA-21 and C (produced by Mitsubishi Chemical Industries, Japan), are used as a support, the present anti-biotic in the filtrate is adsorbed and eluted or example ~ith aqueous solutions containing salts or acids or buers.
Also, the present antibiotic is adsorbed onto supports, such as anion exchange cellulose, e.g. DE-32 (produced by Whatman Co., U.K.) and DEAEkCellulose (produced by Braun Co., West Germany), and anion exchange molecular-sieving resins, e.g.
DEAE- or QAE-Sephadex*(produced by Pharmacia, Sweden), and eluted for example with aqueous solutions containing salts or acids or buffers. For the purpose o~ removing salts, *Trademark

- 18 -coloring matters, etc. in these eluates, there are advan-ta~eously used activated carbon ~or chromatographic uses (produced by Takeda Chemical Industries, Ltd., Japan) or adsorptive resins, such as Diaion*HP-20 or SP-207 (produced by ~i~subishi Chemical Industries, Japan) and Amberlite XAD-II (produced by Rohm & ~aas Co., U.S.A.). The eluted fraction is pulverized through the steps of concentration, lyophilization, etc. In cases in which the resulting powder shows a deteriorated degree of purity, the preparative HPLC
method is advantageously applied to further purification.
The support, which is usable for this purpose, includes, for example, TSK Gel (produced by Toyo Soda Mfg. Co., Japan) and YMC Gel (produced by Yamamura Chemical Laboratories, Japan), while as the mobile phase, there are used mixed solutions of methanol or acetonitrile, etc. with aqueous acid solutions or buffers, etc. With reference to the quaternary alkyl ammonium halide which is employed in the above-mentioned ion paired extraction method, there are used, for example, tri-n-octylmethylammonium chloride, tetra-n-pentylammonium chloride or n-tetradecyldimethylbenzylammonium chloride, and as the organic solvent, there are normally used, for example, methylene chloride, chloroform or dichloroethane.
Furthermore, as the N-deacetyl TAN-588 has a basic characteristics although it is amphoteric, when cation exchange resins, such as Dowex*50W (Dow Chemical Co., U.S.A.), AmberlikekIP~-120B (Rohm and Haas, U.S.A.), Diaion SK-llO ~Mitsuhishi Chemical Industries~ Japan), are used as a support, the present antibiotic in the filtrate is adsorbed, and eluted for example with aqueous solutions or buffers containing salts, acids or alkalis.
Also, the present antibiotic is adsorbed onto supports, such as cation exchange molecular-sieving resins, such as CM-Sephadexk(Pharmacia, Sweden)~ and is eluted for example with aqueous solutions or buffers containing salts.
For the purpose of removing the salts, coloring *Trademarks ~23859~

matters~ etc. in these el-lates, there are advantageously used activated carbon for chromatographic uses or adsorptive resins~ such as Diaion*SP-207 or HP-20.
The eluated fractions are concentrated, lyophilized and then precipitated. When the resulting powder shows a inferior degree of purity, the preparative HPLC method is advantageously applied to further purification. The support and mobile phase is as the same as those of TAN-588.
TAN-588l in the course of purification, exists in the forms having cations in the used salts or buffers, such as sodi~n, potassium, lithium, calcium and ammonium ions, attached thereto. In such cases, TAN-588 is isolated as the corresponding salt, when the eluate with its pH remained as such is chromatographed on activated carbon, and is obtained as a free form, when the eluate is adjusted to a pH
of 5 to 2, preferably a pH of 4.5 to 3, and chromatographed on activated carbon.
The N-deacety TAN-588 is obtalned as an amphoteric substance when it is chromatographed on activated carbon with the eluate of neutral regions. Furthermore, as N-deacetyl TAN-588 has a basic characteristics although it is amphoteric, it can form salts with strong acids. Such strong arids ~nay be mentioned hydrochloric acid, phosphoric acid and trifluoroacetic acid.
T~N-588 as obtained in this manner has two peaks on the reverse-phase HPLC. These peaks are temporarily referred to as ~ and B, respectively, whereby the phenomena to be described in the following a-e observed. Collection of the individual peaks A and B by HPLC for ractionation gives A
and B in a fairly single form, respectively, but when these are allowed to stand in buffers o~ pH 3~ 5 and 7 at room temperature, A turns into B, with B turning into A, about one hour later at any pH values, thus allowing the once isolated A and B to change into an equilibrium mixture with A:B =about 1:1. Therefore, it is thought impossi~le to separate TAN-588 itself into A and B by means of the presently known separation techniques.
*Trademark However, the conversion o~ TAN-588 into the p-nitrobenz~l ester or benzhydryl derivative makes it possible for such a compound to be separated into the A type and B type of the compound , respectively.
As the means of deacetylating AntibiotIc IAN-588 or its salt, there are adopted the known deacetylation reactions.
~y way of example of such procedures, deacetylation is conducted for example by introducing a p-nitrobenzyl or benzhydryl group into IAN--588 and furthermore removing the acetyl group of ~AN-588, followed by elimination of the said p-nitrobenzyl or benzhydryl group, if necessary.
For the purpose of the above-mentioned introduction of a p-nitrobenzyl group, TAN-588 or its salt is reacted with a compound capable of introducing a p-nitrobenzyl group~
Examples of the said compound capable of introducing a p-nitrobenzyl group include, for example, p-nitrobenzyl bromide and p-nitrobenzyl chloride.
Ihe amount of the compound capable of introducing a p-nitrobenzyl group to be used ranges from about 1 to 5 equivalents, preferably from about 1 to 2 equivalents. The reaction is preferably carried out in a solvent, and examples of the said solvent include dimethylformamide (D~), dimethylacetamide (DMAA) and tetrahydrofurane (THF). In the said reaction, for example, triethylamine (Et3N) and pyridine may be added in quantities o~ about 0.1 to 0.5 e~uivalent, preferably about 0.1 to 0.2 equivalent, for the purpose of promoting ithe reaction.
The reaction temperature is about 0C to 40C, more preferably about 20C to 30C, while the reaction time i5 30 about 0.5 to 8 hours, more preferably about 1 to 4 hours.
The reaction is preferablyconducted under stirring.
For the purpose of the above-mentioned introduction of a benzhydryl group, TA~-588 or its salt is reacted with a compound capable of introducing a benzhydryl yroup. Examples of the said compound capable of introducing a benzhydryl . group include, for example, diphenyldiazomethane and diphenyl-methyl bromide. The amount of a compound capable of introducing a benzhydryl group to be used ranges from about l to 6 equivalents, preferably from about 2 to 4 equivalents~ The reaction is preferabl~ carried out in a solvent, and examples o~ the said solvent include THF, dioxane, ethyl acetate and dichlorometilane. In the said reaction, for example, dilute hydrochloric acid, dilute sulfuric acid and dilute phosphoric acid are preferably added in small amounts, e.g. about O.Ol to l.0 equivalent, to adjust the reaction.solution to a pH
in the.neighborIlood of about l to 3, preferably about l~5 to 2.5, for the purpose of promoting the reaction. The reaction temperature is about -lO to -~50C, more preferably 0C to 30C, while the reaction time is about 30 minutes to about 8 hours, more preferably about l to 3 hours~ The reaction is favorably conducted under s~irring. j The es~er derivative obtained by the above procëdure can be collected by the conventional separation or purification means. By way of example of the said means, the. objective compound is extracted into the organic layer by use of e.g. dichloromethane or chloroform, and the extract is concentrated, followed by addition of the resulting concentrate to ether, hexane, etc. to allow the said ester derivative to separate out in the form of a crystalline powder. This ester derivative is separated into two components by the silica gel chromatographic method, but may be used as a mixture when the subsequent reaction is stepped forward to~
Furthermore, the p~nitrobenzyl ester or benzhydryl derivative of T~N-588 as obtained hy the above procedure is subjected to deacetylation.
Examples of the said deacetylation include the imino ether method, solvolysis method and hydrolysis method by use o~ enzymes~
In cases in which the imino ether method is used, ~or examp].e, the starting compound is reacted with phosphorus pentachloride, phos~ene, phosphorus trichloride, phosphorus oxychloride, etc. The above-mentioned reagents are preferabIyllsed in about 1 to 5 equivalents, more preferably about 1.5 to 3 equivalents. The said reaction is conveniently carried out in the presence of a solvent, such as methylene chloride, dichloroethane, choroform, carbon tetrachloride and trichloro-ethane. For the purpose of promoting the reaction, ît is preferableto use for example pyridine, N,N-dimethylaniline, triethylamine, aniline or toluidine in excessive quantities, e.g. about 3 to 20 equivalents, more preferably about 5 to 10 equivalents.
The said deacetylation reaction is desirably carried out at a reaction temperature of about -30C to 0C, more preferably -15C to -5C, for a length of reaction tlme of about 15 minutes to 8 hours, more preferably about 30 minutes to 2 hours. ~he reaction îs conveniently carried out under stirring.
In order to convert imino chloride formed as an intermediate into imino ether, an excess of methanol is added to the reactlon solution, and the mixture is stirred at a 20 temperature of about -30C to 0C, preferably about -15C
to -5C, for a period of time of about 15 minutes to 2 hours, preferably about 30 minutes to 1 hour, followed by further stirring at about 10C o 40C, preferably about 20C to 30C, for about 30 minutes to 2 hours, for the termination of the reaction~ Furthermore, dilute hydrochloric acid is added to the reaction solution to severe the C-N linkage, whereby the reaction temperature is about 10C to 40C, preferably about 20C to 30C, and the reaction time is about 15 minutes to 2 hours, preferably about 30 minutes to 1 hour.
In employing the solvolysis method, for example, the starting compound is dissolved in methanol, ethanol or a mixture thereof with water, and the reaction is allowed to proceed at about 20C to *he refluxing temperat~re, preferably about 50C to the refluxing temperature, for about 0.5 to 30 hours, preferably about 2 to 8 hours.
The reaction solution thus obtained is neutralized, , and the reaction product is extracted with an organic solvent immiscible with water, for example, methylene chloride, diethyl ether or ethyl acetate, follo~ed by concentration of the extract to yield the p-nitrobenzyl or benzhydryl derivative of deacetylated TAN-58~.
In order to eliminate the ester group as a final step, for example, the acid hydrolysis method, catal~tic reduction method, etc. are employed In cases in which the acid hydrolysis method is employed, an acid, such as trifluoroacetic acid, ~ormic acid and hydrochloric acid, is used at a rate of about 3 to 20 equivalents against the star-ting compound to allow the reaction to proceed. Also, it i5 preferred to add anisole o about 1 to 5 equivalents, preferably 2 to 4 equivalents. In the said reaction, as the solvent, there may be used Eor example methylene chloride, chloroform, THF, ethyl acetate,etc.
The reaction temperature is about -30C to 0C, more preferably -20C to -10C, and the reaction time is about 0.5 to 8 hours, more preferably about 1 to 4 hours~
In employing the catalytic reduction method, as the catalys~, there may be used ~or example palladium, platinum, their oxides, etc. to allow the reaction to proceed.in a stream of hydYogen.
~he reaction temperature is about 0C to 50C, more preferably about 10C to ~0C, while the reaction time is about 0.1 to 6 hours, more preferably about 0.2 to a hours.
~ he Eree carboxylic acid derivative thus produced can be separated, collected and purified by removing impurities in the reaction solution by ~iltration or chromatographlc method, such as tllose utilizing for example activated carbon or adsorptive resins, followed by concentration, lyophili-zation, etc.
In each of the above steps, and when the resulting compound is a mixture of isomers, for example, column chromatograplly, such as the methods utilizing silica gel, Sephadex*L~-20 (produced by Pharmacia Co., Sweden), Diaion*
*Trademarks HP-20, etc. as a support, or recrystallization method and preparative reverse phase chormatography [examples of the support: YMC
Gel, TSK Gel; examples of the mobile phase: buffers or buffers containing methanol or acetonitrile] for fractionation can permit the separation into individual isomeric components~

*Trademarks ~25-The TAN-588 sodium salt (an equilibrium mixture of A and B) as obtained in Ecample 1 to be described hereinafter showed physico-chemicalproperties which are as follows:
1) Appearance: White powder 2) Specific rotation: [~]23 -19.0 + 10 (c=0.5, in water) 3) Elemental analysis for the compound being constituted of the elements, C, H, N, O and Na (%): for a sample being dried over phosphorus pentoxide at 40C for 6 hours.
Found Calcd.*
C, 38.5 + 2.0C, 39.61 H, 4.5 + 1.0 H, 3.99 N, 9.1 1 1.5 N, 9.24 O, 39.58 Na, 6.9 + 1.5Na, 7.58 (*calculated assuming that 0.5 mole of water of adhesion is contained).
4) Content of adhesi~e water: 3.0 + 1.5 ~ (by the the~lavimetric method) 5) The molecular ion peak according to the SIMS method is as follows:
m/e 611(2M~Na)~, 317(M~Na)~, 295(M+H)+
6) Molecular formula:
CloHllN207Na 7) Ultraviolet absorption (UV) spectrum (in water); Fig. 1 ~max 216 nm (Elcm = 130 shoulder) 8) In~rared absorption (IR) spectrum (KBr method):
The infrared absorption spectrum (Fig. 2~, recorded as KBr disc, demonstrates the ollowing principal absorption peaks (in wave number):
3~50, 1780, 1730, 1660, 1550, 1385, 1320, 1290, 1260, 1200, 1120, 1040, 980, 910, 810, 770, 690, 600, 540 cm~l 9) 13C-Nuclear magnetic resonance (NMR) spectrum (lO0 MHz, in deuterium oxide): The following peaks are observed.
182.02(s), 177.30(s), 173.79(s), 173,30(s), 173.25(s), 172.58(S), 96.97(s), 96.92(s), 74.27(t), 72.68(t), 55.57(d), 55.34(d), 31.92(t), 31.08(t), 30.9a(t), 24.58(q), ppm (wherein s;
singlet, d; doublet, t; triplet, and q; quartet).

~23~

10) Circular dichroism (CD) spectrum (in water):
The ne~ative Cotton effect is revealed at 232 + 3 nm.
ll) Solubility:
Soluble in: Water, dimethylsulfoxide.
Sparingly soluble in: Ethyl acetate, chloroform, diethyl ether.
12) Color reaction:
Positive: Ninhydrin reaction Negative: Greig-Leaback reaction, Sakaguchi reaction, Eh~lichreaction, Barton reaction, Dragendorff's reaction.
13) Amino acid analysis: Hydrolysis in 6N-hydrochloric acid at 105C for 20 hours allows the detection of serine as the known amino acid.
14) Stability: Stable in an aqueous solution at p~l 5, slightly stable at pH 3 and 7, and unstable at pH 9.
15) Thin-layer chromatography (C~llulose f, produced by Tokyo Kasei Co. of Japan):
Solvent sYstem I Rf value Acetonitrile:water (4:1~ 0.33 Butanol:acetic acid:water (1:1:1~ 0.77 Acetonitrile:3~ ammonium sulfate (4:1) 0.28 16) Discrimination among acidity, neutrality and basicity:
Neutral substance.
17) EIPLC (support: YMC A-312, produced by Yamamura Chemical Laboratories, Japan, mobile phase: 4 ~ methanol/O.OlM phosphate buffer (pH 6.3~, 2 mQ/min.):
Rt = 4.3 and 4.8 (min).

*Trademark ';' ~

~' ' ~ "' ' ' ' :.
.

, .

~;~3~i~

The physico-chemical properties of TAN-588-P-nitrobenzyl ester (a mixture o~ A type and B type compounds) obtained in Example 4 which appears hereinafter are shown below.
1) Appearance: white powder 2) Specific rotation: [~]D ~ 16.3~5 (C=0~485, in CHC13) 3) Molecular weight: 407 (according to SIMS method) 4) Elemental analysis:
Calcd.: C,50.13; H,4.21; N,10.32; 0,35.35 Found : C,50.26; H,4.32; N,10.31 5) Molecular formula: C17H17N3Og 6) UV spectrum: ~ max nm (Elcm) = 262i2 (281i20), 214~2 (278~20, sho~lder) 7) IR spectrum: KBr method, Fig. 3 3400, 3080, 2960, 1805, 1760, 1680, 1610, 1520, 1450, 1380, 1350, 1270, 1180, 1105, 1050, 1015, 970, 905, 850, 740, 690, 600, 540 cm 8) lH-NMR spectrum: 90 MHz/ in CDC13 ~ ppm J(Hz) 2.05(3H,s), 2.3-3.3(4H,m), 4.10(1H,m), 4.5-5.1(2H,m), 5~35(2H,s), 6~25(1H,d,like), 7.55(2H,dd like), 8.27(2H,d,like) 9) TLC:
Carrier: silica gel (Merck, West Germany) Developping solvent: chloroform: methanol (19 1) Rf value, 0.25 and 0.32 10) Acidic, neutral or basic: neutral substance , ~ 3~i~1~

The physico-chemical properties of TAN-588A-p-nitrobenzyl ester obtained in Example 4 which appears hereina~er are shown below.
1) Appearance: white powder 2) Specific rotation: [~]D + 97.3+15 (C=0.48 in CHCl~) 3) Molecular weight: 407 (according to SIMS method) 4) Elemental analysis:
Calcd.: C,50.13; H,4.21; N,10.32; 0.35.35 Found : C,50.20; H,4.22; N,10.13 5) Molecular formula: C17H17N309 6) UV spectrum: ~ma (El~m) = 262+2 nm (280+30), 214~2nm (276+30, shoulder) 7) 3C-NMR spectrum:(lOOMHz, CDC13), 173.70(s), 171.53 (9), 170.72(s), 165.09(s), 148.06(s), 141.38(s), 128.86(d), 123.91(d), 91.82(s), 71.60(t), 67.29(t), 53.00(d), 29.09~t), 27.49(t), 22.64(q), ppm 8) IR spectrum: KBr method,Fig. 4 3400, 3080, 2950, 1805, 1775, 1760, 1680, 1610, 1530, 1450, 1380, 1350, 1300, 1275, 1190, 1105, 1060, 1020, 980, 910, 850, 740, 700, 600, 540 cm 1 9) TLC:

Carrier: silica gel (Merck, West Germany) Developping solvent: chloro~orm: methanol (].9:1) R~ value: 0.25 10) Acidic~neutral or basic: neutral substance " ' The physico-chemical properties of TAN-588B-p-nitrobenzyl ester obtained in Example 4 which appears hereina~ter are shown below.
1) Appearance: white powder 2) Specific rotation: [~]D0 _ 64.5+15 (C=0.50, in CHC13) 3) Molecular weight: 407 (according to SIMS method) 4) Elemental analysis:
Calcd.: C,50.13; H,4.21; N,10.32; 0,35.35 Found : C,50.10; H,4.21; N,10.15 15 5) Molecular formula: C17H17N3Og 6) UV spectrum: ~maH (ElCm) = 262+2nm (282+30) 214+2nm (280+30, shoulder) 7) C-NMR spectrum: (lOOMHz, CDC13), 173.59(s), 170.86(s), 170.61(s), 165.06(s), 148.12(s), 141.24(s), 128.96(d), 123.96(d), 91.69(s), 74.60(t), 67.39(t), 51.94(d), 29.11(t), 27.38(t), 22.67(~), ppm 8) IR spectrum: KBr method, Fig. S
3400, 3090, 2950, 1805, 1760, 1680, 1610, 1530, 1450, 1380, 1355, 1270, 1180, 1105, 1055, 1015, 965, 910, 835, 740, 695, 600, 540 cm 1 9) TLC: the same conditions as those o p-nitrobenzyl ester o~ TAN-588A
R value, 0.32 10) Acidic, neutral or basic: neutral substance The physico-chemical properties of TAN-588 (~ ~n~w~ ~ A ~ ~) benzh~dryl ester~obtained in Example 5 which appears hereinafer are shown below.
1) Appearance: colorless crystal 2) Melting point: 153-155C (Decomposition) 3) Specific rotation: [~]D t 9.2~+5 (C=0.52 in CHC13) 4) Molecular weight: m/z 438(M )(EI~MS method) 5) Elemental analysis:
Calcd.: C,63.01; H,5.06; N,6.39; 0,25.54 Found : C,62.83; H,5.32; N,6.28 6) Molecular formula: C23~22N207 7) UV spectrum:in methanol ~max 220+2nm (ElCm = 285+50, shoulder) and 1%
250-260nm (ElCm = 28+10, shoulder) 8) IR spectrum: KBr method, Fig. 6 3380, 3080, 3050, 2960, 1800, 1780, 1750, 1705, 1690, 1600, 1590, 1540, 1500, 1460, 1380, 1310, 1280, 1190, 1110, 1060, 980, 920, 880, 750, 710, 700, 650, 630, 610, 570, 550, 470 cm 1 9) lH-NMR spectrum: 90 MHz, in CDC13 , ~ ppm J(Hz) 1.97(3H,s), 2.1-3.5(4H,m), 3.8-4.2(1H,m), 4.5~5.1(2H,m), 6.1-6.4(lH,br), 6.97(lH,s), 7.3-7.4(lOH,m) (m: multiplet, br: broad, H: proton) ].0) TLC: the same conditions as those of A iype compound (mentioned below) Rf value, 0.58 and 0.65 11) Acidic, neutral or basic: nuetral substance 3~

The physico-chemical properties of TAN-588 benzhydryl ester (A type compound) and TAN-588 benzhydryl ester (B
type compound) obtained in Example 5 which appears hereinafter are shown below.
A type compound 1) Appearance: colorless crystal 2) Melting point: 97-135C (gradually foaming and decomposing) 3) Specific rotation: [a]D ~ 44.2~10 (C=0.505, in CHC13) 4) Molecular weight: Molecular ion peak according to EI-MS method m/z 438 (M ) 5) Elemental analysis:
Calcd.: C,63.01; H,5.06; N,6.39; 0,25.54 Found : C,62.62; H,5.06; N,6.32 6) Molecular formula: C23H22N2O7 7) UV speetxum: in methanol ~max 220~2nm (ElCm = 290i50, shoulder) and 250-260nm (El%m = 30~10, shoulder) 8) IR spectrum: KBr method, Fig. 7 3380, 3080, 3050, 1800, 1780, 1760, 16~5, 1540, 1500, 1450, 1380, 1310, 12~0, 1190, 1110, 1050, 980, 920, 880, 750, 710, 6S0, 610, 550 em 1 9) 1H_NMR speetrum: 100 MHz, in a mixed solvent of CDC13 and d6-DMSO, ~ ppm J(Hz) 1.98(3H,s), 2.2-3.4(4H,m), 4.10(1H,dd,J=8~10), 4.4-5.0 (2H,m), 6.93(lH,s), 7.3-7.5(10H,m), 8.27(lH,d,J=7) .

10) TLC: Carrier, silica gel (Merck, West Germany) Developping solvent, ethyl acetate R~ value, 0.58 11) Acidic, neutral or basic: neutral substance B type com~ound 1) Appearance: colorless crystal 2) Melting point: 157-160C (Decomposition) 3) Specific rotatlon: [~]D -28.8~10 (C=0.5, in CHC13) 4) Molecular weight: m/z 438(M )(EI-MS method) 5) Elemental analysis:
Calcd.: C,63.01; H,5.06; N,6.39; 0.25.54 Found : C,63.11; H,5.13; N,6.30 6) Molecular formula: C23H22N207 7) W spectrum: in methanol ~max 220~2nm ~El~m = 300~50, shoulder) 250-260nm (ElCm = 26~10, shoulder) 8) IR spectrum: KBr method, Flg. 8 3400, 3080, 3050, 1815, 1780, 1735, 1705, 1540, 1460, 1380, 1290, 1265, 1190, 1060, 980, 920, 880, 760, 715, 610/ 550 cm 1 9) lH-NMR spectrum: 100 MHz, in CDC13 ~ ppm J (Hz), 1.98(3H,s), 2.2-3.4(4H,m), 4.03(1H,dd,J=8/10), 4.6-5.2 (2H,m), 6.32(lH,d,J=5), 6.96(lH,s), 7.2-705(lOH,m) ' ;

, 10) TLC: (the same conditions as those of the A type compound) Rf value, 0.65 11) Acldic, neutral or basic: neutral su~stance The physico-chemical properties of benzhydryl ester (a mixture of A type and B type compounds) of N-deacetyl TAN-588 obtained in Example 6 which appears hereinafter are shown below.
1) Appearance: white powder 2) Specific rotation: [~]D -15.2+5(C--0.5, in CHC13) 3) Molecular weight: m/z 396(M )(EI-MS method) 4) Elemental analysis:
Calcd.: C,63.63i H,5.09; N,7.07i 0,24.22 Found : C,63.63i H,5,05; N,7.02 5) Mdlecular ~ormula: C21H20N206 6) UV spectrum: in methanol ~max 220i2 nm (ElCm = 336i50, shoulder) 250-260nm ~El~m= 32ilO, shoulder) 7) IR spectrum: KBr method, Fig. 9 3400, 3050, 2970, 1800, 1780, 1740, 1600, 1500, 1460, 1305, 1270, 1190, 1110, 1060, 980, 920, 880, 850, 750, 710, 650, 620, 605 cm 1 . .

..

8) l~-NMR spectrum: 90 MHz, in CDC13 ~ppm ~ (Hz), 2.2-3.5(4H,m), 3.7-4.0(2H,m), 4.4-4.6(lH,m), 6.97(lH,s), 7.2-7.4(lOH,m) 9) HPLC:
Model 6000A/660/440 (Waters Assoc., U.S.A.~
Column, YMC-Pack A-312 (Yamamura Chemical Laboratories, Japan) Mobile phase, 65~ methanol/O.OlM phosphate bu~fer (pH 6.3) 2 ml/min, Rt: 5.3, 5.6 min 10) Color reaction:
Positive: Ninhydrin Negative: Ferric chloride 11) Acidic, neutral or basic: basic substance The physico-chemical properties o~ N-deacetyl TAN-588 (a mixture o~ A type and B type compounds) obtained in Example 7 which appears hereinafter are shown below.
1) Appearance: white powder 2) Specific rotation: ~]25-lli5 ~C-O.l, in water) 3) Molecular weight: m/z 231 (M~H)~ (FD-MS method) 4) Elemental analysis:
Found Calcd~*
C,40.42 C,40.17 H,4.36 H, 4.64 N,11.65 N~11.71 0.43.48 35 (*The value is calculated as the sample contains 0.5 moles of water) 5) Molecular formula: C8HloN2O6(0.5 H2O) 6) UV spectrum: in water max 221-2 nm (Elcm= 154+20) 7) IR spectrum: KBr method, Fig. 10 Principal absorption 3450, 3220, 2960, 2900, 1800, 1760, 1740, 1670, 1580, 1420, 1390, 1370, 1310, 1250, 1200, 1120, 1050, 1030, 980, 950, 920, 810, 770, 720, 690, 610, 540 cm 1 8~ H-NMR spectrum: 400 MH2, in D2O
The following signals are observed. ~ppm J (Hz) 2.52(lH,m), 2.72(lH,m), 2.91(lH,m), 3.08(lH,m), 4.35(lH,m), 4.56(lH,m), 4.80(lH,m) 9) CD spectrum: in water The negative eotton e~fect is revealed at 233+3 nm.

10) Solubility:
Soluble: water Sparingly soluble: dimethylsul~oxide, ethyl acetate, diethylethe~

11) HPLC: machine, column and flow rate are the same conditions as those o~ deacetylated benzhydryl ester (a mixture of ~ type and B type compounds) Mobile phase, 0.01 M phosphate bufer ~pH 6.3) R~: 3.1 and 3.3 min 12) Color reaction:
Positive: Ninhydrin, iodine Negati~e: E'erric chloride , :
' ~ :

.~

~:3 51~

13) Acidic, neutral or basic: amphoteric substance The physico-chemical properties of N-deacetyl TAN-588 (A type compound) obtained in Example 8 which appears hereinafter are shown below.

1) Appearance: colorless crystal 2) Melting point: 177-181C (Decomposition) 3) Speci~ic rotation: [a]D +124+20 (C=0.1 in water) 4) Molecular weight: m/z 231 (M+H)~ (FD-MS method) 5) Elemental analysis:
Found Calcd.
C, 41.57 C, 41.75 H, 4.39 H, 4.38 N, 12.11 N, 12.17 O, 41.71 6) Molecular ~ormula: C8HloN2O6 7) UV spectrum:in water ~max 221+2 nm (ElCm = 151~20) 8) IR spectrum: KBr method, Fig. 11 Principal absorption 3450, 3220, 2950, 2900, 1800, 1735, 1660, 1580, 1440, 1~20, 1400, 1360, 13~0, 1310, 1280, 1200, 1160, 1110, 1050, 1025, 980, 940, 920, 810, 700, 710 690, 600, 540 cm 1 .

~L2~5~

9) lH-NMR spectrum: 400 MHz in D2O, The following signals are observed. ~ ppm J (Hz) 2.52(lH,m), 2.72(lH,m), 2.91(lH,m), 3.08(lH,m), 4.34(1H,m), 4.55(1H,m), 4.78(lH,m) 10) CD spectrum:in water The negative Cotton effect is revealed at 238+3 nm 11) Solubility Soluble in: Water Sparingly soluble in: dimethylsuloxide, ethyl acetate, chloroform, diethyl ether 12) HPLC: the same conditions as those of the mixture of A type and B type compounds Rt, 3.3 min.

13) Acidic, neutral or basic: Amphoteric substance The physico-chemical properties of N-deacetyl TAN-588 (B type compound) obtained in Example 9 which appears hereinafter are shown below.

1) Appearance: white powder 2) Molecular: m/z 231 (M-~I) (FD-MS method) 3) Elemental analy~is Found Calcd. *
C, 40.98 C, 40.17 H, 4.88 H, 4.64 N, 12.17 N, 11.71 O, 43.48 (*The value is calculated as the sample contains 0.5 mole of water) ~;~3~

4~ Molecular formula: C8HlON2O6(0.5 ~I2O) 5) UV spectrum: in water ~max 221~2 nm (ElCm= 133~20) 6) IR spectrum: KBr method, Fig. 12 Principal absorption 3~40, 2980, 1800, 1760, 1670, 1570, 1520, 1390, 1290, 1250, 1190, 1090, 1050, 990, 920, 810, 760, 720, 690 cm 1 7) H-NMR spectrum: 400 MHz in D2O
The following signals are observed. ~ ppm J(Hz) 2.52(lH,m), 2.72(lH,m), 2.90(lH"m), 3.08(lH,m), 4.44(lH,m), 4.68(lH,m), 4.86(lH,m) 8) CD spectrum: in water The negative Cotton effect is revealed at 224~2nm 9) Solubility Soluble in: Water Sparingly soluble in: dimethylsulfoxide, ethyl acetate, chloroform, diethyl ether 10) HPLC: The same conditions as those of the mixture of A type and B type compounds Rt, 3.1 min 11) Acidic, neutral or basic: Amphoteric substance Based on said physico-chemical properties and reaction process, it is assumed that TAN-588 has an acetyl group which is bound to a nitrogen atom in its molecule and has a carboxyl group in its molecule.

....

, . ", ~ . . . .

_39- ~3~5~
Thenr the biological characteristics of TAN-588 be described. The TAN-588 sodium salt exhibits the anti-microbial spectrum against various microorganisms as shown in Table 1.
Table 1:
. Minimal inhibitory Test m1croorganlsm concentrationt~g/mQ)(Note 1) Staphylococcus aureus FDA 209P 3~13 Micrococcus luteus IFO 12708 0.39 Bacillus subtilis NIHJ PCI 219 3.13 Bacillus cereus FDA 5 12.5 Escherichia coli ~IHJ JC 2 50 Salmonella typhimurium IFO 12529 50 Citrobacter freundii IFO 12681 100 Klebsiella pneumoniae IFO 3317 100 Serratia marcescens IFO 12648 50 Proteus mirabilis ATCC 21100 25 Proteus vulqaris IFO 3938 25 Proteus morqanii IFO 3168 100 Pseudomonas aeruqinosa IFO 3080 ~100 ,Alcaliqenes faecalis IFO 13111 , 50 Acinetobacter calcoaceticus IFO 13006 25 (Note 1): As the culture rnedium, there was used a medium consisting of 17.5 g of Bacto Antibiotic Medium 3 (produced by Difco Laboratories of U.S.A.), 5.0 g of Bacto Yeast extract (produced by Difco Laboratories of U.S.A.), 2.0 g of Bacto Agar (produced by Difco Laboratories of U.S.A.) and 1000 mQ of distilled water (without adjustment of p~), while as the bacterial inoculation ~olution, there was used about 106 colony forming units/m~.
The TAN-5~8 sodium salt demonstrated the therapeutic effect in the experimental mouse infection as'shown in Table 2.
Table 2:
_ _ Infectious microorganism administration l, 50 ( g/ g) _ _ ~taphylococcus ,aureus 308A-1 Subcutaneous 2S.0 Furthermore, the TAN-588 sodium salt, even when administered subcutaneously at a dose of 400 mg/kg, was ' ' found to produce no acute toxicity.

,. ~

3~

Described below are the biological characteristics of N-deacetyl TAN-588 (a mixture of the A type and B type compounds). I-t is added that a mixture of the said A type and B type compounds is identical in biological characteristics to the A type and B type compounds.
The antimicrobial spectrum of N-deacetyl TAN-588 against various microorganisms is as shown in Table 3 Table 3:
. .Minimal inhibitory Test mlcroorganlsm concentration (~g/mQ)(2~ote 1) Staphylococcus aureus FDA 209P 50 Micrococcus luteus IFO 12708 6.25 Bacillus subtilis NIHJ PCI 219 12.5 Bacillus, cereus, FDA 5 50 15 Escherichia coli NIHJ JC2 , 25 Salmonella ~Ye~y~ IFO 12529 50 Citrobacter freundiiIFO 12681 50 Klebsiella pneumoniae IFO 3317 100 Serratia marcescens IFO 12648 25 20 Proteus mirab'lis ATCC 21100 100 Proteus vulgalis IFO 3988 100 Proteus morqanii IFO 3i68 ~100 Pseudomonas aeruqinosa IFO 308050 Alc~a]iqenes faecalis IFO 13111100 25 Acinetobact ~ calcoa~Q~ IFO 13006 50 (~ote 1): As the culture medium, there was used a medium consisting of 17.5 g of Bacto Antibiotic Medium 3 (produced by Difco Laboratories, U.S.A.), 50 g of Bacto ~reast extract (produced by Difco Laboratories, U.S.A.), 20 g of Bacto Agar (produced by Dico Laboratories, U.S.A.) and 1000 m~ o di.stilled water (without adjustment of pEI), while as the bacterial inoculation solution, there was employed about 106 colony forming unit/m~.
Also, N-deacetyl TAN-588 is stable to various ~-lactamases; it was examined for the stabilities against 2 ',~, kinds of ~-lactamases, with the use of Escherichia coli PG 8 as a kest microorganism, and the results are shown in Table 4.

Table 4:
Deacetylated ~-Lactamase TAN-588 PCG CPC CMC
Without addition 22.5 22 33 34 Penicillinase*l 24.5 _*3 32 34 Cephalosporinase*2 21.5 _ _ _ The values each denotes the diameter of the resulting inhibition zone. Culture medium: Nutrient agar medium (p~ 7.0) containin~dl~aminopimeric acid (20 ~g~Q)PCG: ~enzyl penicillin.
CPC: Cephalopsorin C. CMC: Cephamycin C. The concentrations of the drugs were lO00 ~g/mQ for deacetylated TAN-588 and lO0 ~g/mQ for other drugs.
*l: Deri~red from Bacillus cereus, produced by Calbio Chemical Co. of U.S.A.). *2: Derived from Enterobacter cloacae. *3: No inhibition zone indicated.
As described in the above, TAN-588, its p-nitrobenzyl or benzhydryl-ester derivative or their deacetylated derivatives [hereinater referred to col~lectively as ~'@ompound 1~ t or salts thereof exhibit antimicrobial activities against Gram-positive and Gram-negative microorganisms, and are low in toxicity. Consequently, the Compounds (I) Oe the presentinvention or their saltscanke used for the treatment of bacterial infections in mammals ~e.g., rats, mice, dogs, cats, domestic animals (horses, etc.), human, etc.], fowls, etc.
In using the Compounds (I) oE the present in~ention or their salts as a therapeutic agent for microbial infections, they can be administered, as admixture with pharmacologically acceptable carriers, excipients, diluents, etc., orally as tablets, capsules, etc. or parenterally as injectable solutions, etc. Examples Oe the diluent which is usable in formulating into injectable solutions include isotonic saline solution, etc.
Examples o the carrier which is useful in formulating into capsules include for example lactose, etc. Their dosage leuels are about 5 to 50 mg/k~/day as the Compound (I), preferabIy about lO to 25 mg/kg/day, for the preparations for oral administration, and about 2.5 to 25 mg/kg/day as ~3~4 the Compound (I), preferably about 5 to 20 mg/kg/day, for the preparations for parenteral administration.
Also, the Compounds (I) as obtained according to the present invention or their salts can be employed as an antimicrobial agent and disinfectant~ They can be utilized, for example, as a solution preparation formulated by dissolving in distilled water to a concentration of 0.01 to O.1 W/V % as the Compound (I~ or as an ointment containing 0.2 to 20 mg as the Compound (I) per g, preferably 1 to 10 mg, formulated with white petrolatum or lanolin used as a base, for the sterilization of disinfection of hands, feet, eyes, ears, etc. of man and animals.
The Compounds (I) as obtained by the method according to the present invention are highly promising compounds as an intermediate for tlle synthesis of new drugs.
The above-described physico-chemical properties and biological characteristics llas led the present inventors to the conclusion that the Compounds (I) are novel antibiotics~
Brief Description of the Drawing:
Fig. 1 5hows the absorption spectrum in the ultraviolet region of TAN-588 (an equilibrium mixture of A and B), Figs.
2, 3, ~, 5, 6, 7, 8, 9, 10, 11 and 12 show the absorption spectra in the Infrared region of TAN-588 (an equilibrium mixture of A and B), p-nitrobenzyl ester of TAN-588 (a mi-~ture of A and B), p-nitrobenzyl ester (A type~ of TAN-588, p-nitrobenzyl ester (B ~ype) of TAN-588, benzhydryl ester (a mixture of A and B) of TAN-588, benzhydryl ester (A type) of TAN-588, benzhydryl ester (~ type) of TAN-588, benzhydryl ester (a mixture of A and B) of N-deacetyl TAN-588, benz-hydryl ester (A type) of N-deacetyl TAN-588, benzhydryl ester (B ~ypel of N-deacetyl TAN-588, N-deacetyl TAN-588 (a mixture of A and B), N-deacetyl TAN-588 (A type~ and N-deacetyl TAN-588 (B type~, respectively.

~l23~

The examples and reference axample are described in the following to illustrate the contents of the present invention in more particular, but it is to be understood that the present inv~ntion should not be limited by these. The term "percent" in the culture medlum, unless otherwise specified, denotes "weight/volume%'l.

Example l The strain Empedobacter lactam~enus YK-258 (IFO 143~2, FERM BP-699) grown on a nutrient agar slant was inoculated into a Sakaguchi ~las]~ of a 2-Q capacity charged with 500 mQ of a culture medium which comprises an a~ueous solution (pH 7.0) containing 2 % of glucose, 3 ~ of soluble starch, l % o~ raw soybean flour, 0.5 % of Polypepton*
(produced by Daigo Nutritive Chemicals, Japan) and 0.3 % of sodium chloride being supplemented with 0.5 %
of precipitating calcium carbonate, followed by incubation on a reciprocating shaker at 2~C for 48 hours. The whole volume of the resulting culture broth was inoculated into a a tank of a 50-Q capacity charged with 30 Q of a culture medium consisting of the above culture medium being supplemented with 0.05 ~ of Actcol (produced by Takeda Chemical Ind~stries, Ltd., Japan), an antifoam, followed by incubation at 24C under the conditions of aeration at a rate of 50 Q/min~ and agitation at a rate of 200 r.p.m. for 48 hours. 6 Q of the resulting culture broth was inoculated into a tank of a 200-Q capacity charged with 120 Q of a culture medium which comp~ises an aqueous solution (pX 6.5) containing3 % of dextrin, 1.5 % of raw soybean flour, 1.5 % of corn gluten meal, 0.2 % of Poly-pepton and 0.1 ~ of sodium thiosulfatebeing supplemented with 0.05 % of Actcol, followed by incubation at 17C under the conditions of aeration at a rate of 200 Q/min and agitation at a rate of 150 r.p.m. for 66 hours.
After repeating the said cultivation procedure twice, the culture broth (230 Q) was adjusted to pH 8, and filtered by theuse of 9 kg of Hyflo Super Cel* (produced hy Johns Manville Co., U.S.A.). The filtrate (200 Q) was adjusted to pH 6 and chromatographed on a column of Amberlite IRA-402 (CQ type, lO Q, produced by Rohm ~ Haas Co., U.S.A.J. The antibiotic was eluted with 2 % aqueous sodium chloride solution, and the eluate (53 Q~ was then adjusted to pH 6 and chromatographed on a column of activated carbon (5 Q, produced by Takeda Chemical Industries, Ltd., Japan). The antibiotic was eluted *Trademarks ~3~q~
-~5-with 8 % isobutanol, and the eluate (14 ~) was concentrated to 5 Q under reduced pressure. The concentrate was ad~usted to pH 6, and extracted with 2 ~ tri-n-octylmethylammonium chloride/methylene chloride solution (2.5 Q x 2)~ The ~xtract was treated with 1.6 % aqueous sodium iodide solution (2.5 Q) to conduct phase-transfer of the antibiotic into the watex layer. The water layer was concentrated, and the concentrate was chromatographed on a colurLm of activated carbon (500 m~, followed by eltuion with 8 % isobutanol. The elute was concentrated, and the concentrate was lyophilized to give 1.41 g of a crude powder. The crude powder (14 g) was dissolved in water (100 mQ), and the solution was chromatographed on a column o-f 200 mQ of QAE-Sephadex A-25*(Cl type, produced by Pharmacia Co., Sweden), followed by elu~ion for fractionation with 0.03M aqueous sodiumlchloride solution. The fractions were collected (600 mQ), adjusted to pH 5.1 and desalted b~
chromatography on activated carbon. Lhe eluate was concentrated and the concentrate was lyophilized to give a powder (384 mg~.
The powder was dissolved in water, and the solution was subjected to the preparative HPLC
with use as a support of YMC-Pack SH-343*(produced by Yamamura Chemical Laboratories, Japan), followed by elution with O.OlM phosphate buffer ~p~ 6~3~. The eluates containing the antibiotic were collected and desalted by chromatography on acti~ated carbon, and the eluate was concentrated and lyophilized to give a white powder 1141 mg) of the TAN-588 sodium salt.
Example 2 The strain Empedobacter lactamqenus YK-258 (IFO 14322, FERM BP-~99) grown on a nutrient agar medium was inoculated into two Sakaguchi flasks oE a 2 Q capacity charged with 500 mQ of a culture medium which comprises an aqueous solution (p~I 7.0) containing 2 % of glucose, 3 ~
of soluble starch, 1 % of raw soybean flour, 0~5 % of Poly-pepton* and 0.3 ~ of sodium chloride being supplemented with 0.5 ~ of precipitating calcium carbonate, followed by incubation on a reciprocating shaker at 24C for 48 hours.
*Trademarks ~;~3~S~

The whole volume of the resul~ing culture broths was inoculated into a tank of a 200-Q capacity charged with 120 Q of a culture medium consisting of the above culture medium being supplemented with 0.05 ~ of Actcol, an atifoam, followed by incubation at 24C under the conditions of aeration at a rate of 200 Q/min and agitation at a rate of 150 r;p.m. for 48 hours. 60 Q of the resulting culture broth was inoculated into a tank of a 2000-Q capacity charged with 1200 Q of a culture medium which consisted of an aqueous solution (pH 6.5 containing 3 % of dextrin, 1.5 % of raw soybean flour, 1.5 %
of corn gluten meal, 0.2 % of Pol~pepton* and 0.1 % of sodium thiosulfate being supplemented with 0.05 % of Ac-tcol, followed by incubation at 17C under the conditions of aeration at a rate of 2000 Q/min and agitation at a rate of 120 r.p.m. for 90 hours.
The resulting culture broth was filtered by use of Hyflo Super Ce~.
The filtrate (1150 Q) was chromatographed on a column of 40Q of Amberlite IRA-402*(CQ type). The antibiotic was eluted with 2 % aqueous sodium chloride solution (200 Q), and the eluate was chromatographed on a column of activated carbon (20 Q).
The eluate of 8 % isobutanol solution (81 Q) was chromatographed on a column of 10 Q of Amberlite IR~-68*(CQ type), followed by eltuion with 1 % aqueous sodium chloride solution. The eluate (54 Q) was again chromatographed on a column o activated carbon (10 Q), and the antibiotic was eluted with 8 % aqueous isobutanol. The eluate (~0 Q) was concentrated under reduced pressure, and the concentrate (5 Q~ was adjusted to pH 4.5 and extracted with 2 ~ tri-n-octylmethylammonium chloride/
methylene chloride solution (2.5 Q x 2~. The extract was treated with 1.6 % aqueous sodium iodide solution to conduct phase-transfer of the antibiotic into the water layer, and the water layer was concentrated~ The concentrate (1.5 Q) was subjected to a desalting procedure by means of chromatography on activated carbon (0.5 Q~, and the eluate was concentrated.
The concentrate was chromatographed on a column of 200 mQ of *Trademarks -47^

QAE-Sephadex (CQ type), followed by elution for fractionation with 0.03M aqueous sodium chloride solution to give an active fraction (1.3 Q). The active fraction was subjected to a desalting procedure by means of chromatography on activated carbon (500 mQ), and the eluate was concentrated, followed by lyophilization to give a white powder (3.56 g) of TAN-588.
Since the extraction waste water layer was found to contain about 50 % of the antibiotic remained, the water layer (5 Q) was chromatographed on a column of 1 Q
of QAE-Sephadex*(CQ type). The antibiotic was eluted with 0.03M and 0.05M aqueous sodium chloride solutions, and the eluate was chromatographed on a column of activated carbon (2 Q). The sodium-chloride removal solution (2 Q~ was again extracted (1 Q x 2) with 2 % tri--n-octylmethylammonium chloride/
methylene chloride solution. Ihe extract was treated wi-th aqueous sodium iodide solution, followed by subjecting to adesalting step with activated carbon to give a white powder (3.18 g) of the TAN-588 sodium salt.

*Trad~marks .

5~

Example 3 The strain Lysobacter albus sp~ nov. YK-422 (IFO
14384, FERM BP-698) grown on a nutrient agar was inoculated into a Sakaguchi flask of a 2-Q capacity charged with 500 m~
of a culture medium (without pH adjustment) containing 2 %
of glucose, 3 % of soluble starch, 1 % of raw soybean flour and 0.5 ~ of Polypepton, followed by incubation on a reciprocating shaker at 24C for 48 hours. The whole volume of the resulting culture broth was inoculated into a tank of a 200-Q capacity charged with 120 Qof a culture medium which comprises the above culture medium being supplemented with 0.0~ ~ of ~ctcol, an antifoam, followed by incubation at 28C under the conditions of aeration at a rate of 120 Q/min and agitation at a rate o~ 180 r.p.m. for 48 hours~
120 Q of the resulting culture broth was inoculated into a tank o~ a 6000-Q capacity charged with 4000 Q of a~culture medium which comprises an aqueous solution (without p~
adjustment) containing 3 % of dextrin, 3 c~ of raw soybean flour and 0.2 % of Polypeptonk being supplemented with 0.05 %
of Actcol, followed by incubation at 22C under the conditions aeration at a-rate of 4000 Q/min and agitation at a rate of 120 r.pm. for 66 hours.
The thus-obtained culture broth was filtered by use of Hylo Super Cel-The ~iltrate (4360 Q~ was chromatographed on a column of400 Q of Amberlite IR~-402*(C]. type). The antibiotic was eluted with 2 % aqueous sodium chloride solution (2000 Q~, and the eluate was chromatographed on a column of activated carbon (160 Q)~ The 8 ~ isobutanol solution eluate (640 Q) was chormatographed on a column o 40 Q of Amberlite IR~-68 (Cl type), ~ollowed by elution with 1 % aqueous sodium chloride solution. The eluate (200 Q~ was again chromatographed on a column of activated carbon (80 Q), and the antibiotic was eluted with 8 % aqueous isobutanol solution. The eluate (400 Q) was cQncentrated under reduced pressure, and the concentrate was lyophilized. The lyophilized product was *Trademarks treated with acetone to yield the sodium salt (620 g) of TAN-588 as a precipitate. The HPLC analysis showed that the powder contained 57 % of the sodium salt of TAN-588.
The powder (5 g) thus obtained was dissolved in water, and the solution was chromatograpned on a column of 200 mQ
of QAE-Sephade~*(Cl type), followed by elution for fractionation with 0.03;1 aqueous sodium chloride solution to give the active fraction (1.2 Q). The active fraction was sub~ected to a desalting procedure by means of chromatography on activated carbon (500 Q), and the eluate was concentrated, followed by lyophilization of the concentrate to give a white powder (2.50 g~ of TAN-588.
The Rf value of TLC, Rt value of ~P~C and IR, UV, CD
and N~R spectra as well as antimicrobia] spectrum indicated that the purified powder of TAN-588 is identical to the sodium salt of TA~-588 as obtained in the above Example 1.
Example 4 The sodium salt (~00 mg) of TAN-588 was dissolved in D~F (4 mQ), and triethylamine (100 ~Q) and p-nitrobenzyl bromide (800 mg) were added to the solution, followed by stirring at room tem~erature for 3 hours~ O.OlM phosphate buffer (p~ 6.3, 50 m~) was added to the reaction solution, and the mixture was extracted with two portions of ethyl acetate (50 mQ). The extract~was washed with water and concentrated, and the resulting oily material was converted into a powder (507 mg) with ethyl acetate-petroleum ether to give a mixture of TAN-588-A-p-nit~obenzyl ester and TAN-588-B-p-nitrobenzyl ester~ The resulting powder was chromatographed on a column of Sephadex L~-20k, with ethyl acetate:methanol = 19:1 used as a mobile phase to give TAN-588-A-p-nitrobenzyl ester (105 m~)TAN-588-B-p-nitrobenzyl ester (67 mg~ and a mixture ~280 mg ) of both compounds.
Example 5 In 500 m~ of CH2C12 were dissolved 58.8 g of benzo-phenone hydrazone, 42 mQ of 1,1,3,3-tetramethylguanidine and 150 mg of iodine, and after the mixed solution was cooled to *Trad~mark ' ~23~

0C to -5C, 74 g of m-cllloroperbenzoic aci~ (with a purity oE 70 ~) was added, follot7ed by stirring at 0C for 40 minutes. The reaction solution was washed with water and dehydrated over sodium sulfate, and the solvent was distilled off to give diphen~ldiazomethane.
31 g of TAN-588 was suspended in THF, and a solution of the whole diphenyldiazomethane as obtained in the above in 150 mQ of T~F was added to the suspension. After the mixed solution was cooled to 0C, 60 mQ of 2N E~Cl was added dropwise to the solution, followed by stirring at room temperature for 1 hour. 10 mQ of 2N HCl was added to the reaction solution, and stirring was effected for another 1 hour, followed by addition of 3 Q of CH2C12. The resulting solution was washed with water and concentrated, and ether was added to the residue to give 28 g of a white crystalline powder of TAN-58~ benz-hydryl ester (a mixture of the A *~e and B type).
The above mixture (1.8 g) was chromatographed on a column of silica gel (180 mQ), and elution was carried out witn a solvent system of chloroform:methanol (97:3), whereby the B type compound was first eluted,~with the A type compound being then eluted. ~ach of the fractions was concentrated to gi~e the A type compound (433 mg) and B type compound (400 mg) of TAN-588 benzhydryl ester and a mixture (476 mg) of the A type and B type compounds in the forms ofcolorless crystals.
Example 6 In 1.2 Q of CH2C12 was suspended 26 g (59 mmole~ of I'AN-588 benzhydryl ester (a mixture of the A type and B type) and the suspension was cooled to -20C. 49 mQ of pyridine and 37.6 g of phosphorus pentachloride were added to the suspension, followed by stirring at -10 to -15C for 50 minutes. After the temperature was lowered to -30C, 180 mQ
of MeO~ was added to the mixture, followed by stirring at -5 to -15C for 30 minutes and at room temperature for 1 hour. After the addition of 300 mQ of lN HCl, stirring was efected at xoom temperature for 45 minutes, and 100 mQ of ~3~

of 50 % sodium phosphate and 2N NaO~I (ca. 5Q0 mQ) were added to the reaction solution to adjust the pH of the aqueous layer to 8~0. The mixed solution was separated into the C~2C12 layer and the aqueous layer, and the aqueous layer was ~urther extracted with CH2C12 (600 mQ). The CH2C12 layers were combined and concentrated, and ether was added to the residue to give 17.9 g of a powder of benzhydryl ester (a mixture of the ~ t~pe and B ~pe) of N-deacetyl TAN-588.
Example 7 In 10 mQ of C~2C12 was suspended 396 mg of benzhydryl ester (a mixture of the A type and B type~ of N-deacetyl TAN-588, and the suspension was cooled to -20C, followed by addition of 434 ~ of anisole and 924 ~Q of trifluoroacetic acid and stirring at -20 to -10C for 40 minutes. 280 mQ of CH2C12 was added to reaction solution, followed by extraction with 0.1M H3PO4-Na2HPO4 solution (pE 7.3) (420 mQ)~ The extract was adjusted to pH 5.5, and concentrated, and the concentrate was passed through a column of packed with Diaion ~P-20*(50 to 100 mesh, 100 mQ), followed by washing with water and elution for fractionation with 40 ~ aqueous ~eOH. The fractions exhibiting antimicrobial activity were collected and concentrated, and the concentrate was lyophilized to give 143 mg of a white powder of N-deacetyl TAN-588 (a mixture of the A type and B type~
Example 8 In water was dissolved 100 mg of a white powder of N-deacetyl TAN-588 ( a mixture of the A type and B type), and colorless crystals separated out, upon standing at 7C
overnight. The crystals which separated out were recovered by filtration to give ~0 mg of N-deacetyl TAN-588 (the A type).
Example 9 A 657 mg of TAN-588 benzhydryl ester (B
type) was subjected to the same reaction and treatment as described in Example 6 to give 200 mg of benzhydryl ester (B type~ of N-deacetyl TAN-588. 180 mg of the said compound *Trademark was dissolved in 18 mQ of TE~F:water (1:1), and 90 mg of 10 % palladium~carbon was added to the solution, followed by stirring under a stream of hydrogen. After the reaction solution was filtered, the filtrate was concentrated, and the aqueous layer was washed Witil diethyl ether, concentrated and lyophilized to give 77 mg of a powder of N-deacet~l TAN-588 (B type).

E~ample 10 The strain Empedobacter lactamgenus YX-258 (IFO 14322, . _ FE~ BP-699) grown on a nutrient agar slant was inoculated into a Sakaguchi flask of a 2-Q capacity charged with 500 ml of a culture medium which comprises an aqueous solution (p~l 7.0) containing 2% of glucose, 3% of soluble sta~ch, 1% of raw soybean flour, 0.5% of Polypepton*and 0.3% of sodium chloride being supplemented with 0.5% of precipitat-ing calcium carbonate, followed by incubation on a recipro-cating shaker at 24C for 48 hours. The whole volume ofthe resulting culture broth was inoculated into a tank of a 50-Q capacity charged with 30 Q of a culture medium comprising the above culture medium being supplemented with 0.05% of Actcol, an antifoam, followed by incubation at 24C under the conditions of aeration at a rate of 50 Q/min. and agitation at a rate of 200 r.p.m. for 48 hours. 6 Q of the resulting culture broth was inoculated into a tank of a 200-Q capacity charged with 120 Q of a culture medium which comprises an aqueous solution (pH 6.5) containing 3% of dextrin, 1.5~ of raw soybean flour, 1.5%
of corn yluten meal, 0.2% of Polypepton*and 0.1% of sodium thiosulfate being supplemented with 0.05~ o~ Actcol, followed by incubation at 17C under the conditions of aeration at rate of 200 ~min and agitation at a rate of 150 r.p.m. for 24 hours.
It was detected by means of TLC-bioautography method using Pseudomonas aeruginosa C-141 that N-deacetyl TAN-588 (a mixture of the A type and B type) was contained in the cultured broth.
~ e~
The strain Lysobacter albus sp. nov. YK-422 (IFO 14384, FERM BP-698) grown on a nutrient agar slant was inoculated into a Sakaguchi Elask of a 2-~ capacity charged with 500 ml of a culture medium which comprises an aqueous solution (pH 7.0) containing 2% of glucose, 3% of soluble starch, 1% of raw soybean flour, 0.5% of Polypepton*and 0.3% of *Trademarks ~;~3~g~

sodium chloride being supplemented with 0.5% of precipitat-ing calcium carbonate, followed by incubation on a recipro-cating shaker at 24C for 48 hours. The whole volume of the resulting culture broth was inoculated into a tank of a 50-Q capacity charged with 30 ~ of a culture medium comprising the above culture medium being supplemented with 0.05% of Actcol, an antifoam, followed by incubation at 24C under the conditions of aeration at a rate of 50 Q/
min. and agitation at a rate of 200 r~p.m. for 48 hours.
6 Q of the resulting culture broth was inoculated into a tank of a 200-~ capacity charged with 120 Q of a culture medium which comprises an aqueous solution (pH 6.5) contain-ing 3% of dextrin, 1.5~ of raw soybean flour, 1.5% of corn gluten meal, 0.2% of Polypepton*and 0.1% of sodium thio-sulfate being supplemented with 0.05~ of Actcolk, followedby incubation at 17C under the conditions of aeration at a rate of 200 ~/min and agitation at a rate of 150 r.p.m. for 24 hours.
It was detected by means of TLC-bioautography method using Pseudomonas aeruginosa C-141 that N-deacetyl TAN-588 (a mixture of the A type and B type) was contained in the culture broth.
Example 12 The strain Empedobacter lactamagenus YK-258 (IFO 14322, FERM BP-699) grown on a nutrient agar slant was inoculated into a Sakaguchi 1ask o~ a 2-Q capacity charged with 500 ml of a culture medium which comprises an aqueous solution (pH 7.0) containing 2% of glucose, 3~ o~ soluble starch, 1% of raw soybean flour, 0.5% oP Polypeptonkand 0.3% of sodium chloride being supplemented with 0.5% o precipitat-ing calcium carbonate, Pollowed by incubation on a recipro-cating shaker at 24C Por 48 hours. The whole volume of the resulting culture broth was inoculated into a tank of a 50-Q capacity charged with 30 Q o a culture medium comprising the above culture medium being supplemented with 0.05~ of Actcol*, an antifoam, Pollowed by incubation *Trademarks ~23~5~

at 24C under the conditions of aeration at a rate of 50 Q/
min. and agitation at a rate of 200 r.p.m. for 48 hours.
The strain Acinetobacter sp. YK-504 (IFO 14420, FERM
BP-709) grown on a nutrient agar slant was inoculated into a Sakaguchi flask of a 2~Q capacity charged with 500 ml of a culture medium which comprises an aqueous solution (pH 7.0) containing 2% of glucose, 3% of soluble starch, 1% of raw soybean flour, 0.5% of Polypepton*and 0.3% of sodium chloride being supplemented with 0.5~ of precipitat-ing calcium carbonate, followed by incubation on a recipro-cating shaker at 24C for 48 hours. The whole volume of the resulting culture broth was inoculated into a tank oi a 50-Q capacity charged with 30 Q of a culture medium compris ing the above culture medium being supplemented with 0.05% of Actcol, an antifoam, followed by incubation at 24C under the conditions of aeration at a rate of 50 Q/
min. and agitation at a rate of 200 r.p.m. for 48 hours.
Three liters of said culture broth of Empedobacter lactamgenus YK-258 and three liters of said culture broth of Acinetobacter sp. YK-504 were inoculated into a tank of a 200-Q capacity charged with 120 Q of a culture medium which comprises an aqueous solution tpH 6.5) containing 3%
of dextrin, 1.5~ of raw soybean flour, 1.5% of corn gluten meal, 0.2% of Polypepton and 0.1% of sodium thiosulfate being supplemented with 0.05~ of Actcol*, followed by incuhation at 24C under the conditions of aeration at a rate of 200 Q/min and agitation at a rate of 150 r.p.m. for 24 hours, and furthermore, the cultivation was continued at 20C for 42 hours.
To the culture broth thus obtained is added Hyflo Super Cel, and filtration was carried out, to give 100 Q
of filtrate. The filtrate was adjusted to pH 5 and chromatographed on a column of Amberlite IRA-402 (Cl kype, lO Q). After washing the column, elution was carried out with 2% NaCl aqueous solution (50 Q). In the eluate, TAN
588 was contained at a concentration of 32 ~g/ml. The *Trademarks effluent containing N-deacetyl TAN-588 was subjected to column chromatography of Dowex 50WX 2*(H type, 10 Q) to adsorb N-deacetyl TAN-588. The eluates (50 Q) with water and 0.2 N aqueous ammonia was concentrated and the concen trate (13 Q) was charged to column chromatography of activated carbon (2 Q) at pH 5.
The eluate (20 Q) of 8% isobutanol was concentrated, and the concentrated solution (1 Q) was subjected again to column chromatography of Dowex 50WX 2 (H type, 50 to 100 mesh, 0.4 Q) with the elution of 0.2 N aqueous ammonia.
The active fractions (1.4 Q) was desalted by activated carbon column chromatography. The eluate was concentrated, and to the concentrate was added acetone, whereby crude powder was obtained.
This crude powder was subjected to the preparative HPLC with the use as a support of YMC-Pack, ODS, SH-343*
[produced by Yamamura Chemical Laboratories, Japan, mobile phase: 0.02 M phosphate buffer (pH 6.3)]. The eluates containing active substance were collected, and the inorganic salt in the eluate was removed by chromatography on activated carbon. The eluates containing the antibiotic were concentrated and lyophili~ed, and to the lyophilizate was added acetone, whereby powders (50 mg) of N-deacetyl T~N-588 was obtained.
This powder was identified with the standard sample of N-deacetyl TAN-588 (a mixture of the A type and B type) by TLC-bioautogram, HPLC-biohistogram, IR and PMR spectra.
E ~
The strain Lysobacter albus sp. nov. YK-422 (IFO
14384, FERM BP-698) grown on a nutrient agar slant was inoculated into a Sakaguchi flask of a 2-~ capacity charged with 500 ml o~ a culture medium which comprises an aqueous solution (pH 7.0) containing 2% of glucose, 3% of soluble starch, I% of raw soybean flour, 0.5% of Polypepton*and 0.3% of sodium chloride being supplemented with 0.5% of precipitating calcium carbonate, followed by incubation on *Trad0marks ~2~

a reciprocating sha~er at 24C for 48 hours. The whole volume of the resulting culture broth was inocula-ted into a tank of a 50-Q capacity charged with 30 Q of a culture medium consisting of the above culture medium being supple-mented with 0.05% of Actcol*, an antiform, followed byincubation at 24C under the conditions of aeration at a rate of 50 Q/min. and agitation at a rate of 200 r.p.m. for 48 hours.
The strain Acinetobacter sp. ~K-504 (IFO 14420, FERM
. .
BP-709) grown on a nutrient agar slant was inoculated into a Sakaguchi flask of a 2-Q capacity charged with 500 ml of a culture medium which comprises an aqueous solution (pH
7.0) containing 2~ of glucose, 3% of soluble starch, 1~ of raw soybean flour, 0.5~ of Polypepton*and 0.3% of sodium chloride being supplemented with 0.5% of precipitating calcium carbonate, followed by incubation on a reciprocating shaker at 24C for 48 hours. The whole volume of the resulting culture broth was inoculated into a tank of a 50-Q
capacity charged with 30 Q of a culture medium comprising the above culture medium being supplemented with 0.05~
of Actcol, an antifoam, followed by incubation at 24C
under the conditions of aeration at a rate of 50 Q/min. and agitation at a rate of 200 r.p.m. for 48 hours.
Three liters of said culture broth of ~ albus sp. nov. YK-422 and three liters o said culture broth of Acinetobacter sp. YK-504 were lnoculated into a tank of a 200-Q capacity charged with 120 Q of a culture medium which comprises an aqueous solution ~pH 6.5) containing 3% of dextrin, 1.5~ o raw soybean 1Our, 1.5% of corn gluten meal, 0.2% of Polypepton* and 0.1% o sodium thiosulfate being supplemented with 0.05% of Actco~, followed by incubation at 24C under the conditions o aeration at a rate of 200 ~/min and agitation at a rate of 150 r.p.m. for 24 hours, and furthermore, the cultivation was continued at 20C for 42 hours.
It was detected by means of TLC-bioautography method *Trademarks using Ps_udomonas aeruginosa C-141 that N-deacetyl TAN-588 (a mixture of the A type and B type) was contained in the culture broth.
Example 14 ._ From the results of the physico-chemical properties, decomposition and spectral studies, the structures of the compounds obtained in the foregoing Examples are elucidated to be as follows:
(1) A type compounds:
H

RlNH_~o O ~ = O
1`5 COOR2 Compound Rl R
Sodium salt of TAN-588-ACH3CO- Na TAN-588-A-p-nitrobenzyl ester CH3CO- -CH2 ~ -No2 /~
. TAN-588-A-benzhydryl esterCH3CO- -CH\ ~
,'~3 Benzhydryl ester of H -CH
N-deacetyl TAN-588-A \

N-deacetyl TAN-588-A H H

(2) B type compounds:
,H
RlNH ~0 N

C oOR2 ..

S~

l 2 Comp~und . R R
Sodium salt of TAN-588-BCH3CO- Na TAN-588-B-p-nitrobenzyl ester CH3CO~ -CH2 ~ -No2 /~
TAN-588-B-benzhydryl ester CH3CO- -CH

Benzhydryl ester of H -CH
N-deacetyl TAN-588-B

N-deacetyl TAN-588-B H

.,

Claims (40)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for producing Antibiotic TAN-588, N-deacetyl Antibiotic TAN-588, p-nitrobenzyl or benzyhydryl ester derivative of Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588 or a pharmaceutically acceptable salt of Anti-biotic TAN-588 or N-deacetyl Antibiotic TAN-588, which is represented by the formula (I) [wherein R1 is H or CH3CO- and R2 is H, or a pharmaceutically acceptable salt-forming cation], which method comprises:
(A) for producing Antibiotic TAN-588 [Formula I, R1 is CH3CO- and R2 is H) and/or N-deacetyl Antibiotic TAN-588 (Formula I, R1 is H and R2 is H), cultivating a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588 in a culture medium to have Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588 elaborated and accumulated in the culture medium, recovering the said antibiotic, (B) for proclucing N-cleacetyl Antibiotic TAN-588, carrying out the mixed cultivation of a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or its N-deacetylated derivative and of a microorganism which belongs to the genus Acinetobacter and is capable of having said microorganism of the genus Empedobacter or Lysobacter produce N-deacetylated Antibiotic TAN-588 in a culture medium to have N-deacetylated Antibiotic TAN-588 elaborated and accumulated in the culture broth, and recovering the said antibiotic, (C) for producing N-deacetyl Antibiotic TAN-588, deacetylating Antibiotic TAN-588 or its salt produced in variant (A), (D) for producing the p-nitrobenzyl or benzhydryl ester derivative of Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588, p-nitrobenzylating or benzhydrylating Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588, or (E) for producing the p-nitrobenzyl or benzhydryl ester derivative of N-deacetyl Antibiotic TAN-588, deacetylating p-nitrobenzyl or benzhydryl ester derivative of Antibiotic TAN-588 produced in variant (D), and if desired, converting Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588 produced by any of the above process variants into a pharmaceutically acceptable salt thereof.

2. Antibiotic TAN-588, N-deacetyl Antibiotic TAN-588, p-nitrobenzyl or benzhydryl ester derivative of Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588 or a pharmaceutically acceptable salt of Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588, as defined in claim 1, whenever prepared or pro-duced by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A method for producing Antibiotic TAN-588 and/or N-deacetyl Anti-biotic TAN-588 or their Pharmaceutically acceptable salt, of the formula wherein R1 is H or CH3CO- and R2 is H or a pharmaceutically acceptable salt-forming cation, which method comprises cultivating a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588 in a culture medium to have Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588 elaborated and accumulated in the culture medium, recovering the said antibiotic, and if desir-ed, converting thus produced compound into a pharmaceutically acceptable salt thereof.

4. Antibiotic TAN-588 and/or N-deacetyl Antibiotic TAN-588, or a pharma-ceutically acceptable salt thereof as defined in claim 3, whenever prepared or produced by the process of claim 3 or by an obvious chemical equivalent thereof.

5. A method as claimed in Claim 3, wherein the microorganism is Empedobacter lactamgenus.

6. A method as claimed in Claim 3, wherein the microorganism is Empedobacter lactamgenus YK-258 (IFO 14322).

7. A method as claimed in Claim 3, wherein the microorganism is Lysobacter albus sp. nov.

8. A method as claimed in Claim 3, wherein the microorganism is Lysobacter albus sp. nov. YK-422 (IFO 14384).

9. A process as claimed in Claim 3, wherein Antibiotic TAN-588 is iso-lated, and if desired, it is converted into a pharmaceutically acceptable salt thereof.

10. A process as claimed in Claim 5 or 7, wherein Antibiotic TAN-588 is isolated, and if desired, it is converted into a pharmaceutically acceptable salt thereof.

11. A process as claimed in Claim 6 or 8, wherein Antibiotic TAN-588 is isolated, and if desired, it is converted into a pharmaceutically acceptable salt thereof.

12. A process as claimed in Claim 3, wherein Antibiotic TAN-588 is isolated and thereafter converted to its sodium salt.

13. A process as claimed in Claim 5 or 7, wherein Antibiotic TAN-588 is isolated and thereafter converted to its sodium salt.

14. A process as claimed in Claim 6 or 8, wherein Antibiotic TAN-588 is isolated and thereafter converted to its sodium salt.

15. A process as claimed in Claim 3, wherein deacetyl Antibiotic TAN-588 is isolated.

16. A process as claimed in Claim 5 or 7, wherein deacetyl Antibiotic TAN-588 is isolated.

17. A process as claimed in Claim 6 or 8, wherein deacetyl Antibiotic TAN-588 is isolated.

18. Sodium salt of Antibiotic TAN-588, whenever prepared or produced by the process of claim 12 or by an obvious chemical equivalent thereof.

19. A method for producing N-deacetyl Antibiotic TAN-588 of the formula:

which method comprises:
carrying out the mixed cultivation of a microorganism which belongs to the genus Empedobacter or the genus Lysobacter and is capable of producing Antibiotic TAN-588 and/or its N-deacetylated derivative and of a microorganism which belongs to the genus Acinetobacter and is capable of having said micro-organism of the genus Empedobacter or Lysobacter produce N-deacetylated Antibiotic TAN-588 in a culture medium to have N-deacetylated Antibiotic TAN-588 elaborated and accumulated in the culture broth, and recovering the said antibiotic.

20. A method as claimed in Claim 19, wherein the microorganism belong-ing to the genus Acinetobacter is Acinetobacter sp. YK-504 (IFO 14420).

21. A method as claimed in Claim 19, wherein the microorganism belong-ing to the genus Empedobacter is Empedobacter lactamgenus.

22. A method as claimed in Claim 19 or 20, wherein the microorganism belonging to Empedobacter is Empedobacter lactamgenus YK-258 (IFO 14322).

23. A method as claimed in Claim 19, wherein the microorganism belong-ing to Lysobacter is Lysobacter albus sp. nov.

24. A method as claimed in Claim 19 or 20, wherein the microorganism belonging to Lysobacter is Lysobacter albus sp. nov. YK-422 (IF0 14384).

25. A method for producing N-deacetylated Antibiotic TAN-588 or its pharmaceutically acceptable salt, of the formula:

wherein R2 is H or a pharmaceutically acceptable salt-forming cation, which method comprises:
deacetylating Antibiotic TAN-588 or its salt, and if desired, converting the deacetyl product into a pharmaceutically acceptable salt thereof.

26. A method as claimed in Claim 25, wherein the deacetylation com-prises (i) first reacting Antibiotic TAN-588 or its salt with a compound cap-able of introducing a benzhydryl group or p-nitrobenzyl group to convert Antibiotic TAN-588 into the benzhydryl ester derivative or the p-nitrobenzyl derivative of Antibiotic TAN-588, (ii) subjecting the benzhydryl ester deriva-tive or the p-nitrobenzyl derivative to deacetylation, and (iii) removing the benzhydryl or p-nitrobenzyl group from thus obtained compound.

27. A method as claimed in Claim 26, wherein diphenyldiazomethane or diphenylmethyl bromide is used as the compound capable of introducing a benz-hydryl group; or p-nitrobenzyl bromide or p-nitrobenzyl chloride is used as the compound capable of introducing a p-nitrobenzyl group.

28. A method as claimed in Claim 26, wherein the deacetylation (ii) com-prises (a) reacting the benzhydryl or p-nitrobenzyl ester derivative of Anti-biotic TAN-588 with a member selected from the group consisting of phosphorus pentachloride, phosgene, phosphorus trichloride and phosphorus oxychloride, to form an imino chloride derivative; adding methanol to the thus-prepared imino chloride derivative to form an imino ether derivative; and adding dilute hydro-chloric acid to the imino ether derivative, (b) treating the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 in an aqueous solution of methanol, ethanol or a mixture thereof, or (c) hydrolyzing the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 using an appropriate enzyme.

29. A method as claimed in Claim 26, wherein the removal of the benz-hydryl or p-nitrobenzyl group (iii) comprises an acid hydrolysis or a cata-lytic reduction.

30. A method as claimed in Claim 25 or 26, wherein the starting Anti-biotic TAN-588 or its salt is produced by the process of claim 9.

31. A method for producing the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588, of the formula:

wherein R1 is H or CH3CO- and R2 is benzhydryl or p-nitrobenzyl, which method comprises:
(i) for producing the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588, benzhydrylating or p-nitrobenzylating Antibiotic TAN-588 or its salt, and (ii) for producing the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588, deacetylating the product of step (i).

32. A method as claimed in Claim 31, wherein diphenyldiazomethane or diphenylmethyl bromide is used for the benzhydrylation, or p-nitrobenzyl chlo-ride or p-nitrobenzyl bromide is used for the p-nitrobenzylation.

33. A method as claimed in claim 31, wherein the deacetylation (ii) comprises (a) reacting the benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 with a member selected from the group consisting of phos-phorus pentachloride phosgene, phosphorus trichloride and phosphorus oxychloride, to form an imino chloride derivative; adding methanol to the thus-prepared imino chloride derivative to form an imino ether derivative; and adding dilute hydrochloric acid to the imino ether derivative, (b) treating the benz-hydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 in an aqueous solution of methanol, ethanol or a mixture thereof, or (c) hydrolyzing the benz-hydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 using an approp-riate enyme.

34. A method as claimed in Claim 31 or 32 wherein the starting Antibiotic TAN-588 is produced by the method of Claim 9.

35. A method for producing the benzhydryl ester derivative of N-de-acetylated Antibiotic TAN-588 which method comprises (i) reacting Antibiotic TAN-588 or its salt with a compound capable of introducing a benzhydryl group to convert Antibiotic TAN-588 into the benzhydryl ester derivative of Anti-biotic TAN-588 and (ii) deacetylating the thus-obtained benzhydryl ester derivative.

36. Benzhydryl ester of N-deacetyl Antibiotic TAN-588, whenever prepared or produced by the process of Claim 35 or by an obvious chemical equivalent thereof.

37. A method as claimed in Claim 35, wherein the starting Antibiotic TAN-588 is produced by the method of Claim 9.

38. A method as claimed in Claim 25, wherein the deacetylation product is not converted to its salt.

39. N-Deacetyl Antibiotic TAN-588 as defined in Claim 19, whenever prepared or procluced by the process of Claim 15, 19 or 38 or by an obvious chemical equivalent thereof.

40. Benzhydryl or p-nitrobenzyl ester derivative of Antibiotic TAN-588 or N-deacetyl Antibiotic TAN-588 as defined in Claim 31, whenever prepared or produced by the process of Claim 31 or by all obvious chemical equivalent thereof.