CA1205405A - Substances potentiating the activity of antibiotics and their production - Google Patents
Substances potentiating the activity of antibiotics and their productionInfo
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- CA1205405A CA1205405A CA000421538A CA421538A CA1205405A CA 1205405 A CA1205405 A CA 1205405A CA 000421538 A CA000421538 A CA 000421538A CA 421538 A CA421538 A CA 421538A CA 1205405 A CA1205405 A CA 1205405A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
Abstract of the Disclosure Novel compound having the formula and salts thereof:
Description
~ubstances Potentiating the Activity of Antibiotics and their Production ~echnical ~ield This invention relates to novel substances which potentiate the activity of antibiotics, salts thereof, and -their production Background Art Until now, the protein SP127 has been known as a substance which potentiates the activity of an-tibiotics (M Kikuchi e-t al.; ~he Journal of Antibiotics ~0, 209-214, 1977), and it potentiates the antibacterial activity of macrolide antibiotics (ibid. ~0, 215-~20, 1977), On the other hand, clavulanic acid is known as a substance which prevents inactivation of ~-lactam an-tibiotics by ~-lactamases and synergistically potentiates i-ts activity agains-t ~-lactamase producing bac-teria ~D. A. ~eigh et al., ~ournal of Antimicrobial Chemotherapy, (1981)7, 229-236)o Disclosure of the Invention The present inventors isolated from soil samples a large number of microorganisms for the purpose of developing new antibiotic substances and searche~ for antibiotics in the metabolites elaborated by these microorganisms. ~his exploration led them to the finding that certain strains among these isola-tes would produce novel substances which potentiate antibacterial act~vity, ',~
~s~os that the strains belong to the genus Pseudomonas, and that the substances capable of potentiating the antibacterial activity of ~-lactam an-tibiotics against gram-negative bacteria can be caused to accumulate in a culture medium by growing such a strain of microorganism therein. ~he present inventors isolated the substances, studied their physical, chemical and biological characteristics and, based on these characteristics, confirmed that these substances which potentiate the antibacterial activity are novel active substances.
~urthermore, it has also been found that the partial hydrolyzates of above substances have the action which potentiates the activity of antibiotics.
It is the principal object of the present invention to provide a compound, inclusive of salts thereof, which has the formula (I):
CH20H . O
R20 ~ ~DO~E~ CEl20E CRl (I) wherein Rl is NHCH2CH2S03H or OH and R2 is S03H or H.
~nother object is to pro~ide a method for producing a compolmd, which has the formula (II):
.
CH 0~
~ _ _ CRl (II) wherein Rl is NHCH2CH2S03H or OH, by means of microbial fermentation using a microorganism which belongs to the genus Psudomonas.
~s~os that the strains belong to the genus Pseudomonas, and that the substances capable of potentiating the antibacterial activity of ~-lactam an-tibiotics against gram-negative bacteria can be caused to accumulate in a culture medium by growing such a strain of microorganism therein. ~he present inventors isolated the substances, studied their physical, chemical and biological characteristics and, based on these characteristics, confirmed that these substances which potentiate the antibacterial activity are novel active substances.
~urthermore, it has also been found that the partial hydrolyzates of above substances have the action which potentiates the activity of antibiotics.
It is the principal object of the present invention to provide a compound, inclusive of salts thereof, which has the formula (I):
CH20H . O
R20 ~ ~DO~E~ CEl20E CRl (I) wherein Rl is NHCH2CH2S03H or OH and R2 is S03H or H.
~nother object is to pro~ide a method for producing a compolmd, which has the formula (II):
.
CH 0~
~ _ _ CRl (II) wherein Rl is NHCH2CH2S03H or OH, by means of microbial fermentation using a microorganism which belongs to the genus Psudomonas.
2~5~0S
Further object is to provide a method for producing a compound, inclusive of sal-ts thereof, which has the formula (III):
I _ o 11 H'~ ~NHCOaH~ ~ ~ ~ CRl (III) wherein Rl is NHCH2CH2S03H or OH, which compris subjecting a compound of the formula (IV), inclusive of salts thereof,:
il o~ 11 ~ O ~ ~ CRl (IV) HO3SO NHCOCH3 C~20 wherein Rl is NHCH2CH2S03H or OH, to desulfation.
It should first be noted tha-t in this specification four compounds represented by the formula (I) will sometimes be referred. to briefly as F2, F3, FL~ or F5 as 25 shown in the following Table.
Abbrebiations of ~ -compounds 1 2 . ~HCH2~H2S03H. 3
Further object is to provide a method for producing a compound, inclusive of sal-ts thereof, which has the formula (III):
I _ o 11 H'~ ~NHCOaH~ ~ ~ ~ CRl (III) wherein Rl is NHCH2CH2S03H or OH, which compris subjecting a compound of the formula (IV), inclusive of salts thereof,:
il o~ 11 ~ O ~ ~ CRl (IV) HO3SO NHCOCH3 C~20 wherein Rl is NHCH2CH2S03H or OH, to desulfation.
It should first be noted tha-t in this specification four compounds represented by the formula (I) will sometimes be referred. to briefly as F2, F3, FL~ or F5 as 25 shown in the following Table.
Abbrebiations of ~ -compounds 1 2 . ~HCH2~H2S03H. 3
3 F3 OH S03H
F~ NHCH2CH2S03H H
.. _ OH _ 35A compound of the formula (II)~ F2 and F3 inclusive, of the present invention may be produced by means of ?54~)S
_ L~ _ microbial ~ermentation. F2- and F3-producing strain may be any species of microorganism which belongs to the genus Pseudomonas and is capable of elaborating a compound of the formula (II). For example, Pseudomonas acidophila G-6302 and Pseudomonas mesoacidophila SB-72310 may be mentioned.
'Ihe strain G-6302 and the strain SB-72310 have been deposited at the Fermentation Research Institu-te, the Agency o~ Industrial Science and '~echnology (FRI, 1-3, Yatabecho higashi l-chome, '~sukuba-gun, Ibaraki, Japan), Institute for Fermentation, Osaka (IFO, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka, Japan) and 'Ihe American '~ype Culture Collection (A'~CC, 12301 Parklawn Drive, Rockville, Mar~land 20852 U.S.A.), respectively, as follows:
_ _ FRI IFO A'rCC
. _ _. ._ . ~
Deposit FERM-P No.4344 IFO 13774 ATCC 31363 '~he strain number G-6302 Date of The 20th day '~he 20th day December 20, deposit of December of December 1977 '~he 52nd year '~he 52nd year of Showa of Showa (1977) (1977) - ...... ___ _ - -- . , .... _ Deposit FERM-P No.4653 IFO 13884 A'ICC 31433 25 ~e strain number _ _ _ __ SB-72310 Date of '~he 13rd day '~he 13rd day September 27, deposi-t of September of September 1978 '~he 53rd year '~he 53rd year of Showa of Showa (1978) (1978) . __ ------- . . ... __ _ _ ..
'~he morphological characteristics of the strain G-6302 are described in Japanese Patent Application Laid-open No. 163501/1979 and U.S. Patent 4,229,436 (Patented on October 21, 1980), and those of the strain SB-72310 in Japanese Patent Application ~aid-open No.49394/1980 and U.S. Patent 4,225,586 (Patented on s~s September 30, 1980).
The strains belonging to the genus Pseudomonas which are employed in accordance with this invention are generally liable to vary their characteristics and behaviors and tend to undergo mutation when exposed to ultraviolet light, X-rays, chemical mutagens and other ar-tificial agents.
However, even such mutants or variants can be employed only if they are able to elaborate a compound of the formula (II) falls within -the range of this invention.
In cultivating the above strains, mutants and variants, there are employed such assimilable carbon sources as glucose, sucrose, maltose, spent molasses, glycerol, oils (e.g. soybean oil, olive oil, etc.), organic acids (e.g. citric acid, succinic acid, gluconic acid, etc.). The nitrogen sources that may be employed include various organic and inorganic nitrogen compounds such as soybean meal, cottonseed meal, corn steep liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, etc. There are also employed such inorganic salts as are commonly used in the cultivation of bacteria, such as sodium chloride, calcium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, disodium phosphate, etc. l-t was found tha-t sulfur compounds which the strains can u-tilize, i.e.
inorganic sulfur compolmds such as sodium sulfate, sodium thiosulfate, etc. and organic sulfur compounds such as " cystine, cysteine, methionine, etc. would lead to an increased yield of a compound of the formula (II).
Particularly preferred are cysteine and sodium thiosulfate The concen-tration of such sulfur compounds is 0.01 to 1.0 w/v % and preferably 0.02 to 0.5 w/v %. The addition of a sulfur compound to the medium results in an increased yield of a compound of the formula (II) and is therefore very advantageous in commercial-scale production.
If necessary, -there are also incorporated in the s~o~
heavy metal compounds such as ferrous sulfate, tin sulfate, etc. and vitamins such as vitamin ~1, biotin, etc. It is also possible or advantageous to add an antifoam or/and a surfactant, such as silicone oil, polyalkylene glycol ether, etc. In addition, organic or/and inorganic materials that will promote production of a compound of the formula (II) may also be incorporated in suitable amounts.
~he cultivation can be carried out either in solid medium or in liquid medium by a conventional antibiotics production method. While the cultivation in liquid medium may be conducted under standing still or with stirring, shaking or aeration, spinner culture under aeration is especially preferred. ~he cultivation is performed preferably at a temperature within the range of about 15C-35C, at a pH of the medium within the range of about
F~ NHCH2CH2S03H H
.. _ OH _ 35A compound of the formula (II)~ F2 and F3 inclusive, of the present invention may be produced by means of ?54~)S
_ L~ _ microbial ~ermentation. F2- and F3-producing strain may be any species of microorganism which belongs to the genus Pseudomonas and is capable of elaborating a compound of the formula (II). For example, Pseudomonas acidophila G-6302 and Pseudomonas mesoacidophila SB-72310 may be mentioned.
'Ihe strain G-6302 and the strain SB-72310 have been deposited at the Fermentation Research Institu-te, the Agency o~ Industrial Science and '~echnology (FRI, 1-3, Yatabecho higashi l-chome, '~sukuba-gun, Ibaraki, Japan), Institute for Fermentation, Osaka (IFO, 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka, Japan) and 'Ihe American '~ype Culture Collection (A'~CC, 12301 Parklawn Drive, Rockville, Mar~land 20852 U.S.A.), respectively, as follows:
_ _ FRI IFO A'rCC
. _ _. ._ . ~
Deposit FERM-P No.4344 IFO 13774 ATCC 31363 '~he strain number G-6302 Date of The 20th day '~he 20th day December 20, deposit of December of December 1977 '~he 52nd year '~he 52nd year of Showa of Showa (1977) (1977) - ...... ___ _ - -- . , .... _ Deposit FERM-P No.4653 IFO 13884 A'ICC 31433 25 ~e strain number _ _ _ __ SB-72310 Date of '~he 13rd day '~he 13rd day September 27, deposi-t of September of September 1978 '~he 53rd year '~he 53rd year of Showa of Showa (1978) (1978) . __ ------- . . ... __ _ _ ..
'~he morphological characteristics of the strain G-6302 are described in Japanese Patent Application Laid-open No. 163501/1979 and U.S. Patent 4,229,436 (Patented on October 21, 1980), and those of the strain SB-72310 in Japanese Patent Application ~aid-open No.49394/1980 and U.S. Patent 4,225,586 (Patented on s~s September 30, 1980).
The strains belonging to the genus Pseudomonas which are employed in accordance with this invention are generally liable to vary their characteristics and behaviors and tend to undergo mutation when exposed to ultraviolet light, X-rays, chemical mutagens and other ar-tificial agents.
However, even such mutants or variants can be employed only if they are able to elaborate a compound of the formula (II) falls within -the range of this invention.
In cultivating the above strains, mutants and variants, there are employed such assimilable carbon sources as glucose, sucrose, maltose, spent molasses, glycerol, oils (e.g. soybean oil, olive oil, etc.), organic acids (e.g. citric acid, succinic acid, gluconic acid, etc.). The nitrogen sources that may be employed include various organic and inorganic nitrogen compounds such as soybean meal, cottonseed meal, corn steep liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, etc. There are also employed such inorganic salts as are commonly used in the cultivation of bacteria, such as sodium chloride, calcium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, disodium phosphate, etc. l-t was found tha-t sulfur compounds which the strains can u-tilize, i.e.
inorganic sulfur compolmds such as sodium sulfate, sodium thiosulfate, etc. and organic sulfur compounds such as " cystine, cysteine, methionine, etc. would lead to an increased yield of a compound of the formula (II).
Particularly preferred are cysteine and sodium thiosulfate The concen-tration of such sulfur compounds is 0.01 to 1.0 w/v % and preferably 0.02 to 0.5 w/v %. The addition of a sulfur compound to the medium results in an increased yield of a compound of the formula (II) and is therefore very advantageous in commercial-scale production.
If necessary, -there are also incorporated in the s~o~
heavy metal compounds such as ferrous sulfate, tin sulfate, etc. and vitamins such as vitamin ~1, biotin, etc. It is also possible or advantageous to add an antifoam or/and a surfactant, such as silicone oil, polyalkylene glycol ether, etc. In addition, organic or/and inorganic materials that will promote production of a compound of the formula (II) may also be incorporated in suitable amounts.
~he cultivation can be carried out either in solid medium or in liquid medium by a conventional antibiotics production method. While the cultivation in liquid medium may be conducted under standing still or with stirring, shaking or aeration, spinner culture under aeration is especially preferred. ~he cultivation is performed preferably at a temperature within the range of about 15C-35C, at a pH of the medium within the range of about
4-8, generally for about 8-168 hours, preferably for 24-144 hours.
~he thus-produced compound of the formula (II), which is present for the most part in the culture broth filtrate, is advantageously isolated and purified from the supernatant following separation of the culture broth into a supernatant and bacterial cells by cen~trifugation or filtration It is also possible to isolate and purify a compound of the formula (II) directly from the cul-ture broth.
~ he potency of compound of the formula (II) can be determined, for instance, by the cup method of paper disc method using bouillon agar containing 0.05 ~g/ml of cefmenoxime with scherichia coli IF0 12734 as the test organism.
~ he compound of the formula (II) can be harvested by any means commonly used for harvesting metabolites produced by microorganisms. ~hus, for example~ bacterial cells are removed by centrifugation and the filtrate is subjected to a common process known for-isolation and purification of effective substances~ ~hus, there are 12~?S~O~
utilized solubility or solubility di~ference in an adequate solvent, difference in mamler or rate of precipitation from a solution, difference in affinity with a variety of adsorbents, ion exchange chromatography : 5 with an ion exchanger, concentration under reduced pressure, lyophili~ation, crystallization, recrystallization, drying and other means, either alone or in combination in an optional order, either singly or repeatedly~
A specific example of recovering the metabolites is given as follows. ~'he culture broth after completion of the cultivation is filtered, the filtrate is passed through an activated carbon column, and the thus-adsorbed compound (II) is eluted with a hydrophilic organic solvent system. ~he hydrophilic organic solvent system is, for example, a mixed solution composed of water and one or more of lower ketones, such as acetone, methyl ethyl ketone and methyl isobutyl ketone, and lower alcohols, such as methanol, ethanol, isopropanol, propanol and butanol. Since the desired compound is an acidic substances, the ion exchange resin is advantageously a Cl-type one, such as an . ~ anion exchange resin (Amberlite~IRA-400, 402, Rohm &
Haas, USA; Dowex-l~ Dow Chemical, U~A; Diaio ~ A-21~, Mitsubishi Chemical, Japan). ~he thus-adsorbed active substance is eluted with an aqueous solution of sodium chloride, for instance. ~or desalting, the eluate is again subjected to activated carbon column chromatography.
~he eluate containing the active substance is concentrated, then acetone or the like. is.added, and the precipitate is collected by filtration, washed with acetone, ether or the like and dried to give a pale brown powder For further purification of the powder, column chromatography with ~4E Sephadex~(Pharmacia, Sweden) is advantageous ~hus, after washing QA~ Sephadex A-25 is washed ~ith phosphate buffer (pH 6.6), an aqueous solution of the above powder is passed through the co:Lumn for adsorption, and the column is washed with M/25 phosphate buffer and r~e ~Q~k.
~ZQ540S
then eluted with the same buffer containing 0.5 w/v % of sodium chloride to obtain F3 active fractions. In the next place, the elution is carried out by using the same buffer containing 1.0 w/v % of sodium chloride to obtaining F2 active fractions.
The thus obtained F3 active fractions are pooled, adjusted to lower than pH 6 and again passed through an activated carbon column. After washing with water, elution is performed with 50 v/v % methanol-water. ~he active fractions are concentrated under reduced pressure, and then F3 is obtained in powdery form by lyophilization.
On -the other hand, F2 active fractions are pooled, adjusted to lower than pH 3 and again passed through an activa-ted carbon column. After washing, elution is performed with aqueous acetone or 7 v/v % isobutanol-water.
~he active fractions are concentrated under reduced pressure. Upon addition of acetone, these is obtained F2.
F2 and F3 can form a metal or ammonium salt, respectively. ~he metal salt is, for example, the sodium, potassium or littium salt. ~hese salts can be used as pharmaceutical salts.
~ he physicochemical properties of ~2 and F3 are as follows:
~ he physicochemical properties of the monosodium salt of F2 as obtained in Example 1 are as follows:
1. Melting point: 216-219C (decomposition) 2. Appearance: Colorless crystalline powder 3. Elemental analysis (for the sample dried over phosphorus pentoxide at 40C under reduced pressure ~or 6 hours):
~ 32.11 (%) H 5.38 N 7.27 S 10.67 ~a 3.8 4. Molecular weight: 605 (calculated as the monosodium "` ~LZ~5~05 salt).
~he thus-produced compound of the formula (II), which is present for the most part in the culture broth filtrate, is advantageously isolated and purified from the supernatant following separation of the culture broth into a supernatant and bacterial cells by cen~trifugation or filtration It is also possible to isolate and purify a compound of the formula (II) directly from the cul-ture broth.
~ he potency of compound of the formula (II) can be determined, for instance, by the cup method of paper disc method using bouillon agar containing 0.05 ~g/ml of cefmenoxime with scherichia coli IF0 12734 as the test organism.
~ he compound of the formula (II) can be harvested by any means commonly used for harvesting metabolites produced by microorganisms. ~hus, for example~ bacterial cells are removed by centrifugation and the filtrate is subjected to a common process known for-isolation and purification of effective substances~ ~hus, there are 12~?S~O~
utilized solubility or solubility di~ference in an adequate solvent, difference in mamler or rate of precipitation from a solution, difference in affinity with a variety of adsorbents, ion exchange chromatography : 5 with an ion exchanger, concentration under reduced pressure, lyophili~ation, crystallization, recrystallization, drying and other means, either alone or in combination in an optional order, either singly or repeatedly~
A specific example of recovering the metabolites is given as follows. ~'he culture broth after completion of the cultivation is filtered, the filtrate is passed through an activated carbon column, and the thus-adsorbed compound (II) is eluted with a hydrophilic organic solvent system. ~he hydrophilic organic solvent system is, for example, a mixed solution composed of water and one or more of lower ketones, such as acetone, methyl ethyl ketone and methyl isobutyl ketone, and lower alcohols, such as methanol, ethanol, isopropanol, propanol and butanol. Since the desired compound is an acidic substances, the ion exchange resin is advantageously a Cl-type one, such as an . ~ anion exchange resin (Amberlite~IRA-400, 402, Rohm &
Haas, USA; Dowex-l~ Dow Chemical, U~A; Diaio ~ A-21~, Mitsubishi Chemical, Japan). ~he thus-adsorbed active substance is eluted with an aqueous solution of sodium chloride, for instance. ~or desalting, the eluate is again subjected to activated carbon column chromatography.
~he eluate containing the active substance is concentrated, then acetone or the like. is.added, and the precipitate is collected by filtration, washed with acetone, ether or the like and dried to give a pale brown powder For further purification of the powder, column chromatography with ~4E Sephadex~(Pharmacia, Sweden) is advantageous ~hus, after washing QA~ Sephadex A-25 is washed ~ith phosphate buffer (pH 6.6), an aqueous solution of the above powder is passed through the co:Lumn for adsorption, and the column is washed with M/25 phosphate buffer and r~e ~Q~k.
~ZQ540S
then eluted with the same buffer containing 0.5 w/v % of sodium chloride to obtain F3 active fractions. In the next place, the elution is carried out by using the same buffer containing 1.0 w/v % of sodium chloride to obtaining F2 active fractions.
The thus obtained F3 active fractions are pooled, adjusted to lower than pH 6 and again passed through an activated carbon column. After washing with water, elution is performed with 50 v/v % methanol-water. ~he active fractions are concentrated under reduced pressure, and then F3 is obtained in powdery form by lyophilization.
On -the other hand, F2 active fractions are pooled, adjusted to lower than pH 3 and again passed through an activa-ted carbon column. After washing, elution is performed with aqueous acetone or 7 v/v % isobutanol-water.
~he active fractions are concentrated under reduced pressure. Upon addition of acetone, these is obtained F2.
F2 and F3 can form a metal or ammonium salt, respectively. ~he metal salt is, for example, the sodium, potassium or littium salt. ~hese salts can be used as pharmaceutical salts.
~ he physicochemical properties of ~2 and F3 are as follows:
~ he physicochemical properties of the monosodium salt of F2 as obtained in Example 1 are as follows:
1. Melting point: 216-219C (decomposition) 2. Appearance: Colorless crystalline powder 3. Elemental analysis (for the sample dried over phosphorus pentoxide at 40C under reduced pressure ~or 6 hours):
~ 32.11 (%) H 5.38 N 7.27 S 10.67 ~a 3.8 4. Molecular weight: 605 (calculated as the monosodium "` ~LZ~5~05 salt).
5. Ultraviolet absorption spectrum:
End absorption only (no characteristic absorption at wave-length over 210 nm)
End absorption only (no characteristic absorption at wave-length over 210 nm)
6. Infrared absorption spectrum:
A.n absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 1.
A.n absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 1.
7. Specific rotation: (a~26 + 6.85(c=0.38,`N-CH3COOH) (~)D 7.72(c=0.54~, pH 7 phosphate buffer)
8. ~uclear magne-tic resonance spectrum (100 MHz, in dimethyl sulfoxide):
A -CH3 signal is observed at ~1.84.
A -CH3 signal is observed at ~1.84.
9. Solubility:` Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate and chloroform;
sparingly soluble in ethanol, pyridine and acetone; soluble in methanol and dimethyl sulfoxide;
readily soluble in water.
sparingly soluble in ethanol, pyridine and acetone; soluble in methanol and dimethyl sulfoxide;
readily soluble in water.
10. Color reactions: Positive Greig-~eiback and potassium permanganate reactions;
negative ninhydrin, Sakaguchi and Molisch reactions~
negative ninhydrin, Sakaguchi and Molisch reactions~
11. Stability: S-table in aqueous solution at pH
3-9 against heating at 60C for 3o 10 minutes.
3-9 against heating at 60C for 3o 10 minutes.
12. Acidity/basicity: An acid substance The physicochemical properties of the disodium sal-t of F2 as obtained in ~xample 2 are as follows:
1. Melting point: ~o distinct melting poi.nt 2. Appearance: White powder 3. ~lemental analysis (for the sample dried over --`` 12~5~
phosphorus pentoxide a-t 40C under reduced pressure for 6 hours):
C31.35 (%) H 5.24 5 N 6~78 S 10.38 Na 7.1 4. Molecular weight: 647.6 on the supposition that each molecule contains 2 Na atoms.
5. Ultraviolet absorption spectrum:
End absorption only.
6. Infrared absorption spectrum:
An absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 2.
7. Specific rotation: ~)26 + 5.50 (c=0.56, N-CH3COOH) 8. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform and acetone; sparingly soluble in methanol, ethanol and pyridine;
soluble in dimethyl sulfoxide;
readily soluble in water~
9. Color reactions: Positive ~reig-~eaback and potassium permanganate reactions; negative ninhydrin, Sakaguchi and Molisch reactions.
10. Stability: Stable in aqueous solu-tion at pH
3-9 against heating at 60C for 3o 10 minutes.
~he physico-chemical properties of the monosodium salt of ~2 as obtained in ExampIe 3 are as follows:
1. Melting point: 207-208C (decomposition) 2. Appearance: Colorless crystalline powder 3~ 3. Elemental analysis (for the sample dried o~er phosphorus pentoxide at 40C under reduced pressure S~05 for 6 hours):
C32.59 (%) H 5.22 N 6.93 S10.14 Na3.7 4. Molecular weight: 621.6 (calculated as the monosodium salt).
5.Ultraviolet absorption spectrum:
End absorption only (no characteristic absorption at wavelengths over 210 nm) 6.Infrared absorption spectrum:
An absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 3.
7speclfiC rotation: ~a)D6 ~ 6 0 (c=0.47, N-cH3cOoH) 8.Nuclear magnetic resonance spectrum (100 MHz, in dimethyl sulfoxide):
A -CH3 signal is observed at ~ 4 9. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl aceta1 e and chloroform;
sparingly soluble in ethanol, pyridine and acetone; solubla in methanol and dimethy] sulfoxide;
readily soluble in water.
10. Color reactions: Positive Greig-~eaback and potassium pe~anganate reactions;
negative ninhydrin, Sal{aguchi and Molisch reactions.
11. Stability: Stable in aqueous solution at pH 3-9 against heating at 60C for 10 minutes.
12. Acidity/basicity: An acid substance.
5~L0S
~he physico-chemical properties of the disodium salt of F2 as obtained in Example 4 are follows:
l. Melting poin-t: No distinct melting point 2. Appearance: White powder 3. Elemental analysis (for the sample dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours):
C 30083 (%) H 5.35 N 6.91 S 9.68 Na 7.3 4. Molecular weight: 629.9 on the supposition that each molecule contains 2 Na atoms.
5. Ultraviolet absorption spectrum:
End absorption only.
6. Infrared absorption spectrum:
An absorption spec-trum as obtained by the potassium bromide disc method is shown in Fig 4.
7. Speciflc rotation: ~)D6 + 6 1 (c=0.~9~ N-CH3COOH) 8. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform and acetone; sparingly soluble in methanol, ethanol and pyridine; soluble in dimethyl sulfoxide; readily soluble in wa-ter.
9. Color reactions: Positive Greig-~eaback and potassium permanganate reactions; negative ninhydrin, Sakaguchi and Molisch reactions.
10. Stability: Stable in aqueous solution at pH
3-9 against heating at 60C for lO minutes ~ :~Z~54~)5
1. Melting point: ~o distinct melting poi.nt 2. Appearance: White powder 3. ~lemental analysis (for the sample dried over --`` 12~5~
phosphorus pentoxide a-t 40C under reduced pressure for 6 hours):
C31.35 (%) H 5.24 5 N 6~78 S 10.38 Na 7.1 4. Molecular weight: 647.6 on the supposition that each molecule contains 2 Na atoms.
5. Ultraviolet absorption spectrum:
End absorption only.
6. Infrared absorption spectrum:
An absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 2.
7. Specific rotation: ~)26 + 5.50 (c=0.56, N-CH3COOH) 8. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform and acetone; sparingly soluble in methanol, ethanol and pyridine;
soluble in dimethyl sulfoxide;
readily soluble in water~
9. Color reactions: Positive ~reig-~eaback and potassium permanganate reactions; negative ninhydrin, Sakaguchi and Molisch reactions.
10. Stability: Stable in aqueous solu-tion at pH
3-9 against heating at 60C for 3o 10 minutes.
~he physico-chemical properties of the monosodium salt of ~2 as obtained in ExampIe 3 are as follows:
1. Melting point: 207-208C (decomposition) 2. Appearance: Colorless crystalline powder 3~ 3. Elemental analysis (for the sample dried o~er phosphorus pentoxide at 40C under reduced pressure S~05 for 6 hours):
C32.59 (%) H 5.22 N 6.93 S10.14 Na3.7 4. Molecular weight: 621.6 (calculated as the monosodium salt).
5.Ultraviolet absorption spectrum:
End absorption only (no characteristic absorption at wavelengths over 210 nm) 6.Infrared absorption spectrum:
An absorption spectrum as recorded by the potassium bromide disc method is shown in Fig. 3.
7speclfiC rotation: ~a)D6 ~ 6 0 (c=0.47, N-cH3cOoH) 8.Nuclear magnetic resonance spectrum (100 MHz, in dimethyl sulfoxide):
A -CH3 signal is observed at ~ 4 9. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl aceta1 e and chloroform;
sparingly soluble in ethanol, pyridine and acetone; solubla in methanol and dimethy] sulfoxide;
readily soluble in water.
10. Color reactions: Positive Greig-~eaback and potassium pe~anganate reactions;
negative ninhydrin, Sal{aguchi and Molisch reactions.
11. Stability: Stable in aqueous solution at pH 3-9 against heating at 60C for 10 minutes.
12. Acidity/basicity: An acid substance.
5~L0S
~he physico-chemical properties of the disodium salt of F2 as obtained in Example 4 are follows:
l. Melting poin-t: No distinct melting point 2. Appearance: White powder 3. Elemental analysis (for the sample dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours):
C 30083 (%) H 5.35 N 6.91 S 9.68 Na 7.3 4. Molecular weight: 629.9 on the supposition that each molecule contains 2 Na atoms.
5. Ultraviolet absorption spectrum:
End absorption only.
6. Infrared absorption spectrum:
An absorption spec-trum as obtained by the potassium bromide disc method is shown in Fig 4.
7. Speciflc rotation: ~)D6 + 6 1 (c=0.~9~ N-CH3COOH) 8. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate, chloroform and acetone; sparingly soluble in methanol, ethanol and pyridine; soluble in dimethyl sulfoxide; readily soluble in wa-ter.
9. Color reactions: Positive Greig-~eaback and potassium permanganate reactions; negative ninhydrin, Sakaguchi and Molisch reactions.
10. Stability: Stable in aqueous solution at pH
3-9 against heating at 60C for lO minutes ~ :~Z~54~)5
13 -~he physico-chemical properties of ~3 monosodium salt as obtained in ~xample 5 are as follows, 1. Appearance of the substance:
White powder 2. Elemental analysis:
As dried in vacuo over phosphorus pentoxide at 40C for 6 hrs, (%) C 32,08 ~ 5.33 N 5.17 S 6.28 Na 4.60 3. Molecular weight: 520 + 60 as calculated from the sodium content of monosodium salt.
4. Ultravlolet absorption spectrum:
En~ a~sorptions only (no characteristic a-t wavelength over 210 nm).
5. Infrared absorption spectrum (~ig. 5):
~he dominant peaks on the absorption spectrum (KBr disk) are as follows, 3350, 3100(sh), 2950, 1660(sh), 1640, 1560(sh), 14101 1375, 1320(sh), 1260, 1240, 1110 1070(sh), 1030, 98~, 950(sh), 820, 610, 5~5~
6. specific rotatiOn :~a~D5 - 2,8 (c=0.5, 0,1 M-Na2Hpo4) 7. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate and chloroform; only sparingly soluble in ethanol, pyridine and acetone; soluble in methanol and dimethyl su]foxide; and readily soluble in water.
8. Color reactions: Positive Greig-Leabac~, ninhydrin and l~S405
White powder 2. Elemental analysis:
As dried in vacuo over phosphorus pentoxide at 40C for 6 hrs, (%) C 32,08 ~ 5.33 N 5.17 S 6.28 Na 4.60 3. Molecular weight: 520 + 60 as calculated from the sodium content of monosodium salt.
4. Ultravlolet absorption spectrum:
En~ a~sorptions only (no characteristic a-t wavelength over 210 nm).
5. Infrared absorption spectrum (~ig. 5):
~he dominant peaks on the absorption spectrum (KBr disk) are as follows, 3350, 3100(sh), 2950, 1660(sh), 1640, 1560(sh), 14101 1375, 1320(sh), 1260, 1240, 1110 1070(sh), 1030, 98~, 950(sh), 820, 610, 5~5~
6. specific rotatiOn :~a~D5 - 2,8 (c=0.5, 0,1 M-Na2Hpo4) 7. Solubility: Insoluble in petroleum ether, hexane, diethyl ether, benzene, ethyl acetate and chloroform; only sparingly soluble in ethanol, pyridine and acetone; soluble in methanol and dimethyl su]foxide; and readily soluble in water.
8. Color reactions: Positive Greig-Leabac~, ninhydrin and l~S405
- 14 -potassium permanganate reactions.
Negative ferric chloride-potassium ferricyanide reactions, 9. Stability: Stable in aqueous solution at pH
3 to 9 against heating 60C for 10 minutes In the present invention, a compound of -the formula (III), inclusive of salts thereof, can be produced by subjecting a compound of the formula (IV), inclusive of salts thereof, to desulfation A conventional method for cleavage of sulfuric ester bond, not to be limited, can be employed in the above reaction. ~or example, the reaction is conducted by using an acid, especially an inorganic acid such as sulfulic acid or hydrochloric acid 7 a base such as sodium hydroxide, potassium hydroxide or barium hydroxide, or ion-exchange resin such as Dowex-50 (Dow Chemical~ U.S.A.) or Amberlite IP.-120 (Rohm and Xaas Co , U.S A ).
In case that an acid is used, the reaction is carried out generally in acid concentration of about 0.005N to 5N, preferably 0.05N to lN, in the presence of a sulvent such as alcohol (e.g methanol, ethanol, propanol), dio~ane, acetic acid, water or a mixture thereof ~he reac-tion is generally performed at about 20C to 200C :~or about 30 minutes to 48 hours.
In case that a base is used, the reaction is carried out generally in base concentration of about 0.005N to 5N, preferably 0.05N to lN, in the presence of alcohol (e.g. methanol, ethanol, propanol), water or a mixture thereof, at about 20C to 200C for about 30 minutes to 48 hours, In case that ion-exchange resin is used~ the reaction is carried out by using the resin which is suspended in alcohol (e.g methanol, ethanol, propanol), water or a mixture thereof, at abou-t 30C to 200C for ~¢~405i
Negative ferric chloride-potassium ferricyanide reactions, 9. Stability: Stable in aqueous solution at pH
3 to 9 against heating 60C for 10 minutes In the present invention, a compound of -the formula (III), inclusive of salts thereof, can be produced by subjecting a compound of the formula (IV), inclusive of salts thereof, to desulfation A conventional method for cleavage of sulfuric ester bond, not to be limited, can be employed in the above reaction. ~or example, the reaction is conducted by using an acid, especially an inorganic acid such as sulfulic acid or hydrochloric acid 7 a base such as sodium hydroxide, potassium hydroxide or barium hydroxide, or ion-exchange resin such as Dowex-50 (Dow Chemical~ U.S.A.) or Amberlite IP.-120 (Rohm and Xaas Co , U.S A ).
In case that an acid is used, the reaction is carried out generally in acid concentration of about 0.005N to 5N, preferably 0.05N to lN, in the presence of a sulvent such as alcohol (e.g methanol, ethanol, propanol), dio~ane, acetic acid, water or a mixture thereof ~he reac-tion is generally performed at about 20C to 200C :~or about 30 minutes to 48 hours.
In case that a base is used, the reaction is carried out generally in base concentration of about 0.005N to 5N, preferably 0.05N to lN, in the presence of alcohol (e.g. methanol, ethanol, propanol), water or a mixture thereof, at about 20C to 200C for about 30 minutes to 48 hours, In case that ion-exchange resin is used~ the reaction is carried out by using the resin which is suspended in alcohol (e.g methanol, ethanol, propanol), water or a mixture thereof, at abou-t 30C to 200C for ~¢~405i
- 15 -about 30 minutes to 30 hours.
A compound of -the formula (III) can be obtained from thus prepared reactant by any means commonly used for isolation and purification of effective substances 5 Thus, there are utilized ion exchange chromatography wi-th an ion exchanger, adsorption, concentration, crystallization, recrystallization and other means, either singly or repeatedly.
A practical example of the purification process is as follows. The reactant is passed -through alkaline ion exchange resin column and then eluted with acetic acid or hydrochloric acid e-tc.
The fractions, which show positive Greig-Leabac~
reaction, are pooled and, if necessary, purified by subjecting to gel filtration using Sephadex LH-20 (Pharmacia, Sweden) as carrier and aqueous alcohol as an eluent, respectively.
The biological properties of the compound of the formula (I) are described below The compound of the 20 formula (I) sho~Sthe action which potentiates the antibacterial activi-ty of ~-lactam antibiotics as illus-trated from the following data.
1) The antibiotics-potentiatin~ activity data for F2 It is evident from the data given in Table 1 and Table 2 that F2 exhibits an antibac-terial activity against ~scherichia coli in the copresence of cefmeno~ime or mecillinam in a subbactericidal concentration Table 1: Cefmenoxime- and mecillinam-potentiating activity data for the F2 sample obtained in ~xample 1 (tes-t organism: ~scherichia coli IF0 1273L~) ~2~S~
.Table 1 , F2 concentration Diameter of growth inhibi-ti.on zone (mm)*
in test solution**
(~g/ml) No addition 0~05 ~g/ml 0,03 ~g/ml cefm.enoxime mecillinam O <8 < 8 <8 1 <8 10 ~8 <8 15 10 < 8 24 18 10 125 < 8 32 24 1000 < 8 40 30 _ * The test was performed using a bouillon agar medium containing the antibiotic as speci~ied and a paper disc, 8 mm in diameter.
** A paper disc, 8 mm in diameter, was impregnated with 25 ~1 of the test solution and placed on the agar medium Table 2 Cefmenoxime- and mecillinam-potentiating activity data for the F2 sample obtained in Example 3 (test organism: scherichia coli IFO ] 2734) Table 2 _ F2 concentration Diameter of growth inhibition.~one (mm)*
in test solution** n __ _ _ (~g/ml) No addition0~05 ~g/ml 0~03 ~g/ml .~ef~enoxime mecillinam , " ,. . __ _ . . _ . . . _ . __ . .
O <8 <8 ~8 1 <8 9 <8 < 8 14 9 30 25 < 8 23 19 125 <8 31 23 1000 <8 39 - 29 _ * and **: The same as noted for Table 1.
It is also evident from the data given in Table 3 and Table 4 that F2 is capable of po-tentiating the anti-~2~S4~S
bacterial activity of cefmenoxime an~ mecillinam against Proteus mirabilis ATCC 21100.
Table 3: Cefmenoxime- and mecillinam~potentiating activity data for the ~2 sample obtained in Example 1 (test organism: Proteus mirabilis ~TCC 21100)*
Table 3 ~2 concen-tration Diameter of growth inhibition zone (mm)*
in test solution (~g/ml) No addition 0.01 ~g/ml 0.05 ~g/ml cefmenoxime mecillinam 1 0 . -~
0 < 8 ~ 8 < 8 1 < 8 9 9 5 < 8 13 11 25 < 8 23 19 15 125 < 8 30 26 1000 < ~ 38 32 .. .. _ _ _ _ . . . . . _ * The test was performed under the same conditions as noted for Table 1 except for the test organism and t'ne concentration of each coexisting antibiotic.
Table 4: Cefmenoxime- and mecillinam-potentiating activity data for the ~2 sample obtained in Example 3 (-test organism: Proteus mirabilis ATCC 21100)*
Table 4 ----- ~ - _ . . _ ~2 concentration Diameter of growth inhibition zone (mm)~
in test solution ~o addition 0~01 ~g/ml 0 05 ~g/ml cefmenoxime mecillinam . . . _ 0 < 8 < 8 < 8 301 < 8 9 9 5 ~ 8 12 12 25 < 8 22 19 125 ~ 8 29 25 1000 < 8 38 32 _ * The test was performed under the same conditions as noted ~s~s for Table l except for the test organism and the concentration of each coexisting antibiotic.
Furthermore, F2 strongly inhibits the growth of Escherichia coli in synergy with cephalexin or cefmenoxime.
5 In ~able 5 and ~able 6, there are shown degrees of growth of Escherichia coli under the influence of F2 in the copresence of 5 llg/ml of cephalexin or l ~g/ml of cefmenoxime ~able 5: Inhibition of growth of Escherichia coli IF0 12734 in the copresence of F2 (obtained in :Example l) lO and cephalexin or cefmenoxime.
~able Final F2 Absorbance at 600 nm concen-tration No addition 5 ~giml l llg/ml cephalexin cefmenoxime 0 2.6 2035 1.95 2.65 1.80 0.60 2.65 l.00 0.25 lO0 2,70 0.95 0.30 20 ~ -- -~o 8 ml of YAB medium (1.75% Difco antibiotic medium ~o. 3, 0.5% Difco yeast extract) containing 12% (w/v) of sucrose, there were added 1 ml of water9 50 llg/ml cephalexin solution or lO llg/ml cefmenoxime solution, and the medium 25 was inoculated with 1 ml of a preculture in YAB medium at an early logarithmic growth phase and incubated with shaking at 37C for 2 hours. ~hereafter, the absorbance ` of the culture broth was measured at 600 nm using a ~himadzu-Bausch & Lomb Spectronic 20 colorimeter.
~able 6: Inhibition of growth of Escherichia coli IF0 1273L~ in the copresence of F2 (obtained in ~xample 3) and cephalexin or cefmenoxime*
S~OS
able 6 ~inal ~2 Absorbance at 600 nm concentration . .
(~g/ml) No addlt1on 5 ~g/ml 1 ~g/ml cephalexin cefmenoxime 0 2.4 2.15 1 9o 1 2.45 1.75 0.55 2 50 1.90 0.25 100 2.55 0.75 0.25 1 0 -- ' -' ---* The test was performed in the same manner as noted for Table 5.
As is evident from ~able 5 and ~able 6, ~2 inhibits the growth of Escherichia coli in the presence of cephalexin or cefmenoxime, as evidenced by the decrease in absorbance~
Observation of the culture broths under a phase contra~t microscope revealed that, when ~2 was not added, cells were in the much elongated form under the influence of cephalexin or cefmenoxime but that, when ~2 was added, the elongation was inhibited in dependence on the addi.tion level and at the same time partial swelling occurred and bacteriolysis proceeded.
b) ~he antibiotics-potentiating activity data for ~
~he action of ~3, which potentiates the activity of antibiotics, is shown in ~ab].e 7 -to 9.
~able 7 ~he action of ~3 of Example 5 to potentiate the antibacterial action of cefmenoxime and mecillinam.
(~est organism: Escherichia coli I~O 12734) ~able 7 -~
Concentration Diameter of growth inhibi-tion zone (mm)*
of ~3 in test --~
solution** No addition 0 05 ~g/ml of 0 03 ~g/ml of (~g/ml) cefmenoxime mecillinam _ _ o < 8 < 8 < 8 1 < 8 10 < 8 < 8 1205 < 8 `` ~2~S~
-Concentration Diameter of growth i.nhibition zone (mm)*
of F3 in test solution** No addition 0.05 ~g/ml of 0.03 ~g/ml of (~g/ml) cefmenoxime mecillinam < 8 15 9 5 125 < 8 26 17.5 1000 < 8 38 28.5 .
*, ** ; The test was performed under the similar me-thod as noted for Table l.
Table 8 The ac-tion of F3 of Example 5 to potentiate the antibacterial action of cefmenoxime and mecillinam*
(tes-t organism: Proteus m rabilis ATCC 21100) able 8 ~ _ .
Concentration Diameter of growth inhibition zone (mm)*
of F3 in test --solution No addition 0.01 ~g/ml of 0.05 ~g/ml of (~g/ml) cefmenoxime mecillinam _ 0 ~ 8 < 8 ~ 8 1 < 8 9 < 8 ~ < 8 12 9 < 8 21 13.5 125 ~ 828.5 19 1000 < 832.5 30 * Except for the test organism and the concentration of antibiotics to be potentiated, all other assay conditions are the same as those mentioned for Table l.
Table 9 Inhibition of growth of Escherichia coli IF0 _. _ 12734 in -the co-presence o~ F3 (Example 5) and cefmenoxime ~26~5~)5 Table 9 .
~inal concentration Absorbance at 600 r~
of ~3 ~
(~g/ml) No addition 1 ~g/ml of cefmenoxime . _ 0 3.7 2.9 1 3.7 2.2 3.7 0.6 100 3,7 0.6 '' ' ~
The test was performed in a manner similar to that described for ~able 5.
~ s is evident from Table 9, ~3 inhibits the growth of ~scherichia coli in the presence of cephalexin or cefmenoxime, as evidenced by the decrease in absorbance.
Observation of the culture broths under a phase contrast microscope revealed that, when ~3 was not added, cells were in the much elongated form under the influence o~
cephalexin or cefmenoxime but that, when ~3 -was added, the elongation was inhibited in dependence on the addition level and at the same time partial swelling occurred and bacteriolysis.
c) Antibiotics-potentiating activit~_data for ~4 Table 10 Cefmenoxime- and mecillinam-potentiating activity data for the ~4 sample obtained i.n E~ample 7 (test organism: ~scherichia coli I~O
12734) Table 10 .
. . ~. . . ._ _ . _ . . ~ . _ ~
o ~4 concentration Diameter of growth inhibition zone (mm)*
3 in tes-t solution~* -- ----- - -(~Ig/ml) No addition 0.05 ~g/ml 0 03 ~g/ml cefmenoxime mecillinam . . .
O ~ 8 < 8 ~ 8 < 8 < 8 < 8 35 100 < 8 15 13 1000 < 8 26 23 *, ~*: ~he test was performed under a method similar to that described for ~able 1 ~ urther, the compound of the formula (I) showed a marked antibacterial activity in synergy with cefmenoxime or cephalexin in Escherichia coli- or Proteus mirabillis-.
infected mice. ~`herefore, -the compound of the formula (I) can be used in combination with such an-tibiotics in the treatment of infectious diseases caused by the above bacteria in ma~mals (e.g. mouse, rat, dog, human) and in domestic fowls (e.g. chicken, duck) ~ or the treatment of Escherichia coli infection, for instance, a solution of 0.5-2.0 g of, for example, cefmenoxime and 0.5-10 g of the compound of the formula (I) in 10-100 ml of physiological saline is administered to human adults two to three times a day in admixture with an injectable solution for intravenous drip infusion.
~he compound of the present invention is also a very promising intermediate for the synthesis of novel drugs.
When the compound of the present invention was intravenously administered to mice, the LD50 was more than 1 g/kg.
~he following examples illustrate the invention in more detail However, they should by no means be construed as limiting the present invention~ ~nless otherwise sta-ted, each percentage value is on an weight/volume basis.
Example 1 ~wo 2-liter Sakaguchi flasks each containing 500 ml of a mediu~ containing 1~ glucose, 0.5% Polypepton (Daigo Nutritive Chemicals, Ltd.), 0 5% meat extract and 0.5%
sodium chloride (pH 7.0) were inoculated with cells of Pseudomonas acidophila G-6302 strain (~ERM-P ~o. 4344; I~0 13774; A~CC 31363) grown on a slant nutrient agar and incubated at 28C with reciprocal shaking for 48 hours to give a seed culture~
s A 200-liter stainless steel fermentor was charged with 120 liters of a medium containing 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5% meat extrac-t, 0.5/0 NaCl and 0 1%
cys-teine, and the medium was adjusted to pH 7 0 with 30%
sodium hydroxide solution, then steam-sterilized at 120C
for 20 minutes, and inoculated with the above seed culture.
Incuba-tion was carried out at a temperature of 28C, a rate of aeration of 120 liters/minute and a rate of stirring of 180 rpm for 78 hours ~he culture broth was centrifuged on a Sharples centrifuge for separation of bacterial cells.
~here was obtained 110 liters of superna-tant, which, ~7 after adaustment to pE 4 . 2 1 was passed through a column ~:~ packed with 15 liters of activated carbon ("Shirasagi'~for chromatographic use, ~akeda Chemical Industries, ~-td.) for adsorption of the active substance. After washing with 45 liters of water, elution was conducted with 45 liters of 50 v/v % acetone-water. An aliquot of each 10-liter eluate fraction was tested for the active substance using a bouillon agar medium containing 0.05 ~g/ml of cefmenoxime with ~scherichia coli IF0 12734 as the test organism. As a result, fractions Nos. 2 and 3 were pooled and, after addition of 20 liters of water, passed through a column packed with 10 liters of Dowex-l (C1) (Dow Chemical, USA).
After washing with 25 liters of water, elution was carried out with 50 liters of 5% sodium chloride solution in water.
~he active fractions were pooled and, after adjustment to pH 4.0, passed through a column packed with ac-tivated carbon (~ liters). After washing with 24 liters of water, elution was performed with 20 v/v % methanol-water. ~he active fractions were collected and concentrated under reduced pressure to 50 ml, 200 ml of acetone was added, and the resulting precipitate was collected by fil-tration, washed with 50 ml of acetone and 100 ml of ether and dried under reduced pressure to give 25 g of crude F2.
In 500 ml of M/25 phosphate buffer (pH 6.6) was dissolved 10 g of the crude ~2 and the solution was passed ~ ~r~ Mc~
5~3'5 through a 200-ml column of QAE Sephadex A-25 (Pharmacia, Sweden) buffered with the same buffer as above, for adsorption of ~2. The column was washed with 400 ml of the same buffer and then with 400 ml of the same buffer with 0.5% of sodium chloride added thereto. Thereafter, elution was carried out with the same buffer with 1% of sodium chloride added thereto. The active fractions were pooled and, after adjustment to pH 3.0 with 1 N hydrochloric acid, passed through a 60-ml column of activated carbon.
The column was washed with 200 ml of water and then with 100 ml of 50 v/v % methanol-water, Elution was carried out-with 8.0 v/v % isobutanol-water. The active eluate fractions were pooled and, after ad~justment to pH 5.5, concentrated under reduced pressure. The concentrate was dissolved by addition of 50 ml of methanol, and the solution was filtered and allowed to stand in a cool place.
The resulting crystalline precipi-tate was filtered off, washed with ether and dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours. There was thus obtained 2. 8 g of the monosodium salt of ~2 as crystals.
Its infrared absorption spectrum is shown in ~ig. 1, Elemental analysis: C, 32.11; H, 5.38; N~ 7.27; $~ 10.67;
Na, 3.8%
Example 2 In 90 ml of water was dissolved 3.0 g of the monosodium salt of ~2 obtained in Example 1. After 4.5 ml of 1 N sodium hydroxide was added thereto under cooling, the solution was carefully adjusted -to pH 7.0 with 1 N
sodium hydroxide while measuring the pH and then was lyophilized to give 3.1 g of the disodium salt of F2 as a white powder. An infrared absorp-tion spectrum for this product after drying at 40C under reduced pressure for 6 hours in shown in ~ig. 2. Elemental analysis gave the following results: C, 31.35; H~ 5.24; N, 6.78;
S, 10.38; Na 7.1%.
~2~S~
xample 3 Two 2-liter Sakaguchi flasks each containing 500 ml of a medium containing 1% glucose, 0. 5% Polypepton (Daigo Nutritive Chemicals, Ltd.), 0.5% meat extract and 0.5%
sodium chloride (pH 7.0) were inoculated with cells of Pseudomonas mesoacidophila ~B-72310 (FERM-P No. 4653; IF0 13884; ATCC 31433) grown on a slant nutrient agar and incubated at 28C wi-th reciprocal shaking for L~8 hours to give a seed culture.
A 200-liter stainless steel fermentor was charged with 120 liters of a medium containing 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5/0 meat extract, 0 5% NaCl and 0.1% cysteine, and -the medium was adjusted to pH 7.0 with 30% sodium hydroxide solution, then steam-sterilized at 120C for 20 minutes and inoculated with the above seed cul-ture. Incubation was then carried out at a temperature of 2~C, a rate of aeration of 120 liters per minute and a ra-te of stirring of 180 rpm for 78 hours. The culture broth was centrifuged on a Sharples centrifuge Eor separation of bacterial cells. ~here was obtained 110 liters of supernatant, which~ after adjustment to pH 4.2, was passed through a column packed wit'n 15 liters of activated carbon ("Shirasagi" for chromatography, Takeda Chemical In Industries, Ltd.) for adsorption of the active substanceO
Af-ter washing with ~5 liters of water, elu-tion was performed with LL5 liters of 50 V/V % acetone-water An allquo-t of each 10-liter eluate fraction was -tested :Eor -the active substance using a bouillon agar medium containing 0.05 ~g/ml of cefmenoxime with Escherichia coli IF0 1273L~ as the test organism. Fractions Nos. 2 and 3 were pooled and, after addi-tion of 20 liters of water, passed through a column packed with 10 liters of Dowex-l (Cl) (Dow Chemical, USA). Af~ter washing with 25 liters of water, elution was performed with 50 liters of 5% sodium chloride solution in water. The active fractions were pooled, adausted to pH 4.0 and again passed through an activated carbon column (8 liters). Following washing with 24 liters of water, elution was performed with 20 v/v %
methanol-water. ~he active fractions were pooled and concentrated to 50 ml under reduced pressure, 200 ml of acetone added, and the resulting precipi-tate was collec-ted by filtration, washed with 50 ml of acetone and 100 ml of ether and dried under reduced pressureO
There was obtained 21 g of crude product.
In 500 ml of M/100 phosphate buffer (pH 6.6) was dissolved 10 g of the crude product and the solution was passed through a 200-ml column of QA~ Sephadex A-25 (Pharmacia, ~weden) buffered with the same buffer as above, for adsorption of F2. ~he column was washed with 400 ml of the same buffer and then with 400 ml of the same buffer with 0.5% of sodium chloride added thereto. ~hen, elution was performed with the same buffer with 1% of sodium chloride added there-to. ~he active fractions were pooled, adjusted, to pH 3.0 with lN hydrochloric acid and passed through a 60 ml column of activated carbon~ The column was washed with 200 ml of water and elution was carried out with 8 v/v % isobutanol-water. The active fractions were pooled, adjusted -to pH 5.5 and concentrated under reduced pressure. ~he concentrate was dissolved by addi-tion of 50 ml of me-thanol and the solution was allowed to stand in a cool place. ~he resulting crystalline precipitates were collected by filtration, washed with ether and dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours ~'here was obtained 1.5 g of -the monosodium salt of F2 as crystals. Its illfrared absorption spectrum 30i is shown in Fig. 3. ~lemental analysis: C, 32.59; H, 5.22;
N, 6.93; S, 10~14; Na, 3.7%.
EYample 4 In 30 ml of water was dissolved 1.0 g of t~e monosodium salt of F2 as obtained in EYample 3. Following addition of about 1.5 ml of 1 N sodium hydroxide under cooling, the solution was carefully adjusted to pH 7.0 while measuring the pH. The thus-neu-tralized solution was lyophilized to give 1.05 g of the disodium salt of ~2 as white powder. An infrared spectrum of this product after drying at 40C for 6 hours is shown in Fig. 4 Elemental analysis gave the following data: C, 30.83; H, 5.35; N, 6.91;
S, 9.68; ~a, 7.3%.
Example 5 10 Cells of Pseudomonas mesoacidophila SB-72310 (~.RM-P No. 4653; I~0 13884; ATCC-31433) were inoculated into two 2-liter Sakaguchi flasks each containing 500 ml of a medium composed of 1% glucose, 0.5% Polypepton (Daigo Eiyo Chemical Co., Ltd.), 0.5% meat extrac-t and 0.5% sodium chloride (pH 7.0) and the inoculated flasks were incubated on a reciprocating shaker at 28C for 48 hours to prepare a seed inoculum.
Then a 200-liter stainless steel fermentor was charged with 120 ~ of a medium composed of 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5% meat extrac-t, 0.5%
NaCl and 0.1% cysteine, the pH of which was adjusted to pH 7 with 30% sodium hydroxide. The fermentor was steam-sterilized at 120C for 20 minutes, after which the medium was inoculated with the seed inoculum, Cul-tivation was conducted at 28C with 120 ~/min. aera-tion and at 180 r.p,m. for 78 hours. The culture broth was centrifuged with a Sharples centrifuge to separate the cells to recover 110 ~ of a supernatant. It was adjusted to p~ 4.2 and passed through a column of 15 ~ ac-tivated carbon (Chromatography grade Shirasagi, Takeda Chemical Industries, ~td.) to adsorb the active compounds. The column was rinsed with 45 ~ of water and elution was carried out with 45 ~
of 50 v/v % aqueous acetone. The eluate was collected in 10 ~ fractions and -the active fractions were detected ~5 using plates of broth agar containing 0.05 g/ml of cefmenoxime and, as the test organism, Escherichia coli ~s~
I~0 12734. ~raction Nos. 2 and 3 were pooled, diluted with 20 ~ of water and passed through a column of 10 Dowex-l(Cl) (Dow and Chemical, U.S.A.). After the column was rinsed with 24 ~ of water, elution was carried out with 20 v/v % aqueous methanol. The active fraction was concentrated under reduced pressure to 50 ml, followed by addition of 20~ ml of acetone. The resultant precipitate was recovered by filtration~ washed with 50 ml of acetone and 100 ml of ether, and dried in vacuo to give 21 g of a crude product.
Ten (10) grams of this crude product was dissolved in 500 ml of 1/100 M-phosphate buffer (pH 6.6) and passed through a column of 200 ~1 QAE-Sephadex A-25 buffered with the same buffer as above. The column was washed with lOOOml of the same buffer as above and eluation was carried out with 2000 ml of the same buffer containing 0.5% of sodium chloride. The fractions showing F3 activity were pooled, adjusted to p~I 3 0 with lN-HCl, and passed through a column of 40 ml activated carbon.
The column was rinsed with 200 ml of water and elution was carried out with 50 v/v % aqueous methanol. The active fractions were pooled, concentrated under reduced pressure and lyophilized. The white powder thus obtained was dried over phosphorus pentoxide under reduced pressure at 40C for 6 hours to give 0.85 g of a powder (sodium salt of ~3). The infrared absorption spectrum (KBr) of this powder is shown in ~ig. 5.
Elemental analysis: C, 32.08; H9 5.33; N, 5.17; S, 6.28;
Na, 4 60%
3o EXample 6 Cells of Pseudomonas acidophila G-6302 (~ERM-P
No~ 4344; I~0 13774; ATCC-31363) grown on a nutrient broth agar slant were used to inoculate two 2-liter Sakaguchi flasks each containing 500 ml of a medium composed of 1% glucose, 0.5% Polypepton, 0.5% meat extract and ~?S~OS
0.5% sodium chloride (pE 7.0) and the inoculated medium was incubated on a reciprocating shaker at 28C for 48 hours to give a seed inoculum.
Separately, a 200-liter stainless steel fermentor was charged with 120 ~ of a medium composed of 3%
glycerol, 0.1/0 glucose, 0,5/0 Polypepton, OD50/O meat extract, 0.5% ~aCl and 0,1% cysteine and after adjustment to pH 7.0 with 30% sodium hydroxide was s-team-sterilized at 120C for 20 minutes. '~he fermentor was then inoculated with the above seed inoculum and incubation was carried ou-t at 28C and 180 r.p m. with 120 ~/min. aeration for 78 hours. ~he resulting broth was centrifuged with a Sharples centrifuge to separate the cells and give 110 ~ of a supernatant ~his supernatant was adjusted 15 to pH 4 2 and passed through 15 ~ of activated carbon (Chromatograph~ grade Shirasagi, ~a~eda Chemical Industries, ~td.). After an aqueous rinse with 45 ~ of water, elution was carried ou-t with 45 ~ of 50 v/v % aqueous acetone.
The eluate was collected in lO~liter fractions which were assayed for activity using plates of broth-agar containing O, 05 ~g/ml of cefmenoxime and, as the test organism, ~scherichia coli IF0 12734. Fraction Nos. 2 and 3 were pooled, diluted with 20 ~ of water and passed through a column of 10 ~ Dowex l(Cl) (Dow and Chemicai, U~S.A.).
The column was rinsed with 25 ~ of water and elution was performed with 50 ~ of 5% aqueous sodium chloride. ~'he active fractions were pooled, adjusted to pH 4.0 and passed again -through a column (8 ~) packed with ac-tivated carbon. After the column was rinsed with 24 ~ of water, elution was carried out with 20 v/v % aqueous methanol.
~he active fractions were pooled and concentrated under reduced pressure to 50 m1. ~0 the residue was added 200 ml of acetone and the resultant precipirate was recovered by filtration, washed with 50 ml of acetone 35 and 100 ml of ether, and dried in vacuo to give 25 g of a crude product.
- 3o -A 10 g portion of the crude product was dissolved in 500 ml of M/100 phosphate buffer (pH 6.6) and the solution was poured on a column of 200 ml QA~-Sephade~
A-25 buffered with the same buffer as above to have the product adsorbed thereon~ The column was washed with 1000 ml of the same buffer as above and elution was carried out with 2000 ml of the same buffer containing 0 5% of sodium c'nloride. ~ractions containing F3 activi-ty were pooled, adjusted to pH 3.0 with lN-HCl, and passed through a column of ac-tivated carbon The column was rinsed with 200 ml of water and elution was carried out with 50 v/v %
aqueous methanol The active fractions were pooled, concentrated under reduced pressure, and lyophilized The resulting white powder was dried in vacuo over phosphorus pentoxide at 40C for 6 hours to give 1.3 g of a powder (Sodium salt of ~3).
~lemental analysis: C, 32.28; H, 5 53; N, 5 18; S, 6 25;
Na, 4.52%
Example 7 Monosodium salt of ~2 (5.9 g), which was obtained by the method of Example 1, was dissolved in 150 ml of lN-hydrochloric acid~methanol, and then treated at gooc for 4 hours After methanol was distilled ou-t, the residue was dissolved in 100 ml of water and passed -through a column (100 ml) of Amberlite IR-45(OH ). The solution which passed through the column, together with washings, was again passed through a column (100 ml) of Amberlite IRC-50 (H+). The solution which was -thus passed the column, together with washings, was diluted to 500 ml wi-th water, and then passed through a column (100 ml) of Amberlite IRA-68 (OH ) The col~n was washed with 100 ml of water, elution was carried out with lN-aqueous acetic acid. The elua-te, together with washings, was concentrated. The residue was dissolved in 10 ml of water, and the solution was subjected to a column chromatography of ~ephadex ~H-20 (3.3 x 50cm). Elution was carried out with 10/c methanol-water, fractions of 210 to 230 ml were pooled and concentrated under reduced pressure, and lyophilized. The resulting powder (1.0 g) was treated with ethanol. ~hus, 0.78 g of ~4 was obtained as crystaline solid.
Specific rotation: (~)23 + 1.4 (c=l.O, N-CH3COOH) Elemental analysis, for C16H29N3011S H20 :
Calcd. (/0): C, 39.25j H, 6.38; ~, 8.59; ~, 6.55.
Found (%): C, 38.91; H, 6.L~2; N, 8.47; S, 6.16.
Infrared absorption spectrum:
An absorption spectrum as ob-tained by the potassium bromide disc method is shown in ~ig. 6.
Amino acid analysis (hydlysis with 6N-HCl at 110C for 8 hours):
D-glucosamine, 0.92; taurine, 1.00; 4-hydroxy-5-hydroxymethylproline, O.85 (average recovery 85%).
Example 8 ~odium salt of ~3 (70 mg), which was obtained by the method of Example 5, was dissolved in 17 ml of 0.2N-hydrochloric acid, and then was allowed to stand at 90C for 2 hours. ~he solution was neutrarized with 3.4 ml of lN-sodium hydroxide, and passed through a column (50 ml) of activated carbon. ~he column was washed with 150 ml of water, and then elution was carried out with 8% aqueous iso-butanol. ~he fractions of 100 to 250 ml were pooled and concentrated under reduced pressure, the obtained residue was dissolved in a small amount of wa-ter. ~he solution was subjected to a col~lmn chromatography of 50 ml QAE-~ephadex A-25 buffered with 0.05M-phosphate buffer (pH 6.1). ~lution was carried out with the same buffer as above, the fractions of 19 to 54 ml were pooled and the solution was passed through a ~5 column (20 ml) of activated carbon. ~he column was washed wi-th 60 ml of water, and the elution was carried out ~2~5~
with 8% aqueous iso-butanol. ~he fractions of 40 to 70 ml were pooled, and iso-butanol was ditilled out, the residue was lyophilized. ~h-us, 28 mg of F5 was ob-tained as white powders.
Elemental analysis, for C14H240~N2 H20 :
Calcd~ (%) : C, 43.97; H, 6.85; N, 7.~2.
Found (%) : C, 44 05; H, 6.93; N, 7.10.
Infrared absorption spectrum:
An absorption spectrum as obtained by the pottasium bromide disc method is shown in Fig 70 ~LC Rf : ~he Rf values on the silica gel plate (Merc~, 60~ 254) Rf = 0.14 (n-propanol : water = 4 : 1) Rf = 0.22 (n-propanol : acetic acid : water = 2 : 1 : 1) Amino acid analysis (hydrosis with 6N-HCl at 110C for 14 hours) :
Glucosamine, 1.00; 4-hydroxy-5-hydroxymethyl proline, 0.93 (average recovery 82%).
Brief Description of the Drawings Fig. 1, 2, 3, 4, 5, 6 and 7 show infrared absorption spectra of the monosodium salt of F2 as obtained in Example 1, the disodium salt of F2 as obtained in Example 2, the monosodium salt of ~2 as obtained in Example 3, the disodium salt of F2 as obtained in Example 4, the sodium salt of F3 as obtained in Example 5, the F4 as obtained in E~ample 7 and -the F5 as obtained in E~ample 8, respectively.
A compound of -the formula (III) can be obtained from thus prepared reactant by any means commonly used for isolation and purification of effective substances 5 Thus, there are utilized ion exchange chromatography wi-th an ion exchanger, adsorption, concentration, crystallization, recrystallization and other means, either singly or repeatedly.
A practical example of the purification process is as follows. The reactant is passed -through alkaline ion exchange resin column and then eluted with acetic acid or hydrochloric acid e-tc.
The fractions, which show positive Greig-Leabac~
reaction, are pooled and, if necessary, purified by subjecting to gel filtration using Sephadex LH-20 (Pharmacia, Sweden) as carrier and aqueous alcohol as an eluent, respectively.
The biological properties of the compound of the formula (I) are described below The compound of the 20 formula (I) sho~Sthe action which potentiates the antibacterial activi-ty of ~-lactam antibiotics as illus-trated from the following data.
1) The antibiotics-potentiatin~ activity data for F2 It is evident from the data given in Table 1 and Table 2 that F2 exhibits an antibac-terial activity against ~scherichia coli in the copresence of cefmeno~ime or mecillinam in a subbactericidal concentration Table 1: Cefmenoxime- and mecillinam-potentiating activity data for the F2 sample obtained in ~xample 1 (tes-t organism: ~scherichia coli IF0 1273L~) ~2~S~
.Table 1 , F2 concentration Diameter of growth inhibi-ti.on zone (mm)*
in test solution**
(~g/ml) No addition 0~05 ~g/ml 0,03 ~g/ml cefm.enoxime mecillinam O <8 < 8 <8 1 <8 10 ~8 <8 15 10 < 8 24 18 10 125 < 8 32 24 1000 < 8 40 30 _ * The test was performed using a bouillon agar medium containing the antibiotic as speci~ied and a paper disc, 8 mm in diameter.
** A paper disc, 8 mm in diameter, was impregnated with 25 ~1 of the test solution and placed on the agar medium Table 2 Cefmenoxime- and mecillinam-potentiating activity data for the F2 sample obtained in Example 3 (test organism: scherichia coli IFO ] 2734) Table 2 _ F2 concentration Diameter of growth inhibition.~one (mm)*
in test solution** n __ _ _ (~g/ml) No addition0~05 ~g/ml 0~03 ~g/ml .~ef~enoxime mecillinam , " ,. . __ _ . . _ . . . _ . __ . .
O <8 <8 ~8 1 <8 9 <8 < 8 14 9 30 25 < 8 23 19 125 <8 31 23 1000 <8 39 - 29 _ * and **: The same as noted for Table 1.
It is also evident from the data given in Table 3 and Table 4 that F2 is capable of po-tentiating the anti-~2~S4~S
bacterial activity of cefmenoxime an~ mecillinam against Proteus mirabilis ATCC 21100.
Table 3: Cefmenoxime- and mecillinam~potentiating activity data for the ~2 sample obtained in Example 1 (test organism: Proteus mirabilis ~TCC 21100)*
Table 3 ~2 concen-tration Diameter of growth inhibition zone (mm)*
in test solution (~g/ml) No addition 0.01 ~g/ml 0.05 ~g/ml cefmenoxime mecillinam 1 0 . -~
0 < 8 ~ 8 < 8 1 < 8 9 9 5 < 8 13 11 25 < 8 23 19 15 125 < 8 30 26 1000 < ~ 38 32 .. .. _ _ _ _ . . . . . _ * The test was performed under the same conditions as noted for Table 1 except for the test organism and t'ne concentration of each coexisting antibiotic.
Table 4: Cefmenoxime- and mecillinam-potentiating activity data for the ~2 sample obtained in Example 3 (-test organism: Proteus mirabilis ATCC 21100)*
Table 4 ----- ~ - _ . . _ ~2 concentration Diameter of growth inhibition zone (mm)~
in test solution ~o addition 0~01 ~g/ml 0 05 ~g/ml cefmenoxime mecillinam . . . _ 0 < 8 < 8 < 8 301 < 8 9 9 5 ~ 8 12 12 25 < 8 22 19 125 ~ 8 29 25 1000 < 8 38 32 _ * The test was performed under the same conditions as noted ~s~s for Table l except for the test organism and the concentration of each coexisting antibiotic.
Furthermore, F2 strongly inhibits the growth of Escherichia coli in synergy with cephalexin or cefmenoxime.
5 In ~able 5 and ~able 6, there are shown degrees of growth of Escherichia coli under the influence of F2 in the copresence of 5 llg/ml of cephalexin or l ~g/ml of cefmenoxime ~able 5: Inhibition of growth of Escherichia coli IF0 12734 in the copresence of F2 (obtained in :Example l) lO and cephalexin or cefmenoxime.
~able Final F2 Absorbance at 600 nm concen-tration No addition 5 ~giml l llg/ml cephalexin cefmenoxime 0 2.6 2035 1.95 2.65 1.80 0.60 2.65 l.00 0.25 lO0 2,70 0.95 0.30 20 ~ -- -~o 8 ml of YAB medium (1.75% Difco antibiotic medium ~o. 3, 0.5% Difco yeast extract) containing 12% (w/v) of sucrose, there were added 1 ml of water9 50 llg/ml cephalexin solution or lO llg/ml cefmenoxime solution, and the medium 25 was inoculated with 1 ml of a preculture in YAB medium at an early logarithmic growth phase and incubated with shaking at 37C for 2 hours. ~hereafter, the absorbance ` of the culture broth was measured at 600 nm using a ~himadzu-Bausch & Lomb Spectronic 20 colorimeter.
~able 6: Inhibition of growth of Escherichia coli IF0 1273L~ in the copresence of F2 (obtained in ~xample 3) and cephalexin or cefmenoxime*
S~OS
able 6 ~inal ~2 Absorbance at 600 nm concentration . .
(~g/ml) No addlt1on 5 ~g/ml 1 ~g/ml cephalexin cefmenoxime 0 2.4 2.15 1 9o 1 2.45 1.75 0.55 2 50 1.90 0.25 100 2.55 0.75 0.25 1 0 -- ' -' ---* The test was performed in the same manner as noted for Table 5.
As is evident from ~able 5 and ~able 6, ~2 inhibits the growth of Escherichia coli in the presence of cephalexin or cefmenoxime, as evidenced by the decrease in absorbance~
Observation of the culture broths under a phase contra~t microscope revealed that, when ~2 was not added, cells were in the much elongated form under the influence of cephalexin or cefmenoxime but that, when ~2 was added, the elongation was inhibited in dependence on the addi.tion level and at the same time partial swelling occurred and bacteriolysis proceeded.
b) ~he antibiotics-potentiating activity data for ~
~he action of ~3, which potentiates the activity of antibiotics, is shown in ~ab].e 7 -to 9.
~able 7 ~he action of ~3 of Example 5 to potentiate the antibacterial action of cefmenoxime and mecillinam.
(~est organism: Escherichia coli I~O 12734) ~able 7 -~
Concentration Diameter of growth inhibi-tion zone (mm)*
of ~3 in test --~
solution** No addition 0 05 ~g/ml of 0 03 ~g/ml of (~g/ml) cefmenoxime mecillinam _ _ o < 8 < 8 < 8 1 < 8 10 < 8 < 8 1205 < 8 `` ~2~S~
-Concentration Diameter of growth i.nhibition zone (mm)*
of F3 in test solution** No addition 0.05 ~g/ml of 0.03 ~g/ml of (~g/ml) cefmenoxime mecillinam < 8 15 9 5 125 < 8 26 17.5 1000 < 8 38 28.5 .
*, ** ; The test was performed under the similar me-thod as noted for Table l.
Table 8 The ac-tion of F3 of Example 5 to potentiate the antibacterial action of cefmenoxime and mecillinam*
(tes-t organism: Proteus m rabilis ATCC 21100) able 8 ~ _ .
Concentration Diameter of growth inhibition zone (mm)*
of F3 in test --solution No addition 0.01 ~g/ml of 0.05 ~g/ml of (~g/ml) cefmenoxime mecillinam _ 0 ~ 8 < 8 ~ 8 1 < 8 9 < 8 ~ < 8 12 9 < 8 21 13.5 125 ~ 828.5 19 1000 < 832.5 30 * Except for the test organism and the concentration of antibiotics to be potentiated, all other assay conditions are the same as those mentioned for Table l.
Table 9 Inhibition of growth of Escherichia coli IF0 _. _ 12734 in -the co-presence o~ F3 (Example 5) and cefmenoxime ~26~5~)5 Table 9 .
~inal concentration Absorbance at 600 r~
of ~3 ~
(~g/ml) No addition 1 ~g/ml of cefmenoxime . _ 0 3.7 2.9 1 3.7 2.2 3.7 0.6 100 3,7 0.6 '' ' ~
The test was performed in a manner similar to that described for ~able 5.
~ s is evident from Table 9, ~3 inhibits the growth of ~scherichia coli in the presence of cephalexin or cefmenoxime, as evidenced by the decrease in absorbance.
Observation of the culture broths under a phase contrast microscope revealed that, when ~3 was not added, cells were in the much elongated form under the influence o~
cephalexin or cefmenoxime but that, when ~3 -was added, the elongation was inhibited in dependence on the addition level and at the same time partial swelling occurred and bacteriolysis.
c) Antibiotics-potentiating activit~_data for ~4 Table 10 Cefmenoxime- and mecillinam-potentiating activity data for the ~4 sample obtained i.n E~ample 7 (test organism: ~scherichia coli I~O
12734) Table 10 .
. . ~. . . ._ _ . _ . . ~ . _ ~
o ~4 concentration Diameter of growth inhibition zone (mm)*
3 in tes-t solution~* -- ----- - -(~Ig/ml) No addition 0.05 ~g/ml 0 03 ~g/ml cefmenoxime mecillinam . . .
O ~ 8 < 8 ~ 8 < 8 < 8 < 8 35 100 < 8 15 13 1000 < 8 26 23 *, ~*: ~he test was performed under a method similar to that described for ~able 1 ~ urther, the compound of the formula (I) showed a marked antibacterial activity in synergy with cefmenoxime or cephalexin in Escherichia coli- or Proteus mirabillis-.
infected mice. ~`herefore, -the compound of the formula (I) can be used in combination with such an-tibiotics in the treatment of infectious diseases caused by the above bacteria in ma~mals (e.g. mouse, rat, dog, human) and in domestic fowls (e.g. chicken, duck) ~ or the treatment of Escherichia coli infection, for instance, a solution of 0.5-2.0 g of, for example, cefmenoxime and 0.5-10 g of the compound of the formula (I) in 10-100 ml of physiological saline is administered to human adults two to three times a day in admixture with an injectable solution for intravenous drip infusion.
~he compound of the present invention is also a very promising intermediate for the synthesis of novel drugs.
When the compound of the present invention was intravenously administered to mice, the LD50 was more than 1 g/kg.
~he following examples illustrate the invention in more detail However, they should by no means be construed as limiting the present invention~ ~nless otherwise sta-ted, each percentage value is on an weight/volume basis.
Example 1 ~wo 2-liter Sakaguchi flasks each containing 500 ml of a mediu~ containing 1~ glucose, 0.5% Polypepton (Daigo Nutritive Chemicals, Ltd.), 0 5% meat extract and 0.5%
sodium chloride (pH 7.0) were inoculated with cells of Pseudomonas acidophila G-6302 strain (~ERM-P ~o. 4344; I~0 13774; A~CC 31363) grown on a slant nutrient agar and incubated at 28C with reciprocal shaking for 48 hours to give a seed culture~
s A 200-liter stainless steel fermentor was charged with 120 liters of a medium containing 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5% meat extrac-t, 0.5/0 NaCl and 0 1%
cys-teine, and the medium was adjusted to pH 7 0 with 30%
sodium hydroxide solution, then steam-sterilized at 120C
for 20 minutes, and inoculated with the above seed culture.
Incuba-tion was carried out at a temperature of 28C, a rate of aeration of 120 liters/minute and a rate of stirring of 180 rpm for 78 hours ~he culture broth was centrifuged on a Sharples centrifuge for separation of bacterial cells.
~here was obtained 110 liters of superna-tant, which, ~7 after adaustment to pE 4 . 2 1 was passed through a column ~:~ packed with 15 liters of activated carbon ("Shirasagi'~for chromatographic use, ~akeda Chemical Industries, ~-td.) for adsorption of the active substance. After washing with 45 liters of water, elution was conducted with 45 liters of 50 v/v % acetone-water. An aliquot of each 10-liter eluate fraction was tested for the active substance using a bouillon agar medium containing 0.05 ~g/ml of cefmenoxime with ~scherichia coli IF0 12734 as the test organism. As a result, fractions Nos. 2 and 3 were pooled and, after addition of 20 liters of water, passed through a column packed with 10 liters of Dowex-l (C1) (Dow Chemical, USA).
After washing with 25 liters of water, elution was carried out with 50 liters of 5% sodium chloride solution in water.
~he active fractions were pooled and, after adjustment to pH 4.0, passed through a column packed with ac-tivated carbon (~ liters). After washing with 24 liters of water, elution was performed with 20 v/v % methanol-water. ~he active fractions were collected and concentrated under reduced pressure to 50 ml, 200 ml of acetone was added, and the resulting precipitate was collected by fil-tration, washed with 50 ml of acetone and 100 ml of ether and dried under reduced pressure to give 25 g of crude F2.
In 500 ml of M/25 phosphate buffer (pH 6.6) was dissolved 10 g of the crude ~2 and the solution was passed ~ ~r~ Mc~
5~3'5 through a 200-ml column of QAE Sephadex A-25 (Pharmacia, Sweden) buffered with the same buffer as above, for adsorption of ~2. The column was washed with 400 ml of the same buffer and then with 400 ml of the same buffer with 0.5% of sodium chloride added thereto. Thereafter, elution was carried out with the same buffer with 1% of sodium chloride added thereto. The active fractions were pooled and, after adjustment to pH 3.0 with 1 N hydrochloric acid, passed through a 60-ml column of activated carbon.
The column was washed with 200 ml of water and then with 100 ml of 50 v/v % methanol-water, Elution was carried out-with 8.0 v/v % isobutanol-water. The active eluate fractions were pooled and, after ad~justment to pH 5.5, concentrated under reduced pressure. The concentrate was dissolved by addition of 50 ml of methanol, and the solution was filtered and allowed to stand in a cool place.
The resulting crystalline precipi-tate was filtered off, washed with ether and dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours. There was thus obtained 2. 8 g of the monosodium salt of ~2 as crystals.
Its infrared absorption spectrum is shown in ~ig. 1, Elemental analysis: C, 32.11; H, 5.38; N~ 7.27; $~ 10.67;
Na, 3.8%
Example 2 In 90 ml of water was dissolved 3.0 g of the monosodium salt of ~2 obtained in Example 1. After 4.5 ml of 1 N sodium hydroxide was added thereto under cooling, the solution was carefully adjusted -to pH 7.0 with 1 N
sodium hydroxide while measuring the pH and then was lyophilized to give 3.1 g of the disodium salt of F2 as a white powder. An infrared absorp-tion spectrum for this product after drying at 40C under reduced pressure for 6 hours in shown in ~ig. 2. Elemental analysis gave the following results: C, 31.35; H~ 5.24; N, 6.78;
S, 10.38; Na 7.1%.
~2~S~
xample 3 Two 2-liter Sakaguchi flasks each containing 500 ml of a medium containing 1% glucose, 0. 5% Polypepton (Daigo Nutritive Chemicals, Ltd.), 0.5% meat extract and 0.5%
sodium chloride (pH 7.0) were inoculated with cells of Pseudomonas mesoacidophila ~B-72310 (FERM-P No. 4653; IF0 13884; ATCC 31433) grown on a slant nutrient agar and incubated at 28C wi-th reciprocal shaking for L~8 hours to give a seed culture.
A 200-liter stainless steel fermentor was charged with 120 liters of a medium containing 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5/0 meat extract, 0 5% NaCl and 0.1% cysteine, and -the medium was adjusted to pH 7.0 with 30% sodium hydroxide solution, then steam-sterilized at 120C for 20 minutes and inoculated with the above seed cul-ture. Incubation was then carried out at a temperature of 2~C, a rate of aeration of 120 liters per minute and a ra-te of stirring of 180 rpm for 78 hours. The culture broth was centrifuged on a Sharples centrifuge Eor separation of bacterial cells. ~here was obtained 110 liters of supernatant, which~ after adjustment to pH 4.2, was passed through a column packed wit'n 15 liters of activated carbon ("Shirasagi" for chromatography, Takeda Chemical In Industries, Ltd.) for adsorption of the active substanceO
Af-ter washing with ~5 liters of water, elu-tion was performed with LL5 liters of 50 V/V % acetone-water An allquo-t of each 10-liter eluate fraction was -tested :Eor -the active substance using a bouillon agar medium containing 0.05 ~g/ml of cefmenoxime with Escherichia coli IF0 1273L~ as the test organism. Fractions Nos. 2 and 3 were pooled and, after addi-tion of 20 liters of water, passed through a column packed with 10 liters of Dowex-l (Cl) (Dow Chemical, USA). Af~ter washing with 25 liters of water, elution was performed with 50 liters of 5% sodium chloride solution in water. The active fractions were pooled, adausted to pH 4.0 and again passed through an activated carbon column (8 liters). Following washing with 24 liters of water, elution was performed with 20 v/v %
methanol-water. ~he active fractions were pooled and concentrated to 50 ml under reduced pressure, 200 ml of acetone added, and the resulting precipi-tate was collec-ted by filtration, washed with 50 ml of acetone and 100 ml of ether and dried under reduced pressureO
There was obtained 21 g of crude product.
In 500 ml of M/100 phosphate buffer (pH 6.6) was dissolved 10 g of the crude product and the solution was passed through a 200-ml column of QA~ Sephadex A-25 (Pharmacia, ~weden) buffered with the same buffer as above, for adsorption of F2. ~he column was washed with 400 ml of the same buffer and then with 400 ml of the same buffer with 0.5% of sodium chloride added thereto. ~hen, elution was performed with the same buffer with 1% of sodium chloride added there-to. ~he active fractions were pooled, adjusted, to pH 3.0 with lN hydrochloric acid and passed through a 60 ml column of activated carbon~ The column was washed with 200 ml of water and elution was carried out with 8 v/v % isobutanol-water. The active fractions were pooled, adjusted -to pH 5.5 and concentrated under reduced pressure. ~he concentrate was dissolved by addi-tion of 50 ml of me-thanol and the solution was allowed to stand in a cool place. ~he resulting crystalline precipitates were collected by filtration, washed with ether and dried over phosphorus pentoxide at 40C under reduced pressure for 6 hours ~'here was obtained 1.5 g of -the monosodium salt of F2 as crystals. Its illfrared absorption spectrum 30i is shown in Fig. 3. ~lemental analysis: C, 32.59; H, 5.22;
N, 6.93; S, 10~14; Na, 3.7%.
EYample 4 In 30 ml of water was dissolved 1.0 g of t~e monosodium salt of F2 as obtained in EYample 3. Following addition of about 1.5 ml of 1 N sodium hydroxide under cooling, the solution was carefully adjusted to pH 7.0 while measuring the pH. The thus-neu-tralized solution was lyophilized to give 1.05 g of the disodium salt of ~2 as white powder. An infrared spectrum of this product after drying at 40C for 6 hours is shown in Fig. 4 Elemental analysis gave the following data: C, 30.83; H, 5.35; N, 6.91;
S, 9.68; ~a, 7.3%.
Example 5 10 Cells of Pseudomonas mesoacidophila SB-72310 (~.RM-P No. 4653; I~0 13884; ATCC-31433) were inoculated into two 2-liter Sakaguchi flasks each containing 500 ml of a medium composed of 1% glucose, 0.5% Polypepton (Daigo Eiyo Chemical Co., Ltd.), 0.5% meat extrac-t and 0.5% sodium chloride (pH 7.0) and the inoculated flasks were incubated on a reciprocating shaker at 28C for 48 hours to prepare a seed inoculum.
Then a 200-liter stainless steel fermentor was charged with 120 ~ of a medium composed of 3% glycerol, 0.1% glucose, 0.5% Polypepton, 0.5% meat extrac-t, 0.5%
NaCl and 0.1% cysteine, the pH of which was adjusted to pH 7 with 30% sodium hydroxide. The fermentor was steam-sterilized at 120C for 20 minutes, after which the medium was inoculated with the seed inoculum, Cul-tivation was conducted at 28C with 120 ~/min. aera-tion and at 180 r.p,m. for 78 hours. The culture broth was centrifuged with a Sharples centrifuge to separate the cells to recover 110 ~ of a supernatant. It was adjusted to p~ 4.2 and passed through a column of 15 ~ ac-tivated carbon (Chromatography grade Shirasagi, Takeda Chemical Industries, ~td.) to adsorb the active compounds. The column was rinsed with 45 ~ of water and elution was carried out with 45 ~
of 50 v/v % aqueous acetone. The eluate was collected in 10 ~ fractions and -the active fractions were detected ~5 using plates of broth agar containing 0.05 g/ml of cefmenoxime and, as the test organism, Escherichia coli ~s~
I~0 12734. ~raction Nos. 2 and 3 were pooled, diluted with 20 ~ of water and passed through a column of 10 Dowex-l(Cl) (Dow and Chemical, U.S.A.). After the column was rinsed with 24 ~ of water, elution was carried out with 20 v/v % aqueous methanol. The active fraction was concentrated under reduced pressure to 50 ml, followed by addition of 20~ ml of acetone. The resultant precipitate was recovered by filtration~ washed with 50 ml of acetone and 100 ml of ether, and dried in vacuo to give 21 g of a crude product.
Ten (10) grams of this crude product was dissolved in 500 ml of 1/100 M-phosphate buffer (pH 6.6) and passed through a column of 200 ~1 QAE-Sephadex A-25 buffered with the same buffer as above. The column was washed with lOOOml of the same buffer as above and eluation was carried out with 2000 ml of the same buffer containing 0.5% of sodium chloride. The fractions showing F3 activity were pooled, adjusted to p~I 3 0 with lN-HCl, and passed through a column of 40 ml activated carbon.
The column was rinsed with 200 ml of water and elution was carried out with 50 v/v % aqueous methanol. The active fractions were pooled, concentrated under reduced pressure and lyophilized. The white powder thus obtained was dried over phosphorus pentoxide under reduced pressure at 40C for 6 hours to give 0.85 g of a powder (sodium salt of ~3). The infrared absorption spectrum (KBr) of this powder is shown in ~ig. 5.
Elemental analysis: C, 32.08; H9 5.33; N, 5.17; S, 6.28;
Na, 4 60%
3o EXample 6 Cells of Pseudomonas acidophila G-6302 (~ERM-P
No~ 4344; I~0 13774; ATCC-31363) grown on a nutrient broth agar slant were used to inoculate two 2-liter Sakaguchi flasks each containing 500 ml of a medium composed of 1% glucose, 0.5% Polypepton, 0.5% meat extract and ~?S~OS
0.5% sodium chloride (pE 7.0) and the inoculated medium was incubated on a reciprocating shaker at 28C for 48 hours to give a seed inoculum.
Separately, a 200-liter stainless steel fermentor was charged with 120 ~ of a medium composed of 3%
glycerol, 0.1/0 glucose, 0,5/0 Polypepton, OD50/O meat extract, 0.5% ~aCl and 0,1% cysteine and after adjustment to pH 7.0 with 30% sodium hydroxide was s-team-sterilized at 120C for 20 minutes. '~he fermentor was then inoculated with the above seed inoculum and incubation was carried ou-t at 28C and 180 r.p m. with 120 ~/min. aeration for 78 hours. ~he resulting broth was centrifuged with a Sharples centrifuge to separate the cells and give 110 ~ of a supernatant ~his supernatant was adjusted 15 to pH 4 2 and passed through 15 ~ of activated carbon (Chromatograph~ grade Shirasagi, ~a~eda Chemical Industries, ~td.). After an aqueous rinse with 45 ~ of water, elution was carried ou-t with 45 ~ of 50 v/v % aqueous acetone.
The eluate was collected in lO~liter fractions which were assayed for activity using plates of broth-agar containing O, 05 ~g/ml of cefmenoxime and, as the test organism, ~scherichia coli IF0 12734. Fraction Nos. 2 and 3 were pooled, diluted with 20 ~ of water and passed through a column of 10 ~ Dowex l(Cl) (Dow and Chemicai, U~S.A.).
The column was rinsed with 25 ~ of water and elution was performed with 50 ~ of 5% aqueous sodium chloride. ~'he active fractions were pooled, adjusted to pH 4.0 and passed again -through a column (8 ~) packed with ac-tivated carbon. After the column was rinsed with 24 ~ of water, elution was carried out with 20 v/v % aqueous methanol.
~he active fractions were pooled and concentrated under reduced pressure to 50 m1. ~0 the residue was added 200 ml of acetone and the resultant precipirate was recovered by filtration, washed with 50 ml of acetone 35 and 100 ml of ether, and dried in vacuo to give 25 g of a crude product.
- 3o -A 10 g portion of the crude product was dissolved in 500 ml of M/100 phosphate buffer (pH 6.6) and the solution was poured on a column of 200 ml QA~-Sephade~
A-25 buffered with the same buffer as above to have the product adsorbed thereon~ The column was washed with 1000 ml of the same buffer as above and elution was carried out with 2000 ml of the same buffer containing 0 5% of sodium c'nloride. ~ractions containing F3 activi-ty were pooled, adjusted to pH 3.0 with lN-HCl, and passed through a column of ac-tivated carbon The column was rinsed with 200 ml of water and elution was carried out with 50 v/v %
aqueous methanol The active fractions were pooled, concentrated under reduced pressure, and lyophilized The resulting white powder was dried in vacuo over phosphorus pentoxide at 40C for 6 hours to give 1.3 g of a powder (Sodium salt of ~3).
~lemental analysis: C, 32.28; H, 5 53; N, 5 18; S, 6 25;
Na, 4.52%
Example 7 Monosodium salt of ~2 (5.9 g), which was obtained by the method of Example 1, was dissolved in 150 ml of lN-hydrochloric acid~methanol, and then treated at gooc for 4 hours After methanol was distilled ou-t, the residue was dissolved in 100 ml of water and passed -through a column (100 ml) of Amberlite IR-45(OH ). The solution which passed through the column, together with washings, was again passed through a column (100 ml) of Amberlite IRC-50 (H+). The solution which was -thus passed the column, together with washings, was diluted to 500 ml wi-th water, and then passed through a column (100 ml) of Amberlite IRA-68 (OH ) The col~n was washed with 100 ml of water, elution was carried out with lN-aqueous acetic acid. The elua-te, together with washings, was concentrated. The residue was dissolved in 10 ml of water, and the solution was subjected to a column chromatography of ~ephadex ~H-20 (3.3 x 50cm). Elution was carried out with 10/c methanol-water, fractions of 210 to 230 ml were pooled and concentrated under reduced pressure, and lyophilized. The resulting powder (1.0 g) was treated with ethanol. ~hus, 0.78 g of ~4 was obtained as crystaline solid.
Specific rotation: (~)23 + 1.4 (c=l.O, N-CH3COOH) Elemental analysis, for C16H29N3011S H20 :
Calcd. (/0): C, 39.25j H, 6.38; ~, 8.59; ~, 6.55.
Found (%): C, 38.91; H, 6.L~2; N, 8.47; S, 6.16.
Infrared absorption spectrum:
An absorption spectrum as ob-tained by the potassium bromide disc method is shown in ~ig. 6.
Amino acid analysis (hydlysis with 6N-HCl at 110C for 8 hours):
D-glucosamine, 0.92; taurine, 1.00; 4-hydroxy-5-hydroxymethylproline, O.85 (average recovery 85%).
Example 8 ~odium salt of ~3 (70 mg), which was obtained by the method of Example 5, was dissolved in 17 ml of 0.2N-hydrochloric acid, and then was allowed to stand at 90C for 2 hours. ~he solution was neutrarized with 3.4 ml of lN-sodium hydroxide, and passed through a column (50 ml) of activated carbon. ~he column was washed with 150 ml of water, and then elution was carried out with 8% aqueous iso-butanol. ~he fractions of 100 to 250 ml were pooled and concentrated under reduced pressure, the obtained residue was dissolved in a small amount of wa-ter. ~he solution was subjected to a col~lmn chromatography of 50 ml QAE-~ephadex A-25 buffered with 0.05M-phosphate buffer (pH 6.1). ~lution was carried out with the same buffer as above, the fractions of 19 to 54 ml were pooled and the solution was passed through a ~5 column (20 ml) of activated carbon. ~he column was washed wi-th 60 ml of water, and the elution was carried out ~2~5~
with 8% aqueous iso-butanol. ~he fractions of 40 to 70 ml were pooled, and iso-butanol was ditilled out, the residue was lyophilized. ~h-us, 28 mg of F5 was ob-tained as white powders.
Elemental analysis, for C14H240~N2 H20 :
Calcd~ (%) : C, 43.97; H, 6.85; N, 7.~2.
Found (%) : C, 44 05; H, 6.93; N, 7.10.
Infrared absorption spectrum:
An absorption spectrum as obtained by the pottasium bromide disc method is shown in Fig 70 ~LC Rf : ~he Rf values on the silica gel plate (Merc~, 60~ 254) Rf = 0.14 (n-propanol : water = 4 : 1) Rf = 0.22 (n-propanol : acetic acid : water = 2 : 1 : 1) Amino acid analysis (hydrosis with 6N-HCl at 110C for 14 hours) :
Glucosamine, 1.00; 4-hydroxy-5-hydroxymethyl proline, 0.93 (average recovery 82%).
Brief Description of the Drawings Fig. 1, 2, 3, 4, 5, 6 and 7 show infrared absorption spectra of the monosodium salt of F2 as obtained in Example 1, the disodium salt of F2 as obtained in Example 2, the monosodium salt of ~2 as obtained in Example 3, the disodium salt of F2 as obtained in Example 4, the sodium salt of F3 as obtained in Example 5, the F4 as obtained in E~ample 7 and -the F5 as obtained in E~ample 8, respectively.
Claims (25)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing a compound of the formula I or a pharmaceutic-ally acceptable salt thereof:
(I) wherein R1 is MHCH2CH2SO3H or OH and R2 is SO3H or H which process comprises cultivating a microorganism which belongs to the genus Pseudomonas and is capable of producing a compound of formula II
(II) Wherein R1 is NHCH2CH2SO3H or OH, in a culture medium and harvesting from the resulting culture broth the compound of said formula II thus produced as a metabolite, and where required subjecting a compound of formula II or a salt thereof, to desulfation to obtain a compound of formula I in which R2 is H, and where required forming a pharmaceutically accept-able salt of a compound of formula I or II.
(I) wherein R1 is MHCH2CH2SO3H or OH and R2 is SO3H or H which process comprises cultivating a microorganism which belongs to the genus Pseudomonas and is capable of producing a compound of formula II
(II) Wherein R1 is NHCH2CH2SO3H or OH, in a culture medium and harvesting from the resulting culture broth the compound of said formula II thus produced as a metabolite, and where required subjecting a compound of formula II or a salt thereof, to desulfation to obtain a compound of formula I in which R2 is H, and where required forming a pharmaceutically accept-able salt of a compound of formula I or II.
2. A process according to claim 1, wherein the microorganism is Pseudomo-nas acidophila.
3. A process according to claim 1, wherein the microorganism is Pseudomo-nas acidophila G-6302 (ATCC 31363, FERM-P No. 4344, IFO 13774).
4. A process according to claim 1, wherein the microorganism is Pseudomo-nas mesoacidophila.
5. A process according to claim 1, wherein the microorganism is Pseudomo-nas mesoacidophila SB-72310 (ATCC 31433, FERM-P No. 4653, IFO 13884).
6. A compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof whenever prepared by a process according to claim 1, 2 or 3 or by an obvious chemical equivalent thereof.
7. A compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof whenever prepared by a process according to claim 4 or 5 or by an obvious chemical equivalent thereof.
8. A process for preparing the monosodium salt of the compound F2 of the formula (F2) which comprises cultivating Pseudomonas acidophila G-6302 strain (FERM-P No. 4344;
IFO 13774; ATCC 31363) in a suitable culture medium and harvesting the monosodium salt of the compound F2.
IFO 13774; ATCC 31363) in a suitable culture medium and harvesting the monosodium salt of the compound F2.
9. The monosodium salt of the compound F2 as defined in claim 8 whenever prepared by a process according to claim 8 or by an obvious chemical equivalent thereof.
10. A process according to claim 8 further comprising the step of adding sodium hydroxide to the monosodium salt of the compound F2 so obtained to obtain the disodium salt of compound F2.
11. The disodium salt of the compound F2 as defined in claim 8 whenever prepared by a process according to claim 10 or by an obvious chemical equivalent thereof.
12. A process according to claim 8, wherein the microorganism Pseudomonas mesoacidophila SB-72310 (FERM-P No. 4653; IF0 13884; ATCC 31433) is used instead of Pseudomonas acidophila G-6302 strain (FERM-P No. 4344; IFO 13774; ATCC 31363).
13. The monosodium salt of the compound F2 as defined in claim 8 whenever prepared by a process according to claim 12 or by an obvious chemical equivalent thereof.
14. A process according to claim 12 further comprising the step of adding sodium hydroxide to the monosodium salt of the compound F2 so obtained to obtain the disodium salt of compound F2.
15. The disodium salt of the compound F2 as defined in claim 8 whenever prepared by a process according to claim 14 or by an obvious chemical equivalent thereof.
16. A process for preparing a sodium salt of the compound F3 of the formula (F3) which comprises cultivating Pseudomonas mesoacidophila SB-72310 (FERM-P No. 4653;
IFO 13884; ATCC-31433) in a suitable culture medium and harvesting the monosodium salt of the compound F3.
IFO 13884; ATCC-31433) in a suitable culture medium and harvesting the monosodium salt of the compound F3.
17. A sodium salt of the compound F3 as defined in claim 16 whenever prepa-red by a process according to claim 16 or by an obvious chemical equivalent thereof.
18. A process according to claim 16, wherein the microorganism Pseudomonas acidophila G-6302 (FERM-P No. 4344; IFO 13774; ATCC-31363) is used instead of Pseudomonas mesoacidophila SB-72310 (FERM-P No. 4653; IFO 13884; ATCC-31433).
19. A sodium salt of the compound F3 as defined in claim 16 whenever prepa-red by a process according to claim 18 or by an obvious chemical equivalent thereof.
20. A process according to claim 8 further comprising the step of desulfat-ing the monosodium salt of compound F2 as defined in claim 8 to obtain the com-pound F4 of the formula (F4)
21. A process according to claim 20, wherein the desulfation is effected by means of an ion-exchange resin.
22. The compound F4 as defined in claim 20 whenever prepared by a process according to claim 20 or 21 or by an obvious chemical equivalent thereof.
23. A process according to claim 16 further comprising the step of desulfa-ting the sodium salt of the compound F3 as defined in claim 16 to obtain the com-pound F5 of the formula (F5)
24. A process according to claim 23, wherein the desulfation is effected with hydrogen chloride.
25. The compound F5 as defined in claim 23 whenever prepared by a process according to claim 23 or 24 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23099/1982 | 1982-02-15 | ||
| JP57023099A JPS58141786A (en) | 1982-02-15 | 1982-02-15 | Bactericidal activity promoting substance f3 and its preparation |
| JP57232055A JPS59118799A (en) | 1982-12-24 | 1982-12-24 | Substance for enhancing antibacterial action and its preparation |
| JP232055/1982 | 1982-12-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1205405A true CA1205405A (en) | 1986-06-03 |
Family
ID=26360395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000421538A Expired CA1205405A (en) | 1982-02-15 | 1983-02-14 | Substances potentiating the activity of antibiotics and their production |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1205405A (en) |
-
1983
- 1983-02-14 CA CA000421538A patent/CA1205405A/en not_active Expired
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