Note: Descriptions are shown in the official language in which they were submitted.
-- 1 --
~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
-- 2 --
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)
3 CH20H
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):
CH20H O
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,:
CH2H
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
5 -
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~
7 --
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
-- 8 --
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)
6. Infrared absorption spectrum:
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.
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.
10. Color reactions: Positive Greig-~eiback and
potassium permanganate 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.
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~
-- 10 --
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
-- 11 --
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
-- 12 --
~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
- 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
- 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~
-- 16 --
.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
-- 17 --
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
- 18 -
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
- 19 -
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~
- 20 -
-
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
- 21 -
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
- 22 -
*, ~*: ~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
- 23 -
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
24 -
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~
- 25 -
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
- 26 -
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
- 27 -
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~
- 28 -
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
- 29 -
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~
- 32 -
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.
