CA1055415A - Enzymatic production of 7-(3-alkanesulfonamido phenyl-d-glycinamido)-3-desacetoxy cephalosporanic acid - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Novel 7-(3-alkanesulfonamidophenyl-0-glycinamido)-3-methyl-3-cephem-4-carboxylic acid and salts thereof of formula (I):

Description

~(~5541S
'~ '.

~ NEW ACYL DE NV~TIVES OF 7-AMINO-3-- ~ESACETOXYCEPHALOSPORANIC ~CID
' The present invention relates to a ~ew process for enzymatic preparation of new acyl derivatives o 7-amino~
3-desacetoxycephalsporanic acid. . :
- More particularly, it relates to a new process for ~enzymatic preparation of 7-(3-alkanesulfonamidophenyl-D-!- . . .
glycinamido)-3-methyl-3-cephem-4-carboxylic acid from 3-a1kanesulfonamidophenylglycine and 7-amino-3-methyl-3-:
cephem-4-carboxyllc acid by an enzymatic N-acylation.
The object compound of the present invention, 7-:(3-alkanesulfonamidophenyl-0-glycinamido~-3-methyl-3-cephem-4-carboxylic acid, is novel and can be represented by the following formula (I)~

CHCOH ~ C~

whéreln R is an alkyl group.
-There has been publicly known an enzymatic N-acylation . ~ , :, , : .
~ ;; of:7-aminocephem compound with.an organic acid or its~
-1 ~reactive derivative to provide the corresponding 7-acylated i;~
cephem compound. As a example of such enzymatic N-acylation,:
Japanese Patent Early Publication~No. 47-25388 disclcses a . prior knowledge of an enzymatic N-acylation by which a -,.
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10554~5 ~'~' ' ' , cephalosporin compound can be produced by reacting 7-amino cephem compound with ~-substituted~-amino acid in the presence of an enzyme derived from a microorganism.
As a result of the research made on a variety of microorganisms, the inventors of the present invention have newly founded that there widely exist microorganisms which has the activity for acylating 7-amino-3-methyl-3,cephem-4-carboxylic acid with 3-alkanesulfonamidophenylglycine to provide the object compound (I).
Accordingly, it is an object of the present invention .. . ~
to provide a new acyl derivatives of 7-amino-3-desacetoxy ~;~ cephalosporanic acid (I).
Another object of the present invention is to provide ` a new process for the preparation of new acyl derivatives -~ of 7-amino-3-desacetoxycephalosporanic acid;(I~ by an enzymatic N-acylation.
Still another object of the present invention is to provide new process which is simpler, more convenient in operation and industrially more economical for the preparation 5'~ of new acyl ~erivatives of 7-amino-3-desacetoxycephalo-spoxanic acid (I).
';` In the present invention the object compound (I) can be prepared by reacting 7-amino-3-methyl-3-cephem-4-carboxylic acid with 3-alkanesulfonamidophenylglycine in the presence of a cultured broth or its processed material derived from a microorganism.
The reaction of the present invention may be illustrated ~i3 ~ by the following scheme. ~ , ,; . . ' ' ' ~ 2 - ~

.:, .. . . .
.
:
.: ~ . . .

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r ; , ~ i ~ " ~

~s~s COOH

: .
NIIS02R : -.. , 1- .

~3~H-COOII (III) J ~:.
,' . NH2 .. ~ - , .
' - Enzymatic N-acylatlon in the presence of a cultured broth of microorganism or its processed . ~ ~ material.
N ~ 2R

(/ ~ CH-COHN ~ ~
IH2 o~ - N~ CH3 COO

wherein R is ,an alkyl group. ~ -7-amino 3-methyl-3-cephem-4-carboxylic acid of the ormula ~II) (7-ADCA), one of the starting compound to be .. . . . .
used as a substrate is a known compound.
Alkanesulfonamidophenylglycine of the formula (III)~ ;
~is a new compound and can be prepared, for example, by the following processes.

NH

CHO

HS03R or reacti~e derivatives . ~ .--. .... ...
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NIISo R
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. ~ , NH3, ~ICN
N~IS02R

~lCN
, ¦ 2 : :: ' Hydrolysi.s J
. NHSO2R

. . . ~ f~lCOOlI

: wherein R is an alkyl yroup. - ~-Suitable exampIes of an alkyl group for R may be ones having l to 6 carbon atom(s)~, preferably 1-4 carbon atom(s~
including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, i.i - ~ ~ . . ., . : - .
pentyl,~hexyl and the like..

In the reaction of the present invention, the starting ..
: ~ ~ . ~ , - . .
compound of:the formula (II) may~be used in the form of ts salt. And, the starting compound of the formula (III) may be used in the-form of its derivative at thP carboxy~
group. Accordingly, the object compound of the formuia (I) ~ :~

aIso may include its salt, which is also included within S~ . ; the scope of the present invention.
Suitable examples of the said derivative at the carboxy group of the compound (m) May inclu~e the derivative conventionally used in ~reparation of cephalosporanic acid derivatives, ~or example,the acid amide (e.g., acid amide with imidazole, `.

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. ____ _ : ~ - . . ... ,_,_. .... ...

; ` ,: , l~SS~5 acid amide witll 4-substituted imidazole etc.); the acid ester (e.g., substituted or unsubstituted al};yl ester,for ex~nple, mcthyl estcr, ethyl es-ter, propyl estcr, butyl ester, tert.-buts~l estel, cyanomethyl ester, aryl ester , ~or ex~nple, p-nitrophenyl est~r, etc.); and the salt, for exampl~, an inoryanic salt,e.g., al~ali metal salt (~.g., sodium salt, potassium salt, etc.), alkaline earth metal salt ~e.~., ma~nesium salt, etc.) and a~r.onium salt, and an organic salt, for ex~nple~dicyc]ohexylami~ne salt, pyridine salt, ethanolamine :. .
salt, etc.). Among the derivative at the carboxy~roup, .`
an ester may be preferably used as the derivative at the carboxy group of the startin~ compound of the formula (III).
Suitable examples~of salts of the compound (I) and .
(II~ may include the same salt as illustrated in the aboYe.
.,~ , . ~ .
- In the enzymatic N-acylation of the compound (II) with ; ~ the compound (II), the reaction is conducted in the presence .7, ~ ` of a cultur~d ~roth of a microorganism or its processed material.
Such microorganisms exist widely in nature and there may be used all of the microorganisms which have the -; activity for N-acylating the compounds (II) with the compound (III) to produce the object compound (I), which may be , widely distributed in nature or in the culture collections among fungi, yeasts and bacteria. Accordingly, said ~ microorganisms can bé easily isolated from soils or other~
I sources by conve~tional means, or can also be easily selected from the collected cultures available in public facillities for culture collection.

As the representative of suitable examples of said : , . . . .
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Pseudomonas fragi WB2013, Pseudomonas maltophila WB2714, -~ Pseudomonas melophthora W~3113, Xanthomonas citri IFO3835 and ~ Acetobacter acetosus IFO 3129. The former three strains ., _ .
'~ , ,were newly isolated from soil samples by the inve~tors of ~ the present invention and deposited in Fermentation Research '~ , Institute, Agency of Industrial Science & Technology, Japan under FERM-P No. 2703, 2704 and 2705, xespectively.
t~ The latter two strains were selected rom one of said culture collections, Institute for Fermentation (IFO), Osaka, Japan.
3~ ~ ; Microbiological characteristics of the former three strains newly isolated from soil sample by the inve~tors are shown in the following table.

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The present invention may aiso include, within its ~cop~, the use of mutants produced from these microorganisms by conventional means such as X-rays, ultraviolet radiation, treatment with chemicals such as-nitric acid, nitrogen mustard gas, azaserine, 2-aminopurine, N-methyl-N'-nitro-N-nitrosoguanidine ~NTG) and the li~e.
~ owever, at any rate, it is to be understood that a microorganism used in the present invention is not limited to the genus, the species and the mutant thereof as stated above.
According to the present invention, microorganisms can be preferably used in a form of a cultured ~roth, obtained by culturing the microorganism in a suitable manner, or its ., processed material.

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55~15 Cultivation of said microorganisms can be generally conducted in a conventional manner, and it may be advantageously carried out under stirring with aeration. As a culture medium to be used, there may be used a nutrient one containing sources of assimilable carbon and nitrogen and inorganic salts. The preferred sources of carbon are, for example, glucose, sucrose, lactose, glycerol and starch. The preferred sources of nitrogen are, for example, meat extract, peptone, gluten meal, corn meal, cotton-seed meal, soybean meal, -. ~, .
corn steep liquer, yeast extracts, casaminic acid and amino acids, as well as inorganic and organic nitrogen, for example, ammonium salts (e.g., ammonium nitrate, ammonium phosphate, etc.). If desired, mineral salts, for example, calcium ~ carbonate, sodium or potassium phosphate, magnesium salts `6 and copper salts, and various vitamins can be also used.
~ ~ .
~ Suitable pH of the culture media, suitable cultivation .1 , .
temperature and suitable cultivation time vary with the kind of the microorganisms to be used. A desixable pH usually lie~ in a range of pH 6 to 9. The temperature is usually l ~ 20 selected from about 15C to about 38~C, preferably from ;s 27C to 33C. The cultivation time is usually selected ~rom 15 hours to 72 hours, preferably from 24 hours to 50 hours.
~ The cultured broth per se thus obtained and its process-c~ ed material may be employed for the preparation of 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-s ~ carboxylic acid ~ ere, the processed material of cultured broth, means any preparation which contains enzymatic N-acylation activity and has been processed by conventionally 1,~ ~ . .
:, -., ' ~
. - -' ', ' . ' ' ',:' ' . '; :
,,,., . ' , . . , ,, : , -" 105~;5 suitable moans for hcightening ~aid N~acylation activity, can be usedO
The N-acylation activity may be usually present in cells (intracellularly) ancl out of cells (extracellularly).
When the activity exists mainly in cells, the following preparation or example, may be used as a processed material of the cultured broth.
(1) raw cells; separated from the cultured broth in A, ~ conventional manners such as filtration and centrifugation, (2~ dried cells; obtained hy drying said raw cells in conventional manners such as lyophilization and vaccum drying, J
~, (3) a cell~free extract; obtained b~ destroying said raw or dried cells in conventional manners (e.g., grinding the celis with alunina, sea sand, etc. or treating the cells with super sonic waves), or (4~ an enzyme solution; obtained by purification or partial , ~ , , , , , ! . ' ' ~purification of said cell-free extract in a convention manner.
When the ?ctivity exists mainly out of cells, the ~ -following preparation for example, may be used as a processed matOEial.
(1) a supernatant solution or a filtrate; obtained from the cultured broth in a conventional manner, or

2) an-enzyme solution; obtained by purification or~
partial purification of said supernatant or filtrate .
~ in a conventional manner , , . :

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~ ~)55~15 . .
The reaction of the process of the present invention may be conducted by making the starting compounds (II) and ~III1 as substrates contacted with the cultured broth or ; its processed material. The reaction may be conducted in water or a bu~fer solution, that is, it may be usually conducted by di.ssolving or suspending the cultured:broth or i its processed material in water or a buffer solution and - by adding the starting compounds thereto.
Preferable pH of ~he reaction mixture, concentration of substrates, reaction time and reaction temperature may vary with characteristics, especially the activity of a ~-~ cultured broth or its processed materlal to be used. -j Generally, the reaction is carried out at pH between 4 and 9, preferably between S and 7, at temperature between 20C ~

3 ~ and 45C, preferably between 35 and 42C for 1 to 50 hours. ~ -, ~ In the reaction mixture, the concentration of the :
i starting compound (II) as a substrate may be preferably selected from a range of 0.1 to 20 mg/ml and that of the starting compound ~III) as a substrate may be from a range~
of 0.1 to 40 mg/ml.
-The object compound thus produced in the reaction mixture may be isolated and purified in a conventional manner used in cephalosporin synthesis. As the manner, there are exémplified methods of purification with appropriate solvents, i~
concentration under reduced pressure, lyophillzation, -crystalizationj recrystalization and~*reatmen~ with anionic or cationic exchange resin or macroporous nonionic adsorption resin and the like. Generally, the use o~ macroporous 13 ;~
:

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nonionic adsorption resins as a step for purification gives a good result.
The process for the purification using macroporous nonionic adsorption resins are explained in detail as follows.
Suitable examples of such resins may include Ambelite XAD-l, XAD-2 and XAD-4 (trade mark, maker; Rohm &~Haas Co.), Diaion HP10~ HP20, HP30, HP40 and HP50 (trade mark, ~litsubishi Kasei Co., Ltd.).

, ~ .
In this purification operation, when insoluble materials are contained in the reaction mixture comprising the object compound, they may be removed off in conventional manners such as centrifugation, filtration and precipitation with ... ..
solvents. Subsequently, the reaction mixture, which may -~
be purified to some degree, is applied to macroporous , nonionic adsorption resin by batch or column operation.
. .~ . - - .:
Treatment of the reaction mixture with macroporous nonionic :` adsorption resin is advantageously oonductéd at~p~ of neutrality to acidity.
The object compound in the reaction mixture can be ; adsorbed in macroporous adsorption resin. ~he adsorbed - objéct compound in the resin can be eluted with a hydrophilic .;
solvent system.

~ The preferred hydrophilic solvent systems used or ! -eluting the object compound from the resin may include a lower dialkyl ketone ~e.g., acetone and methyl ethyl ketone), a lo~er alkanol (e.g., methanol, ethanol, n-propanol and isopropanol). An appropriate mixtures of the above solvents i::
~ ~ may be also used. ~ ~

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)554~S

The above solvents may be also used in an admixture with water or a lower alkyl ester of a lower alkanoic acid (e.g., ethyl acetate and butyl acetate3.
The eluate thus obtained is treated by conventional means such as concentration, pH adjustment, lyophilization and recrystallization to give crystals of the object compound - (I)o ~ he object compound (I) thus obtained is a new compound and characterized in the following points' 1) an antimicrobial agent for oral use and superior in its .; . .
effectiveness to cephalexin known as an antimicrobial agent for oral use, 2) stronger activities against pathogenic microorganisms, especially Klebslella pneumoniae and Proteus mirabilis than those of cephalexin known as an antimicrobial agent for oral use, and ;
3) superior~characteristics to cephalexin as an antimicrobial agent for oral use in the points of serum level, tissua distribution, biliary excretion and protecting effect 20~ in experimental mouse infection.
In order to demonstrate such a superiority of the object compound o~ the present invention, the pharmacologically comparative tests of one of the object compound (I) with ~ ;
the known cephalexin are shown in the followlnq.
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~5~4~L5 Test 1 (a) Antibiotics tested .7-(3-~5esylaminophenyl-D-~lycinamido)-3-methyl-3-cephem-

4-carboxylic acid.

'. ,~Cephalexin (b) Test animal : ' Animal used in tne test was male rats of SD-strain, weighin~ 180 - 230 g. -All test rats were fasted for 24 hours prior to use. -(cj Method for assay of the antibiotics (~ioassay) :
~-- Each levels of the test antibiotics in the serum, -.. . .
bile and tissues was deterr~lined by ~he following bioassay.
That is, a test agar plate was prepared by pouring 10 ml of sodium Gitrate agar containing 105 spores per ml of Bacillus subtilis ATCC 6633 into a Petri dish.
~Each paper disc (diameter: 8mm) was dipped in the standard solution or test solution of the test antibiotics. After running off the excess solution o the discs, the discs were placed on the surface of the testing agar plate and then incubated at 37C for 20 hours. Thereafter, the diameter of inhibitory zone was measured, fr~m the results of which an~amount of ~he test antibiotic iD test solution was calculated.

~ ~ (1) Determination of serum levels after oral administration of antibiotics in rat .
~ ` 7-~3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-i ~.'. - . : . . :

j. .~, :.:

,~ . ' , ' ' ' " ' ~'; ' . ' ' ` .

:

`\
`- 1055~5 4-carboxylic acid (or Cephalexin) was each orally administered to lO rats at a single dose of lOO mq/kg. Blood specimens were taken at l, 2, 4 and 6 hours a~ter the administration.
The concentrations of the test anti~iotics in the serum were determined by the bioassay. The results are shown in - -the following table.
' , '.

~Iean Serum Level (~'g/~.l) Test antibiotic l hour 2 hours 4 hours 6 hours ~, : _ . , . 7-(3-Mesylaminophenyl ~
~D-glycinamido)-3- - -methyl-3-cephem-4- 49.8 44.8 4l.l 33.0 carboxylic acid ~
~' : - .. ._ ~ . .~ . . .
Cephalexin - 23.l 12.3 6.6 3.9 ;
. ~ ~: '- ' ,' :: , ,~: . ~ . : :- .
:,~ ~ :,. .:
,, -~ (2) Determination of tissue distribution after oral administration of the te t antibiotics in rat 7-(3-mesylaminopheny-l-D-glycinamido)-3-methyl-3-cephem~ ;
,~ ~ . : : - . ~ . , .:
- ~ 4-carboxylic acid ~or Cephalexin) was each orally ad~inistered to 30 rats at a single dose of lOO mg/kg. At l, 2 and 4 hours after the administration, lO rats in each group were i ~ , bleeded to death. The liver, kidneys, lungs, heart and spleen-were each removed, washed with normal saline and then homogenized-~with 2-fold volumes of 99% ethanol. The homogenates / ~
were centrifuged at 10,000 G for lO minutes to obtain the supernatants. The concentrations of the test antibiotics ` ' in the supernatants were determined by the bioassay. The results are shown in the following table.

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5S4~L5 (3) Determination of biliary excretion after oral administration of antibiotics in rats: `
After 10 rats were respectively anesthetized with ether and cannulated with a polyethylene tube by a standard laboratory procedure, 7-(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid (or Cephalexin) was each orally administered to the rats at a single dose of 100 mg/kg. ; `
Bile specimens were collected at the following intervals over a period of 24 hours after administration~ The concentrations of the test antibiotics in the biles were determined by the ~ bioassay. The results are shown in the following table.

., ~.... . _ - _ _ __ ___ . _ . _ ' ~,:
Mean Bile Level (%) -Test antibiotic ~
0-3 hours 3-6 hours 6-24 hours 0-24 hours ~, ~ ___ . _, ~ .
3 - ~ 7 - (3-Mesylaminophenyl ;~~ ; ; -D-glycinamido~-3-nethyl ~ 26.6 15.6 25.4 6706 -3-c-phem-4-carboxylic . _ ._ _ - ~ _ Cephalexin 5.5 2~0 4.6 12.1 ; . , . . , , . _ 20~ ~est 2 (protecting effect in experimental mice infections~
; ` (a) Antibiotics tested~
.7-(3-Mesylaminophenyl-D-glycinamido~-3-methyl-; - , 3-cephem-4-carboxylic acid. ~`-~ Cephalexin. ~ -;1 ~ (b) Test animal :

Male mice of ICR-strain, weighing 20 - 25 g and divided into groups of 10 animals each were used ,, ~ All mice were fasted 24 hours prior to use~
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A prescribed amount of pathogenic bacteria, suspended in ; a prescribed medium, was intraperitoneally injected. One hour after the injection, 7-(3-mesylaminophenyl-D-glycinamido) -3-methyl-3-cephem-4-carboxylic acid (1) [or Cephalexin(2)]
was orally given. These animals were observed for survival or death for 2 weeks, and the ED50 values were calculated by the probit method. The results are s~own in thé following table.
.

.; _ Injected Injected amount of Injection ED50: mg/mouse microorganisms (number of cells/mouse) medium (1) (2) _ _ _ _ _ Staphyloccocus aureus 226 1.8 x 10 B* 1.00 3.40 ': . . . :
~- Escherichia.
~ coli 312 4.7 x 103 M* 0.04 0.10 :' _ _ `~ Klebsiella 3 ~ pneumonlae 6.5 x 10 M* 0.04 0.20 :~ ~ _ _ ' ~ Proteus ~ ~
b~ 6.~ x 106 _ _ _ _ ~.14 L . ~ ~--- B*: Brain heart infusion medium.

M*: 2~ Mucin.

M**: 5% Mucin.

7-~3-masylaminophenyl-D-glycinamido)-3-methyl 3-cephem-4-carboxylic acid was shown superior protective effects to Cephalexin in any test condition mentioned above ~significant difference: P ~0.05). i,-.

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i~S54~5 .
The ~ollowincJ Lxamples are given for the purpose of illustrating the present invention.
' : -Example l A fermentation medium was prepared from the followingin~redients:
Ryokuto powdery Bouillon - 2% by wt.
(Trade mark: Kyokuto pharmaceutical Co., Ltd ~ -~' Yeast extracts 0.3~ by wt.
. - . .
Tap water ~ q,~s.
100 ml of the medium (pH 7.-0~ in each of twenty Sakaguchi-flasks (capacity: 500 ml) were sterilized in a conventional manner and then inoculated with a loopful of 2-day slant ~
cultures of Pseudomonas fragi FERM-P 2703. The organism ~ -was grown in the medium at 30C for 24 hours on a shaker, whereafter cells were collected by centrifugation. The cells wére washed with 500 ml of 0.2 M phosphate buffer solution (pH 5.6) and then suspended in the same buffer solution (pH 5.6~. To the suspension 5100 ml) were added 100 ml of 0.2M phosphate buffer solution (pH 5.6) comprising 2~ methyl ester of 3-mesylaminophenyl-D-glycine and 1% 7-amino-3-: 1: ~ ~ , - . : , , methyl-3-cephem-4-carboxylic acid. The reaction mixture was incu~ated at 37C~for 4~h~urs~on a shaker. In the reaction mixture, 100 ml o~ acetone were added to a part of the reaction mixture (lqO ml). The mixture was stirred for a while and then insoluble materials were filtered off.
The fiitrate was ooncentrated under reduced pressure. The conaentrate (35 ml) was washed twice wi h 10 ml of ethyl acetate. The aqueous layer was separated and concentrated at 35C under reduced pressure. ~he concentrate ~15 ml) '1 , . ...

~ - 21 -",,, ~ , . ~ , .

" , ,, :. - , .
, .. .. . . . .

55~L5 .
was adjusted to pl~ 3.5 with 10% hydrochloric acid. ~rom the solution, unrea~ted starting materials, 7-amino-3-methyl-3-cephem-4-carboY~ylic acid was precipitated and then filt~red off. The filtrate was adjusted to pH 2 with lO~
hydrochloric acid and then passed through a column packed with macroporous nonionic adsorption resin, Diaion HP 20 (25 ml), 100 ml of water, 100 ml of 5% iso~ropanol~aqueous solution, 50 ml of 6.7% isopropanol aqueous solution, 80 ml of lO~ isopropanol aqueous solution, respectively in turn was passed through thevcolumn. Active fractions (150 ml), containing 7-(3~mesylaminophenyl-D-glycinamido)-3-methyl-, , .
3-cephem-4-carboxylic acid, were collected.

The active fractions were concentrated under reduced pressure at 35~C and then crystals were precipitated under ice-cooling.

The crystals were collected by filtration, ~ashed with water and then dried to give crystals of 7-(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid (105 mg).

Ro absorption spectrum (nujol) 3600, 3500, 3350, 1750, 1700, 1600 cm 1 NMR absorption spectrum ~solvent: DCl-D20) ; (PP~

2 . 10 ( 3H ~ s ) ~?~ : 3.20 (3H~ s) J ~ 2H ABqr J=18Hz~

: 5.08 (lH~ d~ J--4.5Hz)

5.52 (lH~ 8) .- 5.70 (lH~ d~
7.53 (4H~ broad s) .
~ `~ 22 -. ~ .
. ~ - ,: .. :
, ' ~ ~ ' ' ' ' .
. ~ .,., , . - , . , ,, , ~F.
~,,. . , ~ .. -., : .

. .

\
1CJ 55~15 l'hin l~er chromato~raphy Carrier: Eastman.cl~romagr~m cellulose sheet No. 6065 (containing fluorescent agent) (trade mark, maker: Eastman-Kodak Company) Developing solvent: a mixture of n-butanol: acetic acid:
- water ~3: 1: 1) Detection: W absorption and bioassay using Bacillus subtilis PCI 219.
Rf value: nearly 0.6.

Example 2 ~ - -, A ermentation medium was prepared from the following ., .
~- 5 - ': ingredients:

Kyo~uto powdery Bouillon - ~ ~ 2% by wt. ~ -~, Yeast extracts 0.3% by wt.

'~ Tap water q.s.

~ ~ Each 100 ml of the medium tpH 7.0) in a Saka~uchi-flask -.
(capacity: 500 ml) were sterilized in a conventional manner and then inoculated with a loopful of each of 2-day slant cultures of Pseudomonas fragi FERM-P 2703! Pseudomona~
maltophila FERM-P 2704, Pseudomonas melophthora FERM-P 2705 and Xanthomonas citri IFO 3835. These~ organisms were grown in the medium at 30C for ~0 hours on a shaker, whéreafter each cells were collected by centrifugation.
The cells were washed with 30 ml of 0.5 M phosphate buffer solution (pH 5.6) and then suspended in 0.2 M phosphate ~buffer solution tp~I 5.6~. To the suspension (3 ml), there .
were added 3 ml of 0.2 M phosphate buffer solution tPH 5.6) comprising 2% methyl ester of 3-mesylaminophenyl-D-glycine A
~ ' , I . , .
i .
~ - - 23 :,~' ' : ' .' lOS59L1 ~ji and 0.4% 7-amino-3-methyl-3-cephem-4-carboxylic acid.
The mixture was incubated at 37~C for 2 hours on a shaker.
In the reaction mixture, production of 7-(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid was confirmed by Rf value showing nearly 0.6 in thin layer chromatography rcarrier: Eastman~chromagram cellul3se sheet No. 606S trademark of Eastman-Kodak Company (containing fluorescent agent), developing solvent: a mixture of n-butanol :
acetic acid ~ water t3 : 1 : 1), detection: W absorption and ~ioautography using Bacillus subtilis PCI 219]. 7-~3-mesyl-aminophenyl-D-glycinamido~-3-methyl-3-cephem-4-carboxylic acid produced in the reaction mixture was detrmined by bioassay using Bacillus subtilis PCI 219. The result was shown in the following table.

:~ , ~ .
, ~ Yield (~g/ml) of 7-(3-mesylaminophenyl-D- -~
Microorganism glycinamido)-3-methyl--~ 3-cephem-4-carboxylic ~!1 ' : - -- . ! _._ -- - , ~ . .
20 ~ Pseudomonas~ fragi FERM-P~2703 1000 Pseudomonas maltophila FERM-P 2704 30 Pseudomonas melophthora FERM-P 2705 1000 ~;~ Kanthomonas citri IF0 3835 200 Example 3 . A fermentation medium was prepared from the following , ingredients:

`I ~ , ~ 2~ -.-~ 7 . . .
':

1055~5 Kyokuto powdery Bouillon 2% by wt.
Yeast extracts 0.3~ by wt.
Glucose 0.5~ by wt.
Glycerol 1.5~ by wt.
Tap water q~s.
100 ml of the medium (plI 7.0) in a Sakaguchi-fl~sk (capacity:
500 ml) were sterilized in a conventional manner and then inoculated with a loopful of 2-day slant cultures of Acetobacter acetosus IF0 3129. The organism was grown in the medium at 30~C for 40 hours on a-shaker, whereafter cells were-collected by centrifugation. The cells were washed with 30 ml of ~-0.05 M phosphate buffer solution (pH 5.6) and then suspended in 0.2 ~I phosphate buffer solution (plI 5.6). To the suspens-on (3 ml), there were added 3 ml o~ 0.2 M phosphate buffer -1 - :
solution (plI 5.6) comprising 2~ methyl ester of 3-mesyl~
aminophenyl-D-glycine and 0.4~t 7-amino-3-methyl-3-cephem-4 carboxylic acid. The mixture was incubated at 37C for ~2 hours on a shaker. I~ the reaction mixture~production of ~
7-(3-mesylaminophenyl-D-glycinamido)-3-methyl~3-cephem- ~-4-carboxylic acid was confirmed by the Rf value showing nearly 0.6 in thin layer chromatography carrie~ out in substantially the same manner as described in Example 2.
7-~3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-4- ~`
carboxylic acid, produced in the reaction mixture, was t~ determined by bioassay using Bacillus subtilis PCI 21~.
,~ Tha yield : 30~ /ml.
~t Example 4 A fermentation medium was prepared from the following ingredients:

. ~
, . , , . ~

- , , . , . : ~

` ~ll0~i5415 Kyokuto pGWdel-y Bouillon 2% by wt.
; Yeast extracts 0.3~ by wt.
100 ml of the m~dium (pH 7.0) in a Sa~aguchi-flas~ (capacity:
500 ml) were sterilized in a conventional manner and then ~ -inoculated with a loopful of 2-day slant cultures of Pseudomonas fra~i FE~M-P 2703; The organism was grown in the medium at 30C for 40 hours on a shaker, whereafter cells ~ -were collected by centrifugation. The cells were washed with 30 ml of 0.05 M phosphate buffer solution (pH 5.6) and , ~ . I
then suspended ln 0.2 ~ phosphate buffer solution (p~ 5.6).
To the suspension (3 ml), there were added 3 ml of 0~2 M
~` ~phosphate buffer solution (pH 5.6) comprising 2% 3-mesyl-aminophenyl-D-glycine and 0.4% 7-amino-3-methyl-3-cephem-4 carboxylic acid. The mixture was incubated a~ 37C for ;~ 2 hours on a shaker. In the reaction mixture~production : ,~ . ..
of 7-(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem--4-carboxylic acid was confirmed by the Rf value showing , t: . : : .
nearly 0.6 in thin layer chromatography carried out in substantially the same manner as described in Example 2.
7-(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem-4-i carboxylic acid, produced in the reaction mixture, was 3~
determined by bioassay using Bacillus subtilis PCI 219.

The yield : 2~ y/ml.

;~ - - Example 5 i :
. i , . .
A fermentation medium was prepared from the following ingredients:

Kyokuto powdery Bouillon 2~ by wt.

Yeast extracts 0.3~ by wt~

. .

.: ; ' ' ' , , ' ,~ ' ~ , ':
;, ~ . ,, :~ . . . .
- , ~ . ' , . . ' . .:

Each 100 ml of the medium (pl~ 7.0) in a Sakaguchi-flask (capacity: 500 ml) were sterilized in a conventional manner and then inocula~ed with a loopful of each of 2-day slant cultures of Pseudomonas fra~i FERM-P 2703 or Pseudomonas melophthora FERM-P 2705. These organisms were grown in the medium at 30C for 40 hours on a shaker, whereafter each -~
cells were removed off by centrifugation. To 3 ml of the supernatant thus obtained, there were added 3 ml;of 0.2 M
, , phosphate buffer solution (pH 5.6) comprising 2% methyl ~
ester of 3-mesylaminophenyl-D-glycine and 0.~ 7-amino-3- -methyl-3-cephem-4-carboxylic acid. The mixture was incubated at 37C for 2 hours on a shaker. In the reaction mixture, production of 7-(3-mesylaminopheny1-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid was confirmed by the R~ value showing nearly 0.6 in thin layer chromatography carried out in substantially the same manner as described in Example 2.
.
, 7-.(3-mesylaminophenyl-D-glycinamido)-3-methyl-3-cephem~4- : .
carboxylic acid, produced in the reaction mixture, was determined by bioassay using Dacillus subtilis PCI 219.
The-result was shown in the following table.

-i, - ~ : , , ; ~ Yielcl (~g/ml) of 7-(3-. mesylaminophenyl-D-Microorganism glycinamido)-3-methyl-3-cephem-4-carboxylic __ _ _ acid Pseudomonas fragi FERM-P 2703 30 -: : .. _ __ _ .. .. . _ .. -~ 2705 30 :
: - ,, ~ 27 , : .. . .

.
,.: ,, ' ,, ' ;
.'~ ., - .~, - , .,. ,,:
';, ", " " ,. ., ,, ' ', .,

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for preparing 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a salt thereof, of formula (I) (I) wherein R is alkyl of 1 to 6 carbon atoms which comprises reacting 7-amino-3-methyl-3-cephem-4-carboxylic acid or a salt thereof with 3-alkanesulfonamidophenylglycine or a derivative thereof at the carboxy group in the presence of a cultured broth, or its processed material, of a microorganism selected from the genus Pseudomans, Xanthomonas and Acetobacter having an activity effective to N-acylate 7-amino-3-methyl 3-cephem-4-carboxylic acid or a salt thereof with 3-alkanesulfonamido-phenylglycine or a derivative thereof at the carboxy group to produce7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a salt thereof.

2. A process according to claim 1, wherein the processed material is a filtrate.

3. A process according to claim 1, wherein the processed material is a supernatant.

4. A process according to claim 1, wherein the processed material is a cell suspension.

5. A process according to claim 1, wherein the microorgan-ism is Pseudomonas fragi FERM-P 2703.

6. A process according to claim 1, wherein the micro-organism is Pseudomonas maltophila FERM-P 2704.

7. A process-according to claim 1, wherein the micro-organism is Pseudomonas melophthora FERM-P 2705.

8. A process according to claim 1, wherein the micro-organism is Xanthomonas citri IFO 3835.

9. A process according to claim 1, wherein the micro-organism is Acetobacter acetosus IF0 3129.

10. 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a salt thereof, whenever prepared by the process of claim 1, or by an obvious chemical equivalent.

11. 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a salt thereof, whenever prepared by the process of claim 5, 6 or 7 or by an obvious chemical equivalent.

12. 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a-salt thereof, whenever prepared by the process of claim 8 or by an obvious chemical equivalent.

13. 7-(3-alkanesulfonamidophenyl-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid or a salt thereof, whenever prepared by the process of claim 9, or by an obvious chemical equivalent.