CA1177832A - ISOMERISATION OF .beta.-LACTAM COMPOUNDS AND NOVEL PRODUCTS OBTAINED THEREBY - Google Patents

Abstract

Isomerization of .beta.-Lactam Compounds and Novel Products Obtained Thereby Abstract of the disclosure Compounds of the formula (I):
(I) wherein R is an ethyl group which may optionally be sub-stituted and n is 0 or 1, or a physiologically acceptable salt thereof, can be produced by subjecting to isomerization a compound of the formula (II):

(II) wherein R and n have the same meaning as defined above, or a salt thereof, by using a quaternary ammonium halide and are useful as a bactericide or disinfectant, and also a synergistic effect with penicillin and/or cephalosporin antibiotic agents.
Among the compounds of the formula (I), compounds shown by the formula:

Description

7~3,~

Isomerization of ~-Lactam Compounds and . . _ _ . . ~ . .
Novel Products Obtained Thereby The present invention relates to a method of producing compounds of the Eollowing formula (I), or pharmaceutically acceptable salts thereof, which have high antimicrobial acitivity and ~-lactamase inhibitory activity.

H~ ~1 C = C
, ~ ' ` (I) N
O COOH
wherein R is an ethyl group which may optionally be substituted and n is O or 1.
In accordance with the present invention, there is provided a process fortha production of a compound of the formula:

H H
C = C
J ~1 ( ) n (I) O COOH

wherein R is an ethyl group or a group of formula III:

R _ C (III) wherein Rl is H or methyl; R2 is H, OH, R3COO- or R503SO-; R3 is R4 or -NHR4; R4 is lower alkyl, lower alkenyl, phenyl-substituted lower alkyl~ phenyl or phenyloxy, wherein phenyl in the last three groups is unsubstituted or substituted by lower alkyl, lower alkoxy ~1~'7~32 or halogen; and R5 is H or lower alkyl or a pharmaceutically acceptable salt thereof, which comprises subjecting to the cis-trans isomerization a compound of the formula:

H~ ,NHCOC 3 R ~ ~ ' ~ (O) H (II) COOH
wherein R and n have the same meaning as defined above, or a salt thereof, by using a quaternary ammonium halide, and if necessary, converting the resulting compound into a pharmaceutically accept-able salt thereof.
A physiologically acceptable salt of the compound of formula I will also be referred to sometimes as compound I here-inafter and a salt of the compound of formula II will also be re-Eerred to sometimes as compound II hereinafter. The isomerization is preferably carried out in an organic solvent. However, solvents of strong polarity, for example methanol and dimethylformamide, are not suitable for the reaction. The compounds of formula I
includes compounds of formula IV hereinafter mentioned.
The present invention also provides a process for the production of a compound of the formula:

,CH3 ~C = C ~
CH3 - C ~ - ~ S~ o NHCOCH3 (IV) R6 - O ~ N

o COOH

';~.
~4., - 2 -~783~

wherein R6 is H or -S03H, or a pharmaceutically acceptable salt thereof, which process comprises: (i) subjecting to the cis-trans isomerization a compound of the formula:

,CH3 H\ -NHcoc 3 R6 /~ N n ~ H
O COOH

wherein R6 is as defined above, or a salt thereof, in an organic solvent except for a strong polar solvent by using a stoichiometric-ally excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, and (ii) if necessary, converting the resulting compound into the free acid or to a pharmaceutically acceptable salt thereof, and the novel compound of formula IV or a pharmaceuticaliy acceptable salt there-of when prepared by the above process.
The present invention includes a process for the pro-duction of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6-(l-hydroxy-l-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, which process comprises: (i) subjecting to the cis-trans isomer-ization [5R, 6R~-3-r(E)-2-acetamidoethenyl-(R)-sulfinyl]-6-(l-hydroxy-l-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-

2-carboxylic acid or a salt thereof, in a water-immiscible organic solvent by using a stoichiometrically excessive amount of quater~ary ammonium chloride or bromide having 13 to 30 carbon atoms for the four substi-tuents, (ii) extracting a quaternary ammonium salt of - 2a -~L77~3~

the resulting compound in tlle organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into -the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt, and the product when prepared by this process.
The present invention also includes a process for the production of [5R, 6R]-3-[(~-2-acetamidoethenyl-(R)-sulfinyl]-6-(l-hydroxysulfonyloxy-l-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid or a pharmaceutically acceptable salt thereof, which process comprises: (i) subjecting to the cis-trans isomerization [5R,6R]-3-[(~-2-acetamidoethenyl-(R)-sulfinyl]-6-(l-hydroxyl-l-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid or a salt thereof, in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, (ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt, and tne product when prepared by this process.

- 2b -~1~77~3~

The present invention further includes a process for the production of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, which process comprises: (i) subjecting to the cis-trans isomeriz-ation [5~, 6R]-3-[(E~-2-2acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxy-l-methyl-ethyl)-7-oxo-l-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid or a salt thereof, in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, (ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the.desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt, and the product when prepared by this process.
The present invention still further includes a process for the production of [5R, 6R]-3-[(Z)-2-acetamido.ethenyl-(S)-sulfinyl]
-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2~carboxylic acid, or a pharmaceutically acceptable salt thereof, which process comprises: (i) subjecting to the cis-trans isomerization [5R, 6R]-3-[(E)-2-acetamidoethenyl-(S)-sulfinyl]-6-(l-hydroxysulfonyloxy-l-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid or a salt thereof, in a water-immiscible . . 2c -7~33;~

organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, (ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolat-ing an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt ofthe desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt, and the product when prepared by this process.
The principle of the above isomerization reaction may be explained as follows. The compound (II) is invariably a water-soluble substance and as such cannot be extracted into a nonpolar solvent by the usual extraction procedure. However, if the com-pound (II) exists together with an excess of a quaternary ammonium halide, the carboxy group and the sulfonyloxy group (if present) which are responsible for the water solubility of compound (II) form quaternary ammonium salts, whereby the lipophilicity of the whole molecule increases to the extent that the compount (II) migrates into a nonpolar medium. And the excess ammonium halide acts selectively on the double bond in the side chain so that the compound (II) of E(trans-)configuratlon is transferred in good yield into the compound (I) of Z(cis-)configuration. As to the addition reaction of the ammonium halide to the sulfonyloxy group and carboxy group, the addition to the sulfonyloxy group tak~s place preferentially owing to the relative - 2d -.

~l~L7~~3Z

acidity of these groups.
The quaternary ammonium halide useful for the purposes of the present invention includes compounds which are commonly used in ion-paired extraction, i.e. the chlorides or bromides of quaternary ammonium compounds having a total of about 18 to 30 carbon atoms for the four substituents (e.g. alkyls of 1 to 25 carbon atoms, benzyl, e~c ), such as tetra-n-pentylammonium, tetra-n-hexyl-ammonium, tri-n-octylmethylammonium, di-n-octylmethyl-ammonium, di-n-decyldimethylammonium, n-hexadecylbenzyldi-methylammonium, n-tetradecylbenzyldimethylammonium, etc. although the chlorides are more satisfactory in the efficiency of extraction and the yield of the reaction.
Such quaternary ammonium halide is used in stoichiometric excess, generally in a proportion of 3 to 1000 molar equivalents based on starting compound (II), ? - 2e -~77~3~

preferably in a proportion of 100 to 1000 molar equivalents based on compound (II) which does not possess sulfonyloxy group and in a proportion of 3 -to 100 molar equivalents based on starting compound (II) possessing sulfonyloxy group.
The organic solvent mentioned above includes chloro-form, dichloromethane, 1,2-dichloroethane, l,l,l-trichloro-ethane, benzene, toluene, ethyl acetate, diisopropyl ether, etc., although such halogenated hydrocarbon as chloroform, dichloromethane and 1,2-dichloroethane are most advantage-ous. In general, a nonpolar solvent or solvent of weak polarity is preferably employed. However, the reaction is not interfered with by the presence of a small proportion of solvents of strong polarity such as methanol, dimethyl-formamide, tetrahydrofuran, etc. in said organic solvents.The organic solvent is generally used in such a proportion that the concentration of said quaternary ammonium halide in the organic solvent is about 0.5 to 3 percen-t. While the reaction is carried out at a tempexature near the boiling point of the solvent, it proceeds even at room temperature (ca. 15C)o The range of 40 to 70C is especially advantageous. The reaction goes to completion in about 30 minutes to 3 days, although the time varies with the temperature and the type of solvent used.
To isolate the product compound (I) from the reaction mixture, the quaternary ammonium salt of compound (I) and the quaternary ammonium halide in the reaction mixture are extracted, for example with an aqueous solution of sodium iodide or potassium iodide, whereby the sodium or potassium salt of compound (I) and the quaternary ammonium iodide are separated into the aqueous layer and the organic solvent layer, respectively. When a quaternary ammonium chloride is employed, it is possible to employ sodium bromide or potassium bromide, for instance.
The aqueous layer containing the compound (I) can then be separated from the starting compound (II), for ~77~3Z

example by chromatography. Suitable chromatographic systems for this purpose may involve the use of a high porous resin HP-20 (Mitsubishi Chemical Industries, Japan), a basic ion exchange resin Sephadex QAE-25 (Cl-form, Pharmacia, Sweden) or activated carbon (Takeda Chemical Industries, Ltd., Japan) as a support in conjunction with water, a suitable aqueous solution of,methanol or butanol, or an aqueous solution containing a suitable inorganic salt as an eluent.
Referring to the formulas (I) and (II), R is an ethyl group or a group of the-formula (III): - -C~3 Rl C ( {Rl is H or methyl; R2 is H, OH, R3COO-[R3 is R4 or -NHR4 (R4 is alkyl, alkenyl, phenyl, or alkyl substituted by phenyl or phenyloxy; provided that said phenyl may option-ally be substituted by lower alkyl, alkoxy or halogen)] or R503SO-(R5 is H or lower alkyl)}. The alkyl R4 is prefer-ably a group of 1 to 6 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl, etc. The lower alkyls R4 and R5 each is preferably a group of up to 3 carbon atoms, such as methyl and ethyl. The alkenyl R4 is a group of up to 6 carbon atoms, such as vinyl, propenyl, butenyl, pentenyl, hexenyl, etc. The alkoxy R4 is a group of up to

3 carbon atoms, i.e. methoxy, ethoxy and propoxy. The halogen R4 is preferably chloro or fluoro.
These starting compounds are described in the speci-fication of Japanese Unexamined Patent Laid-Open No.
144394/1979 and the literature listed in Table 1, or can be prepared from the compounds listed in Table 1 by the method described in the specification of Japanese Unexamined Patent Laid-Open No. 144394/1979.

.

~L~a7~32 Table 1 Code Name of Compound Structural Formula Reference .._ ._ _ . _ IIa C-19393H2 OH 3 (1) IIb C-19393S2 OSO3H CH3 l (2) IIc C-19393E5 OH H
IId MM4550 OS03H H 1 (4) IIe Epithienamycin B OH H O (5) IIf MM13902 OSO3H H O (4) IIg C-19393H2M-1 OH CH3 0 IIh C-19393S2M-1 OSO3H CH3 O
IIi Epithienamycin D OH H O (5) IIj C-19393 iso-H2 OH 3 LIk C-1939l iso-52 0503H CH3 1 References (1) Japanese Unexamined Patent Laid-Open No. 5496/1981 (2) Japanese Unexamined Patent Laid-Open No. 104296/1980

(4) The Journal of Antibiotics 32 295 (1979)

(5) Japanese Unexamined Patent Laid-Open No. 131596/1977 ~77~32 The structural formulas of compounds (I) obtainable by isomerization of compounds (II) are sometimes designated by the codes given in Table 2.

Table 2 Structural Formula _ Code R2 Rl n Reference __ . .... _ Ia OH C~I3 Ib OSO3H CH3 Ic OH H 1 -(8) Id OSO3H H 1 (8) Ie OH H (8) If OSO3H H O (8 Ig OH CH3 o Ih OSO3H CH3 o Ii OH H (8) Ij OH CH3 Ik OSO3H CH3 1 ~ _ 25References:
(8) Japanese Unexamined Patent Laid-Open No. 38371/1980 The above-mentioned compounds (I) and (II) can be fractionally assayed by high-performance liquid chromato-graphy (briefly, HPLC) using Microbondapak C18 or Radial Pak A (Waters Associates Inc.,U.S.A.) as a support, methanol-0.02M phosphate buffer (pH 6.3) as a mobile phase, and a UV (254 nm) detector. The Rt values are shown in the examples which appear hereinafter.
In the context of the present invention, the salts of 11';~7~83~
-- 7 ~

compounds (I) and (II) include the salts of alkali metals such as sodium, potassium, etc. and of alkaline earth metals such as magnesium, calcium, etc.
Among the compounds obtainable by the method of this invention, compounds which may be represented by the formula (IV):
H` ,H
CH3-C ~ ~' \NHCOCH (IV) R60 ~ N COOH

wherein R6 is H or -SO3H, as well as salts thereof, are novel compounds.
The product compounds (I) of this invention are valuable compounds which are active against various gram-positive and gram-negative bacteria and have a strong ~-lactamase-inhibitory activity. These compounds are remarkably superior to the starting compounds in stability in mouse kidney homogenate and, thanks to their increased stability in body fluids, can be expected to display potent therapeutic effects. The antimirobial spectra and in-hibitory activities of the compounds according to this invention are shown below.

(1) Antimicrobial spectrum [MIC (~g/ml)]
. Compound Test Organlsm a Ib Ig Ih Escherichia coli 0.7~ 20 2.5 40 Proteus vulgaris 6.25 40 5.0 >40 IFO 12529 1.56 20 2.5 20 Staphylococcus aureus 0 39 2.5 0.625 2.5 ~.7783~

(2) beta-Lactamase-inhibitory activity [lD50 (~g/ml)]

producing Compound Strain Ia Ib Ig Ih ... . _ _ .. _ Staphylococcus aureus 0.055 0.24 0.36 1.0 Escherichia coli 0.0016 0.000175 0.010 0.017 Enterobacter 0 0050 0.0152 0.030 0.0032 cloacae TN 1282 Proteus vulgaris 0.00105 0.00055 0.017 0.0038 The compounds (I) obtained according to the present invention, as is obvious from the above antimicrobial spectrum, exhibit antimicrobial activity against gram-positive and gram-negative bacteria. Therefore, the compounds (I) can be used for the treatment of bacterial infections in mammals (e.g. mouse, rat, dog, human being, etc.) and domestic animals (e.g. domestic fowl, duck, etcO) To use the compounds (I) as an agent for treating, for example, E-coli infections, the compound (I) is dis-solved in physiological saline solution to prepare an injectable solution which can be administered parenterally, e.g., subcutaneously or intramuscularly at a dose of 0.1 to 200 mg/kg/day, preferably 1 to 50 mgfkg/day. Also, for oral administration, the compound (I) is blended with lactose and encapsulated to prepare a capsule preparation which can be administered at a dose of 1 to 500 mg/kg/day, preferably 5 to 200 mg/kg/day.
Further, the compounds (I) obtained in accordance with the present invention can be used as a disinfectant.
For example, a liquid preparation which can be prepared by dissolving the compound (I) in distilled water at a concen-tration of 0.01 to 1.0 w/v % or an ointment containing 0.5 to 50 mg, preferably 2 to 20 mg, the compound (I) per 1 g of white petrolatum or lanolin as a base can be used as a ~1~7~3~

g bactericide or disinectant for hands, legs, eyes, ears, etc. of the above animals.
The compounds (I) exhibit a beta-lactamase inhibitory activity and, thereEore, markedly increase the sensitivity of penicillin- or cephalosporin-resistant bacteria to ampicillin or cefotiam due to its ability to produce beta-lactamase. Accordingly, the compounds (I) can be used for treatment of infections in mammals (for example, mouse, rat, dog, human being) and avian species (for example, domestic fowl, duck), in particular, bacterial infections due to beta-lactam antibiotic-resistant bacteria, in combination with penicillin or cephalosporin antibiotics.
When the compound (I) is used in combination with other beta-lactam type ag~nts for the treatment of in-fections by, for example, beta-lactam antibiotic-resistant E. coli, equal amounts of the compound (I) and ampicillin are dissolved in physiological saline to prepare an inject-able solution which can be administered parenterally, e.g., subcutaneously or intramuscularly, at a dose of 0.1 to 20 20 mg/kg/day, preferably 0.5 to 5 mg/kg/day. The compound (I) can also be administered orally at a dose of 1 to 200 mg/kg/day, preferably 5 to 100 mg/kg/day as capsules each containing an equal proportion of the compound (I) and cephalexin.
When the compound (I) is used as disinfectant, a liquid preparation, for example, an aqueous solution con-taining the compound (I) at a concentration of 0.1 to 10 W/V ~ and benzylpenicillin at a concentration of 0.1 to 1.0 W/V ~, or an ointment containing 5 to 20 mg of the compound (I) and 5 to 20 mg of benzylpenicillin per 1 g of white petrolatum or lanolin as a base can be used as a bactericide or disinfectant for hands, legs, eyes, ears, etc. of the above animals.
The compounds (I) are also expected to be very useful as an intermediate for the synthesis of novel types of pharmaceuticals. The compounds of the present invention ~7~3~2 are stable in aqueous solution in a neutral pH region.
The following production example and example illus-trate in more particular the practice of -the present invention, but are not intended to limit it. The term "percent" in the production example designates weight/volume ~, unless otherwise specified.

Production Example 1 Streptomyces sp. C-19393 (FERM-P No. 4774, IFO 13886, ATCC 31486) was grown on 200 ml of T culture medium [the medium comprises 2 ~ oatmeal, 2 ~ tomato paste, 0.2 ~
bovril (manufactured by Bovril, England) and 2 % agar and has a pH of 7.0] in a one-liter conical flask to cause sporulation. The spores were suspended in sterile water to a viable count of 1.2 x 108/ml. The spore suspension was diluted 10-fold with sterile water and 1 ml of the dilution was used to inoculate 40 ml of a seed medium in a 200-ml conical flask, which was incubated at 28C on a rotary shaker for 2 days. The resulting culture fluid was trans-ferred to a 2-liter Sakaguchi shake flask containing 500 ml of the same seed medium as above and cultivated at 28C on a reciprocating shaker for 2 days. The culture was further transferred to a 200-liter stainless steel fermenter containing 100 liters o said seed medium supplemented with 50 ml of Actocol (Takeda Chemical Industries, Ltd., Japan)and cultivated at 28C, 70 liters/min aeration and 150 r.p.m. for 2 days.
Then, the culture was transferred to a 6-m3 fermenter containing 4 m3 of a main culture medium and grown at 30C, 2800 liters/min aeration and 150 r.p.m. for 3 days. The seed medium mentioned above contains per liter 20 g of glucose, 30 g of soluble starch, 10 g of raw soybean 1Our, 10 g of corn steep liquor, 5 g of Polypepton (Daigo Nutritive Chemicals, Ltd.), 3 g of sodium chloride and 5 g of precipitated calcium carbonate (adjusted to pH 7.0 before sterilization~. The main culture medium contained per liter 30 g of glucose, 30 g of soluble starch, 15 g of raw soybean flour, 15 g of ~h rk 77~3Z

cottonseed flour, 0.25 g of potassium dihydrogen phosphate, 0.6 g of potassium monohydrogen phosphate, 0.002 g of cobalt chloride and 0.5 g of Actoccol (adjusted to pH 7.0 before sterilization). These media were all steam-sterilized at 120C for 20 minutes. The fermentation broth thus obtained was filtered with Hyflo-Supercel (Johns Manville Co., U.S.A.) and the filtrate (40Q3 Q) was adjusted to pH 6.3 and passed through a column of Amberlite IRA-402 (Cl -form, Rohm and Haas Co., U. S. A.). After washing the column with 200 Q of 0.02 M
aqueous NaCl, elution was carried out with 1000 Q of 1.5 M
NaCl. The eluate was passed through a column of HP-20 (70 Q) and the antibiotic activity was eluted with 280 Q
o~ water. The eluate was passed through a column of activated carbon (15 Q) and after washing the column with 45 Q of water, the antibiotic activity was eluted with 60 Q
of 7% isobutanol. The eluate was concentrated to 10 Q and 500 g of NaCl was added to the residue. The mixture was passed through a column of HP-20 (6 Q) and elution was carried out with 36 Q of 5% NaCl. The eluate was passed through a column of activated carbon (3 Q). The column was washed with 7.5 Q of water and, then, elution was carried out with 8% isobutanol. The eluate was concentrated to 8 Q
under reduced pressure and, then, passed through a column of Diaion WA-30 (acetate-form, Mitsubishi Chemical Industries, Japan) (500 mQ). The column was washed with 2.5 Q of 0.2 M
acetic acid-sodium acetate buffer and the activity was eluted with 5 Q of 1 M NaCQ in the same buffer.
The eluate was passed through an activated carbon column (500 ml). After the column was washed with 5% NaCl, elution was carried out with 2.5 Q of 5% aqueous NaCl-methanol (4:1). The methanol was distilled off under reduced pressure and the residue was passed again through a carbon column (200 ml). The column was washed with 600 ml of H2O and 600 ml of 20% aqueous methanol, followed by elution with 600 ml of 8% isobutanol. The elua-te was concentrated under reduced pressure, the residue was treated m ~ ~k 1~77F~32 with acetone and the resultant powder was collected (580 mg). The powder was dissolved in a small amount of water and passed through a column of Amberli-te XAD-II
(100-200 mesh,Rohm and Haas Co., U.S.A.) (360 ml).
Fractional elution was carried out with water and the active fractions were pooled, concentrated to dryness, and treated with acetone to give 100 mg of powder. The powder was dissolved in a small amount of water and passed through a column of Sephadex QAE-25 (C1 -form) (40 ml). Elution was carried out with 0.04 M phosphate buf~er and the active fractions were pooled and subjected to liquid chromato-graphy as described hereinbefore. The fractions giving a single peak were pooled and passed through a column of 10 ml activated carbon. The carbon column was washed with 30 ml of water and the activity was eluted with 50 ml of 8% isobutanol. The eluate was concentrated to dryness and acetone was added to the concentrate. The above procedure gave 20 mg of C-19393 E5 sodium salt.
, Production Example 2 Crude powder (30% of purity, 8 mg) of Antibiotic C-19393 H2 sodium salt was dissolved in 10% aqueous methanol (10 ml), and the solution was added to a mixture of 10%
aqueous methanol (10 ml~ and 10% palladium-carbon (20 mg) into which hydrogen had been introduced in advance for 30 minutes. Then, hydrogen was introduced into the resultant mixture at room temperature under 1 atmospheric pressure for 3 hours to carry out reduction, and the catalyst was then filtered out, followed by concentrating the filtrate under vacuum to 2 ml of volume. The concentrated solution was passed through a column (10 ml) of XAD-II(100 to 200 mesh), and the objective compound was adsorbed on the adsorbent. After the column was washed with water (50 ml), elution was carried out with 20% methanol-water, and the fractions containing the objective compound were collected. Methanol of the active fractions was distilled -~ k ~

~L7~3~2 off and the residue was freeze dried, thereby yielding 1.0 mg of powder of sodium [5R, 6R]-3-[(E)-2-ace-tamide-ethenylthio]-6-[1-hydroxy-1-methylethyl]-7-oxo-1-azabicyclo [3,2,0]hept-2-en-2-carboxylate.
VV: ~max (H20) 229 and 310 nm IR: vma~ (KBr) 1760, 1620 cm Thin layer chromatography [Cellulvse f (Tokyo Kasei Co.
Ltd.)]: Rf = 0.87 (Solvent system: propanol : water = 4 : 1) High performance liquid chromatography (Manufactured by Waters Associates Inc., U.S.A.): Rt = 8.2 min. [micro-bondapak C18/14% methanol-0.02M phosphate buffer (pH 6.3), 2 ml/min/cm (2000 psi)], wherein Rt of the starting compound under the same conditions was 4.3 min.
15 NMR: ~(lOOMHz, D20, TMS): 1.33(3H,s,C8-CH3), 1.44(3H,s, C8-CH3), 2.10(3H,s,COCH3), 3.03(1H,dd,J=lO,l9,C4-H), 3.85(lH,dd,J=10.5,19,C4-H), 3.72(1H,d,J=6,C6-H), 4.28(lH, m,C5-H), 6.10(1H,d,J=14,S-CH=), 7.20(1H,d,N-OEI=) Production Example 3 Crude powder (30% of purity, 60 mg) of Antibiotic C-19393 S2 disodum salt was dissolved in 10% aqueous methanol (20 ml), and the resulting solution was added to a mixture of 10% aqueous methanol (10 ml) and 10% palladium-carbon (20 mg) into which hydrogen had been introduced in advance for 30 minutes. Then, hydrogen was introduced into the resultant mixture at room temperature under 1 atmos-pheric pressure for 3 hours to carry out reduction, and the catalyst was then filtered out, followed by concen-trating the filtrate under vacuum to 2 ml of volume. Theconcentrated solution was flown through a column (50 ml~ of XAD-~ (100 to 200 mesh), and the objective compound was adsorbed on the adsorbent and then eluted with water.
The fractions from 45 ml to 150 ml which contained the objective compound were coilected and lyophilized, whereby there was obtained 7.3 mg of powder of [5R, 6R~-3-~:l77~3Z

[(E)-2-acetamidoethenylthio]-6-[1-(hydroxysulfonyloxy)-1-methylethyl]-7-oxo-1-azabicyclol3,2,0]hept-2-ene-2-carboxylic acid disodium salt.
o W: ~max(H2O) 228 and 309 nm o IR: vmax(KBr) 1760, 1620, 1240, 1050 cm o Thin layer chromatography [Cellulose f (Tokyo Kasei Co., Ltd.)]: Rf = 0.65 (sovent system: propanol:water = 4:1) o High performance liquid chromatography (Waters Associates Inc.): Rt = 4.4 min. [Microbondapak C18/14% methanol-0.02M-phosphate buffer (pH 6.3), 2 ml/min/cm (200 psi)], wherein Rt of the starting compound wnder the same conditions was 2.2 min.
o NMR; ~(100 MHZ, D2O, TMS): 1.63(3H,S,C~-CH3), 1.70(3H, S,C8-CH3), 2.10(3H,S,COCH3), 3.05(lH,dd,J=10,19,C4-H), 3.82(1H,dd,10.5,19,C4-H), 3.88(1H,d/~=6,C6-_), 4.20(1H,m, C5-H), 6.10(1H,d,J=14,S-CH=), 7.20(1H,d,N-CH=).

Production Example ~
(1) The compound obtained according to Production Example 3 (IIh, 100 mg) was dissolved in methanol (50 ml) followed by addition of m-chloroperbenzoic acid (78 mg).
The mixture was stirred at 0-5C for 30 minutes. The reaction mixture was then added to 0.02 M phosphate buffer (pH 6.3, 100 ml) and concentrated. The concentrate (50 ml) was washed with ethyl acetate (50 ml) and the water layer was chromatographed on an HP-20 column (100-200 mesh, 100 ml) pretreated with a 5% aqueous solution of sodium chloride using methanol-5% NaCl (5:95) as an eluent. The fraction containing the product compound was detected by HPLC, desalted by carbon chromatography and lyophilized to give [5R, 6R]-3-[(E)-2-acetamidoethenyl-(S)-sulEinyl]-6-[l-hydroxysulfonyloxy-l-methylethyl]-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid disodium salt (IIk, 37.2 mg).
(2) The compound obtained according to Production Example 3 (IIh, 2 mg) was dissolved in acetonitrile (6 ml) ~77~3Z

followed by addition of 12% aqueous hydrogen peroxide (4 ml). The mixture was stirred at room temperature for 2 hours. HPLC of the reaction mixture revealed a peak of product compound (IIk) according to (1) in a yield of 22~.
Thin-layer chromatography (hereinafter, briefly TLC) [cellulose f (Tokyo Kasei, Japan)]: Rf = 0.38 (solvent system: propanol-water =
HPLC: Rf= 1.6;min.
[Radial Pak A, 2 ml/min. (the same applies hereinafter), 8~ methanol-0.02M phosphate buffer (pH 6.3; hereinafter briefly, P.B.)]
W: ~manm (Elcm) = 250(298) & 288.5(251) CD: [~]H2 240(E-56900) & 290(+32900) IR: vmaBxr 1760, 1700, 1255, 1050 cm PMR: ~(lOOMHz, D20, TMS-; the same applies herein-after; 166, 173(3Hx2,s,8-CH3), 215(3H,s,NHCOCH3), 3.19 &
3.91(lHx2,dd,H4), 3.93(1H,d,H6), 6.44(lH,d,S-CH=), 7.65(lH,d,N-CH=) ppm.

Production Example 5 The compound obtained according to Production Example 2 (IIg, 76 mg) was dissolved in methanol (38 ml) followed by addition of m-chloroperbenzoic acid (70 mg). The mix-ture was stirred at 0-5C for 30 minutes. To this re-action mixture was added water (40 ml) and the mixture was adjusted to pH 6.3 with phosphate bufferr concentrated and washed with ethyl acetate. The water layer was chromato-graphed on an HP-20 (100 ml) column and eluted with water.
The fraction containing the product compound was concen-trated and lyophilized to give sodium [5R, 6R]-3-~(E)-2-acetamidoethenyl-(S)-sulfinyl]-6-[1-hydroxy-1-methylethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (IIj, 25 mg~.
TLC: Rf = 0.64 HPLC: Rt = 3.8 min. [8~ methanol-P.B.]

~L77~33~:
~ 16 -W: ~maxnm (Elcm) = 249(476) & 285(354) IR: vmKaxr 1770, 1700, 1630cm CD: [~]nm 240(-55000) & 287(-~10100) PMR: 1.34, 1.45(3Hx2,s,8-CH3), 2.15(3H,s,NHCOCH3), 3.14, 3.83(lHx2,dd,H4), 3.84(1H,d,H6), 6.31(1H,d,S-CH=), 7.56(lH,d,N-CH=) Example 1 (1) Compound IIb (50 mg) was dissolved in water (20 ml) and extracted with 1% tri-n-octylmethylammonium chlo-ride in dichloromethane (20 ml). The extract was allowed to stand at room temperature for 3 days, after which the product compound was re-extracted into a 0.375% solution of sodium iodide (20 ml). The water layer was concentrated and chromatographed on an HP-2 (100-200 mesh, 30 ml) column pretreated with 5% a~ueous NaC1, elution being carried out with 5% aqueous NaCl and water~ The product compound was detected by HPLC and the fractions giving a single peak were pooled, concentrated and lyophilized to give [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(RI-sulfinyl]-6-(l-hydroxy-sulfonyloxy l-methylethyl)-7-oxo-1-azabicyclo (3,2,0)-hept-2-ene-2-carboxylic acid disodium salt (Ib, 24.4 mg~.
HPLC: 8% methanol-0.02M phosphate buffer (pH 6.31, 2 ml/min. (Except for the concentration of methanol, -the same conditions apply hereinafter): Rt = 5.9 min. (Rt of IIb = 3.1 min.).
UV: ~ma2Xnm (ElCm) = 241.5(310) & 292-5(215) IR: vmBax 1770, 1630, 1250, 720cm~l PMR(lOOMHz): ~pDpm 1.66 & 1.73(8-(CH3)2,each 3H), 5.90(S-CH=, lH,d,J=8Hz), 7.39(N-CH-,lH,d,J=8Hz) (2) Compound IIb (42 mg) was dissolved in water (10 ml) and extracted with 1% tri-n-octylmethylan~onium chloride in dichloromethane (20 ml). The extract was refluxed for 8 hours and after cooling, the product compound was re-extracted into a 0.75% solution of sodium iodide (20 ml). The water layer was worked up as above (1) 1~7783Z

to give the disodium salt of compound Ib (19.3 mg).
(3) Compound IIb (100 mg) was dissolved in water (50 ml) and extracted with 1~ tri-n-octylmethylammonium chloride in chloroform (50 ml). The extract was refluxed for 1.5 hours. HPLC showed a peak of the product compound in a yield of 88%. The product compound was re-extracted into a 0.75~ solution of sodium iodide ~25 ml). The water layer was concentrated, the concentrate was chromatographed on an HP-20 (100-200 mesh, 125 ml) column, and elution was carried out with water. The fraction containing the product compound was concentrated and the residue was treated with acetone to give the disodium salt of Ib as powders (56.5 mg).
(4) Compound IIb was reacted using the different combinations of quaternary ammonium halide and solvent under various conditions as set forth in Table 3 and the reaction mixtures worked up in the same manner as above (1). In all cases, the product compound (Ib) was assayed by HPLC. The results are shown in Table 3. (In all cases, the concentration of starting compound IIb was 200 ~g/ml).

Table 3 .. _ _ . ., .. , .. . . ... . _ Halide* Solvent P(C) T(hrmse ) Yi(~e)d . _ . _ . . .. _ .
(1) Dichloromethane 40 8 93 " Chloroform 61 1 85 " 1,2-Dichloroethane60-62 0.5 61 " l,l,l-Trichloroethane 74 1 43 " Benzene 81 1 23 " Ethyl acetate 60-62 3 25 (2) Dichloromethane 40 6 77 (3) " 40 16 76 (4) Chloroform 61 2 55 (5) " 61 2 40 _ . ... .. . _ . .

~7715~3Z

*(1): Tri-n-octylmethylammonium chloride (2): Tetra-n-pentylamrnonium chloride (3): n-Hexadecylbenzyldimethylammonium chloride (4): n-Tetradecylbenzyldimethylammonium chloride-dihydrate (5): Tetra-n-pentylammonium bromide Example 2 (1) Compound IIa (30 mg) was dissolved in dimethyl-formamide (3 ml) followed by addition of 2% tri-n-octyl-methylammonium chloride in dichloromethane (300 ml). The solution was refluxed for 8 hours and, after cooling, the product compound was extracted into a 3% solution of sodium iodide (150 ml). HPLC showed a peak of product compound in a yield of 82%. The water layer was concentrated, the concentrate was chromatographed on an HP-20 (100-200 mesh, 30 ml) column, and elution was carried out with water.
The fraction containing the product compound was concen-trated and lyophilized to give sodium [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6-[1-hydroxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (Ia, 19.8 mg).
HPLC: 15% methanol, Rt = 7.5 min. (Rt of IIa = 3.6 min.).
W ~maxnm (Elcm) 239(367) & 295(255) IR: VmaBx 1765, 1630, 1260, lOOOcm~l PMR: ~p2m 1.34 & 1.45(8-(CH3)2, each 3H), 5.88 (S-CH=,lH,d,J=8Hz), 7.40(N-CH=,lH,d,J=8Hz) (2) The above reaction (1) was repeated except that methanol was used in lieu of dime~hylformamide. After 9 hours of reaction, HPLC revealed a peak of Ia in a yield of 85%.

Example 3 Compound IId (12.8 mg) was dissolved in water (10 ml) and extracted with 1% tri-n-octylmethylammonium chloride in di-chloromethane (10 ml). The extract was ref]uxed for ~L17'7~32 8 hours. HPLC showed a peak of product compound in a yield of 88%. The reaction mixture was re-extracted three times with a 0.375% aqueous solution of sodium iodide (10 ml).
The second extract only was concentrated, the concentrate was chromatographed on an HP-20 (100-200 mesh, 60 ml) column, and elution was carried out with water. The eluate was concentrated and lyophilized to give [5R, 6R, 8S]-3-[(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6-[1-hydroxysulfonyl-oxyethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid disodium salt (Id, 3.2 mg).
HPLC: 4% methanol, Rt = 4.8 min. (Rt of IID = 2.3 min.).
UV: ~max (ElCm) = 242(281) & 290(196) IR: vmax 1770, 1700, 1260, 1040Cm-l PMR: 1.55(8-CH3,d,J-6Hz), 5.90(S-CH=,d,J=8Hz), 7.39(N-CH=,d,J=8Hz) Example 4 Compound IIc (15 mg) was dissolved in water (200 ml) and extracted with 2% tri-n-octylmethylammonium chloride in chloroform (200 ml). The extract was refluxed for 2 hours, after which the reaction product was re-extracted into a 3% aqueous solution of sodium iodide (50 ml). HPLC showed a peak of reaction product in a yield of 78%. The water layer was worked up in the same manner as Example 2-(1) to give sodium [5R, 6R, 8S]-3-~(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6-[1-hydroxy-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (Ic, 6.6 mg).
HPLC: 8% Methanol, Rt = 5.5 min. (Rt of IIc = 2.8 min.).
UV: ~maxnm (Elcm~ = 238(330) & 291(228) IR: vmaX 1770, 1630, 1260cm~l PMR: ~pp 1.38(8-CH3,d,J=6Hz), 5.90(S-CH=,d,J=8Hz), 7.40(N-CH=,d,J=8Hz) ~77~3Z

Example 5 Compound IIe (64 mg) was dissolved in water (1.28 Q) and extracted with 2.5% tri-n-octylmethylammonium chloride in chloroform (1.28 Q). The extract was refluxed for an hour and, after cooling, -the product compound was re-extracted into a 7.5% solution of sodium iodide (160 ml).
HPLC showed a peak of reaction product in a yield of 95%.
The water layer was worked up in the same manner as Example 2-(1) to give sodium [5R, 6R, 8S]-2-[(Z)-2-acetamido-ethenylthio]-6-[1-hydroxy-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (Ie, 33 mg).
HPLC: 6% methanol, Rt = 11.6 min. (Rt of IIe = 6.8 min.) W: ~maXnm (ElC~,) = 226(320) & 306(268) IR vmaxr 1755, 1630, 1265Cm~l PMR: ~ppm 1.35(8-CH3,d,J=6Hz), 5.74(S-CH=,d,J=7.5Hz), 7.20(N-C_=,d,J=7.5Hz) - Example 6 Compound IIf (20.9 mg) was dissolved in water (10 ml) and extracted with 1% tri-n-octylmethylammonium chloride in dichloromethane (10 ml). The extract was refluxed for 9 hours and after cooliny, the reaction product was re-extracted into a 0.375% solution of sodium iodide (10 ml).
HPLC showed a peak of reaction product in a yield of 95%.
The water layer was concentrated, the concentrate was chromatographed on an HP-20 (100-200 mesh, 130 ml) column, and elution was carried out with water and 5% aqueous methanol. The fraction containing the product compound was concentrated and lyophilized to give ~5R, 6R, 8S]-3-~(Z)-2-acetamidoethenylthio]-6-~1-hydroxysulfonyloxyethyl]-7-oxo-l-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid disodium salt (If, 8.8 mg).
HPLC: 4% methanol, Rt = 5.2 min. (Rt of IIf = 4.1 min.).

7~332 UV ~maxnm (Elcm) = 228(291) ~ 307(179) IR: vm3xr 1750, 1620, 1260, 1035cm~l PMR ~ppm 1.50(8-CH3,d,J=6Hz), 5.74(S CH=,d,J=8Hz), 7.21(N-CH=,d,~=8Hz) Example 7 Compound lIg (10 mg) was dissolved in water (200 ml) and extracted with 2% tri-n-octylmethylammonium chloride in dichloromethane (200 ml). The extract was refluxed for 8 hours and the product compound was re-extracted into a 3% solution of sodium iodide (50 ml). The water layer was worked up in the same manner as Example 2-(1) to give sodium [5R, 6R]-3-[(Z)-2-acetamidoethenyl-thio]-6-[1-hydroxy-l-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene~2-carboxylate (Ig, 7 mg). In TLC, HPLC, W, IR, and PMR, this compound Ig was identified with the authentic sample.

Example 8 Compound IIh (40 mg~ was dissolved in water (20 ml) and extracted with 1% tri-n-octylmethylammonium chloride in chloroform (2Q mll. The extract was refluxed for 2 hours and the reaction product was re-extracted into a 0.75%
solution of sodium iodide (10 ml). The water layer was worked up in the same manner as Example 1-(3) to give ~5R, 6R]-3-[(Z)-2-acetamidoethenyl-thio]-6-[1-hydroxysulfonyloxy-l-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)~hept-2-ene-2-carboxylic acid disodium salt (Ih, 31 mg). In TLC, HPLC, UV, IR and PMR, this compound Ih was identified with the authentic sample.

Example 9 Compound IIi (130 mg) was dissolved in water (2.0 Q) and extracted with 2% tri-n-octylmethylammonium chloride in chloroform (20 ml). The extract was refluxed for 1.5 hours and after cooling, the product compound was re-extracted li77~3Z

into a 6.6% solution of sodium iodide (375 ml). HPLC
showed a peak of product compound in a yield of 93~. The water layer was worked up in the same manner as Example 2-(1) to give sodium [5R, 6S, 8S~-3-[(Z)-2-acetamido-ethenyl-thio]-6-[1-hydroxy-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (Ii, 64 mg).
HPLC: 10% methanol, R-t = 14.0 min. (Rt of IIi = 6.7 min.).
UV: ~m2xnm (Elcm) = 231(390) & 308(344) IR vKBx 1755, 1620, 1400, 1265cm~l PMR ~ppm 1.70(8-CH3,d,J=6Hz), 5.70(S-CH=,d,J=8Hz), 7.16(N-CH=,d,J=8Hz) Example 10 The compound obtained in Production Example 5 (IIj, 7 mg) was dissolved in water (140 ml) and extracted with 2~ tri-n-octylmethylammonium chloride in chloroform (140 ml). The extract was refluxed for 3 hours and after cool-ing, the product compound was re-extracted into a 1.5%
solution of sodium iodide (100 ml). HPLC showed a peak of reaction product in a yield of 83%. The water layer was worked up in the same manner as Example 2-(1) to give sodium [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-[l-hydroxy-l-methyl-ethyl]-7-oxo~l-azabicyclo(3,2,0)-hept-2-ene-2-carboxylate (Ij, 5 mg).
TLC: Rf = 0.64 HPLC: Rt = 4.2 min. [8% methanol-P.B.]
UV: ~maXonm (ElCm) = 237(408) & 290(262) IR: vmar 1765, 1705, 1635, 1385, 1270, 1020cm~
CD: [9]nm 235(~-22700) h 286(+32800) PMR: 1.32, 1.41(3Hx2,s,8-CH3), 2.15(3H,s,NHCOCH3), 3.22, 3.83(lHx2,dd,H4), 3.82(1H,d,H6), 5.71(lH,d,S-CH=), 7.27(lH,d,N-C_=) ~`

~1~7f~32 Example_ll The compound obtained in Production Example 4 (IIk, 10 mg) was dissolved in water (10 ml) and extracted with 1 tri-n-octylmethylammonium chloride in chloroform (10 ml).
The extract was refluxed for 3 hours and after cooling, the product compound was re-extracted into a 0.75~ solution of sodium iodide (10 ml). HPLC showed a peak of reaction product in a yield of 88%. ~he water layer was chromato-graphed on an HP-20 (100-200 mesh, 20 ml) column pretreated with 5% NaCl and elution was carried out with 5% aqueous NaCl and methanol-5~ aqueous NaCl (3:97). The fractions containing the product compound were pooled and concen-trated. The concentrate was chromatographed on a column of activated carbon (5 ml) and the product compound was eluted with 8% aqueous isobutanol and N/50 aqueous ammonia-8%
aqueous isobutanol (2:98). The eluate was concentrated and lyophilized to give [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl~-6-[1-hydroxysulfonyloxy-1-methyl-ethyl]-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid disodium salt (Ik, 8.4 mgl.
TLC: Rf = 0.30 HPLC: Rt = 1.8 min. ~3% methanol-P.B.) UV: ~max(Elcm) = 237(276) & 292(188) CD: [~]nHm 232(~-12900) & 282(+31400) IR vmaBx 1765, 1700, 1260, lQ50cm 1 PMR: ~ 1.65, 1.68(3Hx2,s,8-CH3), 2.17(3H,s,NHCOCH3~, 3.26 & 3.92(1Hx2~dd,H~), 3.98(1H,d,H6), 5.85(1H,d,S-CH=), 7.30(1Hrd,N-CH=

Claims (20)

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

1. A process for the production of a compound of the formula:

(I) wherein R is an ethyl group or a group of formula (III):

(III) wherein R1 is H or methy;;
R2 is H, OH, R3COO- or R5O3SO-;
R3 is R4 or -NHR4;
R4 is lower alkyl, lower alkenyl, phenyl-substituted lower alkyl, phenyl or phenyloxy, wherein phenyl in the last three groups is unsubstituted or substituted by lower alkyl, lower alkoxy or halogen; and R5 is H or lower alkyl; or a pharmaceutically acceptable salt thereof, which comprises subjecting to the cis-trans isomerization a compound of the formula:
(II) wherein R and n have the same meaning as defined above, or a salt thereof, by using a quaternary ammonium halide, and if necessary, converting the resulting compound into a pharmaceutically accept-able salt thereof.

2. The process as claimed in claim 1, wherein the quaternary ammonium halide is used in a stoichiometric excess based on the starting compound.

3. The process as claimed in claim 2, wherein the isomeriz-ation is carried out in an organic solvent except for a strong polar solvent.

4. The process as claimed in claim 3, wherein the organic solvent is a halogenated hydrocarbon.

5. The process as claimed in claim 1, 2 or 3, wherein the quaternary ammonium halide compound has a total of about 18 to 30 carbon atoms for the four substituents and chlorine or bromine atom as the halogen atom.

6. The process as claimed in claim 3, wherein the quaternary ammonium halide is used 3 to 1000 molar equivalents based on the starting compounds, and in the formulae, R is a group of formula (III) wherein R1 is H or methyl, R2 is H, OH, R3COO- or R503SO-, R3 is R4 or -NHR4, R4 is an alkyl group with 1 to 3 carbon atoms, an alkenyl group with up to 6 carbon atoms, a phenyl-substituted alkyl group with 1 to 3 carbon atoms in the alkyl moiety, phenyloxy or phenyl, wherein phenyl in the last three groups is unsubstituted or substituted by alkyl with 1 to 3 carbon atoms, alkoxy with 1 to 3 carbon atoms, chloro or fluoro.

7. The process as claimed in claim 1, 2 or 3, which further comprises extracting a quaternary ammonium salt of the resulting compound in the organic solution with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution.

8. A process for the production of a compound of the formula:

(IV) wherein R6 is H or -SO3H, or a pharmaceutically acceptable salt thereof, which process comprises:
(i) subjecting to the cis-trans isomerization a compound of the formula:

wherein R6 is as defined above, or a salt thereof, in an organic solvent except for a strong polar solvent by using a stoichiometric-ally excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, and (ii) if necessary, converting the resulting compound into the free acid or to a pharmaceutically acceptable salt thereof.

9. The process as claimed in claim 8, which as a step after the isomeriza-tion further comprises extracting a quaternary ammonium salt of the resulting com-pound in the organic solution with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solu-tion.

10. A compound of the formula:

(IV) wherein R6 is H or -SO3H, or a pharmaceutically acceptable salt thereof whenever prepared or produced by the process of claim 8 or 9 or by an obvious chemical equivalent thereof.

11. A process for the production of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(R)--sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2--carboxylic acid, or a pharmaceutically acceptable salt thereof, which process comprises:
(i) subjecting to the cis-trans isomerization [5R, 6R]-3-[(E)-2-acetamidoethenyl--(R)-sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene--2-carboxylic acid or a salt thereof, in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents;
(ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt.

12. A process for the production of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(R)-sulfinyl]-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, which process com-prises:
(i) subjecting to the cis-trans isomerization [5R, 6R]-3-[(E)-2-acetamidoethenyl-(R)-sulfinyl]-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid or a salt thereof in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents;
(ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt.

13. A process for the production of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-(l-hydroxy-1-methyl-ethyl)-7-oxo-l-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid, or a phaarmaceut-ically acceptable salt thereof, which process comprises:
(i) subjecting to the cis-trans isomerization [5R, 6R]-3-[(E)-2-acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-l-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid or a salt thereof in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, (ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt.

14. A process for the production of [5R, 6R]-3-[(Z)-2-acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxysulfonyloxy-I-methyl-etnyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, which process comprises:
(i) subjecting to the cis-trans isomerization [5R, 6R]-3-[(E)-2-acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid, or a salt thereof, in a water-immiscible organic solvent by using a stoichiometrically excessive amount of a quaternary ammonium chloride or bromide having 18 to 30 carbon atoms for the four substituents, (ii) extracting a quaternary ammonium salt of the resulting compound in the organic solvent with an aqueous solution of an alkali metal iodide thereby isolating an alkali metal salt of the desired compound into the aqueous solution and the iodide of the quaternary ammonium compound into the organic solution, and (iii) if desired, converting the alkali metal salt of the desired compound to the free acid or to a pharmaceutically acceptable salt thereof other than the alkali metal salt.

15. The process as claimed in claim 11 or 12 wherein the quaternary ammonium chloride or bromide is selected from the group consisting of tri-n-octylmethyl ammonium chloride, tetra-n-pentylammonium chloride, n-hexadecylbenzyldimethylammonium chloride, n-tetradecylbenzyldimethylammonium chloride and tetra-n-pentyl-ammonium bromide.

16. The process as claimed in claim 13 or 14 wherein the quaternary ammonium chloride or bromide is selected from the group consisting of tri-n-octylmethylammonium chloride, tetra-n-pentyl-ammonium chloride, n-hexadecylbenzyldimethylammonium chloride, n-tetradecylbenzyldimethylammonium chloride and tetra-n-pentylammonium bromide.

17. [5R, 6R]-3-[(Z)-2-Acetamidoethenyl-(R)-sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, whenever prepared or produced by the process of claim 11, or by an obvious chemical equivalent thereof.

18. [5R, 6R]-3-[(Z)-2-Acetamidoethenyl-(R)-sulfinyl]-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, whenever prepared or produced by the process of claim 12, or by an obvious chemical equivalent thereof.

19. [5R, 6R]-3-[(Z)-2-Acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxy-1-methyl-ethyl)-7-oxo-1-azabicyclo(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, whenever prepared or produced by the process of claim 13, or by an obvious chemical equivalent thereof.

20. [5R, 6R]-3-[(Z)-2-Acetamidoethenyl-(S)-sulfinyl]-6-(1-hydroxysulfonyloxy-1-methyl-ethyl)-7-oxo-1-azabicyclo-(3,2,0)-hept-2-ene-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, whenever prepared or produced by the process of claim 14, or by an obvious chemical equivalent thereof.