CA1338670C - .beta.-lactam antibiotics - Google Patents

.beta.-lactam antibiotics

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Publication number
CA1338670C
CA1338670C CA000370320A CA370320A CA1338670C CA 1338670 C CA1338670 C CA 1338670C CA 000370320 A CA000370320 A CA 000370320A CA 370320 A CA370320 A CA 370320A CA 1338670 C CA1338670 C CA 1338670C
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amino
oxo
acid
salt
solution
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Richard Brook Sykes
William Lawrence Parker
Christopher Michael Cimarusti
William Henry Koster
William Allen Slusarchyk
Alan William Fritz
David Mack Floyd
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ER Squibb and Sons LLC
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ER Squibb and Sons LLC
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    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
    • C07D205/085—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams with a nitrogen atom directly attached in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/0803—Compounds with Si-C or Si-Si linkages
    • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
    • C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

.beta.-Lactams having a sulfonic acid substituent in the 1-position and an amine function in the 3-position and having the formula: wherein R1 is H or acyl derived from a carboxylic acid; R2 is H or alkoxy; R3 and R4, the same or different, are H, alkyl, cycloalkyl, phenyl or substituted phenyl, or one of R3 and R4 is H and the other is alkoxycarbonyl, alken-1-yl, alkyn-1-yl, 2-phenylethenyl or 2-phenylethynyl; and X is H or SO3X is SO3?M? wherein M?is a cation, and the use of said .beta.-lactams as antibiotics.

Description

- GC155f B-LAcTAM ANTIBIOTICS
This invention is directed to a novel family of B-lactam antibiotics, and to the use of such compounds as antibacterial agents.
It has been discovered that the B-lactam nucleus can be biologically activated by a sulfonic acid salt substituent attached to the nitrogen atom in the nucleus.
B-Lactams having a sulfonic acid, or a salt chereof, substituent in the l-position and an acylamino substituent in tne 3-position exhibit activity against a range of gram-negative and gram-positive bacteria.

15The preferred members of the novel family of B-lactam antibiotics of this invention are those encompassed by the formula I _2 -4 20Rl-NH-C- -C-R3 o In addition to the above described B-lactams having a sulfonic acid, ~r a salt thereof, substi-tuent in the l-position and an acylamino substituent in the 3-position, this invention also encompasses ~-lactams naving a sulfonic acid, or a salt thereof, substituent in the l-position and an amino substi-tuent in the 3-position.

,,, ~

G(~-I55f _ 2 1 338670 The preferred compounds of this type have the formula Ia R2 _4 These compounds are intermediates useful for the preparation of corresponding 3- acylamino compounds.
As used in formulas I and Ia, and through--out the specification, the symbols are as defined below:
Rl is acyl;
R2 is hydrogen or alkoxy of 1 to 4 carbons;
R3 and R4 are the same or different and each is hydrogen, `alkyl, cycloalkyl, phenyl or sub-stituted phenyl, or one of R3 and R4 is hydrogen and the other is alkoxycarbonyl, alken-l-yl, alkyn-l-yl, 2-phenylethenyl or 2-phenylethynyl;
~4 is hydrogen or a cation.

The terms "alkyl" and "alkoxy" refer to both straight and branched chain groups. Those groups having 1 to 10 carbon atoms are preferred.
The terms "cyclalkyl" and "cycloalkenyl" refer to cyclo-alkyl and cycloalkenyl groups having 3, 4, 5, 6, or 7 carbonatoms.
The term "alkenyl" refers to both straight and branched chain groups. Those groups having 2 to 10 carbon atoms are preferred.
The term "halogen" refers to fluorine, chlorine, bromine and iodine.
The term "substituted phenyl" refers to a phenyl group substituted with one, two or three amino, halogen, hydroxyl, trifluoromethyl and lower alkyl or alkoxy groups.
The term "protected carboxyl" refers to a carboxyl group which has been esterified with a conventional ester protecting group. These groups are well known in the art; see, for ex-ample, United States patent 4,144,333, issued ~larch 13, 1979.
The preferred protected carboxyl groups are benzyl, benzhydryl and t-butyl esters.
The term "acyl" includes all organic radicals derived from an organic acid (i.e., a carboxylic acid) by removal of the hydroxyl group. Certain acyl groups are, of course, pre-ferred, but this preference should not be viewed as a limita-tion of the scope of this invention. Exemplary acyl groupsare those acyl groups which have been used in the past to acylate ~-lactam antibiotics including 6-aminopenicillanic acid and derivatives and 7-aminocephalosporanic acid and der-ivatives; see, for example, Cephalosporins and Penicillins, edited by Flynn, Academic Press (1972), German Offenlegungs-schrift 2,716,677 published October 10, 1978, Belgian patent 867,994, published December 11, 1978, United States patent 4,152,432, issued -May 1, 1979, United States patent 3,971,778, issued July 27, 1976, United States patent 4,172,199, issued October 23, 1979, and British patent 1,348,894, published March 27, 1974.
The following list of acyl groups is presented to further exemplify the term "acyl"; it should not be regarded as limiting that term. Exemplary acyl groups are:
(a) Aliphatic groups having the formula R -C-wherein R5 is alkyl; cycloalkyl; alkoxy; alkenyl;
cycloalkenyl; cyclohexadienyl; or alkyl or alkenyl substituted with one or more halogen, cyano, nitro, amino, mercapto, alkylthio, or cyanomethylthio groups.
(b) Carbocyclic aromatic groups having the formula ~\~(CH2 ) n~C~ ' 6\ ~ f H-CI -Rg 6~CH2_o_C_ GCi55f _5_ 1 338670 6 ~ O-CH2-C-R
~6 ~ S-CH2-C- or R

~ CH -S-C-wherein n is 0, 1, 2 or 3; R6, R7, and R8 each is independently hydrogen, halogen, hydroxyl, nitro, amino, cyano, trifluoromethyl, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms or aminomethyl; and Rg is amino, hydroxyl, a carboxyl salt, protected carboxyl, formyloxy, a sulfo salt, a sulfoamino salt, azido, halogen, hydrazino, alkylhydrazino, phenylhydrazino, or [(alkylthio)thioxomethyl]thio.
Preferred carbocyclic aromatic acyl groups include those having the formula H ~

~ C 2 1 33867~ GC155f HO ~ CH-C- (Rg is preferably a carboxyl salt or sulfo salt) and . ~ CH-C- (Rg lS preferably a carboxyl salt or sulfo salt).
(c) lleteroaromatic groups having the formula O
Rlo (CH2)n O
~oCH~C~
Rg O
Rlo-o-cH2 -C-Rlo-S-CH2-C- , or It 11 Rlo--C--C-wherein n is 0, 1, 2 or 3; Rg is as defined above; and Rlois a substituted or unsubstituted 5-,6-or ~membered heterocyclic ring containing 1, 2, 3 or 4 (preferably 1 or 2) nitrogen, oxygen and sulfur atoms. Exemplary heterocyclic GC155f rings are thienyl, furyl, pyrrolyl, pyridinyl, pyrazinyl, thiazolyl, morpholinyl, pyrimidinyl and tetrazolyl. Exemplary substituents are halogen, hydroxyl, nitro, amino, cyano, trifluoro-methyl, alkyl of 1 to 4 carbon atoms, or alkoxyof 1 to 4 carbon atoms.
Preferred heteroaromatic acyl groups include those groups of the above formulas wherein Rlois 2-amino-4-thiazolyl, 2-amino-5-halo-4-thiazolyl, 4-aminopyrimidin-2-yl, 5-amino-1,2,4-thiadiazol-5-yl, 2-thienyl or 2-furanyl.
(d) 1[(4-Substituted-2,3-dioxo-1-piperazinyl)-carbonyl~amino]arylacetyl groups having the formula l l ~

11 ~
O O

wherein R~ is an aromatic group (including carbocyclic aromatics such as those of the formula and heteroaromatics as included within the definition of Rld;and R12 is alkyl, substituted alkyl (wherein the alkyl group is substituted with one or more halogen, cyano, nitro, amino or mercapto gro-ups), arylmethyleneamino (i.e., -N=CH-Rll wherein Rll is as defined above), arylcarbonylamino O
(l.e., -NH-C-Rll wherein Rllis as defined above) or alkylcarbonylamino.

GC155f _ -8- 1 3 3 8 6 7 0 Preferred [[(4-substituted-2,3-dioxo-1-piperazinyl)carbonyl]amino~arylacetyl groups include those wherein R12 is ethyl, phenylmethylene-amino or 2-furylmethyleneamino.
(e) (Substituted oxyimino)arylacetyl groups having the formula o -C-C=N-O-R13 Rll wherein Rllis as defined above and R13 is hydrogen, alkyl, cycloalkyl, alkylaminocarbonyl, arylamino-o carbonyl (i.e., -C-NH-Rll wherein Rllis as defined above) or substituted alkyl (wherein the alkyl group is substituted with 1 or more halogen, cyano, nitro, amino, mercapto, alkylthio, aromatic group (as defined by Rl~, carboxyl (including salts thereof), amido, alkoxycarbonyl, phenylmethoxycarbonyl, diphenylmethoxycarbonyl, hydr~oxyalkoxyphosphinyl, dihydroxyphosphinyl, hydroxy (phenylmethoxy)phosphinyl, or dialkoxy-phosphinyl substituents).
Preferred (substituted oxyimino)aryl-acetyl groups include those wherein Rllis 2-amino-4-thiazolyl. Also preferred are those groups wherein R13 is methyl, ethyl, carboxy-methyl, or 2-carboxyisopropyl.
(f) (Acylamino)arylacetyl groups having the formula O O
Il ll Rll wherein Rllis as defined above and R14 is - GC155f _g_ R ~ (CH2)n~~, amino, alkylamino, (cyanoalkyl)amino, amido, alkylamido, (cyanoalkyl)-amido, -CH2-NH-C ~ , -CEI-CH2-C-NH-CH3 , ~ so2-N(C~2 CH2 OH)2 ~ ~ CH3 OH

OH OH

~ ~ N~ ~ N N -Cll Preferred (acylamino)arylacetyl groups of the above formula include those groups wherein R14 is amino, or amido. Also preferred are those groups wherein Rllis phenyl or 2-thienyl.

(g) [[[3-Substituted-2-oxo-1-imidazoli-dinyl]carbonyl]amino]arylacetyl groups having the formula o o O C
Il 11 / ~

Rll CH CH

GC155f -lo- 1 3 3 8 6 7 0 wherein Rllis as defined above and R15 is hydrogen, alkylsulfonyl, arylmethyleneamino (i.e., -N=CH-~lwherein Rllis as defined above), o -C-R16 (wherein R16 is hydrogen, alkyl or halogen substituted alkyl), aromatic group (as defined by Rllabove), alkyl or substituted alkyl (wherein the alkyl group is substituted with one or more halogen,cyano, nitro, amino or mercapto groups).
Preferred [[3-substituted-2-oxo-1-imidazoli-dinyl]carbonyl]amino]arylacetyl groups of the above formula include those wherein Rllis phenyl or 2-thienyl. Also preferred are those groups wherein Rls is hydrogen, methylsulfonyl, phenyl-methyleneamino or 2-furylmethyleneamino.
The term "cation", as used throughout the specification, refers to any positively charged atom or group of atoms. The "-SO3M~" substituent on the nitrogen atom of the ~-lactams of this invention encompasses all sulfonic acid salts.
Pharmaceutically acceptable salts are, of course, preferred, although other salts are also useful in purifying the products of this invention or as intermediates for the preparation of pharmaceutically acceptable salts. The cationic portion of the sulfonic acid salts of this invention can be obtained from either organic or inorganic bases. Such cationic portion includes, but is not limited to, the following ions: ammonium; substituted ammonium, such as alkylammonium (e.g., tetra-n-butylammonium, referred to hereinafter as tetrabutylammonium);
alkali metal, such as lithium, sodium and potassium;
alkaline earth metal, such as calcium and magnesium;

- 11 - 1 338~70 pyridinium; dicyclohexylammonium; hydrabaminium; benzathinium;
N-methyl-D-glucaminium.
As set forth in formula I, and in the definitions following formula I, M~ can be hydrogen.
As is described hereinafter, the ~-lactams of this invention can be prepared by synthetic means. The non-alkoxy-lated 4-unsubstituted ~-lactams of formula I, l.e., those compounds of formula I wherein R2, R3 and R4 are hydrogen can be prepared using 6-aminopenicillanic acid or a 6-acylamino-penicillanic acid as a starting material. The ~-lactams of formula I wherein R2 is alkoxy can be prepared from the corres-ponding non-alkoxylated ~-lactam. Some of the compounds of this invention may be crystallized or recrystallized from sol-vents containing water. In these cases water of hydration may be formed. This invention contemplates stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophiliza-tion.
Some of the ~-lactams of formula I have also been prepared by biological means. Cultivation of a strain of the microorganism Chromobacterium violaceum SC 11,378, yields a salt of (R)-3-(acetylamino)-3-methoxy-2-oxo-1-azetidine-sulfonic acid. Cultivation of various acetic acid bacteria e.g., Gluconobacter species SC 11,435, yields a salt of (R)-3-[[N-(D-~-glutamyl)-D-alanyl]amino]-3-methoxy-2-oxo-1-aze-tidinesulfonic acid.

GC155f B-Lactams having a sulfonic acid salt substituent in the l-position and an amino or acylamino substituent in the 3-position contain at least one chiral center the carbon atom (in the 3-position of the B-lactam nucleus) to which the amino or acylaminosubstituent is attached. This invention is directed to those B-lactams which have been described above, wherein the stereochemistry at the chiral center in the 3-position of the B-lactam nucleus is the same as the configuration at the carbon atom in the 6-position of naturally occurring penicillins (e q., penicillin G) and as the configuration at the carbon atom in the 7-position of naturally occurring cephamycins (e.g., cephamycin C).
With respect to the preferred B-lactams of formulas I and Ia, the structural formulas have been drawn to show the stereochemistry at the chiral center in the 3-position. Because of the nomenclature convention, those compounds of formulas I and Ia wherein R2 is hydrogen have the S configuration and those compounds of formulas I and Ia wherein R2 is alkoxy have the R configuration.
Also included within the scope of this invention are racemic mixtures which contain the above-described B-lactams.

- ~ 338670 -12a-The invention as claimed herein is a process for preparing a ~-lactam having a sulfonic acid sub-stituent -SO3M wherein M is hydrogen or a cation in the l-position and an amino substituent -NH2 which may optionally be acylated or protected with a read-ily removable protecting group in the 3-position with the proviso that when the ~-lactam ring is unsubsti-tuted in the 4-position, the second valency at the 3-position is not hydrogen or methoxy, said process being characterized by sulfonating a corresponding ~-lactam which is unsubstituted in the l-position or acylating a corresponding ~-lactam which has the amino group in the 3-position.
A preferred substituent in the 4-position in the above process is an alkyl radical, particularly a methyl radical, and the process is especially val-uable when used to prepare a ~-lactam of the for-mula R ~ - R3 ~ N-SO3H

or a pharmaceutically acceptable salt thereof wherein:
R is an amino group which may optionally be acylated or protected with a readily remov-able amino protecting group;
R2 is hydrogen or alkoxy of 1 to 4 carbon atoms;
R3 and R4 are the same or different and each is Cl-C10 alkyl, C3-C7 cycloalkyl, phenyl or phenyl substituted with 1 to 3 amino, halo-gen, trifluoromethyl or Cl-C4 alkyl or al-koxy groups or one of R3 and R4 is hydrogen and the other is Cl-C10 alkyl, C3-C7 cyclo-, -12b- 1 3 3 ~

alkyl, phenyl, optionally bearing substi-tuents as hereinbefore defined, Cl-C10 al-koxycarbonyl, C2-C10 alken-l-yl, C2-C10 alkyn-l-yl, 2-phenylethenyl or 2-phenyl-ethynyl.
The invention as claimed herein is further de-fined as a process for preparing a compound of the formula R - ~ R3 ~ N-S03H
o or a pharmaceutically acceptable salt thereof wherein:
R is an amino group which may optionally be acylated or protected with a readily remov-able amino protecting group;
R2 is hydrogen or alkoxy of 1 to 4 carbon atoms;
one of R3 and R4 is hydrogen and the other is g , Cl C10 alkyl, C3-C7 cycloalkyl phenyl optionally substituted with 1 to 3 amino, halogen~ trifluoromethyl or Cl-C4 alkyl or alkoxy groups, Cl-C10 alkoxycarbonyl, C2-C10 alken-l-yl, C2-C10 alkyn-l-yl, 2-phenylethenyl or 2-phenylethynyl;
characterized by cyclizing a compound of the formula R .~2 ~ R4 oD NH-SO3H

~ -12c- 1 3 3 8 6 7 0 or a salt thereof wherein:
V is a methanesulfonyl, benzenesulfonyl, toluenesulfonyl, chloro, bromo or iodo type leaving group; and R is an amino group which is acylated or protected and removing said protecting group to form a compound wherein R is amino and acylating said compound to form a product wherein R is acylated amino.
Preferred substituents in the process immed-iately above are those wherein R2 is hydrogen and one of R3 and R4 is hydrogen and the other is an alkyl radical, particularly a methyl radical.

GC155f _ -13-~ -Lactams having a sulfonic acid salt substituent in the l-position of the ~-lactam nucleus and an amino or acylamino substituent in the 3-position of the ~-lactam nucleus have activity against a range of gram-negative and gram-positive organisms. The sulfonic acid salt substituent is essential to the activity of the compounds of this invention. The compounds wherein R3 and/or R4 are - hydrogen or alkyl, especially methyl, exhibit especially useful activity.
The compounds of this invention can be used as agents to combat bacterial infections (including urinary tract infections and respiratory infections~
in m~mm~ 1 ian species, such as domesticated animals (e.g., dogs, cats, cows, horses, and the like) and humans.
For combating bacterial infections in mammals a compound of this invention can be administered to a mammal in need thereof in an amount of about 1.4 mg/kg/day to about 350 mg/kg/day, perferably about 14 mg/kg/day to about 100 mg/kg/day. All modes of administration which have been used in the past to deliver penicillins and cephalosporins to the site of the infection are also contemplated for use with the novel family of ~-lactams of this invention. Such methods of administration include oral, intravenous, intramuscular and as a suppository.

GC155f , The ~-lactam products of this invention are generally prepared by the introduction of a sulfonic acid substituent (a sulfo group -SO3-) onto the nitrogen atom in the l-position of the ~-lactam nucleus. This sulfonation reaction is readily effected by treating the ~-lactam with a sulfur trioxide complex or with an e~uivalent sulfonating reagent such as a chlorosulfonate.
The sulfur trioxide complexes most commonly used are pyridine-sulfur trioxide; lutidine-sulfur trioxide;
dimethylformamide-sulfur trioxide; and picoline-sulfur trioxide. Instead of using a pre-formed complex, the complex can be formed in situ, e.g., using chloro-sulfonyl-trimethylsilyl ester and pyridine as reagents.
Alternatively, sulfonation can be effected by way of an intermediate compound as for example first silylating the nitrogen atom of the ~-lactam nucleus and then subjecting the silated compound to a silyl interchange reaction with trimethylsilylchlorosulfonate or a similar reagent. Exemplary silylating agents are monosilyltri-fluoroacetamide, trimethylsilylchloride/triethylamine and bis-trimethylsilyl trifluoroacetamide.
Generally, the sulfonation reaction is carried out in the presence of an organic solvent such as pyridine or a mixture of organic solvents, preferably a mixture of a polar solvent such as dimethylformamide and a halogenated hydrocarbon such as dichloromethane.
The product initially formed in the sulfonation reaction is a salt of the sulfonated ~-lactam.
When pyridine-sulfur trioxide is the sulfonating complex the product initially formed is the ~-lactam-sulfonated pyridinium salt of the sulfonated ~-lactam wherein M+ in the formula below is the pyridinium ion:

/ ~-SO3 M

_ GC155f -15- 1 3 3 8 6 7 ~

These complexes can be converted to other sulfonic acid salts using conventional techniques (e.g., ion-exchange resins, crystallization or ion-pair extraction.
These conversion techniques are also useful in purifying the products. Conversion of the pyridine salt to the potassium salt using potassium phosphate or potassium ethyl hexanoate; to the tetrabutylammonium salt using tetrabutyl ammonium hydrogen sulfate; or to a zwitterian (M+ = hydrogen) using formic acid; are particularly useful.
It should be appreciated that the sulfonation reaction which introduces the sulfo group onto the nitrogen atom of the ~-lactam nucleus can be effected at various stages of the synthesis, including inser-tion prior to the formation of a ~-lactam nucleus, where such procedure is followed as outlined below.
The sulfonation reaction is effected in the presence of solvents previously described and usually at room temperature. Where the amino function is present it is preferably conducted with the amino function protected.
Using a benzyloxycarbonyl protecting group as an example, the sulfonation reaction can be visualized as follows:

'~2 ~4 ~2 R~
C6H5CH2OCO R3 ~H2N R3 0~ ~ 2)d~L~ ~on ~ N-SO3 M
II III
Other protecting groups can be used to protect the amine function, for example, a t-butyloxycarbonyl group, a simple acyl grou~ such as acetyl or benzoyl of phenyl-acetyl, a triphenylmethyl group, or having the amino function in the form of an azide group. The desired acyl group (Rl) can then be affixed by a conventional acylation xeaction.

- GC155f Exemplary acylation techniques for converting a compound of formula IIIto a product of formula I
include reaction with a carboxylic acid ~Rl-OH), or corresponding carboxylic acid halide or carboxylic acid anhydride. When R2 is alkoxy, acylcation is best effected by use of an acid chloride or acid bromide. The reaction with a carboxylic acid proceeds most readily in the presence of a carbodiimide such as dicyclohexylcarbodiimide and a substance capable of forming an active ester in situ such as N-hydroxybenzo-triazole. In those instances when the acyl group (Rl) contains reactive functionality (such as amino or carboxyl groups) it may be necessary to first protect those functional groups,then carry out the acylation reaction, and finally deprotect the resulting product.
Alternatively, the sulfonation reaction can be effected with the acyl group already in place, i.e., ~2 R4 ~2 R4 Rl-NH - R3 ti ~ R -NH -- R3 sulfona ~ -S03M
O O

IV V
In the case where R2 is lower alkoxy, there can be an additional variation in that the R2-lower alkoxy group can be inserted after the sulfonation as well as before the sulfonation using the conventional procedure of chlorinating the acylated nitrogen atom in the 3-position followed by reaction with a lower alkoxide 1 3 3 8 6 7 0 (;C155f --.17--Cl H R=4 (R2)alkoxide R2 R4 acyl-N l R3 ~ acyl-NH l R3 ~-S03 M s5: S03 VI . . V
The acyl groups in the above reactionalsoin~ easily removable groups which function as a protecting group and which can be removed after the reaction to afford the "deacylated" product (-NH2).
In addition, the ~-lactam ring can be formed via a cyclization reaction and the sulfonation reaction can be éffected prior to cyclization as well as subsequent thereto, i.e., 2 0 R4 > '~
acyl-NH ~R cyclization acyl-NH ~3 VII

wherein V is a leaving group such as methanesulfonyl, benzenesulfonyl, toluenesulfonyl, chloro, bromo or iodo. The acyl group in this reaction can also be an easily removable group which functions as a protect-ing group and which on removal affords the -NH2 pro-duct.

GC155f The azetidinone starting materials wherein R2 is hydrogen and at least one of R3 and R4 is hydrogen can also be prepared from amino acids having the formula ~H
NH2 ¦ / 4 XII H C

~C OH
o (at least one of R3 and R4 is hydrogen). The amino group is first protected with a classical protecting group, e.g., t-butoxycarbonyl (referred to hereinafter as "Boc"). The carboxyl group of the protected amino acid is then reacted with an amine salt having the formula XIII Y-O-NH3Cl wherein Y is alkyl or benzyl, in the presence of a carbodiimide to yield a compound having the formula OIH~ R4 XIV BOC-NH-CH C

¦ 3 O"C NH-O-Y

GC155f -19- 1 3 3 8 6 7 o (at least one of R3 and R4 is hydrogen). The hydroxyl group of a compound of formula XIV is converted to a leaving group (V) with a classical reagent, e.g., methanesulfonyl chloride (methanesulfonyl is referred to hereinafter as "Ms"). Other leaving groups (V) which can be utilized are benzenesulfonyl, toluene-sulfonyl, chloro, bromo and iodo.
The fulIy protected compound having the formula BOC-NH- H
XV ~ R3 O~C NH-O-Y
(at least one of R3 and R4 is hydrogen) is cyclized by treatment with base, e.g., potassium carbonate.
The reaction is preferably carried out in an organic solvent such as acetone, under reflux conditions, and yields a compound having the formula BOC-NH I = R3 XVI
~ N-O-Y

(at least one of R3 and R4 is hydrogen).
Alternatively, cyclization of a compound of formula XIV can be accomplished without first converting the hydroxyl group to a leaving group.
Treatment of a compound of formula XIV with triphenyl-phosphine and diethylazodicarboxylate, yields a compound of formula XVI wherein at least one of R3 and R4 is hydrogen.

GC155f Removal of the protecting group from the 1-position of an azetidinone of formula XVI can be accomplished via sodium reduction when Y is alkyl, and yields an intermediate having the formula XVII
~ - NH

(at least one of R3 and R4 is hydrogen). If Y is benzyl, catalytic (e.g., palladium on charcoal) hydrogenation will initially yield the corresponding N-hydroxy compound, which upon treatment with titanium trichloride yields an intermediate of formula XVII
wherein at least one of R3 and R4 is hydrogen.
Synthesis involving ring closure of the type described above results in inversion of the stereo-chemical configuration of the R3 and R4 substituents.
As previously mentioned, the above azetidinone can be sulfonated to form a com?ound having the formula o~ - N-S03 M

(at least one of R3 and R4 is hydrogen).
An alternative procedure for preparing a compound of formula I wherein R2 is hydrogen and at least one of R3 and R4 is hydrogen, utilizes as a starting material an amino acid amide having the formula OH

NH2-fH C R

o~,C NH2 (at least one of R3 and R4 is hydrogen).

GC155f Protection of the amino group with a classical protecting group, e.g., benzyloxycarbonyl (reffered to hereinafter as Z) or Boc, and conversion of the hydroxyl group to a leaving group (V) such as Ms yields a compound having the formula OMs XX ANH ICH C ~ R4 o~,C NH2 3 (at least one or R3 and R4 is hydrogen), wherein A is a protecting group.
Sulfonation of a compound of formula XX yields a compound having the formula QMs ANH CH C ",R4 XXI ¦ 3 ~C NHSO3 M
(at least one of R3 and R4 is hydrogen).
Cyclization of a compound of formula XXI is accomplished with base, e.g., potassium carbonate. The reaction is preferably carried out in a mixture of water and an organic solvent (e.g., a halogenated hydrocarbon such as 1,2-dichloroethane) under reflux conditions and yields a compound having the formula _4 ANH
XXII
C~ N-S03 M+
(at least one of R3 and R4 is hydrogen).
Deprotection of a sulfonated azetidinone of formula XXII , wherein A is a protecting group, as well as the counterpart compounds previously described having an R2-alkoxygroup by catalytic hydrogenation yields a compoundhaving the formula - GC155f ~ -22- 1 3 3 8 6 7 0 2 _4 (at least one of R3 and R4 is hydrogen), wherein R2 is hydrogen or alkoxy which can be converted to the corresponding zwitterion having the formula R2 _4 XXIV NH3 = 3 O~ ~-SO3 (at least one of R3 and R4 is hydrogen) by treatment with an acid such as formic acid.
Deprotection of a sulfonated azetidinone of formula XXII wherein A is a Boc protecting group using acidic conditions (e.g., using formic acid) yields the corresponding zwitterion of formula XXIV.
An excellent source for the ~lactam starting materials are the 6-aminopenicillanic acids and the 7-aminocephalosporanic acids which can bear an optional 6 alkoxy or 7-alkoxy substituent respectively. These compounds are of the formulae ~2~ CH3 Rl-NH ~ ~ -CH3 XXVa O" C-COOH

~ and XXVb R ~ S ~
N ~ CH3 COOH

_ ~C155f -23~ 1 3 3 8 6 7 0 respectively wherein R2 is hydrogen or alkoxy and Rl is hydrogen or acyl. By adapting procedures described in the literature, 3-amino-2-azetidinones can be prepared; see for example, Chem. Soc. Special Publication No. 28, pg. 288(1977); The Chemistry of Penicillins, Princeton Univ. Press, pg. 257, and Synthesis, 494(1977).
The 6-amino-penicillanic acid or 7-amino-cephalosporanic acid is first desulfurized by reduction using Raney nickel. The reaction can be run in water under reflux conditions; the resulting compound has the structural formula ~2 Rl-NH
XXVI
-fH-CH(CH3)2 COOH
Replacement of the carboxyl group of the compound of formula XXVI with an acetate group followed by hydrolysis yields a 3-amino-3-alkoxy-2-azetidinone of formula IV wherein Rl is hydrogen or acyl, R2 is hydrogen or lower alkoxy and R3 and R4 are hydrogen.
Treatment of a compound of formula XXVI with cupric acetate and lead tetraacetate in an organic solvent (e.g., acetonitrile) replaces the carboxyl group with an acetate group. Hydrolysis of the resul-ting compound can be accomplished using potassium carbonate in the presence of sodium borohydride.
Introduction of a sulfo group in the l-position of the above 3-amino-3-alkoxv-2-azetidinone compound can be accomplished by reacting the intermediate with a complex of dimethylformamide and sulfur trioxide.

GC155f -A 3-azido-2-azetidinone starting material can be prepared by first reacting an olefin having the formula XXVII CH C - R

with a halosulfonylisocyanate (preferably chloro-sulfonylisocyanate) having the formula XXVIII O = C = N - S02-halogen to yield an azetidinone having the formula XxIx ~ N-S02-halogen Reductive hydrolysis of an azetidinone of formula XXIX yields an N-unsubstituted ~-lactam having the formula R4 R
XXX
O" NH
For a more detailed description of the above described reaction sequence reference can be made to the literature; see, for example, Chem. Soc. Rev., 5, 181 (1976) and J. Org. Chem., 35, 2043 (1970).
An azido group can be introduced in the 3-position of an azetidinone of formula XXX (or the sulfonated counterpart) by reaction of the compound with an arylsulfonyl azide (such as toluenesulfonyl azide) to obtain a starting azetidinone having the formula - GC155,f, N3 = R3 XXXI
o~ NH
The reaction proceeds best by first protecting the azeti'dinone nitrogen with a silyl residue (e.g., t-butyldimethylsilyl, or t-butyldiphenylsilyl), then generating the anion at the 3-position of the nucleus with a strong organic base (e.g., lithium diisopropyl-amine) at a low temperature, and then treating the anion with toluenesulfonyl azide. The resulting intermediate is quenched with trimethylsilyl chloride, and subsequent acid hydrolysis or fluoride solvolysis of the N-protecting group yields the compound of formula XXXI.
Alternatively, the compound of Formula XXXI can be obtained by first reacting a primary amine having the formula H2N-CH2 ~ O-alkyl or 2N ~ O-alkyl O-alkyl with an aldehyde of the formula R3CH=O to yield the corresponding Schiff base. A[2+2]cycloaddition with an activated form of -azido acetic acid yields a 3- 5 azido-2-azetidinone having the formula N ~ R3 N - Q

GC155f wherein Q is -CH2 ~ O-alkyl ~ O-alkyl or O-alkyl Oxidative removal of the Q-substituent yields the compound of formula XXXI.
The 3-acylamino-2-azetidinones can be obtained by first reducing a 3-azido-2-azetidinone of formula XXXI to obtain the corresponding 3-amino-2-azetidinone and then acylating the 3-amino-2-azetidinone.
As previously mentioned in the case where R2 is lower alkoxy, the product can be prepared from the counterpart product wherein R2 is hydrogen.
Clorination of the amide nitrogen of a nonalkoxylated compound yields an intermediate having the formula Cll H R4 R -N R

~ N-SO3 M
Reagents and procedures for N-chlorinating amides are known in the art. Exemplary reagents are tert.-butyl hypochlorite, sodium hypochlorite, and chlorine. The reaction can be run in an organic solvent (e.g., a lower alkanol such as methanol) or in a two phase solvent system (e.g., water/methylene chloride) in the presence of a base such as sodium borate decahydrate.
The reaction is preferably run at a reduced temperature.

GC155f -27- 1 33867~

Reaction of an intermediate of formula XXXI with an alkoxylating agent, e.g., an alkali metal alkoxide yields a product of formula I wherein R2 is alkoxy, in combination with its enantiomer. The reaction can be run in an organic solvent, e.g., a polar organic solvent such as dimethylformamide, at a reduced temperature.
An alternative synthesis for preparing the compounds of formula I wherein R2 is alkoxy comprises first alkoxylating an intermediate of formula VI wherein RlNH is a carbamate (e.g., Rl is benzyloxycarbonyl) and R2 is hydrogen and then introducing a sulfo group in the l-position of the resulting compound. Chlorination of a compound of formula VI using the procedure described above (for chlorination of a non-alkoxylated compound of formula I to yield a compound of formula XXXII yields an intermediate having the formula Cl R4 ~ N-Cl Using the alkoxylation procedure described above (for converting a compound of formula XXXII to a product of formula I), and subsequently adding a reducing agent such as trimethylphosphite, the com-pound of formula XXXIII can be converted to an intermediate having the formula O-alkyl R4 C H O-C0-NH = R

~ NH

in a combination with its enantiomer.

GC155f The above procedures yield those products of formula I wherein R2 is alkoxy as a racemic mixture.
If desired the enantiomer having the R configuration can be isolated from the racemic mixture using conventional techniques such as fractional crystallization of a suitable salt with an optically active organic amine or by ion-paired chromatography utilizing an optically active cation.

~C155f _ -29- 1 338670 Biological Production of the Antibiotic ~M5117 Salts of (R)-3-(acetylamino)-3-methoxy-2-oxo.
l-azetidinesulfonic acid (formula I, Rl is acetyl and R2 is methoxy; referred to hereinafter as ~5117) can also be prepared by cultivation of a strain of the microorganism Chro~obacterium violaceum SC 11,378, which has been deposited in the American Type Culture Collèction as A.T.C.C. No. 31532.

The Microorganism The microorganism used for the production of EM5117 is a strain of Chromobacterium violaceum SC 11,378. A subculture of the microorganism can be obtained from the permanent collection of the American Type Culture Collection, Rockville, ~aryland. Its accession number in the repository is A.T.C.C. No. 31532. In addition to the specific microorganism described and characterized herein, it should be understood that mutants of the microorganism (e.g., mutants produced through the use of x-rays, ultraviolet radiation or nitrogen mustards) can also be cultured to produce E~15117.
Chromobacterium violaceum SC 11,378, A.T.C.C.
~o. 31532 can be isolated from a moist soil sample containing the microorganism by first stamping GC155f the soil sample on a medium containing:
Soil Extract - 400 ml Distilled Water- 600 ml Yeast Extract - 5.0 g Glucose - 10.0 g Agar - - 17.5 g The medium is adjusted to p~l 6.0 and sterilized in an autoclave at 121 C for 20 minutes. After 24 to 72 hours incubation at 25C colonies of the Chromobacterium violaceum SC 11,378 are isolated from the plated soil. These isolated colonies are then grown on a medium containing:

Yeast Extract - 1 g Beef Extract - 1 g NZ amine A - 2 g Glucose - 10 g Agar - 15 g Distilled water to 1 liter The medium is adjusted to pH 7.3 and autoclaved at 121C for 30 minutes.
Chromobacterium violaceum SC 11,378 is a gram negative rod often showing barring, bipolar staining and lipid inclusions. It is motile by a single polar flagellum with occassional --GC155f lateral flaqella which are smaller in size.
On nutrient agar Chromobacterium violaceum SC 11,378 produces violet colonies. The pigment is enhanced on media rich in tryptophane and S yeast extract. In nutrient broth it produces a violet ring on the wall of the tube but no confluent pellicle. The violet pigment is soluble in ethanol but insoluble in water and chloroform.
Chromobacterium violaceum SC 11,378 is mesophilic, growing over a range of 15-37C;
no growth occurs at 4C or above 37C. Casein is hydrolyzed strongly by the microorganism, which is oxidase positive. In the presence of Chromobacterium violaceun SC 11,378 glucose, fructose and trehalose are fermented (method of Hugh ~ Leifson, 1953). L-Arabinose is not - utilized by the microorganism either fermentatively or oxidatively. Hydrogen cyanide is produced by 20 the microoryanism and aesculin hydrolysis is negative.
The above described key characteristics provide the basis for the identification of the microorganism as Chromobac terium violaceum as 25 distinguished from Chromobacterium lividum, the only other species of the yenus Chromobacterium recognized in the 8th edition of Bergey's Manual of Determinative Bacteriology.
Additional strains of Chromobacterium 30 . violaceum can also be cultured to produce EM5117.

----ÇC155f The Antibiotic To obtain the antibiotic EMS117, Chromobacterium violaceum SC 11,378 A.T.C.C. No. 31532 is grown at, or about, 25C under submerged aerobic conditions - 5 on an aqueous nutrient medium containing an assimilable carbon and nitrogen source.
The fermentation is carried out until substantial antibiotic activity is imparted to the medium, usually about 18 to 24 hours, preferably about 10 20 hours.
Using the following procedure, EM5117 can be separated from the fermentation medium and purified. After the fermentation-has been completed the broth can be centrifuged to remove the mycelium lS or, alternatively, filtration can be used to remove the mycelium from the broth. After removing the mycelium from the broth E~.5117 can be extracted f-rom the broth. Preferably the broth is extracted at pH 5 by ion-pair extraction with cetyldimethyl-20 - benzylammonium chloride in methylene chloride and back-extracted into aqueous sodium iodide (adjusted to pH 5 with acetic acid). After concentration of the sodium iodide extract in~-vacuo, an aqueous solution of the residue can be washed with 25 butanol. Concentration of the resulting aqueous solution to dryness yields a residue whi~h is dissolved, to the extent possible, in methanol.
Centrifugation can be used to separate insolubles which are washed with methanol and then discarded.
.

1 338670 GC155f Purification of the antibiotic can be accomplished by dissolving the methanol concentrate in methanol/water ~1:1) and applying it to a chromatography column, e.q., one comprising Sephadex G-10 in the same solvent mixture.
After elution with the same mixture active fractions are combined and concentrated to dryness. The residue is mixed with methanol and insoluble material is filtered out and discarded.
Further purification is achieved by applying the methanol soluble material to a column of DEAE cellulose. The column can be eluted with a linear gradient.prepared from pH 5 sodium 0Ø M phosphate buffer and pH 5 sodium 0.1 M phosphate buffer. Active fractions are combined, concentrated and methanol insoluble materials are removed and discarded.
Still further purification of EM5117 is accomplished by dissolving this material in water and placing the solution on a column of *
Sephadex LH-20, a~d eluting the column with water.
Active fractions are combined and concentrated.
Further purification of EM5117 is then acco;nplished by dissolving the material in water, placing the solution on a column of Diaion HP-20AG and eluting with water. Active fractions are combined and concentrated. The concentrate is dissolved in water and passed through a column of ~owex 50W-X2, potassiurn form, washing with water. The effluent can be concentrated to yield a crystalline material *Trade Mark GC155f ~34~ 1 3 3 8 6 7 0 which is relatively pure potassium salt of EM5117.
The above described isolation and purification process yields the potassium salt of EM5117. Other salts can be prepared corresponding to the form of the ion exchange resin utilized in the final purification step.

Biological Pr~oduction of the Antibiotic EM5210 Salts of (R)-3-[[_-(D-y-glutamyl)-D-alanyl]-amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid (formula I, Rl is D-~-glutamyl-D-alanyl and R2 is methoxy; referred to hereinafter as EM5210) can also be prepared by cultivation of various acetic acid bacteria.
The Microorganism The microorganism Gluconobacter species - SC11,435 can be used for the production of EM5210. A subculture of the microorganism can be obtained from the permanent collection of the American Type Culture Collection, Rockville, Maryland. Its accession number in the repository is A.T.C.C. No. 31581. In addition to the specific microorganism described and characterized herein, it should be understood that mutants of the microorganism (e.g., mutants produced through the use of x-rays, ultraviolet radiation or nitrogen mustards) can also be cultured to produce EM5210.

GC155f Gluconobacter species SC11,435, A.T.C.C.
No.31581 can be isolated from ground moss containing the microor~anism by first incubating the ground moss in a 10% aqueous pectin solution (pH 2.5~ for 7 days at 25 C. The organism can - then be isolated ~y plating on a medium containing:
*Extract of Spartina patens Grass 400 ml Distilled Water 600 ml Yeast Extract 5 g Glucose 10 g Crude Flake Agar 17.5 g *Extract of Spartina patens grass is prepared by adding 500 g of chopped, dried grass to 3 liters of tap water, brought to a boil and simmered for 30 minutes.
The medium is adjusted to p~ 6.0 and sterilized in an autoclave at 121C for 20 minutes. After - about 18ho~rs incubation at 25 C, colonies of the Gluconobacter species SC11,435 are isolated from the plated pectin enrichment solution. These isolated colonies are then grown on a medium containing:
Yeast Extract 1 g Beef Extract l-g NZ amine A 2 g Glucose 10 g ~gar 15 g Distilled Water to 1 liter Tne medium is adjusted to pl~ 7.3 and autoclaved at 121C for 30 minutes.
Gluconobacter species SC11,435 is a pleiomorphic gram negative rod motile by means GC155f of one to three polar Flagella. It is obligately aerobic, catalase positive and oxidative. It is differentiated from Pseudomonas in that it is cytochrome oxidase negative and tolerant of extremely acid conditions. These characteristics indicate that the microorganism is more closely related to the acetic acid bacteria than to true Pseudomonads.
On BBL Trypticase soy agar Gluconobacter species SC11,435 growfi a~ a mixture of rough and smooth colony types. The rough type is associated with a faint yellow soluble pigment whereas the smooth type is mucoid and pigmentless.
Dissociation between the two types is influenced by media, temperature and storage conditions.
EM5210 activity tends to decrease in cultures - where the rough component is dominant.
On BBL Wart agar (pH 4.8) Gluconobacter species SC11,435 grows luxuriantly as heaped up mucoid slimy colonies. Similar growth is obtained on ~alt-yeast extract agar (1% each) adjusted to pH 4.5.
On medium containing 1% yeast extract, 10%
glucose, 3~ calcium carbonate and 2.5~ agar, sufficient acid is produced from the glucose to produce a zone of clearing of the calcium carbonate around the growth. This is a characteristic feature of Acetobacter and Gluconobacter.
In the presence of Gluconobacter species SC11,435:(i) a brown soluble pigment is produced on yeast extract-glycerol and yeast extract-calcium GC155f lactate agar plates, (ii) acid is pro~ucea on glucose, fructose, galactose, mannose, xylose, mannitol and arabinose, but no growth or acid production occurs on rhamnose, lactose, sucrose or maltose, when these sugars are incorporated into ~ugh and Leifson's medium.
The biological production of E~15210 is not - limited to Gluconobacter species SC11,435, but is broadly distributed throughout the acetic bacteria. The following cultures can each be used for the production of E~15210:
Acetobacter pasteurianus subsp. pasteurianum A.T.C.C. 6033 Acetobacter aceti subsp. aceti A.T.C.C. 15973 Gluconobacter oxydans subsp. oxydans A.T.C.C. 19357 Gluconobacter oxydans subsp. suboxydans A.T.C.C. 23773 Gluconobacter oxydans subsp. oxydans A.T.C.C. 15178 Acetobacter aceti suvsp. liquefaciens A.T.C.C. 23751 Acetobacter peroxydans A.T.C.C. 12874 Gluconobacter oxydans subsp. suboxydans A.T.C.C. 19441 Acetobacter sp. A.T.C.C. 21780 Gluconobacter oxydans subsp. industralis A.T.C.C. 11894 GC155f -The ~ntibiotic To obtain the antibiotic EM5210, Gluconobacter species S~11,435 A.T.C.C. No. 31581 or one of the microorganisms listed above, can be grown at, or about, 25C under submerged aerobic conditions on an aquèous nutrient medium containing an assimilable carbon and nitrogen source. The fermentation is carried out until substantial antibiotic activity is imparted to the medium, usually ab~ut 16 to 24 hours, preferably about 20 hours.
Using the following procedure EM5210 can be separated from the fermentation medium and purified. After the fermentation has been completed the broth is centrifuged to remove the bacteria and the antibiotic is removed from the broth supernateat pH 3.7 by absorption onto an anion exchange resin e , Dowex l-X2. Antibiotic is eluted from the resin with sodium chloride at about pH 4, and the eluate is concentrated and passed through a charcoal column. EM5210 is then eluted from the charcoal with methanol:water,l:l.
The active fractions are collected and dried, and then placed on an anion exchange column, e g., *
Dowex l-X2, and eluted with a gradient of sodium chloride buffered at pH 4. Active fractions are concentrated and desalted on a macroreticular styrene-divinylbenzene copolymer resin (Dianion HP20AG) column. Active fractions eluted with water are concentrated and freeze dried and this material represents the sodium salt of EM5210.

* Trade Mark GC155f ~39~ 1 3 3 8 6 7 0 The above described purification procedure will yield the sodium salt of EM5210. Other salts can be obtained by changing the salt used to elute EM5210 in the ion-exchange chromatography described above, e.g., using potassium chloride to give the potassium salt.
The following examples are specific embodiments of this invention.

G~155f _ -40-1 338~70 Example 1 (S)-N-(2-Oxo-l-sulfo-3-azetidinyl)-2-phenylacetamide, ootassium 5 alt .lethod I:
A) l-[(lR)-carboxy-2-methyl(propyl)]-2-oxo-(3S)-~phenyl[acet-yl(amino)~azetidine Raney nickel is washed with water by decantation for several hours until the pH of the water (5-6 times volume of that of the Raney nickel)is 7.6.
To a solution of 9.0 g of penicillin G
(Na ) in 500 ml of water is added 54 g (90 ml) of Raney nickel. The flask, fitted with a reflux condenser, is immersed in a bath at 155C. ~7hen refluxing begins, it is conti~e~
for 15 minutes. The flas~ is immediately cooled in an ice-water bath, and the Raney nickel is removed by filtration through Celite. The pH is adjusted to 3 using dilute HCl, and the aqueous solution is concentrated to ca. 150 ml and cooled.
The oily layer crystallizes upon scratching.
After washing with water and drying in vacuo for 3 hours at 50 C there is 3.83 g of the title compound.
B) l-[Acetyloxy-2-methyl(propyl)]-2-oxo-(3S){phenyl-[acetyl(amino)]]azetidine Nitrogen is bubbled for 15 minutes through a stirred suspension of 608 m~ (2 mmol) of the above compound in 20 ml of dry acetonitrile.
A water bath at 40-45C is used for several minutes to dissolve all of the acid. The water ~ GC155f _ ~ -41- 1 338670 bath is removed, and powdered cupric acetate monohydrate (182 mg, 1 mmol~ is added, followed after 1 minute of stirring, by 886 mg (2 mmol) of lead tetracetate. The mixture is stirred at room temperature for 20 minutes. The acetonitrile solution is decanted from the precipitate, and the solids are washed with ethyl acetate. The combined acetonitrile-ethyl acetate solution is evaporated to a residue, which is taken up in ethyl acetate-water. The ethyl acetate layer is washed sequentially with water (3 times), aqueous sodium bicarbonate (pH 7), and water.
The ethyl acetate layer is dried over sodium sulfate and evaporated to a residue (515 mg), lS which is used without further purification in the next reaction.

C) 2-Oxo-(3S)-[phenyl[acetyl(amino)]]azetidine To a solution of 911 mg (2.86 mmol) of the above compound in 21 ml of methanol is added 3.5 ml of water followed by 383 mg - (2.86 mmol) of potassium carbonate. The mixture is stirred under nitrogen for 1 minute, and then 1~0 mg (4.30 mmol) of sodium borohydride is added. The reaction is stirred at room temp-erature for 20 minutes. The methanol is removedin vacuo, and the residue is taken up in ethyl acetate and a small amount of water. This is adjusted to pH 2.5. The ethyl acetate layer is washed at pH 7.0 with aqueous sodium bicar-bonate and then a small volume of water and ~CI-~5~
-~ 338670 finally dried over sodium sulfate and evaporated to give the crude product (493 mg). Addition of a small amount of ethyl acetate gives 250 mg (43% yield) of the desired crystalline product.
Further quantities of product can be obtained by crystallization or chromatography.

D) (S)-N-(2-Oxo-l-sulfo-3-azetidinyl)-2-phenyl-acetamide, potassium salt Pyridine SO3 complex (215 mg, 1.35 mmol) is added to a stirred solution of 251 mg (1.23 mmol) of the above product in 2 ml of dry dimethyl-formamide and 2 ml of dry methylene chloride under nitrogen at room temperature. The mixture is stirred for 3 hours. The solvents are removed _ vacuo, and the residue is taken up in methylene chloride-water. The pH is adjusted to 6.5 using 2N potassium hydroxide. The aqueous layer is washed with methylene chloride (3 times) filtered and evaporated to a residue. The residue is stirred with 20 ml of methanol, and the potassium sulfate is removed by filtration.
The filtrate is evaporated to a residue, which is stirred with 10-15 ml of methanol. The solids are collected to give 49 mg of the title compound, melting point 189C, dec.
Anal. Calc'd for CllHllN2O5SK: C, 40.99; H, 3.44;
N, 8.69; S, 9.93 Found: C, 45.96; H, 3.83;
N, 9.86; S, 8.99 The compound has identical spectral characteristics to the product obtained in Method II.

GC155f Method II:
A solution of 660 mg of (S)-2-oxo-3- tI (phenyl-methoxy)carbonyl]amino]-l-azetidinesulfonic acid, potassium salt (see Example 3) in 13 ml of water is stirred in an atmosphere of hydrogen for 2 hours with 200 mg of 10% palladium on charcoal.
The catalyst is filtered and the filtrate diluted with an equal volume of acetone and cooled in an ice bath. Over 30 minutes, phenylacetyl chloride (eight 40 ~1 portions) and 10% potassium bicarbonate solution are added tpH kept between 5.2-5.8). After 40 minutes, the solution is concentrated in vacuo to remove acetone and applied to a 200 ml HP-20 column. Elution with water and then water-acetone (9:1) gives 160 mg of crude product after T~C
eY~m;n~tion of Rydon positive fractions, followed by pooling and evaporation. Crystallization from methanol-ether gives 101 mg of the title compound, melting point 210 C, dec.
A-nal. Calc'd for CllHllN2O5SK /2 H2O: C, 39.86;
H, 3.65; N, 8.45; S, 9.68; K, 11.80 Found: C, 40.01; H, 3.37; N, 8.59; S, 9.59;
K, 11.98 NMR(D20) 3.66 (s,3), 3.67(d of ~, )=6.4), 3.90 (t,)=)), 4.90 (d of d, )=6.4), 7.36 ppm (m.5).

- - GC155f Method III: 1 338670 To a solution of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (121 mg; see Example 6A) in dry methylene chloride S (3 ml) is added 40 mg of phenylacetic acid and 61 mg of dicyclohexylcarbodiimide. The mixture is stirred for 48 hours at room temperature, and filtered to remove dicyclohexylurea. The solvent is removed in vacuo and the compound residue is taken up in acetone, filtered and the title compound precipitated by the addition of 5 ml of acetone saturated with potassium iodide.
The supernatant is decanted, the residue washed with acetone (3 times) and, after drying, 48 mg of product is obtained,having spectral characteristics in agreement with the products of Methods I and II.
Method IV:
A solution of 2.83 g of (S)-2-oxo-3[~(phenyl-me~hoxy)carbonyl~]amino]-l-azetidinesulfonic acid, pyridine salt (see Example 2) in 36 ml of water is stirred in an atmos~here of hydrogen with 707.5 mq of 10% palladium on charcoal (175 ml of hydroqen taken llp). After 2 hours the slurry is filtered, and the filtrate cooled to 0C
and diluted with 46 ml of acetone (initial pH=
4.25 adjusted to pH 6.7 with cold 10% potassium bicarbonate solution). A solution of 2.4 ml of phenylacetyl chloride in 10 ml of acetone is added dropwise over 15 minutes. The pH is maintained between 5.2 to 5.8 by the simultaneous addition of cold 10% potassium bicarbonate solution. After 45 minutes the slurry is GC155f _45- 1 3 3 8 6 7 0 diluted with 93 ml of O.SM potassium phosphate (pH=4.2) and concentrated to remove acetone.
The slurry is filtered and washed with water.
The filtrate and washings are combined and applied S to a 450 ml HP-20 column. Elution with 1 liter of 0.5M potassium phosphate (pH=4.2), 1 liter of water, and then 2.5 liters of 9:1 water-acetone gives 1.285 g of the title compound in fractions 14-19 (fraction 1-15 were 200 ml, fractions 16-21 were 100 ml). Spectral data is in agreement with that obtained in the foregoing methods.

~C155f Example 2 (S)-2-Oxo-3-~[(phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, pyridine (1:1) salt S Method I:
A) l-~(lR)-Car~oxy-2-methyl(propyl)]-2-oxo-(3SL-~(phenylmethoxy)carbonyl]amino]azetidine A slurry of 6-aminopenicillanic acid (12.98 g, 0.06 mole) in 140 ml of water containing 5.18 g of sodium bicarbonate (stirred for ca.
10 minutes without camplete solution) is added in one portion to a well-stirred (mechanical stirrer) suspension of Raney nickel (washed with water to pH 8.0, 260 ml of slurry=130 g) in a 70 C oil bath. After 15 minutes the slurry is cooled, filtered, and the filtrate treated with 5.18 g of sodium bicarbonate and a solution of 11.94 g (0.07 mole) of benzyl chloroformate in 12 ml of acetone. After 30 minutes, the solution is acidified to pH 2.5 and extracted with methylene chloride. The organic layer is dried, evaporated, and triturated with ether-hexane to give a total of 6.83 g of the title compound.
B) l-~(Acetyloxy)-2-methyl(propyl)]-2-oxo-(3S)-[[(~henylmethoxy)carbonyl]amino~azetidine A solution of 6.83 g (0.0213 mole) of the above acid in 213 ml of acetonitrile is treated -30 with 1.95 g (0.Q107 mole) of cupric acetate monohydrate and 9.5 g (0.0213 mole) of lead GC155f -47~ 1 3 3 8 6 7 0 tetraacetate. The slurry is immersed in a 65C
oil bath and stirred with a stream of nitrogen bubbling through the slurry until the starting material is consumed. The slurry is filtered and the solids washed with ethyl acetate. The combined filtrate and washings are evaporated _ vacuo and the residue taken up in 100 ml each - of ethyl acetate and water and adjusted to pH 7.
The ethyl acetate layer is separated, dried, and evaporated to give 6.235 g of the title compound.

C) (S)-(2-Oxo-3-azetidinyl)carbamic acid, phenylmethyl ester A solution of 3.12 g (0.0093 mole) of the above acetate in 70 ml of methanol and 7 ml of water is cooled to -15C and 1.33 g of potassium carbonate and 349 mg of sodium borohydride are added. The reaction mixture is stirred at -15C - 0C. After the reaction is complete (ca. 2 hours), the mixture is neutralized to pH 7 with 2N HCl and concentrated in vacuo.
The concentrate is adjusted to pH 5.8, saturated with salt and extracted with ethyl acetate (3 times). The organic layer is dried and evaporated in vacuo. The residue is combined wit~ material from a similar experiment and triturated with ether to give 3.30 g of the title compound.

GC155f _ -48-D) (S)-2-Oxo-3-~(phenyLmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, pyridine salt Method I: -A solution of 440 mg (0.002 mole) of the above azetidinone in 2 ml each of dry methylene chloride and dry dimethylform~ e is stirred for 2 hours under nitrogen with 35Q mg (0.0022 mole) of pyridine-sulfur trioxide complex. The bulk of the solvent is then removed in vacuo and the residue triturated with ethyl acetate to give 758 mg of solid, which is primarily the title compound.
NMR(D20-CD30D) 3.63(1H, d of d~ )~6-4)~ 3-90 (lH~ t~
)=6), 4.85 (lH, d of d, )=6.4), 5.10(2~,S) 7.27 (5H,S) 8-0-9.oppm(m's 5H).

Method II:
Chlorosulfonyltrimethylsilyl ester (18.87 g) is added dropwise at -20C to 7.9 g of anhydrous pyridine with stirring under a nitrogen atmosphere.
When the addition is complete, stirring is continued for 30 minutes at room temperature, and trimethylchlorosil-ane is then removed in vacuo. A solution of 20 g of the above azetidinone (Method I, part C) in 120 ml of dimethylform~m;~e and 120 ml of methylene chloride is added and stirring at ambient temperature is continued for 3.5 hours. The solvent is distilled off in vacuo and the oily residue crystallized by addition of ethyl acetate, yielding 31 g of the title compound. NMR data is identical to that of the Method I product.

GC155f _ _49_ Example 3 . ( S ) -2-Oxo- 3-~l(phenylmethoxy)carbonyl]amino~-l-azetidinesulfonic acid, potassium salt Method I:

(S) -2-Oxo-3- ~ I (phenylmethoxy~carbonyl~amino]-l-azetidinesulfonic acid, pyridine salt (13S mg;
see Example 2 ) is dissolved in 2 ml of 0 . 5 M
monobasic potassium phosphate (adjusted to pH S . 5 .
with 2N potassium hydroxide) and applied to a 25 ml HP-20AG column. The column is eluted with 100 ml of buffer, 200 ml of water and 100 ml of 1:1 acetone-water. Fractions (25 ml) 14-15 are highly Rydon positive. Evaporation yields lS 80 mg of material which is primarily the title compound(spectral data identical to that prepared below).

Method II:
(S ) -2-Oxo-3- [ [ (phenylmethoxy)carbonyl~amino]-20 - l-azetidinesulfonic acid, pyridine salt (600 mg;
see Example 2 ) is dissolved in 2 ml of water and mixed with 15 ml of pH 5 . S monobasic potassium phosphate buffer. A solid forms and the slurry is cooled to 0C, filtered, washed with cold buffer, cold 50% ethanol, ethanol and ether to give 370 mg of the title compound (containing excess potassium ion by analysis). A solution of 280 mg of the salt in 10 ml of water is applied to a 100 ml HP-20 column. The column is eluted with 200 ml of water and then water-acetone (9:1).

GC155f ~5~-Fractions (5Q ml) are collected; evaporation of fraction 7 gives a solid. Trituration with acetone, filtration, and drying in vacuo gives 164 mg of the title compound, melting point 193-196 C.
Anal. Calc'd for Cl~HllN2O6SK 1/2H2O: C, 38.02;
H, 3.48; N, 8.06; S, 9.23; K, 11.25 Found: C, 38.19; H, 3.24; N, 8.15; S, 2.12;
K, 11.53 NMR(D20) 3.69(1H, d, of d, )=6.4), 3.91(1~, t, J=6)~
4.76(lH, m), 5.16(2H,S), 7.43ppm (5H,S) Method III:
(S)-(2-Oxo-3-azetidinyl)carbamic acid, phenylmethyl ester (20.0 g, see Example 2C) is suspended in 200 ml of acetonitrile, 21.6 ml of monotrimethylsilyltrifluoroacetamide (25.3 g) is added and the mixture is heated to 50C with stirring for 1 hour. After cooling in an ice bath to 0 C, 17.2 g of trimethylsilyl chlorosul-fonate is dropped in and the solution is stirred at ambient temperature for 6 hours. To the solution is added 24.2 g of potassium ethyl hexanoate in 100 ml of butanol and stirring is continued for an additional 1 hour. The slurry is poured into 1 liter of dry diethyl ether and the precipitate is filtered off and dried in vacuo. The compound is dissolved in 500 ml of water, the pH is adjusted to 5.0 with potassium carbonate, insoluble material is filtered off and the mother liquor is freeze dried. The yield of crude compound is 19.4 g. The compound contains small amounts of potassium chloride which is removed by chromatography,spectral data ide~tical to that of Method II.

~ GC155f Example 4 (S)-2-Oxo-3-~[(phenylmethoxy)carbonyl]amino~-l-azetidinesulfonic acid, tetrabutylammonnium salt (1:1) S ..
Method I:
(S)-2-Oxo-3-t I (phenylmethoxy)carbonyl~amino]-l-azetidinesulfonic acid, pyridine salt (1:1~
(34.3 g; see Example 2) is dissolved in 800 ml of water. The solution is cleared with charcoal, 30.7 g of tetrabutylammonium hydrogen sulfate in 80 ml water is added and the pH is adjusted to 5.5 with lN-potassium hydroxide. The solvent is removed in vacuo until a volume of about 200 ml lS is reached. The precipitated tetrabutylammonium salt is filtered off and dried in vacuo. The compound can be recrystallized from water or dissolved in methylene chloride, filtered and precipitated by addition of ether. Yield 34.3 g melting point 108-110C.

Method II:
(S)-2-Oxo-3-l~(phenylmethoxy)carbonyl]amino~-l-azetidinesulfonic acid, potassium salt (1:1) (20.2 g; see Example 3) is dissolved in 500 ml of water, filtered and 20.3 g of tetrabutyl-ammonium hydrogen sulfate in 100 ml of water is added. The pH is brought to 5.5 with lN potassium hydroxide.~ The volume is reduced in vacuo to about 100 ml and the precipitated tetrabutyl-ammonium salt is filtered off. The compound is GC155f dissolved in 30 ml of methylene chloride, filtered and precipitated by addition of ether, yielding 21 g of the title compound, melting point 109-111C.

- Example 5 (3S~ t(2-Oxo-l-sulfo-3-azetidinyl)amino~-carbonyl]benzeneacetic acid, phenylmethyl ester, - potassium salt (1:1) A) (S)-3-~mino-2-Azetidinone (S)-(2-Oxo-3-azetidinyl)carbamic acid, phenylmethyl ester (3 g; see Example 2C) is hydrogentated in 100 ml of methanol in the presence of 1 g of palladium on charcoal catalyst.
When the theoretical amount of hydrogen is absorbed, the catalyst is filtered off and the filtrate evaporated to dryness. On st~n~ing, 1.1 g of the title compound crystallizes.

B) (3S)--[~(2-Oxo-3-azetidinyl)amino~carbonyl]-~20 benzeneacetic acid, phenylmethyl ester The above azetidinone (3.0 g) is dissolved in 100 ml of dimethylformamide. The solution is cooled to 0C and 4.5 g of N-methylmorpholine is added followed (dropwise) by 10.8 g of a-(chlorocarbonyl)benzeneacetic acid, phenyl-methyl ester in 50 ml of acetonitrile withstirring. The mixture is stirred for about 16 hours at 5C. The solvent is distilled off in vacuo and 100 ml of water is added to the residue. The aqueous suspension is extracted twice with 100 ml portions of methylene chloride.

GC155~
~53~ 1 3 3 8 6 7 0 The organic layers are combined, washed with sodium bicarbonate, 2N phosphoric acid and water, dried with sodium sulfate, filtered and evaporated to dryness. The residue is crystallized with ethyl acetate and petroleum ether, yielding 8.7 g, melting point 164-166C.

C) (3S)-a-11(2-Oxo-l-sulfo-3-azetidinyl)amino~-carbonyl]benzeneacetic acid, phenylmethyl ester, potassium salt (1:1~
The aboYe compound (6.9 g3 is suspended in 150 ml of acetonitrile. Monotrimethylsilyl-trifluoroacetamide (5.7 g)is added and the solution is heated for 30 minutes at 50C with stirring. The solution is cooled to 0C and 3.9 g ~rimethylsilyl chlorosulfonate is added dropwise. When the addition is complete the mixture is heated to 50C for 5 hours.
After cooling to 20 C, 7.6 g of potassium ethyl hexanoate in 10 ml of butanol is added and stirring is continued for 30 minutes. On addition of 300 ml of ether the ti~tle compound precipitates and is filtered off. The crude product is stirred with 100 ml of dry acetonitrile for 30 minutes and filtered off, yielding 4.5 g of the title compound, melting point 118-120C.
Further purification of the crude product by HP20 chromatography-followed by freeze-drying yields pure material havinq a meltina point of 188-190C.

- GC155f Example 6 (S)-3-~[(2-Amino-4-thiazolyl)acetyl]amino~-2-oxo-l-azeti~;nesulfonic acid,- potassium salt A~ (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (S)-2-Oxo-3-[~(phenylmethoxy)carbonyl~amino~-l-azetidinesulfonic acid,tetrabutylammonium salt (2 g; see Example 4) is dissolved in 100 ml o~
di~ethylforr~ e and hydrogenated for about 30 minutes with 1 g of palladium on charcoal (10%) as catalyst. The catalyst is filtered off and the dimethylformamide is removéd leaving the title compound as an oil. NMR (CDC13) 3.82 (lH, t,)=
5.5),4.05(d. lH, d of d, J= 5.5, 2.5 cps).
B) (S)-3-[[(2-Amino-4-thiazolyl)acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt The above compound (2 g), 0.5 g of amino-thiazole acetic acid and 0.4 g of hydroxybenzo-triazole are stirred at 0 C in 100 ml of dry dimethylformamide, while a solution of 0.7 g of dicyclohexylcarbodiimide in 10 ml of dimethyl-forr-mi~e is added dropwise. After the addition is complete, stirring is continued for 12 hours at 20C. Insoluble urea is- filtered off and the solvent is evaporated in vacuo. The oily residue is treated with a solution of potassium perfluorobutane sulfonate in 20 ml of acetone at room temperature for 15 minutes. After the addition of 200 ml of dimethyl ether the title compound precipitates, and is filtered off, dried and purified via a 300 ml HP-20 chroma-tography column using water as eluent. The yield ~C155f is 8 sa mg of the title compound, melting point >300C.

Example 7 [3S(~)]-3-Lt(Formyloxy)phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dimethylformamide and 2 ml Qf propylene oxide are cooled to 0C.
A solution of O-formylmandelic acid chloride in 10 ml of acetonitrile is added dropwise with stirring. The temperature is maintained for 1 ho-lr and the solvent is then distilled off in vacuo. The oily residue is treated with a solution of 2 g of potassium perfluorobutane sulfonate in 15 ml of acetone. After adding 200 ml of ether, the title compound crystallizes and is filtered off yielding 1.5 g of product. The product is purified by HP-20 chromatography, m.p.
180-185C. with decomposition.
Example 8 [3S(+)]-3-tt(Formyloxy)phenylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of F.~mpl e 7, but substituting D-O-formylmandelic acid chloride for O-formylmandelic acid chloride, yields the title compound, melting point 120-125C, after freeze drying.

GC155f -56- ~ 3 3 8 6 7 0 Example 9 [3S(-)~-3-[~(Formyloxy)phenylacetyl]amino-2-oxo-l-azetidinesulfonic acid, potassium salt ~ollowing the procedure of Example 7, but substituting L-O-formylmandelic acid chloride for O-formylmandelic acid chloride, yields the title compound, containing 1 mole of water, melting point, 203-205C. After careful drying the product melts at 228-230C.
Example 10 (S)-2-Oxo-3- I (l-oxooctyl)amino]-l-azetidine-sulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dimethylformamide and 2 ml of propylene oxide are cooled to 0C. At this temperature a solution of 0.8 g of caprylic acid chloride in 20 ml of dry acetone is added dropwise and stirring is continued for 30 minutes. The solvent is evaporated in vacuo, and the oily residue is treated with 2 g of potassium perfluoro-butane sulfonate in 15 ml of acetone. The acetone is distilled off in vacuo. The residue is dissolved in S ml of water and chromatographed using 300 ml of HP-20 resin and water/acetone (9:1) as eluent, yielding 0.9 g of the title compound, melting point 173-180C, after freeze-drying.

GC155f ~57~ 1 3386 70 Example 11 13S(Z)]-3-[[~2-Amino-4-thiazolyl)[[[hydroxy(phenyl-methoxy)phosphinyl]methoxy]imino]acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt S (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (0.8 g; see Example 6A) in 30 ml of dimethylfo~r~ e~ 0.9 g of (Z)-2-amino-a-[[[hydroxy(phenylmethoxy)phosphinyl]-methoxy]imino]-4-thiazoleacetic acid, 0.3 g of hydroxybenzotriazole and 0.7 g of dicyclohexyl-carbodiimide are stirred for 24 hours at room temperature. The precipitated urea is filtered off and the solvent is removed in vacuo. The reamining oil is treated with an equivalent amount of potassium perfluorobutane sulfonate in 10 ml of acetone. The title compound is filtered off and purified using HP-20 resin and water as eluent, yielding 500 mg, melting point 210-215C,dec.

Example 12 [3S(Z)]-3-[~(2-Amino-4-thiazolyl)(ethoxyimino)-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dimethylfor~ e, 0.6 g of hydroxy-benzotriazole, 1 g of dicyclohexylcarbodiimide and 0.8 g of (Z)-2-amino-~-(ethoxyimino)-4-thiazoleacetic acid are stirred at room temperature for 24 hours. The solvent is distilled off and the residue is dissolved in 30 ml o~ acetone.

- 58 33 8 6 70 GC155f Urea is filtered off and the mother liquor is treated with a solution of 2 g of potassium perfluorobutane sulfonate in 20 ml of acetone.
After the addition of 200 ml of ether the title S compound precipitates, is filtered off and dried. Purification is accomplished by chroma-tography using anHP-20 column and water as eluent, yielding 1.1 g of the title compound, melting point 180-185C, dec.

Example 13 [3S(E)]-3-[l(2-Amino-4-thiazolyl)(ethoxyimino)-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 12, but substituting (E)-2-amino--~ethoxyimino)-4-thiazoleacetic acid for (Z)-2-amino-a-(ethoxy-imino)-4-thiazoleacetic acid, yields the title compound, melting point 160-170C after freeze drying.

Example 14 [3S(Z)-3-[[(2-Amino-4-thiazolyl)](2,2,2-trifluoro-ethoxy)imino~acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 12, but substituting (Z)-2-amino-a-[(2,2,2-trifluoro-ethoxy)imino]-4-thiazoleacetic acid for (z)-2-amino--(ethoxyimino)-4-thiazoleacetic acid, yields the title compound melting point 160-170C after freeze-drying.

....

- . 1 3 3 8 6 7 ~

Example 15 (S)-2-Oxo-3-l(l-oxopropyl)amino]-1-azetidine-sulfonic acid, potassium salt Method I:
(S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformAm;~e and 4 ml of propylene oxide are cooled to 0C with stirring.
At this temperature 0.5 g of propionic acid chloride in 10 ml of acetonitrile is added dropwise and the mixture is stirred for 2 hours.
The solvent is distilled off in ~acuo and the oily residue is treated with an equivalent amount of potassium perfluorobutane sulfonate in 5 ml of acetone. On addition of ether the title compound crystallizes and is filtered off, yielding 0.8 g of product, melting point 135-140 C after freeze-drying.
Method II:
(S)-2-Oxo-3-tl(phenylmethoxy)carbonyl~amino~-l-azetidinesulfonic acid, tetrabutylammonium salt (4 g; see Example 4) is hydrogenated in 100 ml of diglyme with 1 g of palladium on charcoal.
The hydrogenation is complete after 2.5 hours.
The catalyst is filtered off and 2 ml of propylene oxide is added. After cooling to 0 C,0.5 g propionic acid chloride in 10 ml dry diglyme is added with stirring. After 30 minutes the solvent is removed in vacuo and the oily residue is treated GC155f with an equivalent amount of potassium perfluoro-butane-sulfonate in 20 ml of acetone. After the addition of ether, the title compound crystallizes, is filtered off and recrystallized from water/acetone, yielding 0.9 g of product, melting point 156-160C (dec.).

Example 16 [3S(+)~-3-[(Hydroxyphenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is stirred for about 16 hours with 1.5 g of 15- dicyclohexylcarbodiimide, 0.5 g of hydroxy-benzotriazole and 0.6 g of mandelic acid. The solvent is removed in vacuo and the residue is dissolved i~ 20 ml of acetone. The precipitated urea is filtered off and the mother liquor is treated with an equivalent amount of potassium perfluorobutane sulfonate. After the addition of ether the title compound precipitates and is filtered off, yielding 1.4 g of crude product.
After recrystallization from water, the product has a melting point of 138-140C.

Example 17 (S)-3-[[[(Cyanomethyl)thio]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) and 0.72 g of [(cyanomethyl)thio]acetic acid are dissolved in 70 ml of acetonitrile and a solution of 1.04 grams of dicyclohexylcarbodiimide GC155f -in acetonitrile is added dropwise. The mixture is stirred for about 16 hours at 0C and the precipitated dicyclohexylurea is filtered off, the filtrate evaporated and the oily residue dissolved in acetone. On the addition of a saturated solution of potassium-iodide in acetone, the title compound precipitates, yielding 1.1 g of product, melting point 150-155C.

Example 18 (S)-2-Oxo-3-[(lH-tetrazol-l-ylacetyl)amino]-l-azetidinesulfonic acid, potassium salt To a solution of 0.005 mole of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (see Example 6A) in 70 ml of dimethylforr~m;~e is added 0.77 g of lH-tetrazole-l-acetic acid and a solution of 1.13 g of dicyclohexylcarbodiimide in 5 ml of dimethylformamide. The mixture is stirred for about 16 hours at room temperature and the solvent is removed in vacuo. The remaining oil is dissolved in 20 ml of acetone and treated with 0.006 mole of a solution of potassium perfluorobutane sulfonate in acetone, yielding 1.5 g of the title compound, melting point 170-175C, dec.

Example 19 (S)-2-Oxo-3-[(2H-tetrazol-2-ylacetyl)amino]-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 18, but substituting 2H-tetrazole-2-acetic acid for lH-tetrazole-l-acetic acid, yields the title compound, melting point 175-177 C, dec.

GC155f ._ Example 20 (S)-2-Oxo-3-~(2-thienylacetyl)amino]-1-azetidine-sulfonic acid, potassium salt Following the procedure of Example 18, but substituting 2-thiopheneacetic acid for l~-tetrazole-l-acetic acid, yields the title compound, melting point 180-190C, dec.

Exa~ple 21 ~3S(Z)]-3-f[(2-Amino-4-thiazolyl)(methoxyimino)-acetyl~amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azeti~;nesulfonic acid, tetrabutylammonium salt (prepared as described in Example 6A from 7.9 g of (S)-2-oxo-3-~(phenylmethoxy)carbonyl]amino]-1-azetidine-sulfonic acid, tetrabutylammonium salt) is cooled to 0 C and 3.53 g of (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid is added, followed by a - 20 solution of 3.27 g of dicyclohexylcarbodiimide in 10 ml of dimethylformamide. The mixture is stirred for 16 hours at 5C, filtered and the solvent removed in vacuo. The residue is dissolved in acetone and filtered. On addition of 60 ml of a 10% solution of potassium perfluoro-butane sulfonate in acetone, 4.7 g of crude product crystallizes. The crude product is purified by chromatography on HP-20, 100-200 mesh to yield 3.0 g of the title compound, melting point 235C.

GC155f Éxample 22 (3S~-a~ 2-Oxo-1-sulfo-3-azetidinyl)amino~-carbonyl~benzeneacetic acid, dipotassium salt (3S)~ (2-Oxo-l-sulfo-3-azetidinyl)amino~-carbonyl]benzeneacetic acid, phenylmethyl ester, potassium salt (100 mg; see Example 5) is dissolved in 20 ml of anhydrous methanol. Palladium on charcoal (10 mg, 10%) is added and the mixture is treated with hydrogen for 15 minutes. The catalyst is filtered off, and the methanol evap-orated in vacuo. me residue is dissolved in5 ml of water and the pH adjusted to 6 with lN potassium hydroxide. After freeze drying crude product is obtained. The crude material is chromatographed over HP 20 resin (water as eluent), yielding 60 mg of the title compound melting point 80-85C.
Example 23 (S)-3-(Acetylamino)-2-oxo-1-azetidinesulfonic acid, potassium salt Method I:
A) (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, potassium salt (S)-3-(Benzyloxycarbonylamino)-2-oxo-1-azetidinesulfonic ~acid, potassium salt (169 mg;
see Example 3) is dissolved in 4.0 ml water and hydrogenated over 37 mg of 10% palladium on charcoal for 1 hour 40 minutes. The catalyst is removed by filtration and washed with 1 ml of 50% aqueous acetone.

_ ~C155~
-64~ 1 3 3 8 6 7 0 B) (S)-3-(Acetylamino)-2-oxo-1-azetidinesulfonic acid, potassium salt The above solution of the free amine is diluted with 3.5 ml of acetone and stirred in an ice bath. Acetyl chloride (320 ~1) is added over ca. 15 minutes in small portions and alternated with solid potassium bicarbonate to maintain pH
6.5-7.2. After 30 minutes a silica gel TLC in acetone:acetic acid (19:1) and the ~ydon test in~
the reaction to be essentially finished. Six ml of 0.5M pH 5.5 monobasic potassium phosphate buffer are added and the solution is acidified to pH 4.8 with 2N hydrochloric acid. The acetone is removed in vacuo and the aqueous solution thus obtained is passed through a 50 ml HP-20 AG
column to give 2.197 g of solid. This is digested with methanol to yield 282 mg of extractable material which still contains some salt. The product is further purified by passage through an IRC-S0 column, followed by acidification to pH 3.8 with subsequent stripping to dryness in vacuo. Trituration with acetone gives 64 mg of desired product containing ca. 0.5 equivalent of inorganic potassium salts. Final passage through a 200 ml HP-20 AG column, followed by lyophilization from .5 ml of water gives 22 mg of product as amorphous powder which is dried ln vacuo for 2 hours at 50C, melting point 170-180C after softening at 100C.
Anal. for C5H7O5N2SK, Calc'd: C, 24.38; H, 2.87;
N, 11.37; K, 15.9 Found: C, 26.06; H, 3.14;
N, 9.96; K, 18.04 GC155f _ , .

~ethod II:
A solution of 2.0 g of (S)-2-oxo-3-I[(phenyl-methoxy)carbonyl]amino]-l-azetidineuslfonic acid, pyridine salt (see Example 2) in 25 ml of water is hydrogenated over 500 mg of 10% palladium on charcoal. After 2 hours the solution is filtered, cooled to 0C and 40 ml of acetone is added.
The pH of the solution is kept between 5.2-5.8 by simultaneous addition of acetyl chloride and cold 10% potassium bicarbonate solution. The pH of the solution is adjusted to 4.2 with acetyl chloride and the solution is concentrated on the rotary evaporater to remove acetone.
Chromatography on a 300 ml HP-20 AG column lS (water eluant -25 ml fractions) gives 900 mg of the title compound in fractions 13 and 14 contaminated with some potassium acetate.
Rechromatography on HP-20 AG gives an analytical sample, melting point 205-210C.
Anal.calc'd for C5H7N2O5SK: C,24.38; H,2.86;
N,11.38; S,13.02; K,15.88 Found: C,24.23; H,2.81; N,11.25; S,12.86;
K,15.74 - GC155f ~xA~ple 24 (S~-2-Oxo-3-l(phenoxyacetyl)amino]-1-azetidine-sulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is cooled to 0C. Propylene oxide (2 ml) is added and 1 g of phenoxyacetyl chloride is added dropwise with stirring. The reaction is completed within 1 hour. The solvent is removed in vacuo and the residue is treated with an equivalent amount of potassium perfluorobutane sulfonate in 20 ml of acetone. On addition of ether the title compound (1 g) crystallizes and is filtered off and dried. After recrystal-lization from boiling water the title compound has a melting point of 176-180C.

Example 25 [3S(R*)~-3-[[[[[3-~(2-Furanylmethylene)amino]-2-oxo-l-imidazolidinyl]carbonyl]amino]phenylacetyl]-amino]-2-oxo-1-azetidinesulfonic acid, potassium - salt (S)-2-Oxo-3-l[(phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (3 g; see Example 4) is hydrogenated in 100 ml of dimethylformamide with 1.5 g of palladium on charcoal. After 0.5 hours, the catalyst is filtered off and 1.8 g of dicyclo-hexylcarbodiimide, 2 g of (R)-~-lL[3-~(2-furanyl-methylene)amino]-2-oxo-1-imidazolidinyl~carbonyl~-GC155f amino~benzeneacetic acid and 0.9 g of hydroxy-benzotriazole are added. After 3 hours the reaction mixture is evaporated to dryness, dissolved in 50 ml of dry acetone and the precipitated urea remo~ed by filtration~ An equivalent amount of potassium perfluorobutane sulfonate in 20 ml of acetone;is added and the title compound precipitates. Crystallization is completed by addition of 200 ml ether.
After filtration the title compound is recrystal-lized from water, yielding 2 g, melting point -220-225C, dec.

Example 26 [3S(R*)]-2-Oxo-3-[[[~(2-oxo-1-imidazolidinyl)-carbonyl]amino]phenylacetyl]amino]-l-azetidine-sulfonic acid, potassium salt (S)-2-Oxo-3-l[(phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (3 g; see Example 4) is hydrogenated in 100 ml of dry dimethylformamide with 1.5 g of palladium on charcoal; the catalyst is filtered off after 30 minutes. (R)-~-[[(2-Oxo-l-imidazoli-dinyl)carbonyl~amino]benzeneacetic acid (1.8 g), 1.3 g of dicyclohexylcarbodiimide and 0.9 g hydroxybenzotriazole are added and the solution is stirred for 2.5 hours. The solvent is removed in vacuo and theresidue is dissolved in 50 ml of acetone. The precipitated urea is filtered off and the mother liquor is treated with an equivalent GC155~
_ amount of potassium perfluorobutane sulfonate in 20 ml of acetone. The title compound crystal-lizes and is filtered off after the addition of 200 ml of ether, yielding 1.8 g of product, melting point 210-215C after recrystallization from water/acetone.

Example 27 [3S(Z)]-3-~[(Methoxyimino)phenylacetyl]amino~-2-oxo-l-azetidinesulfonic acid, potassium salt A) [3S(Z)~-3-[l(Methoxyimino)phenylacetyl~amino~-2-azetidinone (Z)-a-(Methoxyimino)benzeneacetic acid (3.58 g) is dissolved in methylene chloride, cooled to 5C, and treated with a solution of 4.53 g of dicyclohexylcarbodiimide in 50 ml of methylene chloride. The mixture is stirred for 30 minutes at 5C and a solution of 1.72 g of 3-amino-2-azetidinone (see Example 5A) in 100 ml of methylene chloride is added. The reaction mixture is kept at 5 C for 1 hour and for 2 hours at room temperature. After removal of the dicyclohexylurea by filtration, the filtrate is evaporated, yielding 6.6 grams of crude product.
This material is purified by chromatography on 750 grams of silica gel, using a mixture of methylene chloride/ethyl acetate (7:3) as eluent, yielding 2.9 g of product.

GC155f --69- t 3 3 8 6 7 0 B) [3S(Z))]-3-¦ r (Methoxyimino)phenylacetyl~-amino~-2-oxo-1-azetidinesulfonic acid, potassium salt Pyridine (0.5 ml) is dissolved in 5 ml of absolute methylene chloride, cooled to -3QC
and a solution of 0.93 ml of trimethylsilyl chlorosulfonate in 5 ml of methylene chloride is added. The mixture is stirred at room temperature for 30 minutes and evaporated in vacuo to dryness. The residue is dissolved in 10 ml of dimethylformamide and treated at room temper-ature with a solu~inn of l.23 ~ of the above azetidinone in 10 ml of dimethylformamide. After stirring for two hours at room t~m~rature, the solution is evaporated to dryness to yield 2.1 g of crude [3S(Z)]-3-[[(methoxyimino)phenylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, pyridine salt.
Treatment of the pyridine salt with tetrabutylammonium hydrogen sulfate yields the corresponding tetrabutylammonium salt which is extracted with methylene chloride and remains as an oil after evaporation.
Treatment of the tetrabutylammonium salt with an equimolar amount of potassium perfluorobutane sulfonate in acetone,evaporation, and treatment of the residue with ether, yields 1.6 grams of the title potassium salt which is purified by chromatography on HP-20. Elution is carried out with water/acetone 90:10 and yields a product having a melting point of 220C,dec.

~ GC155f Example 28 [3S(Z)~-3-~[(2-Amino-4-thiazolyl)~2-(diphenyl- -methoxy)-l,l-dimethyl-2-oxoethoxy]imino]acetyl]-amino~2-oxo-1-azetidinesulfonic acid, potassium salt (1:1) A solution of 0.005 mole of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyl-ammonium salt (see Example 6A) and 0.006 mole of (Z)-2-amino-a- I 12-(diphenylmethoxy)-1,1-dimethyl-2-oxoethoxy]imino]-4-thiazoleacetic acid in 60 ml of dimethylformamide is treated with 0.7-g of hydroxybenzotriazole and 1.13 g of dicyclohexyl-carbodiimide. The mixture is stirred for about 16 hours at room temperature, filtered and the filtrate evaporated. The residue is dissolved in 30 ml of acetone, filtered and treated with 20 ml of a solution of 10~ potassium perfluoro-butane sulfonate in acetone. After the addition of petroleum ether the title compound precipitates - 20 and is treated with ether and filtered to yield 3.8 g of product,melting point 190C, dec.

Example 29 [3S(Z)]-3-[[(2-Amino-4-thiazolyl)[(l-carboxy-1-methylethoxy)imino]acetyl]amino]-2-oxo-1-azetidine-sulfonic acid, dipotassium salt (3S(Z)]-3-~(2-Amino-4-thiazolyl)[ r 2-(diphenylmethoxy)-l,l-dimethyl-2-oxoethoxy~imino]-acetyl]amino~-2-oxo-1-azetidinesulfonic acid, potassium salt (2 g; see Example 28) is suspended in 5 ml of anisole and 25 ml of trifluoroacetic GC15~f acid is added at -10C. The mixture is stirred for 10 minutes at -10C. Ether (100 ml) is added slowly at -10C and subsequently ~0 ml of petrol-eum ether is added. Ihe preci?itate is filtered to yield 1.6 g of the trifluoroacetic acid salt. This is suspended in 20 ml of water at 0C, adjusted to pH 5.5 with diluted potassium hydroxide and purified on an HP-20 column. The title compound is eluted with water and has a melting point of 225C,dec.
Example 30 ~3S(Z)~-3-~[(2-Amino-4-thiazolyl)~2-(diphenyl-methoxy)-2-oxoethQxy]imino]acetyl]amino~-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure described in Example 28, but substituting (z)-2-amino-~-[[2-(diphenylmethoxy)-2-oxoethoxy]imino]-4-thiazole-acetic acid for (Z)-2-amino-~-[[2-(diphenylmethoxy~-1,1-dimethyl-2-oxoethoxy]imino]-4-thiazoleacetic - acid, yields the title compound, melting point 180 C, dec.

GC155f -72~

Example 31 [3S(+)~-3-~(Azidophenylacetyl)amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Method I:
A) (+,S)-a-Azido-N-(2-oxo-3-azetidinyl)benzene-acetamide (S)-3-Amino-2-azetidinone (2.15 g; see Example 5A) and 2.1 g of sodium bicarbonate are dissolved in 50 ml of acetone/water (2:1).
(+)-~-Azidobenzeneacetyl chloride (5 g) dissolved in 10 ml of acetone is added dropwise whi~e maintaining the temperature at 0-5C and the pH
at 6.8 with sodium bicarbonate. After stirring for 1 hour, the acetone is distilled off and the r~m~i n; ng aqueous solution is adjusted to pH 8 with sodium carbonate and extracted with three 50 ml portions of methylene chloride.
Evaporation of the sodium sulfate dried organic layers yields 3.6 g of the title compound as an oil which crystallizes after triturating with ether. Recrystallization from methylene chloride/ether, yields the title compound, melting point 97-100C.

~;C155f -73- 1 33867~

B) ~3S (+)]-3- [(Azidiophenylacetyl)amino~-2-oxo-l-azetidinesul fonic acid-, potassium salt A solution of 2.45 g of the above azetidinone and 3 g of monosilyltrifluoroacetamide in 20 ml 5 of acetonitrile are kept for 1 hour at 4ac.
After cooling to 0C the solution is treated with 1.88 g of trimet}~ylsilyl c~lorosulfonate and stirred for 5 hours under argon.
Finally 6.12 ml of a 2N solution of potassium-2--10 ethyl hexanoate in n-butanol are added and stirring is continued for 45 minutes. The - solution is pol~red into 300 ml of ether and the precipitate is filtered off. A filtered solution of 1.2 g of precipitàte in phosphate buffer (pH 5.5) is chromatographed on 100 ml of HP-20.
Elution is performed with: 1) 20 ml buffer;
2) 200 ml H2O; 3) 200 ml water:acetone (9:1);
4) 200 ml water:acetone (3:1). The elution is monitored with TLC (~ydon test on SiO2). 25 ml 20 fractions are taken and from fractions 15 and 16 280 mg of the title compound are obtained. A
second column chromatography of this material yields 120 mg of the title compound, melting point 148C, dec.
Method II:
(S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2.03 g; see Example 6A) and 0.9 g of (+)-~-azido-benzeneacetic acid are 30 dissol~ed in 30 ml of acetonitrile and a solution of 1.03 g of dicyclohexylcarbodiimide in 10 ml GC155f - ~74 1 33867~

of acetonitrile is added. The temperature is maintained for one hour at 0C and for lO`hours at room temperature. After filtering off the resultant precipitate, the solvent is distilled off in vacuo and the oily residue is dissolved in 20 ml of acetone and treated with 1.70 g potassium perfluorobutane sulfonate in acetone.
The addition of 10 ml of ether crystallizes the title compound, melting point 149C, dec.
Method III:
~-Azidiophenylacetyl chloride (2.5 g) is added to a solution of 4.06 g of 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (see Example 6A) and 5 g of propylene oxide in 30 ml of acetonitrile. After stirring for two hours the solvent is distilled off in vacuo and the oily residue is treated with one equivalent amount of potassium perfluorobutane sulfonate in acetone. After the addition o ether the title compound crystallizes and is filtered off, melting point 148-149C dec.

Example 32 [3S(D)] -3-[[~[[(4-Methoxyphenyl)methoxy]carbonyl]-amino]phenylacetyl~amino]-2-oxo-1-azetidinesulfonic acid, potassium salt To a solution of 2.03 g of 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (2.03 g; see Example 6A) and 1.58 g of D-~- r L [ (4-methoxyphenyl)methoxy~carbonyl]amino~benzeneacetic GC155f ~75~ 1 33 8 6 70 acid in 50 ml of acetonitrile, a solution of 1.03 g of dicyclohexylcarbodiimide in 10 ml of acetonitrile is added dropwise. The temperature is maintained at 5C for one hour and for 6 S hours at room temperature. Separation of the formed dicyclohexyl urea and distilling off the solvent yields the title compound as an oily residue. Treating this oil in acetone with potassium perfiuorobutane sulfonate and ether yields 2.4 g of product, melting point 108-111C
dec.

Examples 33-37 Following the procedure of Example 32, lS but substituting the compound listed in column I
for (D)-~-~[~(4-methoxyphenyl)methoxy]carbonyl~-amino]benzeneacetic acid, yields the compound listed in column II.

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GC15~f _77- 1 3 3 8 6 7 3 Example 38 (3S)-3- L [ 11 (Methylthio)thioxomethyl]thio]~henyl-acetyl]amino]-2-oxo-1-azetidinesulfonic acid., tetrabutylammonium salt A solution of 1.03 g of (+)-~ (methylthio)-.
thioxomethyl~thio]benzeneacetic acid and 1.63 g of 3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyl-ammonium salt (see ~xample 6A) in 30 ml of acetoni-trile is treated with 0.8 g of dicyclohexyl-}0 carbodiimide dissolved in 10 ml of acetonitrile at -5C. After stirring for about 16 hours the formed dicyclohexyl urea is filtered off and the mother liquor is evaporated. The remaining oily residue is chromatographed on a column of 400 g of silica (ethyl/acetate/methanol/water (8.5:1:0.5) is the eluent), yielding 1.3 g of the title compound.

Example 39 3(S)~3-~[(Methylthio)thioxomethyl~thio]phenyl-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (3s)-3-t~[[(Methylthio)thioxomethyl]thio]-phenylacetyl]amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.3 g; see Example 38) is dissolved in acetone and treated with an equivalent amount of potassium perfluorobutane sulfonate. The addition of ether c~ystallizes the title compound, which is filtered off, yield 0.18g of product, melting point 157C, dec.

GC155f Example 40 [3S(D)]-3-[[~1~4-Ethyl-2,3-dioxo-1-piperazinyl)-carbonyl]amino]-2-thienylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt S (D)--[1(4-Ethyl-2,3-dioxo-1-piperazinyl)-carbonyl]amino}2-thiopheneacetic acid (3.25 g), 4.20 g of 3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (see Example 6A~ and 1.3 g of N-hydroxybenzotriazole are dissolved in 25 ml of acetonitrile. Dropwise addition of 2.06 g of dicyclohexylcarbodiimide dissolved in 10 ml of acetonitrile at -10C takes 20 minutes and stirring is continued for about 16 hours. Formed urea is filtered off, and the solvent is evaporated in vacuo. The remaining oil is dissolved in 50 ml of acetone and after treatment with an equivalent amount of potassium perfluorobutane sulfonate, the title compound crystallizes, melting point 185-187C, dec.

Example 41 [3S(+)]-3-[(Bromophenylacetyl)amino]-2-oxo-1-azet;~;~esulfonic acid, potassium salt ~-Bromophenylacetyl chloride (1.4 g) in 10 ml of acetonitrile is added dropwise to a solution of 5mM 3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt, and 3 g of propylene oxide in acetonitrile at 0C. After three~hours stirring, the solvent is distilled off and the remaining oily residue is dissolved in 30 ml of acetone. The equivalent amount of potassium perfluorobutane sulfonate in acetone is added.
The addition of ether crystallizes the title 1 338670 GC155f compound, melting point 135-137C dec.

- Example 42 [3S(+)]-3-~[~(Aminocarbonyl)amino~-2-thienylacetyl]-amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Nethod I:
To a solution of 2 g of .(S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt and 1.5 g of propylene oxide in acetonitrile is added 1.1 g of (+)-2-amino-4-(2-thienyl)-5(4H)-oxazolone, hydrochloride. After three hours stirring at 0 C and one hour at room temperature, the solvent is distilled off and the oily residue is dissolved in 30 ml of acetone. By adding the equivalent amount of potassium perfluorobutane sulfonate in acetone, the title compound crystallizes out. Purification by column chromatography on HP-20 using water as eluent yields the title compound, melting point 218-222 C, dec.

GC155f Method II:
To a suspension of 2 g of (+)--~(amino-carbonyl)amino]-2-thiopheneacetic acid, lOmM of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyl-ammonium salt and lOmM of N-hydroxybenzotriazole in 50 ml of acetonitrile at 0C is added (with stirring) lOmM of dicyclohexylcarbodiimide dissolved in 15 ml of acetonitrile. Stirring is continued for one hour at -15C and for about 16 hours at room t~mr~rature. After filtering off the dicyclohexyl urea, the solvent is evaporated and the oily residue is dissolved in 50 ml of acetone. Adding the equi~alent amount of potassium perfluorobutane sulfonate in acetone yields crystalline product. Purification by column chromatography on HP-20, using water as an eluent, yields the title compound, melting point 220-223C.

Example 43 [3S(+)]-3-[[[~(Methylamino)carbonyl]amino]-2-thienylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (+)--[[(Methylamino)carbonyl]amino~-2-thiopheneacetic acid (0.54 g) and 1.00 g of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (see Example 6A) are dissolved in 20 ml of acetonitrile and 0.5 g of dicyclohexylcarbodiimide is added at a temperature of O C. After stirring for 8 hours the dicyclo-hexylurea is filtered off and after distilling off GC155f the solvent the oily residue is dissolved in 50 ml acetone and the equivalent amount of potassium per~luorobutane sulfonate is added.
Crystalline product is filtered off and purification is performed on HP-20 using water as an eluent, melting point 205C, dec.

Example 44 13S (+~ ] -3-1~ mi nooxoacetyl)amino]-2-thien acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 42, method II, but substituting (+)-~-[(aminooxoacetyl)-amino]-2-thiopheneacetic acid for (+)-a-[(amino-carbonyl)amino]-2-thiopheneacetic acid, yields the title compound, melting point 218-222 C.

Example 45 [3s(R*)]-3-[[~[(4-Ethyl-2~3-dioxo-l-piperazinyl)-carbonyl]amino]phenylacetyl]amino]-2-oxo-1-azetidine-sulfonic acid, potassium salt Following the procedure of Example 40, but substituting (R)-a-1~(4-ethyl-2,3-dioxo-1-pipera-zinyl)carbonyl]amino]benzeneacetic acid for (D)-~-[1(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]-amino]-2-thiopheneacetic acid, yields the title compound, melting point 155-157C, dec.

GC155f . , .
-82- 1 3386~

Example 46 3-(Acetylamino)-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt Method I:
A) 3-[~N-Acetyl-N-chloro)amino]-2-oxo-1-azetidine-sulfonic acid, mixed sodium and potassi~m salt To a solution of 3-(acetylamino)-2-oxo-1-azetidinesulfonic acid,potassium salt (172 mg, see Example 23) in methanol (17 ml) containing 4% sodium borate decahydrate cooled to -15 to -10C is added tert-butyl hypochlorite (110 ~1).
The mixture is stirred in the cold for 1 hour, 45 minutes, poured into 0.5 M monobasic potassium phosphate solution (50 ml), and the pH is lowered to 5.5. Solvent is removed in vacuo, and the residue is dissolved in a mi n;mum amount of water and chromatographed on HP-20AG, 100-200 mesh (140 ml).
Elution with water yields an oil (63 mg) which crystallizes on standing. Trituration with methanol/ether and then ether gives a solid (53 mg) melting point 124C,slow dec.

GC155f B) 3-(Acetylamino)-3-methoxy-2-oxo-1-azetidine-sulfonic acid, potassium salt 3-[(N-Acetyl-N-chloro~amino]-2-oxo-1-azetidinesulfonic acid mixed salt (37 mg) is dissolved in l.S ml of dry dimethylformamide and added to a solution of lithium methoxide (50 mg) inl.~L~l (1 ml) at -78&. After stirring for 15 minutes at -78C, a 0.5 M solution of monobasic potassium phosphate (10 ml) is added, and the solution is then acidified to pH 4 with lN hydrochloric acid.
To the solution is added tetrabutylA~mQn;um hydrogen - sulfate (70 mg) and the solution is extracted four times with methylene chloride. The combined extracts are dried ~sodium sulfate) and solvent is removed in vacuo to give 55 mg of an oil.
Chromatography on 5.5 g of silica gel yields the oily product (41 mg) as the tetrabutylammonium salt (eluted with 8% methanol:92% methylene chloride). The oil (31 mg) is dissolved in water and passed through an ion-exchange column (5 ml of AG 50W-X2,K form, 200-400 mesh, 0.6 meq/ml).
Removal of water in vacuo gives an oil which crystallizes from methanol-ether. Trituration twice with ether gives the product as a colorless powder (11 mg); melting point 182-183C, dec.

- GC155f -84- i 338670 Method II:
A) 3-Amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt A 4% sodium borate decahydrate in methanol solution (100 yl) is added to a suspension of 10% palladium on charcoal (30 mg) in methanol (2 ml), and the mixture is stirred under an atmosphere of hydrogen for 15 minutes. 3-Methoxy-2-oxo-3-.[[(phenylmethoxy)carbonyl]amino~-1-azetidinesulfonic acid, tetrabutylammonium salt(60mg;see Example 49) in methanol (2 ml) is added and th.e mixture is vigorously stirred for 15 minutes under a hydrogen atmosphere. Catalyst is removed by filtration through Celite on a millipore filter (0.5 m~), and solvent is removed from the filtrate in vacuo, and the residue is extracted with methylene chloride. Removal of solvent under reduced pressure yields 35 mg of the title compound, as an oil.
B) 3-(Acetylamino)-3-methoxy-2-oxo-1-azetidine-sulfonic acid, potassium salt To a solution of 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (35 mg) in methylene chloride (10 ml) at 0C is added propylene o~ide (2 ml) and acetyl chloride (74 ~1). After 2 hours the solvent is removed under reduced pressure and the residual oil is chromatographed on silica gel (4 g).
Elution with 6-8% methanol in methylene chloride gives an oil (18 mg) which is dissolved in water and passed through an ion-exchange resin (3 ml GC155f _.

of AG 50W-X2, K form, O.6 meq/ml). RemoYal of water from the eluate in vacuo yields the desired product (10 mg).

Example 47 N-.(3-Methoxy-2-oxo-1-sulfo-3-azetidinyl)-2-phenyl-acetamide, tetrabutylammonium salt A) N-Chloro-N-(2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, potassium salt To a solution of (S)-N-(2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, potassium salt (50 mg; see Example 1) in methanol containing 4% sodium borate decahydrate (5 ml) cooled in a -5C bath is added tert-butyl hypochlorite (20 ~1). After stirring for 32 minutes, the mixture is poured into 0.5 M pH 5.5 potassium phosphate buffer at 0C. The resultant solution (pH 5.9) is adjusted to pH 4.5, concentrated _ vacuo to remove methanol, and chromatographed on HP-20AG, 100-200 mesh (100 ml). After washing the column Wit}l 0.5 M pH 5.5 buffer (100 ml) and water the desired product is eluted with 9:1 water:acetone. Concentration in vacuo gi~es 50 mg of the title compound.
B) N-(3-Methoxy-2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, tetrabutylammonium salt To a stirring solution of lithium methoxide (160 mg) in 5 ml of methanol cooled to -78C is added a solution of N-chloro-N-(2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, potassium salt GC155f (149 mg) in 10 ml of dry dimethylformamide. After addition is complete, the mixture is stirred for 15 minutes at -78 C, poured into 0.5 N
monobasic potassium phosphate solution (100 ml), and washed three times with methylene chloride.
Tetrabutylammonium bisulfate (213 mg) is added to the aqueous layer, which is then extracted three times with methylene chloride. The combined extracts are dried (sodium sulfate), and solvent is removed in vacuo giving an oil (271 mg). Chromatography of the oil on silica gel (25 g) and elution with 4% methanol:96%
methylene chloride yields 149 mg of the product as an oil.
Example 48 N-(3-Methoxy-2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, potassium salt N-(3-Methoxy-2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamide, tetrabutylammonium salt (91 mg; see Example 47) is dissolved in water and passed through an ion-exchange column (10 ml of AG 50W-X2, K~ form). The eluate is concentrated in vacuo and the ~sidual oil solid-ifies on scratching with a methanol-acetone-ether mixture. After triturating twice with ether, the product is obtained as a solid (53 mg):
umax1762, 1665 cm ; NMR (CD30D) ~ 3.41 (S, 3H, OCH3), 3.59 (S, 2H, CH2), 3.84 (ABq, J=6.3 Hz, 2H, H4), 7.30 (m, 5H, aromatic).
Analysis: Cal- for C12Hl3~06~l/2H2o Calc.: C,39.88; H,3.62; N,7.75 Found : C, 39.62; H,3.65; N,7.60 GC155f Example 49 3-Methoxy-2-oxo-3- I [ (phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, tetrabutylammonium salt Method I:
A) 2-Oxo-3-~N-chloro-N-~(phenylmethoxy)carbonyl]-amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (S)-2-Oxo-3-[[(phenylmethoxy)carbonyl~- -amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (0.9g; see Example 4) dissolved in 8Q ml of methylene chloride is added to a mixture (cooled to 0-5C) of 3.17 g of sodium borate decahydrate and 11.8 ml of a 5.25% sodium hypochlorite solution in 70 ml of water. The reaction mixture is vigorously stirred for 55 minutes while cooling in an ice bath. After diluting the mixture with 0.5 M monobasic potassium phosphate solution, the product is extracted with methylene chloride (three 150 ml portions). Combination of the extracts, drying (sodium sulfate), and removal of solvent in vacuo yields the title compound as an oil (0.94 g).

B) 3-Methoxy-2-oxo-3-[[(phenylmethoxy)carbonyl]-amino]-l-azetidinesulfonic acid, tetrabutyl-ammonium salt To a stirring solution of lithium methoxide (667 mg) in anhydrous methanol (10 ml) at -78C
is added a solution of 2-oxo-3-[N-chloro-N-[(phenyl-methoxy)carbonyl~amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (0.94 g) in dry dimethyl-GC155f formamide (10 ml). After stirring the mixture at -78C for 1 hour, it is poured into 0.5 M
monobasic potassium phosphate solution and extracted with methylene chloride (three 150 ml portions). The combined extracts are dried (sodium sulfate) and solvent is removed in vacuo to give an oil (0.83 g). The desired product is obtained by chromatographing the oil on silica gel (100 g) and eluting with 4-5~ methanol in methylene chloride to give an oil (513 mg):
(neat) 1767, 1720 cm 1; NMR (CDC13) ~ 3.40 (S,OCH3), 3.03 (ABq, J=6.5 Hz, H4), 5.08 (S,CH2), 6.00 (S,NH), 7.27 (S, aromatic).

Method II:
To a solution of 3-benzyloxycarbonylamino-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt (400 mg) in water is added a 0.1 M tetra-butylammonium bisulfate solution (10.9 ml, adjusted to pH 4.3 with potassium hydroxide). The mixture is extracted three times with methylene chloride, the extracts are combined, dried (Na2S04) and solvent is removed in vacuo to give a foam (625 mg)~
having spectral characteristics approximating those of the product of Method I.

- - GC155f Example 50 3-Methoxy-2-oxo-3-~[(phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, potassium salt Method I:
A)- 2-Oxo-3-[N-chloro-N-[(phenylmethoxy)carbonyl]-amino]-l-azetidinesulfonic acid, potassium salt To a solution of (S)-2-oxo-3-l~(phenyl-- methoxy)carbonyl]amino]-l-azetidinesulfonic acid, potassium salt (1.00 g; see ~YAmple 3) in methanol (90 ml) containing 4% sodium borate decahydrate at -10C is added tert-butyl hypochlorite (420 ~1).
After stirring for 2 hours at -10 C, 0.5 M mono-basic potassium phosphate solution (100 ml) is added and the pH is adjusted to 6 with 1 N hydro-chloric acid. After concentration of the solvent in vacuo to 30 ml, the aqueous solution is chromatographed on HP-20AG, 100-200 mesh (200 ml).
After passage of a solution of monobasic potassium phosphate (50 g) in water (1000 ml), followed by water (2000 ml), the product is eluted with 10% acetone-90~ water. Solvent is removed in vacuo and the title compound is crystallized from water to give a solid (530 mg), melting point 173-175C.

GC155f -go- 1 338 6 70 B) 3-Methoxy-2-oxo-3-[l(phenylmethoxy)carbonyl]-amino]-l-azetidinesulfonic acid, potassium salt To a solution of lithium methoxide (874 mg) in dry methanol (10 ml) at -78 C is added 2-oxo-3-[N-chloro-N-[(phenylmethoxy)carbonyl]amino]-l-azetidinesulfonic acid, potassium salt (857 mg) in dry dimethylformamide (13 ml). After 15 minutes at -78C the mixture is poured into 0.5 M monobasic potassium phosphate solution (200 ml) and the pH is adjusted to 5.5 with lN
hydrochloric acid. The aqueous mixture is washed with methylene chloride (three 100 ml portions), and tetrabutylammonium bisulfate (1.169 g) is added.
The product is extracted with methylene chloride (three 200 ml portions), dried (sodium sulfate), filtered, and concentrated in vacuo. The residual oil is chromatographed on silica gel (150 g) and the product is eluted with 2-4~
methanol in methylene chloride yielding the - 20 tetrabutylammonium salt of the product as an oil (701 mg). A portion of the oil (51 mg)is dissolved in water and passed through an ion-exchange column (3 ml of AG 50W-X2, 200-400 mesh, K~ form, 0.6 meq/ml). Concentration of the eluate in vacuo yields an oil (30 mg), which is crystallized by scratching with acetone:
vmax(KBr) 1760, 1725 cm ; NMR (D20) ~ 3.48 (S, 3H, OCH3), 3.92 (S, 2H, H4); 5.20 (S, 2H, CH2), 7.42 (S, 5H, aromatic), m.p. 196-198.

GC155f Method II: `
A) l-Chloro-3-1N-chloro-N-l(phenylmethoxy)carbonyl]-2-azetidinone A solution of 3-~[(phenylmethoxy)carbonyl]-amino]-2-azetidinone (440 mg.; see Example 2C) in 40 ml of 4% methanolic borax is cooled to 0C and 0.5 ml of t-butyl hypochlorite is added.
After 30 minutes at 0C, the solution is poured into 200 ml of cold water and extracted with two 100 ml portions of ethyl acetate. The combined ethyl acetate layer is washed with water, dried, and evaporated in vacuo to give 546 mg of the title compound as an oil.

B) 3-Methoxy-3-[[(phenylmethoxy)carbonyl~amino~-2-azetidinone A solution of 730 mg (0.0025 mole) of l-chloro-3-[N-chloro-N-[(phenylmethoxy)carbonyl~amino]-2-azetidinone in 5 ml of tetrahydrofuran is cooled to -78C and 4 ml of methanol containing 285 mg of lithium methoxide is added. After 20 minutes at -78 C, 0.6 ml of acetic acid and 0.6 ml of trimethylphosphite are added. The solution is stirred for 5 minutes at -78C, allowed to warm to ambient temperature and stirred for 30 minutes.
The resulting solution is diluted with ethyl acetate, washed with 5% sodium bicarbonate, water, 5% potassium bisulfate, water, saturated salt solution, and dried. Solvent removal gives an oil that is applied to four 20 x 20 cm xl mm Çcl55f silica gel plates. Development with benzene-ethyl acetate (1:1) and isolation of the major W-active band of Rf=0.25 gives 91 mg of oil that crystallizes from ether to give a solid.
Recrystallization from ether gives the title compound, melting point 112-114C.

C) 3-Methoxy-2-oxo-3-[~(phenylmethoxy)carbonyl~-amino~ azetidinesulfonic acid, potassium salt ~ solution of 25 mg of 3-methoxy-3-[[(phenyl-methoxy)carbonyl]amino]-2-azetidinone in 0.175 ml each of dichloromethane and dimethylformamide is stirred for 24 hours with 55.4 mg of a complex of pyridine-sulfur trioxide. The resulting slurry is diluted with 5 ml of cold 0.5 M monobasic potassium phosphate (adjusted to pH 4.5) and extracted with ethyl acetate. The aqueous layer is applied to a 40 ml HP-20 AG column. Elution with additional buffer, water, and water-acetone (9:1~ gives 32 mg of the title compound as an oil that slowly solidifes. Crystallization from acetone gives the title compound, melting point 196-198C, dec.

- G~155f Example 51 3-lll,3-Dioxo-2-phenyl-3-(phenylmethoxy)propyl]amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt S A) 3-[l1,3-Dioxo-2-phenyl-3-(phenylmethoxy)propyl~-amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt Crude 3-amino-3-methoxy-2-oxo-1-azetidine-sulfonic acid, tetrabutylammonium salt (431 mg, see Example 74), containing borax, is dissolved in 30 ml of dry acetonitrile. Dry pyridine ( 317 I~l) is added and the solution stirred well at -lO C under dry nitrogen. ~-(Benzyloxycarbonyl)-phenylacetyl chloride (568 mg) in 3 ml of dry acetonitrile is added dropwise. Thin layer chromatography indicates the reaction to be complete after 15 minutes. Potassium phosphate buffer (0.5 M, pH 5.5, 17 ml) is added and most of the acetonitrile is removed in vacuo. The 2~0 residue is diluted with water and extracted three times with equal volumes of methylene chloride. The extract is dried over anhydrous sodium sulfate and evaporated in vacuo to give 1.032 g of crude product, which is dissolved in 3 ml of methylene chloride and chromatographed on a silica column using methylene chloride-methanol to give 470 mg of the title compound.

- 1 3 3 8 6 70 GC155f B) 3-ltl~3-Dioxo-2-phenyl-3-(phenylmethoxy)-propyl~amino]-3-methoxy-2-oxo-1-azetidinsulfonic acid, potassium salt 3-[[1,3-Dioxo-2-phenyl-3-(phenylmethoxy)-propyl]amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (470 mg) is dissolved in 15 ml of 30% acetone: water and put through a Dowex 50~X2(K form) column using the same solvent as eluent. The total eluate is evaporated in vacuo to yield 345 mg of an amorphous solid which is lyophilized to an amorphous powder, melting point 100-120C.
Anal. for C20H19O8N2SK Calc'd: C,49.37; H,3.94 N,5.76; S,6.59 lS Found: C,49.08; H,4.00; N,5.58; S,6.29 Example 52 (+)-3-tL~(Cyanomethyl)thio]acetyl]amino~-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt.3 To a solution of 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (414 mg, see Example 74) in dry acetonitrile (50 ml) at -20C is added diethylaniline (210 yl) and cyanomethylthioacetyl chloride (169 mg).
After 10 minutes the solvent is removed under reduced pressure and a 0.LM solution of tetrabutyl-ammonium sulfate (22.8 ml) adjusted to pH 4.3 with potassium hydroxide is added and the product extracted with three 50 ml portions of methylene chloride, dried, filtered and concentrated under reduced pressure. The oily residue is purified on silica gel (60 g) and the product (294 mg) is eluted with 4% methanol-methylene chloride.
The purified product is passed through an ion 1 338670 GC155f exchange resin (16 ml AG 50W-X2(K form), 0.6 mequiv./ml 200-400 mesh). Removal of water gives 164 mg of partially purified product.
The product is further purified on Diaion AG HP 20 (100 ml) using water as eluent. The solvent is removed under reduced pressure to give 126 mg of product which is triturated with ether to give 76 mg of the title compound, melting point 110-125C.
Anal. Calc'd for C8HloN3S2O6K: C,27.66; H,2.88;
N,12.10; S,18.44 Found: C,27.25; H,3.00; N,10.84; S,17.53 Examples 53-56 Following the procedure of Example 42, Method II, but substituting the compound listed in Column I for (+)-a-~ (aminocarbonyl)amino]-2-thiopheneacetic acid, yields the compound listed in Column II.

GC155f -I ~ I
0 ~ ~ ~
O ~ _I
O ~ ,~ ,1 ~ ~ ~ .,1 ~
c _I U O E ~ E~ e ,, ~ e ~
. E ~ ~ N ' I~ N ' ~ e u ~ O ~
,1 ~ c a) ~ ~ c E c E
c e o o ~ ~ O
_, o ~ e ,~ ,~ x , - , 1 0 S ~ ~ O N U' '.~ N Ul 1 ~ O I U O I O~
H G
o ,., ~n ~U ~ N a) I~ ''I O ~lf ''I ~
, e ~" N U~ e ~ o ~E C: o e ~,~ ~ ~ o I e --8 C.) --e ~c ~ 1~ ~ ~--I ~-0 --~ ~ C l I~ N
I r e ~ ~ ~
~ N ~0 1 ~N OQ _ ~ O O ~ U N
+1 0 0 U ~ +1 '~ O ~ O ~ ~ ~1 0 JJ
~ e I o -l ~ X ~c ~ O ~ ~ O

c`~ l u c o-~ I a x J
O o ~ o c - _ c) c o o ~ ~1 c .,, ~ nJ e e ~
--I ~ s _~ _ ~ _ ~ >1 _I

H J _I O J ~,, ~11 C
C ' S ~- C ~ o u Y ~ e~ ~ ~
O~ U O I
C ~ : ~ O U C
~ O C.~l C
~ U ~ C,-,, ~
F ~ e ~ ~ ~
~ --e ~u ~u I _ I U

+ l ~ + l U ~ U
_,, _ " -- -- ~a , ~ ~ In ~D
Ul u~ Ln U~

1 3386~0 GC155f Example 57 [3S(+)]-3-[[2-~ethylthio)-1-oxopropyl~amino~-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 31, S Method II, but substituting (+)-2-(methylthio)-propanoic acid for (+)--azido-benzeneacetic acid, yields the title compound, melting point 173C,dec.

Example 58 [3S(~)~]-3-t~[[~2,3-Dioxo-4-I(phenylmethylene)amino]-l-piperazinyl]carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (R)--~[~2,3-Dioxo-4-l(phenylmethylene)amino~-l-piperazinyl]carbonyl]amino]benzeneacetic acid (0.7 g) and (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (0.8 g; see Example 6A) are dissolved in 20 ml of acetonitrile.
While stirring, 0.4 g of dicyclohexylcarbodiimide is added dropwise at 0C. Stirring is continued for 18 hours and, after filtration, the solvent is distilled off leaving an oily residue. The residue is dissolved in acetone and treated with potassium perfluorobutane sulfonate to precipitate the title compound. Purification by column chromatography on HP-20 using water as eluent yields the title compound, melting point 193-194 C.

GC155f Example 59 ~3S(Z)]-3-[[~2-Amino-4-thiazoly~[(2-methoxy-2-oxoethoxy)imino]acetyl]amino]-2-oxo-1-azetidine-- sulfonic acid, potassium salt (z)-2-Amino--t(2-methoxy-2-oxoethoxy)imino]-4-thiazoleacetic acid (1.3 g) and (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (2.03 g; see Example 6A) is dissolved in 50 ml .of acetonitrile and 1.03 g of dicyclohexyl-carbodiimide dissolved in 5 ml of acetonitrile is added dropwise at 0C. A~ter stirring for 15 hours and filtering off dicyclohexyl urea the solvent is distilled off. The remaining oily residue is dissolved in acetone and treated with the equivalent amount of potassium perfluoro-butane sulfonate. The title compound is isolated and purified by column chromatography on HP-20 using water as eluent, yielding the title compound melting point 195-198C.
Example 60 [3S~R*)~-3-1[1[[3-~(4-Chlorophenyl)methylene~-amino]-2-oxo-1-imidazolidinyl]carbonyl]amino]-phenylacetyl]amino]-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of absolute diglyme (diethylene-glycoldimethyl ether), 1.5 g of (R) -a- [ r [3-~(4-chlorophenyl)methylene]amino~-2 oxo-1-imidazolidinyl]-carbonyl]aminolbenzeneacetic acid, an equivalent amount of dicyclohexylcarbodiimide, and Q~5 g of 1 338670 GC155f _99_ hydroxybenzotriazole are stirred together for 12 hours. The solvent is removed in vacuo and the residue is dissolved in 50 ml of acetone and filtered. Acetone is distilled off and the residue is dissolved in 200 ml of methylene chloride. The solution is washed with aqueous sodium bicarbonate and then with aqueous sodium chloride solution. The methylene chloride layer is dried over sodium sulfate, evaporated to dryness and crystallized by the addition of ether. The compound is recrystallized from acetone-ether. The resulting white crystalline powder is dissolved in acetone and treated with an equivalent amount of potassium perfluoro-butane sulfonate in acetone. The title compoundprecipitates and is filtered off, yielding 1.4 g of product melting point 217-222C.

Example 61 [3S(R*)]-3-[[~[12-Oxo-3-[(phenylmethylene)amino]-l-imidazolidinyl]carbonyl~amino]phenylacetyl]-amino]-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylAmmon;um salt (2.25 g; see Example 6A) in 100 ml of dry dimethylformamide (DMF),an equivalent of dicyclohexylcarbodiimide, 2.5 ~ of (R) -a- [ [ [2-oxo-3-[(phenylmethylene)amino~-1-imidazolidinyl]carbonyl]amino]benzeneacetic acid and 0.85 g of hydroxybenzotriazole are stirred together at ambi~nt temperature for 12 hours.
After this time, DMF is removed in vacuo and the residue is dissolved in 50 ml of acetone.

GC155f ._ lOO 1 338670 The precipitated urea is filtered off and the mother liquor is treated with an equivalent amount of potassium perfluorobutane sulfonate in 20 ml of acetone. After addition of 100 ml of ether, the title compound precipitates and is filtered off. Purification is achieved by dissolving the compound in DMF/acetone and precipitating with water, yielding 1.5 g of product, melting point 224-226C, dec.
Example 62 ~3S(R*)]-3-t~[~13-(Methylsulfonyl)-2-oxo-1-imidazolidinyl]carbonyl~amino]phenylacetyl]-amino}2-oxo-1-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2.25 g; see Example 6A) in 60 ml of dimethylformamide is stirred for 12 hours with 1.9 g of (R)-~-[~[3-(methylsulfonyl)-2-oxo-1-imidazolidinyl]carbonyl]amino]benzeneacetic acid, 0.75 g of hydroxybenzotriazole and 2 3 g of dicyclohexylcarbodiimide. The solvent is removed in vacuo and the residue is dissolved in 20 ml of acetone and filtered. The mother liquor is treated with 1.87 g of potassium perfluorobutane sulfonate in 20 ml of acetone.
After the addition of ether, the title compound precipitates, yielding 2.0 g of material. The compound is purified by recrystallization from water and has a melting point of 240-245C, dec.

~ GC155f -lol- I 3 3 8 6 7 0 Example 63 [3S(R*)]-3-[(Hydroxyphenylacetyl)a~inoJ
azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is stirred for about 16 hours with 1.5 g of dicyclohexyl-carbodiimide, 0.5 g of hydroxybenzotriazole and 0.6 g-of R-~-hydrox~benzeneacetic acid.
The solvent is removed in vacuo and the residue is dissolved in 20 ml of acetone. The precipitated urea is filtered off and the mother liquor is treated with an equivalent amount of potassium perfluorobutane sulfonate. After addition of ether, the title compound precipitates and is filtered off, yielding 1.3 g of crude product.
The product is purified by chromatography using HP-20 and water/acetone (9:1) as eluent, and has a melting point of 145-150C, dec.

Example 64 [3S(S*)]-3-~(Hydroxyphenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 63, but substituting (S)-~-hydroxybenzeneacetic acid for (R)--hydroxybenzeneacetic acid, yields the title compound, melting point 195-197 C.

~C15s~
.
-1~2~ 1 33 8 6 70 Example 65 [3S(+)]-2-Oxo-3-[(phenylsulfoacetyl)amino]-1-azetidinesulfonic acid, potassium salt (1:2) (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2.25 g; see ~Y~mrle 6A) in 100 ml of dry diglyme, 2.4 g of triethylamine and 0.3 g of dimethylaminopyridine are cooled to 0C. (R)-~-(Chlorocarbonyl)-benzenemethanesulfonic acid (1.8 g) in 20 ml of diglyme is added dropwise. The temperature is maintained for 2 hours, the solvent is distilled off in vacuo and the residue is dissolved in acetone. The insoluble precipitate is filtered off and the mother liquor is treated with an equivalent amount of potassium perfluoro-butane sulfonate. After the addition of ether, the title compound precipitates and is filtered off, yielding 1.4 g of crude product; melting point 240-245C dec., after recrystallization from water/acetone-Example 66[3S(Z)-3-tt(2-Amino-4-thiazolyl)lt(diethoxy-phosphinyl)methoxy]imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2.25 gJ see Example 6A) in 100 ml of dry dimethylformamide is treated with 1.87 g of (Z)-2-amino-~-[[(diethoxy-phosphinyl)methoxy]imino]-4-thiazoleacetic acid, 0.75 g of hydroxybenzotriazole and 2.29 g of dicyclohexylcarbodiimide for 12 hours with stirring.
The precipitated urea is filtered off and the solvent - GC155f removed in vacuo. The remaining oil is treated with an equivalent amount of potassium perfluoro-butane sulfonate in 20 ml of acetone. After the addition of ether, the title compound precipitates and is filtered off, yielding 2.77 g of crude product. Purification of this crude product by column chromatography using HP-20 and water/
acetone (9:1) as eluent yields the title compound, melting point 155-160C, dec.

Example 67 [3S(Z)]-3-~(2-Amino-4-thiazolyl)[12-(1,1-dimethyl-ethoxy)-2-oxo-1-phenylethoxy]imino~acetyl~amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2.25 g; see Example 6A) in 60 ml of dimethylformamide is stirred at room temperature with 2.4 g of (Z)-2-amino--~[2-(l,l-dimethylethoxy)-2-oxo-1-phenylethoxy]imino~-4-thiazoleacetic acid, 1 g of hydroxybenzotriazole and 1.5 g of dicyclohexylcarbodiimide for 12 hours.
The solvent is removed in vacuo and the residue is dissolved in 50 ml of acetone. The precipitated urea is filtered off and the mother liquor is treated with an equivalent amount of potassium perfluorobutane sulfonate. After the addition of ether, the title compound crystallizes and is filtered off. Purification of the compound is achieved by HP-20 column chromatography using water/acètone (7:3) as eluent, yielding 1 g of product, melting point >250 C,dec.

GC155f Example 68 [3S(Z)]-3-11(2-Amino-4-thiazolyl)[(lH-tetrazol-5-ylmethoxy)imino]acetyl~amino]-2-oxo-1-azetidine-sulfonic acid,potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.9 g; see Example 6A) in 60 ml of dimethylformamide is treated with 1.4 g of (Z)-2-amino-~-[(lH-tetrazol-5-ylmethoxy)imino]-4-thiazoleacetic acid, 0.7 g of hydroxybenzotriazole and 1.4 g of dicyclohexylcarbodiimide with stirring for 24 hours. Afte~ the solvent is removed in vacuo, the residue is dissolved in acetone and the pre-cipitated urea filtered off. The mother liquor is treated with an equivalent amount of potassium perfluorobutane sulfonate in 10 ml of acetone.
The title compound is precipitated by the addition of 200 ml of ether. Purification is achieved by HP-20 column chromatography using HP-20 resin and water as eluent and yields 1.05 g of product, melting point 250C, dec.

Example 69 [3S(Z)]-3-[{(2-Amino-4-thiazolyl)~(phenylmethoxy)-imino]acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (1.5 g; see Example 6A), 1.23 g of (Z)-2-amino-~-[(phenylmethoxy)imino]-4-thiazoleacetic acid, 0.57 g of hydroxybenzo-triazole and 1.14 g of dicyclohexylcarbodiimide are stirred in 60 ml of dimethylformamide at room temperature for 24 hours. The precipitated urea is filtered off, the solvent removed and GC155f the residue treated with an equivalent amount of potassium perfluorobutane sulfonate in 10 ml of acetone. After the addition of 200 ml of ether, the title compound precipitates, is filtered off and is purified by HP-20 column chroma-tography using water/acetone (9:1) as eluent, yielding 1 g of material, melting point 200 C, dec.

- Example 70 [3S(Z)]-3-[~(2-Amino-4-thiazolyl)[(carboxymethoxy)-imino]acetyl]amino~-2-oxo-1-azetidinesulfonic acid, potassium salt (1:1) [3S(Z3]-3-lt(2-Amino-4-thiazolyl)t~2-(diphenyl-methoxy)-2-oxoethDxy]imino]acetyl]amino]-2-oxo-1-lS azetidinesulfonic acid, potassium salt (1:1) (1.3 g;see Example 30) is mixed with 5 ml of anisole.
At -15C 25 ml of trifluoroacetic acid is added and the mixture is stirred for 10 minutes. Ether (100 ml) is added slowly at -10C and subsequen~ly 50 ml of petroleum ether. The precipitate is suspended with cooling in 20 ml of water and adjusted to pH 5.0 with diluted potassium hydroxide. The produc~ is purified by chroma-tography on an HP-20 column, yielding 3.0 g of the title compound, melting point 230-235C, dec.

- GC155f Example 71 [3S(Z)]-3-~[(2-Amino-4-thiazolyl)1[2-oxo-2-(phenyl-methoxy)ethoxy]imino]acetyl]amino]-2-oxo-1-azetidine-sulfonic acid, potassium salt Following the procedure of Example 28, but substituting- (Z)-2-amino-a-[[2-oxo-2-(phenyl-methoxy)ethoxy]imino]-4-thiazoleacetic acid for (Z)-2-amino-~-1[2-(diphenylmethoxy)-1,1-dimethyl-2-oxoethoxy]Lmino]-4-thiazoleacetic acid, yields the title compound, melting point ca. 170C, dec.

Example 72 [3S(Z)]-3-[1~(2-Amino-2-oxoethoxy)imino](2-amino-4-thiazolyl)acetyl]amino}2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure described in Example 28, but substituting (Z)-2-amino-~-[(2-amino-2-oxoethoxy)imino]-4-thiazoleacetic acid for (Z)-2-amino-~-[12-(diphenylmethoxy)-1,1-dimethyl-2-oxoethoxy]imino~-4-thiazoleacetic acid, yields the title compound melting point 205-210C, dec.

Example 73 [3S(Z)]-3-[1(2-Amino-4-thiazolyl)(hydroxyimino)-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt A solution of 0.6 grams of 90% hydroxy-benzotriazole in 100 ml of dimethylformamide is stirred for one hour with 10 grams of 4A
molecular sieves, filtered,and the filtrate added to a solution of 0.004 mole of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium GC155f salt (see Example 6A) in dimethylformAm;~e.
(Z)-2-Amino-a-(hydroxyimino)-4-thiazoleacetic acid (0.89 g) is added, followed by the addition of 0.91 g of dicyclohexylcarbo~ e. The mixture is stirred for about 16 hours, evaporated in vacuo and the residue dissolved in 20 ml of acetone and filtered. The addition of a solution of potassium perfluorobutane sulfonate causes the title compound to Precipitate. Chromatography on HP-20 resin yields 0.44 g of p~oduct, melting point >240C. - ~

Example 74 lS 3-Methoxy-2-oxo-3-[(2-thienylacetyl)amino]-1-azetidinesulfonic acid, potassium salt A) 3-Amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (+)-3-Methoxy-3-[[(phenylmethoxy)carbonyl]-aminoJ-2-oxo-1-azetidinesulfonic acid, tetra-butylammonium salt (143 mg, see Example 49) is - dissolved in 15 ml of dry methanol. Na2B4O7 10 H2O
(12 mg, 0.1 equiv.) is added, followed by 10%
palladium on carbon (72 mg). The mixture is hydrogenated at one atmosphere pressure for 15 minutes. The catalyst is removed by filtration and the filtrate evaporated in vacuo, yielding 114 mg of the title compound.

GC155f -B) 3-Methoxy-2-oxo-3-[(2-thienylacetyl)amino~-1-azetidinesulfonic acid, tetrabutylammonium salt 3-Methoxy-3-amino-2-oxo-1-azetidinesulfonic acid, tetra~utylammonium salt (102 mg) is dissolved S in 10 ml of dry acetonitrile. Dry pyridine (56.~ ~1) is added and the solution is stirred well at -10 C under dry nitrogen. Thienylacetyl chloride (44 ~1) in 1 ml of dry acetonitrile is added dropwise. ~n 15 minutes the reaction is complete as shown by thin layer chromatography.
Potassium phosphate buffer (0.5 M, pH 5.5, 4.2 ml) and tetrabutylammonium sulfa*e (8.5 mg, 0.1 equiv.) are added and most of the acetonitrile is removed _ ~acuo. The residue is diluted with water and extracted with three 20 ml portions of methylene chloride. The extract is dried over anhydrous sodium sulfate and evaporated in vacuo to yield 107 mg of a gum. The crude product is purified by chromatography on silica gel using methylene chloride-methanol, yielding 66 mg of the title compound.

C) 3-Methoxy-2-oxo-3-1(2-thienylacetyl)amino]-l-azetidinesulfonic acid, potassium salt 3-Methoxy-2-oxo-3-[(2-thienylacetyl)amino-l-azetidinesulfonic acid tetrabutylammonium salt (154 mg) is dissolved in 3 ml of 30%
acetone-water, passed through a column of Dowex 50W-X2 (K+ form) and eluted with the 30 same solvent. The total eluate is evaporated in vacuo to yield 95 mg of product which is GC155f lyophilized to gi~e an amorphous powder, melting point 120-135C.
Anal. Calc'd for CloHllN2O6S2K: C,33.51; H,3.09;
N,7.82; S,17.89 Found: C,33.46; H,3.08; N,7.92; S,17.64 Example 75 [3S(Z)]-3-~(2-Amino-4-thiazolyl)[(carboxymethoxy)-imino]acetyl~amino]-2-oxo-1-azetidinesulfonic acid, potassium salt [3S(Z)]-3-[ I (2-Amino-4-thiazolyl)[[2-oxo-2-(phenylmethoxy)ethoxy]imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (0.1 g;
see Example 71) is dissolved in a mixture of 5 ml of ethanol and 5 ml of water and hydrogenated at room temperature in the presence of 0.2 g of 10% palladium on charcoal. After 2 hours the catalyst is filtered off and the remaining solution is freeze-dried yielding the title compound.
m.p. 235(dec.).
Example 76 3-[~(S)-[(Aminocarbonyl)amino~-2-thienylacetyl~-amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt, isomer A
To a solution of 3-amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (277 mg; see Example 46, method II, part A) in 15 ml of dry acetonitrile at -20C is added pyridine (71 ~1) 0.888 mmole) and (D)-2-amino-4-(2-thienyl)-5-(4H)-oxazoline hydrochloride (166 mg).
The mixture is stirred for 10 minutes and a second GC155f -llo- 1 3 3 8 6 7 0 aliquot of pyridine ~71 ~1) and oxazoline (166 mg) is added. After another 10 minutes the solvent is removed under reduced perssure and the residue is dissolved in a water-acetone mixture and passed through an ion-exchange resin (20 ml AG 50W-X2,K+
form, 200-400 mesh). Removal of the water from fractions 2-3 gives a mixture of diastereomers (248 mg).
The product is purified and the diastereomers separated on Diaion AG HP 20 (130 ml) and eluted with water. Isomer A is eluted in fractions 23-28 (30 mg), and isomer B in fractions 35-45 (30 mg) (8 ml fractions).
The middle fractions (10 mg) are combined with fractions from other runs and a total of 35 mg of isomer A and 59 mg of isomer B is isolated.
Anal. for isomer A, m.p. 158-165C.
C CllH13 4 7 2 K / H2O
C, 31.05; H, 3.29; N, 13.18 Found:C, 30.95; H, 2.97; N, 12.98 Anal. for isomer B, m.p. 160-170C(dec).
Calc'd for CllH13N4O7S2 / 2 C, 31.05; H, 3.29; N, 13.18; S, 15.05 Found:C, 31.17; H, 3.09; N, 13.13; S, 15.09 Example 77 3-Methoxy-2-oxo-3-[(phenylsulfoacetyl)amino]-1-azetidinesulfonic acid, dipotassium salt To a stirred solution of crude 3-amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutyl-ammonium salt (366 mg; see Example 74A) and 38 mg of borax in 35 ml of dry acetonitrile at -10C
under nitrogen is added 0.53 ml of dry pyridine followed by a two minute addition of a solution of ~-sulfophenylacetyl chloride monoetherate GC155f (348 mgt in 8 ml of acetonitrile. After 20 minutes the solvent is remo~red in vacuo and the residue is treated with 35 ml of 0.5 ~1 pH 5.5 potassium phosphate buffer. Tetrabutylammonium hydrogen sulfate (383 mg) is added and the mixture is extracted three times with methylene chloride.
The methylene chloride is dried (sodium sulfate) and evaporated to give 685 mg of crude product.
The crude product is combined with 115 mg io of crude from a second run and the combined material is purified on a col-umn of SiliCAR CC-4 using methylene chloride and then 2,4,6,8 and 10% methanol in methylene chloride as eluent. The product, a mixture (about 6:1) of racemic diastereomers in the tetrabutylalr~nonium salt form, is converted to the potassium salt by passage through Dowex 50W-X2 (K+ form) resin using 20~ acetone in water as solvent. The product is lyophilized yielding 171 mg of the title compound, melting point 205-210C, dec.
Anal. Calc'd for C12H12N2OgS2K2 2 N,5.74; S,13.10 Found: C,29.45; H,2.74; N,5.51; S,12.82 Example 78 3-[(Carboxyphenylacetyl)amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, dipotassium salt 3- [[1,3-Dioxo-2-phenyl-3-tphenylmethoxy)-propyl]amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt (39 mg; see Example 51) is dissolved in methanol (5 ml). Anhydrous potassium GC155f carbonate (3.9 mg) and 10% palladium on carbon (19 mg) are added and the mixture is hydrogenated at atmospheric pressure for 20 minutes. The catalyst is removed by filtration and the filtrate is S evaporated in vacuo to yield a glassy residue (34 mg) which is lyophilized to an amorphous powder, melting point 178-190C,dec.
C13H12O8N2S K2- 0 5 H2O: C,35.20; H 3 18;
N,6.32; S,7.23 Found: C,35.51; H,2.96; N,6.29; S,6.92 Example 79 [3S(Z)]-3-[[(2-Amino-4-thiazolyl)[[2-(1,1-dimethyl-ethoxy)-l-(methylthio)-2-oxoethoxy]imino]acetyl]-amino]-2-oxo-1-azet;~;nesulfonic acid, potassium salt Following the procedure of Example 73, but substituting (Z)-2-amino-~-[[2-(1,1-dimethyl-ethoxy)-l-(methylthio)-2-oxoethoxy]imino]-4-thiazoleacetic acid for (Z)-2-amino-a-(hydroxy-imino)-4-thiazoleacetic acid, yields the title compound, melting point 130 C, dec.

GC155f _ Example 80 (+)-3-Butoxy-3-l~(phenylmethoxy)carbonyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt A solution of 185 mg of 2-oxo-3-[N-chloro-N-- 5 [(phenylmethoxy)carbonyl~amino]-l-azetidinesulfonic acid, tetra~utylammonium salt (see Example 49A) in 1 ml of dimethylfor~ e is cooled to -78C and 0.73 N lithium n-butoxide (3.8 ml) in n-butanol is added at -78C. After 15 minutes, 0.5 M monobasic potassium phosphate buffer is ~ and the-product extracted into dichloromethane (three 40 ml portions), dried over sodium sulfate, filtered and concentrated in vacuo to give 179 mg of the corresponding tetrabutylammonium salt of the title compound.
To a solution of 109 mg of the tetrabutyl-ammonium salt in acetone is added perfluoro-butylsulfonic acid, potassium salt (60 mg) in acetone. The solvent is removed in vacuo and ethyl acetate is added. The product . crystallizes and is collected and dried-to yield 66 mg of the title compound, melting point 186.5-187.5C, dec.

Example 81 [3+(E)]-3-~ethoxy-3-[[(methoxyimino)[2-[[(phenyl-methoxy)carbonyl]amino]-4-thiazolyl]acetyl]-amino-2-oxo-1-azetidinesulfonic acid, potassium salt A suspension of 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (Example 46, Method II, part A) (0.175 mmole) and sodium borate (0.0175 mmole) in 2 ml of GC155f ~114~ 1 3 3 8 ~ 70 dichloromethane at 0C is treated with 28 ~1 of pyridine and 0.175 mmole of (E)-~-(methoxy-imino)-2-[l(phenylmethoxy)carbonyl]amino]-4-thiazolylacetyl chloride. After 1 hour, the mixture is diluted with dichloromethane and quenched with water. The organic layer is washed with water, saturated salt, dried, and evaporated in vacuo. The residue is purified on Mallinckrodt SilicAR CC-4 silica gel (20 g) to give 43 mg of the corresro~ing tetrabutylammonium salt of the title compound.
The tetrabutylammonium salt (43 mg) is dissolved in 0.5 ml of acetone and 20 mg of potassium perfluorobutane sulfonate in 0.5 ml of acetone is added. After addition of 3 ml of ether the solid is collected and dried in vacuo to give 28 mg of the title compound melting point 144-146C, dec.

Example 82 I3+(Z)]-3-Methoxy-3-~(methoxyimino)~2-~(phenyl-methoxy)carbonyl~amino~-4-thiazolyl]acetyl]amino~-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 81, but substituting (Z)-~-(methoxyimino)-2-[I(phenyl-methoxy)carbonyl]amino]-4-thiazolylacetyl chloride for (E)-~-(methoxyimino)-2-[~(phenylmethoxy)-carbonyl]amino]-4-thiazolylacetyl chloride, yields the title compound, melting point 168-172C, dec.

GC155f --115- 1 3 3 8 6 7 o Example 83 3-[l(R)--[[(4-Ethyl-2,3-dioxo-1-piperazinyl)-carbonyl]amino]phenylacetyl~amino~-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt To a stirred solution of 0.69 mmole of 3-amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (Example 46, ~ethod II, part A) in 30 ml of dry acetonitrile at -20 C
under nitrogen is added 242 ~1 of dry pyridine followed by a solution of 352 mg of (R)-~-[1(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]-phenylacetyl chloride in 4 ml of acetonitrile.
After 1 hour 84 yl of pyridine is added followed by an additional 117 mg of the above named acid chloride in 1 ml of acetonitrile. The reaction is stirred for 20 minutes, diluted with 24 ml of 0.5 M pH 5.5 monobasic potassium phosphate buffer and concentrated in vacuo to remove acetonitrile. The aqueous remainder is extracted three times with methylene chloride and the combined extracts are dried (Na2SO4) and evaporated leaving 546 mg of residue. Passage of the residue through a silica gel column using methylene chloride and then2%, 4% and 6% methanol in methylene chloride, provides two fractions (285 mg and 173 mg) of the corresponding tetrabutylammonium salt of the title compound.
Passage of the 173 mg fraction through 4.5 g of Dowex 50-X2(K+) resin using acetone-water as eluent yields 119 mg of the title compound. A 104 mg portion of this material is GC155f applied to a column of HP 20-AG re-sin in water.
Sequential elution with water,5% acetone in water and 10% acetone in water yields 60 mg of product as a mixture (ca. 1:1) of diastereomers.
Lyophilization of the 60 mg fraction yields a solid, metling point 171-172C, dec.

Example 84 N-(3-Butoxy-2-oxo-1-sulfo-3-azetidinyl)-2-phenyl-acetamide, tetrabutylammonium salt Following the procedure of Example 80, but substituting N-chloro-N-(2-oxo-1-sulfo-3-azetidinyl)-2-phenylacetamidej tetrabutylammonium salt (see Example 47A for preparation of the corresponding potassium salt) for 2-oxo-3-lN-chloro-N-~(phenyl-methoxy)carbonyl]amino]-l-azetidinesulfonic acid, tetrabutylammonium salt, yields the title compound as an oil: nmr (CDC13) 3.62 (s,2H,C6H5CH2), 4.03 (ABq,2H,v=7cps, C-4 CH2), 6.98 (s,lH,NH) and 7.30 ppm (S,SH,C6H5).

Example 85 ~R)-3-Methoxy-2-oxo-3-[(phenylacetyl)amino]-1-azetidinesulfonic acid, potassium salt A lM solution of dimethylformamide-sulfur trioxide complex is prepared by the slow addition of trimethylsilyl chlorosulfonate to dimethyl-formamide at 0C followed by evacuation at 0.1 mm for 30 minutes at 0-25C. Under an argon atmosphere, 50 mg of (R)-N-(3-methoxy-2-oxo-1-azetidinyl)-phenylacetamide is dissolved in 0.2 ml of anhydrous dimethylformamide and cooled to 0C. Cold lM

_ GC155f dimethylformamide-sulfur trioxide solution (0.428 ml) is added, the mixture is stirred for 2 hours and poured into 15 ml of 0.5 N
monobasic potassium phosphate. The solution is extracted twice with dichloromethane (discard) and 73 mg of tetrabutylammonium bisulfate is added. Extraction with dichloromethane (three 10 ml portions) gives a viscous oil after drying and evaporation in vacuo. Chromatography on Mallincrodt CC-4 silica gel (50:1i~usingi2~ me~hanol in dichloromethane as eluant gives 34 mg of (R)-3-methoxy-2-oxo-3-~(phenylacetyl)amino~-1-azetidinesulfonic acid, tetrabutylammonium salt. Ion exchange on Dowex 50W-X2 (K+, 10 equivalents) give the title potassium salt after lyophilization of the aqueous eluate: melting point 130 C, dec., [~D=+52 (C=0.5, water).

Example 86 (S)-3-[[[[(1-Ethyl-4-hydroxy-3-methyl-lH-pyrazolo-[3,4-b]pyridin-5-yl)-carbonyl]amino]phenylacetyl]-amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 73, but substituting ~-[[(1-ethyl-4-hydroxy-3-methyl-lH-pyrazolo[3,4-b]pyridin-5-yl)carbonyl]amino]benzene-acetic acid for (Z)-2-amino-~-(hydroxyimino)-4-thia-zoleacetic acid, yields the title compound, melting point 233-236C, dec.

_ GC155f Example 87 (R)-3-Acetylamino-3-methoxy-2-oxo-1-azetidine-sulfonic acid, potassium salt ~) 3-Acetylamino-l-~l-carboxy-2-methyl(propyl)]-(3R)-3-methoxy-2-oxoazetidine To a solution of (6R-cis)-7-acetylamino-7-methoxy-3-methyl-8-oxo-5-thia-l-azabicyclo[4.2.o]oct 2-ene-2-carboxylic acid (650 mg) and sodium bicarbonate (191 mg) in water is added a slurry (11 ml) of commercial grade Raney nickel (0.6 g/ml), which has been washed to neutrality with water.
The mixture is lowered into an oil bath preheated to 170 C and proceeds to reflux in 2-3 minutes, while maintaining the bath t~mperature at 150-170 C. After refluxing for 15 minutes, the reaction is quenched by cooling in an ice bath. Catalyst is removed by filtration through Celite and the filtrate (pH 11) is adjusted to pH 2 with 1 N hydrochloric acid. After extracting the aqueous solution five times with ethyi acetate, the combined extracts are dried (Na2SO4) and solvent is removed in vacuo to give an oil (487 mg). Chromatography on silica gel yields the product (eluted in chloroform) as an oil (381 mg).

B) 3-Acetylamino-l-[-l-(acetyloxy)-2-methyl(propyl)]-(3R)-3-methoxy-2-oxoazetidine The above azetidinone (464 mg) is dissolved in dry acetonitrile (15 ml) and the solution is purged with argon for 15 minutes. Copper acetate (359 mg) is added, stirred for one minute to dissolve the salt, and the lead tetraacetate GC155f -- --119-- .

(797 mg) is added. ~hile argon continues to bubble through the mixture, the temperature of the reaction is raised by lowering the flask into an oil bath, preheated and maintained at 55-65C for 15 minutes. The mixture is allowed to cool to room temperature, filtered through Celite and the filter pad is washed well with acetonitrile. Solvent is removed in vacuo from the combined filtrate and w~shings. The residue is taken up in water, and extracted four times with ethyl acetate. The combined extracts are dried (Na2SO4) and solvent is removed in vacuo to yield the desired product as an oil (382 mg).
C) (R)-N-(3-Methoxy-2-oxo-1-azetidinyl)acetamIde The above oil is dissolved in a methanol (10 ml):water (1 ml) mixture, cooled in an ice-methanol bath at -10 to -15C and potassium carbonate (194 mg) is added followed by sodium borohydride (53 mg). After stirring at -15 to -8C for 110 minutes, solvent is removed in vacuo, the residue is taken up in water and the solution is adjusted to pH 6 with 1 N hydrochloric acid.
Exhaustive extraction with ethyl acetate, drying (Na2SO4), and removal of solvent in vacuo yields an oil (224 mg). Chromatography of the oil on silica gel, eluting with 5% methanol:95% methylene chloride giving an oil (169 mg). The title compound crystallizes from ether-pentane to give 131 mg of material, melting point 106-112C
(sintering 103.5C).

GC155f D) (R)-3-Acetylamino-3-methoxy-2-oxo-1-azetidine-sulfonic acid, potassium salt Under an argon atmosphere, S0 mg of (R)-3-acetylamino-3-methoxy-2-oxo-1-azetidine is placed in a flask and cooled to 0C. A 1 M
solution of dimethylf orm~m; ~-sul fur trioxide complex in dimethylformamide (.95 ml) is then added and the solution is stirred for 15 minutes.
The contents of the flask are then poured into 40 ml of 0.5 N K2HP04 soultion and extracted - twice with 10 ml of methylene chloride. Tetrabutyl-ammonium sulfate (1.2 equivalents) is added to the aqueous solution and the resulting mixture is extracted with four 10 ml portions of methylene chloride. The extracts are then dried over Na2SO4 and concentrated to afford 39 mg of product. The tetrabutylammonium salt is converted to the title potassium salt by passing it through a column of Dowex 50-X2 (K+).
Concentration of the aqueous fraction gives 19 mg of the potassium salt, with identical NMR
spectra to that prepared in EX46Ib and by isolation from natural sources (Ex. 165).

GC155f Example 88 (+) -3- ~ (Azidophenylacetyl~ amino-] -3-methoxy-2-o~-1-azetidinesulfonic acid, potassium salt A) (+)-3-~(Azidophenylacetyl~amino]-3-methoxy-2-oxo-l-azet~ esulfohic acid, tetrabutylammonium s-alt 3-Amino-3-methoxy-2-oxo-1-azetidinesulfonic acid tetrabutylammonium salt (.2Q2 mgs; see Example 74A2 is dissolved in 20 ml of dry acetonitrile.
To the well stirred solution at -20C under dry nitrogen are added dry pyridine (167 pl) and a-azidophenylacetyl chloride (96 yl). After 20 minutes, 0.5M pH 5.5 monobasic potassium phDsphate buffer (12 ml) is added and the acetonitrile removed in vacuo. The aqueous residue is extracted three times with methylene chloride.
The extract is dried over anhydrous sodium sulfate and evaporated in vacuo to give 281 mg of crude product as a gum. This is purified by chromatography through a column of silica gel (30 g) using methylene chloride and mixtures of methylene chloride-methanol up to 6% methanol, and yielding 231 mg o the title compound.

B) (+)-3-[(Azidophenylacetyl~amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt (+)-3-~(Azidophenylacetyl~amino~-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (231 mg) in 30~ acetone: water (15 ml) is passed through a column of Dowex 50w-X2 (K form; 3 ml) and eluted with water. The total eluate is evaporated in vacuo to give a colorless glass GC155f _ , (168 mg~ as a 1:1 mixture of diastereomers.
Passage of this through a 6Q ml column of ~P2Q-AG using water and water:10% acetone gives 71 mg of a-l:l mixture of racemic diaste eu crs and 70 mg of a 1:3 mixture of racemic diastereQmers.
The 1:1 mixture is lyophilized and dried in vacuo at 4QC to gi~e t~e dried product as a hemihydrate, melting point dec. 130C.
Analysis for C12H12N56-K 5 H2O Calc~d C~ 35-~;
H, 3.26; N, 17.45; S, 7.98 Found: C, 35.~4; H, 3.Q7; N, 17.24; S, 8.Q2 EXample 89 3-~(Azidophenylacetyl~amino]-3-methoxy-2-oxo-1-azetidinesulfonic, potassi-um salt, isomer A
The 1:3 mixture of racemic diastereomers obt~;ne~ in Example 88B is allowed to stand in deuterated water at room temperature, and isomer A
crystallizes. After refrigeration, the mother liquor is removed, and the crystals (28 mg) are dried in vacuo at 40C, melting point 130C, dec as a mono-deuterate.
Example 90 [3+(R*)]-3-[~1 I (4-Ethyl-2,3-dioxo-1-piperazinyl~-carbonyl]amino]phenylacetyl]amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt To a stirred solution of 3-amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (0.69 mmol; see Example 74A) in 30 ml of dry acetonitrile at -20C under nitrogen is added 242 ~1 of dry pyridine followed by a solution of GC155f 352 mg of (R)~ (4-ethyl-2,3-dioxo-1-piperazinyl)-carhonyl~amino~phenylacetyl chloride in 4 ml of acetonitrile. After 1 hour, 84 ~1 of pyridine is added followed by 117 mg more of the acid chloride in 1 ml of acetonitrile. The reaction is stirred for 20 minutes, diluted with 24 ml of 0.5 M pH 5.5 mono~asic potassium phosphate buffer, and concentrated in vacuo to remove acetonitrile. The aqueous remainder is extracted three times with methylene chloride and t~e combined methylene chloride extract is dried (Na2SO4) and evaporated to a residue (546 m~.
Passage of this material through a column of ~ilicAR CC-4, using methylene chloride and then 2%, 4%, and finally 6% methanol in methylene chloride provides two fractions (285 mg and 173 mg) of purified product in the tetrabutyl-ammonium salt form.
Passage of the 173 mg portion through 4.5 g of Dowex 50-X2 (K ) resin using acetone-water yields 119 mg of potassium salt.~ A 104 mg portion of this material is applied to a column of HP20-AG
resin in water. Sequential elution with water, 5% acetone in water, and finally 10% acetone in water provides 60 mg of product as a mixture (ca. 1:1) of diastereomers and 21 mg of product as a mixture (ca. 9:1) of diastereomers. Lyophili-zation of the 60 mg fraction yields the title compound, melting point 171-172C, dec.
Analysis for Cl9H22NsgSK H2O: Calc~d C, 41.23;
H, 4.37; N, 12.65; S, 5.78 Found: C, 41.39; H, 4.12; N, 12.58; S, 5.63 GC155f Lyophilization of the 21 mg fraction yields the title compound, melting point 171-172 C, dec.
-Analysis for ClgH22NsOgSK-H2O: Calc~d C, 41.23 H, 4.37; N, 12.65 Found: C, 41.43; H, 4.11; N, 12.28 Treatment of the 285 mg fraction of tetra-butylammonium salt with Dowex 50-X2(R ) provides 145 mg of potassium salt, which is combined with the remaining 15 mg of the aforementioned 119 mg portion of Dowex resin derived potassium salt.
Passage of this material through HP2Q-AG, as already described, provides an additional 31 mg of product as a mixture ~ca. 1:11 of d;astereomers and an additional 42 mg of product as a mixture (ca. 9:1) of diastereomers. The total amount of (1:1) mixture of diastereomers is 91 mg, and the total amount of (9:1) mixture of diastereomers is 63 mg.

Example 91 ~[3S(Z)]-3-l~(Methoxyimino)~2-~l(phenylmethoxy)-carbonyl]amino]-4-thiazolyl]acetyl]amino~-2-oxo-l-azet;~;~esulfonic acid, potassium salt To a solution of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (0.170 mmol; see Example 6A) and sodium borate (0.170 mmol) in 2 ml of methylene chloride at 0C is added pyridine (62 ~1) and (Z)-~-(methoxy-imino)-2-[~(phenylmethoxy)carbonyl~amino]-4-thiazoleacetyl chloride (0.51 mmol). The reactionmixture, after 40 minutes, is diluted with methylene chloride and water, followed by 0.1 M

GC155f -tetrabutylammonium suifate buffered to pH 4 ~5.1 ml). The organic layer is separated and washed with water, adjusted to pH 2, water adjusted to pH 7, water saturated with sodium chloride, dried over sodium sulfate, filtered, and concentrated in Ya-cuo. The residue is purified on SilicAR CC-4 silica gel ~10 g) and the product eluted with 10% methanol/methylene chloride.
The tetrabutylammonium salt, after dissolution in acetone/water, is passed through an ion-~YrhAn~e resin (8 ml, AG 50W-X2, R form lQ0-20Q mesh~.
Removal of ~h~ water in v-acuo from fractions 1-2 give 40 mg of the title c~mpound, melting point 172-174C, dec.
Analysis for C17H16N58S2K H2 H, 3.33; N, 12.99; S, 11.87 Found: C, 37.95; H, 3.30; N, 12.73; S, 11.53 Example 92 (+)-3-Butoxy-2-oxo-3-[(phenylacetyl)amino~-1-azeti~;~esulfonic acïd, potassium salt A) 3-tChloro(phenylacetyl)amino]-2-oxo-1-azetidine-sulfonic acid tetrabutylammonium salt A solution of (S)-2-oxo-3-[t(pheny~methoxy)-carbonyl]amino]-l-azetidinesulfonic acid, tetra-butylammonium salt (350 mg; see Example 4) in methylene chloride (3 ml) is added to a suspension of sodium borate (1.27 g) in a 5.25% solution of sodium hypochlorite (4.72 ml) and water (20 ml~

GC155f at 0 C. After 1 hour 0.5 M monobasic potassium phosphate (25 ml2 is added and the mixture is extracted three times with methylene chloride ~50 ml portions). The organic extracts are dried over sodium sulfate, filtered and concentrated in vacuo to give 344 mg of the title compound.

B) (+)-3-Butoxy-2-oxo-3~ henylacetyl).-amino~
azetidinesulfonic aci.d', tetrabu'tylamm~h'ium salt A solution of 3-~chloro(~henylmethoxy2~
carbonyl~amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (344 mg~ in dimethyl-formamide (5 ml2 îs added to 0.73 N n-lithium butoxide in n-butanol ~6 mll in dimethylfonmamide (1 ml) at -78C under an inert atmosphere. After 10 minutes the mixture is diluted with 0.5 M
monobasic potassium phosphate solution (175 ml).
After extracting three times with methylene chloride, the organic extract is dried over sodium sulfate, filtered, and the solvent removed in vacuo.
The residue is purified on SilicAR CC-4 silica gel (80 g) and the title compound (130 mg) is eluted with 4-8% methanol in methylene chloride.

C) (+)-3-Butoxy-2-oxo-3-1(phenylacetyl)amino]-l-azetidinesulfonic acid, potassium salt (+)-3-Butoxy-2-oxo-3-[(phenylacetyl~amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (43 mg) is dissolved in a water-acetone mixture ~9:1) and placed on a cation exchange column (Dowex AGMP 50w-X2, 100-200 mesh, 5 g, K form).

GC155f -127- 1 338 6 ~o The product is eluted with water and the eulate concentrated 'in vacuo to give 2Q mg of the title compound, melting point 122-125C.
- Analysis calc'd for C15H12N2O6SK 1/2 H2O: C, 44.66;
H, 4.96; N, 6.q5; S, 7.94 Found: C, 44.77; H, 4.76; N, 6.76î S, 7.75 EXample g3 (~)-3-Ethoxy-2-oxo-3-~(phen~lacetyllamino3-1-aze-~ n~sulfonic''acid, pot'assium salt 3-IChloro~phenylacet~l)amino~-2-oxo-l-azet;~in~culfonic acid, tetrabutylammonium salt (200 mg;
see~example 92A~ in dimeth~lformamide ~4 ml) is added to Q.5 N lithium ethoxide in ethanol (12.20 ml) at -78C under an inert atmosphere. After 10 minutes the mixture is diluted with 0.5 M
monobasic potassium phosphate solution (15 ml).
After extracting three times with methylene chloride, the organic layer is dried over sodium sulfate, filtered and the solvent removed in vacuo. The residue is purified on SilicAR
CC-4 silica gel (20 g) and the tetrabutylammonium salt of the product (40 mg~ is eluted with 2%
methanol in methylene chloride.
The tetrabutylammonium salt is dissolved in a water-acetone mixture (9:1) and placed on a cation exchange column (Dowex AGMP 50W-X2, 100-200 mesh (5 g, K form)). The product is eluted with water and the eluate concentrated in vacuo to give 25 mg of the title compound, melting point 94-96C.
Analysis calc'd for C13H15N2O6 H, 4.10; N, 7.65; S, 8.74 Found: C, 40.36; H, 3.66; N, 6.77; S, 8.44 GC155f Exa~ple 94 I 3+(Z)]-3-~C2-Amino-4-thiazol~(methoxyimino~_ acetyl~amino~-3-methoxy-2-oxo-1-azetidines~lfonic acid, potassium salt 3-Amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt ~see Example 46, method II, part Al is dissolved in acetonitrile (20 ml~ and pyridine a ml~ and adde~ to a vigorously stirring suspension of (Zl-a-Cmeth imino~-2-amino-4-t~iazolPacetyl chloride in acetonitrile (2a ml~ cooled to Q-5C. After stirring cold for 1 hour the mixture is diluted with Q.5 M mono~asic potassium p~osphate solution (100 ml~ CpH of the mixture is 4.8) and solvent is removed in vacuo. The residue is taken up in a min;mAl amount of water containing a small amount of acetone. Chromatography on ion exchAnge resin (AG 50W-X2, 100-200 mesh, K form 200 ml) gives the crude product as the potassium salt upon elution with water. Further purification on HP-20 resin (200 ml) using water as the eluant gives S9 mg of the product as a powder after trituration with acetonitrile-ether and then twice with ether. The product is an amorphous powder that melts slowly and decomposes above 150C.
Analysis calc'd for CloH12N5O7SK: C, 28.77;
H, 2.90; N, 16.78; S, 15.36; K, 9.37 Found: C, 27.77; H, 2.82; N, 15.87; S, 13.63;
K, 10.11 GC155f .
-129- 1 3 3 ~ 6 7 0 Exa~nple 9S
[3R(R*) and 3S CS*~-3~ '(p~inocArbonyllam-ino~-phenylacetyl] amino~'-3-methoxy-2-oxo-1-azeti'dine-sulfohic acid, potassi~n salt A) (+~-3-~ (Aminopheh~lacety~l~amino~ -3-me-thQX5r-2-oxo-1-azetidinesulf'onic acid, inne~' salt (~)-3-1(AzidopElenylacetyl)amino]-3-methoxy-2-oxo-1-aze~i~inesulfonic acid, potassium salt (209 mg; see Example 88)~is dissolved in 6a ml of dry me~hAnol. Anhydrous trifluoroacetic acid (0.6 ml) and ln% palladilml on carbon ClQ5 mg~
are added and the mixture is hydrogenated for hour. The catalyst is removed by fîltration 15 and the filtrate is evaporated ln vacuo to yield 271 mg of crude product.

B) ~3R(R*) and 3S(S*)~ -3-I[I(Aminocarbonyl)amino]-phenylacetyl]amino~ -3-methoxy-2-oxo-1-azetidine-20 sulfonic acid, potassium salt (+)-3-l(Aminophenylacetyl)amino]-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt (271 mg) is dissolved in 6.5 ml of water.
Potassium cyanate (87 mg) is added and the 25 mixture is stirred at room temperature for 3 hours.
The solution is concentrated in vacuo to approxi-mately 2 ml and chromatographed on a 100 ml column of HP20-AG using water as eluant. Isomer A
(29 mg) is isolated after lyophilization, melting 30 point 160C, dec.
Analysis for C13H15N4O7SK monohydrate: Calc'd: C, 36.44;
H, 3.99; N, 13.07; S, 7.48 Found: C, 36.35; H, 3.79; N, 12.81; -S, 7.32 GC155f Exa~ple ~6 ~3R(S*) and 3s(R*)l-3~ cAminocarbonyl~amih - phenylacetyl]amiho]-3-methoxy-2-oxo-l-azetidine sulfonic acid, po*assium salt Along with l3R(R*) and 3S(S*)~-3-tl~amino-carbonyl~amino]phenylacetyl]amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt produced in Example ~5, there are produced 21 mg of l3R(S*1 and 3SCR*II-3~ (aminocarbonyl~-amino~phenylacetyl]amino]-3-methoxy-2-oxo-1-azeti~inesulfonic acid, potassium salt as a lyophilate, melting point 16QC dec.
Analysis for C13H15N4O7SK sesquihydrate:
Calc'd: C, 35.6q; H, 4.14; N, 12.81; S, 7.33 Found: C, 35.98; H, 3.87; N, 12.5Q; S, 7.32 Example 97 13-(s*)]-3-Methoxy-3-~ 2-oxo-3-I(phenylmethylene) amino~-l-imidazolidinyl]carbonyl]amino]-2-thienyl-acetyl]amino~-2-oxo-1-azetidinesulfonic acid, potassium salt To a stirred solution of 3-amino-3-methoxy-2-oxo-azetidinesulfonic acid, tetrabutylammonium salt (306 mg of crude material assumed to contain 274 mg of organic material, prepared as described in Example 74) in 20 ml of dry acetonitrile at -20C under nitrogen is added 0.30 ml of dry - pyridine (3.72 mmol) followed by 484 mg of (S)-~[[2-oxo-3-phenylmethylene)amino]-1-imidazolidinyl]-carbonyl]amino]-2-thienylacetyl chloride partially dissolved and suspended in 10 ml of dry acetonitrile.

GC155f -131- 1 3 3 8 6 7 o The reaction is stirred and allowed to rise to QC over the course of 1 hour.
The reaction was diluted with a large volume of methylene chloride and then treated with 22 ml of 0.5 M pH 5.5 monobasic potassium phosphate buffer. The aqueous layer is washed with methylene chloride and the combined meth,ylene chloride extract is washed ~ith water, dried Q~a2SO41, and evaporated to a residue (5Q8 mg2. This residue is chromatographed on 5Q g of SilicAR CC-4, using methylene chloride and then 2% and 4% methanol in methylene chloride to give 251 mg of the tetrabutylammonium salt of the title ccYmpound.
To this salt (251 mg) in acetone is added a solution of 107 mg of perfluorobutanesulfonic acid potassium salt in several milliliters of acetone. Ethyl acetate is added, and the precipitate is washed three times with ethyl acetate by centrifugation, and dried in vacuo at 40 C/lmm for 2 hours to give 95 mg of desired potassium salt as a mixture (ca. 1:2) of diastereomers --having a melting point 20ac, dec.
Calc, d for -C21H21N6OgS2K: C, 42.85; H, 3.60;
N, 14.28; S, lQ.87 Found: C, 43.02; H, 3.~4; N, 13.94; S, 10.71 GC155f Ex'ample 98 (+--~L~)-4-Methyl-'2-oxo-3-I'['(phehylmethoxy]carbonyl~-amino~-l-azeti~in~sulfonic'acid'r potassium 'salt A~ N-Benzyloxy-t-~oc*-~llothreonine amide A solution of 6.9 g of d,l-t-boc-allothreonine and the free amine from 5.3 g of o-benzylhydroxyl-amine HCl (~0.033 mole, et~yl acetate-sodium bicarbonate liberation~ in 80 ml of tetrahydro-furan is treated with 4.82 g of N-hydroxy-benzotriazole and 6.5 g of dicyclohexylcar~odiimide in 20 ml of tetra~ydrofuran. After stirring for about 16 hours at room temperature the slurry is fîltered, concentrated in vacuo and chromatographed lS on a 400 ml of silica gel column. Elution with 5-10% ethyl acetate in chloroform gives 6.8 g of the title compound in fractions (200 ml each) 7-22.

B) (+-c ~N-Benzyloxy-3-t-butoxycarbonylamino-4-methylazetidinone A solution of 6.8 g of N-benzyloxy-t-boc-allothreo~ine amide in 200 ml of tetrahydrofuran is stirred for about 16 hours with 5.24 g of triphenylphosphine and 3.2 ml of diethylazodicarboxylate.
The solvents are evaporated in vacuo and the residue is chromatographed on a 500 ml silica gel column. Elution with methylene chloride followed by crystallization from ether gives a total of 2.65 g of azetidinone. Rechromatography of the mother liquors and mixed fractions give an * "boc" is used to describe butoxycarbonyl GC155f additional 0.6 g. Crystallization of a portion twice from ether (-2Q C~ gives the analytical sample of the title compound melting point 140-142C.

Cl (+-cis~3-t-~uLo~ycarbonylamino-l-hydroxy-4-methylazeti~ino~e A solution of 3.2 g of cis-N-benzyloxy 3-t-butoxycar~onylamino-4-methylazeti~;no~e in 200 ml of 95% ethanol is stirred in an atmosphere of hydrogen wit~ 0.7 g of 10% palladium on charcoal. After 4Q minutes the slurry is filtered (uptake 249 ml) and th~ filtrate is evaporated and triturated with ether to give, in two crops, 2.05 g of solid, melting point 134-136C.
D) (+-cis-3-t-~utoxycarbonylamino-4-methylazetidinone A solution of 2.05 g of -c -3-t-butyloxy-carbonylamino-l-hydroxy-4-methylazetidinone in 60 ml of methanol is treated with a total of 90 ml of 4.5 M ammonium acetate (40, 20 and 30 ml portions) and 45 ml of 1.5 M titanium trichloride (20, 10 and 15 ml portions2 the second and third additions are made after 15 and 120 minutes, respectively. After 135 minutes the solution is diluted with an equal volume of 8% sodium chloride and extracted with three 300 ml portions of ethyl acetate. The combined or~anic layer is washed with a mixture of 100 ml each of 5% sodium bicarbonate and saturated salt, dried, and evaporated. Trituration with ether ~ives, in two crops, 1.65 g of solid. A portion of the first crop is recrystallized from ether to give the analytical sample, melting point 176-178.5C.

GC155f _ E) (+ -cis~3-Benz~ carLo~ m;no-4~met~ylazetidinone A solution of 1.55 g of- cis-3-t-butoxy-carbonylamino-4-methylazeti~ino~e in 4 ml each of methylene chloride and anisole is cooled to 0C
and 5Q ml of cold trifluoroacetic acid is added.
After 9Q minutes the solvents are e~aporated in vacuo (~en7ene added and evaporated three timesl.
The residue is dissol~ed in 25 ml of acetone, the initial pH (2.51 i8 raised to 7 wi.th 5%
sodium bicarbonate, and 2 ml of benzylchloroformate is added. The solution is kept at QC and pH 7 for 4 hours and the acetone is r~o~ed in vacuo to give a slurry that is filtered. The filtrate is saturated with salt and extracted ~ith methylene chloride. The solid is dissolved in methylene chloride and dried. The organic layers are combined, concentrated, and the residue chromato-graphed on a 200 ml silica gel column. Elution with 3:1 chloroform, ethyl acetate gives 850 mg of the title compound in fractions (100 ml each) 4-11. Crystallization of a small sample from ether gives the analytical sample, melting point 165-166C.

F) (~-cis)-4 ~e~h~1-2-oxo-3-[~(.phenylmethoxy)-carbonyl]amino]-l-azetidinesulfonic acid, potassium salt To a suspension of -cis-3-benzyloxycarbonyl-amino-4-methylazetidinone (0.75 g) in 7 ml each of dimethylformamide (dried with 4A sieves activated at 320C for 15 hours under argon flow) and methylene chloride (dried through basic A12O3) is added 1.66 g of pyridine-sulfur trioxide complex.
After 3- hours stirring at room temperature under nitrogen, an additional amount of pyridine-sulfur trioxide complex (1.66 g) is added. The reaction mixture is then stirred at room temperature under nitrogen for about 16 hours. The dimethyl-fonmamide is removed in vacuo to give 4.6 g of residue which is ~isFolved in 300 ml of Q.5 ~
monobasic potassium phosphate solution C4QC for 10-15 minutes~. T~e solution is cooled, passed through a column of HP-20 resin (3 cm x 60 cm) with 400 ml of 0.5 M ~n9h~sic potassium phosphate 1 L of distilled water and C14:1l water:acetone to give 280 mg of ~loduct in fractions 13 to 26 ~l~Q ml eachl. Crystallization from MeOH: petroleum ether gives 757.5 mg of an analytical sample, melting point 214-215.5C, dec.
Analysis calc'd for C12H13N2SO6K: C, 40.90; H, 3.72 N, 7.95; S, 9.10; K, 11.10 Found: C, 40.43; H, 3.60; N, 7.89; S, 8.69;
K, 10.82 Example 99 (3 S-trans~-4-Methyl-2-oxo-3-~ I ~pheny~methoxy)car~onyl~-amino]-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 98, but substituting l-t-boc-threonine for d,l-t-boc-allothreonine, yields the title compound, melting point 133-135C.
Analysis calc'd for C12H13N2O6SK: C, 40.90; H, 3.72;
N, 7.95; S, 9.10; K, 11.10 Found: C, 40.72; H, 3.60; N, 7.99; S, 8.80;
K, 10.82 GC155f Example laO
~S-trans~-4-Methyl-2-oxo-3- I (phenylacetyl2amino~-l-azetidinesulfonic acid-, potassium salt A2 (3S~-3-Amino-4-methyl-2-oxo-1-azetidinesulfonic acid, tetrabutyl`ammoni~m salt (4S-*rans~-4-Methyl-2-oxo-3-[ I (phPnylmethoxy2 -carbonyl]amino]-l-azeti~ineculfonic acid, potassium salt (352.4 mg; see Example 92~ is dissolved in 20 ml of distilled water and treated with 373.5 mg (1 mmole) of tetra~ut~l ammonium hydrogen sulfate.
After lQ minutes s~tirring at room temperature, the solution is extracted three times with lQ ml portions of methylene chloride after saturation with sodium chloride. The methylene chloride is dried over sodium sulfate and evaporated in vacuo to give 536 mg of the tetrabutylammonium salt which is hydrogenated with 270 mg of 10% palladium on charcoal in 25 ml of dimethylformamide. The mixture is filtered through Celite and washed twice with 2.5 ml portions of dimethylformamide to yield the title compound in solution.

B) (Bs-tran~4-Met~yl-2-oxo-3-~(phenylacetyl2-amino]-l-azetidinesulfonic acid, potassium salt The crude (3S)-3-amino-4-methyl-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt from part A (the combined filtrate and washings) are treated at 0C with 206 mg of dicyclohexyl- -carbodiimide, 153 mg of N-hydroxybenzotriazole, and 138 mg of phenylacetic acid. The reaction GC155f mixture i5 stirred at QC for 1 hour and then at room temperature for 2 hours. The resulting precipitate is ~iltered, the filtrate is evaporated in vacuo, the residue is dissolved in 10 ml of acetone and filtered. The filtrate is treated with 25 ml of acetone saturated with potassium iodide and then 2Q0 ml of ether. The resulting solid (752.7 mg~ i5 a mixture of the potassium and tetra~utylammonium salts of the title compound.
The solid is dissol~ed in 5Q ml of Q.5 mono~asic potassium phosphate and applied to an HP-2Q column.
Elution with water and t~en water-acetone gi~es several fractions that are combined and evaporated to give the purified tetrabutylammonium salt. An aqueous solution of this material is passed through Dowex 50W-X2 (R form) to give the title potassium salt (121.4 mg). Trituration with acetone-hexane yields lQ4.6 mg of the title compound, melting point 211-213C.
Analysis calc'd from C12H13N2O5SK 1/2 H2O: C, 41.72;
-~ H, 4.09; N, 8.11; S, 9.28; K, 11.32 Found: C, 41.70; H, 4.01; N, 8.Q7; S, 9.01;
K, 11.02 Exa~ple lQl (cis)-4-Methyl-2 o~o 3-~(phenylacetyl)amino~
azetidinesulfonic acid, potassium salt A solution of 320 mg of (+ -cis)-4-methyl-2-oxo-3-~(phenylmethoxy)carbonyl]amino]-l- azetidine-sulfonic acid, potassium salt (see example 98) isErepared in 20 ml of water containing 483 mg of tetrabutyl-ammonium hydrogen sulfate and adjusted to pH 5.5.

GC155f -138- - 1 3 ~ 8 6 7 0 E~ctraction with 8iX 25 ml portions of methylene chloride giv~;517.3 mg of oil. A solution of this material in 15 ml of dimethylfor~ e is stirred with 4QQ mg of 10% palladium on charcoal 5 in an atmosphere of hydrogen for 90 minutes.
The catalyst is filtered and the filtrate stirred with 150 mg of phenylacetic acid, 16~ mg of N-hydroxybenzotriazole and 247 mg of dicyclo}~xyl-carbodiimide for 7.5 hours. The solvent is removed 10 in vacuo and the residue is dissol~ed in 2Q ml of acetone and filtered. The filtrate is treated with 25 ml o~ Q.Q44 M potassium iodide in acetone.
Dilution with an equal volume of ether gives a solid (330 mg~ which is applied to a 5Q ml HP-2Q
15 column in 20 ml of O.Q5 M monobasic potassium phosphate. Elution with 2QQ ml of water followed by 1:9 acetone-water giYes Rydon positive material in fractions (~50 ml) 6-10. Evaporation of fractions 7-9 gives 81 mg of solid. Recrystallization from 20 acetonitrile-water gives 46 mg of the title compound, which decomposes at >205C. A second crop (6 mg) is obtained from the filtrate. A further 5 mg is obtA; n~ from fractions 6 and 10 by e~raporation and recrystallization.
Analysis calc'd for C12H13N2O5SK: C, 42.84; H, 3.89 N, 8.33; S, 9.53; K, 11.62 Found: C, 42.75; H, 3.82; N, 8.32; S, 9.26 K, 11.63 GC155f EXa~ple lQ2 ~3S-~3~ (z),4~]~-3~ 2-Amino-4-thiazolyl) (methoxyimino]-acetyl] amino~-4-met~y'1-2'-oxo-1-azetidine'sulfohic a-cid, potassium salt s A~ (~S-trans~-4-Meth~1--2-oxo--3-~l(pheny-Lmeth~xyl-carbonyl]amino~ azet'id'inesul'fonic ac'id','-'tetra-butylammonium salt (3S-trans~-4-Methyl-2-oxo-3-~1Cphenylmethox~)-10 carbonyl]amino]-l-aze~ neculfonic acid, potassium salt (352.4 mg; see Example 99~ is dissolYed in 20 ml of water and tetral~utylammonium hy~oyen sulfate (373.5 mgl is added. The aqueous solution is extracted three times with methylene c~loride 15 and the combined extracts are dried over sodium sulfate. After removal of the solvent, 534.6 mg of the title compound is obtained.

B) [3S-[3a(Z),4~]]-3-~(2-Amino-4-thiazolyl) (methoxy-20 imino)acetyl]amino3-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt A solution of 534.6 mg of ~3'S-trans)-4-methyl-2-ox,o-3-~1(phenylmethoxy)carE~onyl]amino]-l-azeti~in~-sulfonic acid, tetrabutylamn~onium salt in 20 ml of 25 dimethylformamide is hydrogenated with 220 mg of 10% palladium on charcoal at atmospheric pressure for 2.75 hours; the uptake of hydrogen is 26.3 ml.
The mixture is filtered and washed twice with 2.5 ml of dimethylformamide. The filtrate and washings 30 (.total ca. 25 ml) are stirred under nitrogen with 161 mg of (Z)-~-(methoxyimino)-2-amino-4-thiazole-acetic acid, 136 mg of N-hydroxybenzotriazole GC155f ~~ -140- 1 3 3 8 6 7 0 and 164.8 mg of dicyclohexylcarbodïimide. The mixture is stirred under nitrogen for about 16 hours. The dimethylformamide is removed in vacuo and the gummy residue is dissolved in acetone and filtered to ~ove urea. To the filtrate is added a solution contA; ni ng 272 mg (0.8 mmole) of perfluorobutanesulfonic acid, potassium salt in a .8 ml of acetone. The - slurry is diluted with an equal Yolume of et~er and filtered to give 325.5 mg of crude product which is purified through c~romatography on 75 ml of EP-20AG. Elution with 400 ml of water and 4ao ml of (9:11 water: acetone mixture (5Q ml fractions) gives 33S mg in fractions 3 to 10.
After trituration with acetone-hexane, 97.3 mg of an analytical sample is obtA;ne~ from fractions 3-5. S;m; 1 ~r trituration of fractions 6-lQ gives an additional 90.4 mg of product as a solid.
Analysis calc'd for CloH12N5O6S2K: C, 29.92; H, 3.01;
N, 17.45; S, 15.97; K, 9.74 Found: C, 30.32; H, 3.49; N, 15.82; S, 13.95;
R, 10.45 NMR(D20) 1.57 (3H, d,)=7), 3.97 (3H,S), 4.30(1H, d of q, )=7.3), 4.70(1H, d. )=7), 6.95ppm(1H,S).

~Amp1e lQ3 I~S-~3a (Z), 4~]-3-l[~2-Amino-4-thiazolyl)~ carboxy-l-methylethoxy)imino~acetyl~amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt A) N-Benzyloxy-t-boc-threonine amide A solution of 8.76 g of t-boc-threonine and the free amine from 6.4 g of D-benzylhydroxylamine HCl (ethyl acetate-sodium bicarbonate liberation) in 100 ml of tetrahydrofuran is treated with 6.12 g GC155f of N-hydroxybenzotriazole and 8.24 g of dicyclo-hexylcarbodiimide in 2Q ml of tetrahydrofuran.
The mixture is stirred under nitrogen for 26 hours, filtered, and evaporated in vacuo.
The residue is chromatographed on a 300 g silica gel column ~elution with chloroform and chloroform-ethyl acetate (3:1~1 yielding 7.2 g of com~ound, Crystallization from ether-h~Y~n~ gives 4.18 g of the title compound.
B) (3S-trans) N ~ loxy-3 L ~tox~carbonylamino-4-methylazetidinone A solution of 12.67 g of N-benzyloxy-t-boc-threo~;ne amide, 11.5 g of triphenylphosphine, and 6.23 ml ofd~thylazodicarboxylate in 380 ml of tetrahydrofuran is stirred under nitrogen for about 16 hours. The solution is evaporated - and chromatographed on a 900 gram silica gel column. Elution with chloroform-ethyl acetate (3:1) gives 13.69 g of compound that crystallizes from ether-hexane to yield 9.18 g of the title compound.

C) (3S-trans~)-3-t-B~toxycarbonylamino~ ydroxy-4-methylazetidinone A solution of 9.18 g of (3S-trans)-N-benzyloxy-3-t-butoxycarbonylamino-4-methylazetidinone in 300 ml of 95% ethanol is stirred in an atmosphere of hydrogen with 1.85 g of 10% palladium on charcoal.
After 141 minutes the slurry is filtered and evaporated in vacuo. The residue is recrystallized from ether-hexane to yield 5.12 g of the title compound.

GC155f D) (3S-trans~-3-t-ButoxycarbonylAmino-4-~ethyl-azetidinone A solution of 4.98 of (3S-trans~-3-t-butoxy-carbonylamino-l-hydroxy-4-methylazetidinone in 2QQ ml of methanol is treated with 132 ml of 4.5 M ammonium acetate and then 66 ml of l.S M titanium trichIoride and stirred for 4.5 hours. m e aqueous solution is diluted with an equal volume of 8% sodium chloride and extracted with ethyl acetate to give 3.48 g of crude product. Recrystallization from ether-hexane yields 3.3 g of the title compound.

E~ (3s-trans)-3-Benzyloxycarbohylamino-4-meth azet;~ino~e A solution of 3.3 g of ~3S-trans)-3-t-butoxy-carbonylAmino-4-methylazetidinone in 10 ml each of dichloromethane and anisole is cooled to 0C
and 112 ml of trifluoroacetic acid is added. The solution is stirred for 90 minutes and evaporated in vacuo (benzene added and evaporated three times~.
The residue is dissolved in 70 ml of acetone and the solution is adjusted to pH 7 with 5~ sodium bicarbonate solution. A total of 5.33 g of benzyl chloroformate is added over 1 hour at pH 6.5-7.5.
The mixture is stirred for 30 minutes at pH 7, diluted with lOO ml of saturated salt, and extracted with ethyl acetate (three 400 ml portions).
The residue obtained by evaporation is chromatographed on a 1 liter silica gel column. Elution with chloroform-ethyl acetate (4:1) gives 2.19 g of compound. Crystallization from ether-hexane yields 1.125 g of the title compound.

~143~ 6 G&l55f F) (3S-trans)-4 ;~eL~y~1-2-oxo-3-~l(p~en~methoxy~-carbohyl]amino]-l-azetidinesulfonic acid', tetra-butylammonium salt A solution of 600 mg of ~3S-trans~-3-benzyloxycar~onylamino-4-methylazetidinone in 2 ml of dimethylformamide is cooled to ~C and 4 ml of 0.8 M sulfur trioxide in dimethylformamide is added. The solution is stirred at room temperature under nitrogen for 1 hour and poured.into 8Q ml of cold 0.5 N monobasic potassium phosphate (adjusted to pH 5.5~. The solution is extracted with three 50 ml portions of methylene chloride (discarded) and 868 mg of tetrabutylammonium bisulfate is added. The resulting solution is extracted with four 75 ml portions of methylene chloride. The combined organic layer is washed with 8% aqueous sodium chloride, dried, and evaporated in vacuo yielding 1.54 g of the title compound.
G~ ~3S-~3a(Z),4~]}3-lI(2-Amino-4-thiazolyl)-[(l-diphenylmethoxycarbonyl-l-methylethoxylimino~-acety~-amino~-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt A solution of 1.54 g of (3S-trans)-4-methyl-2-oxo-3-~I(phenylmethox,y)carbonyl]amino~-l-azetidine-sulfonic acid, tetrabutylammonium salt in 45 ml of dimethylformamide is stirred in an atmosphere of hydrogen with 800 mg of 10% palladium on charcoal for 2 hours. The catalyst is filtered and the filtrate stirred for about 16 hours with GC155f ~144- 1 3 3 8 6 7 0 1.24 g of (Z)-2-amino-~-~(1-diphenylmethoxycarbonyl-1-methylethoxy)i~ino]-4-thiazoleacetic acid, 0.4 g of N-hydroxybenzotriazole, and 580 mg of dicyclo-hexylcarbodiimide. The slurry i8 evaporated in vacuo and the residue is triturated with 20 ml of acetone and filtered. The filtrate ~plus 2 ml of w~shings~ is treated with 868 mg of potassium perfluoro~utanesul~onate in 3 ml of acetone. Dilution wi~h 75 ml of ether gives a solid thatis isolated ~y decantation of the mother liquor, trituration with ether, and filtration to give 0.91 g of the title oompound. The mother liquor is diluted with a further 100 ml of ether to give a second crop, Q.45 g, of the title compound.

H~ [3S-[31(z),4~]-3-I~(2-Amino-4-thiazolyl)~(1-carboxy-l-methylethoxy)imino]-acetyl]amino~-4-methyl-2-oxo-1-azetidinesulfonic acid, dipotassium salt A slurry of 140 mg of ~3S-r3~(z),4~]-3-~1(2-amino-4-thiazolyl)[(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-aze~i~in~sulfonic acid, potassium salt (first crop) in 0.5 ml of anisole is stirred at -12C
under nitrogen and 2.5 ml of cold (-10C) trifluoro-acetic acid is added. After 10 minutes, 10 ml of ether and 5 ml of hexane are added and the resulting slurry is stirred for 5 minutes at -12C, and allowed to warm to room temperature. The solid is isolated by centrifugation and washed twice with ether. A
solution of this solid in 5 ml of cold water is GC155f immediately adjusted to pH S.5 with 0.4 N potassium hydroxide and then applied to an 80 ml HP-20AG
column. Elution with water gives 72 mg of the title compound in fractions (10 ml~ 7-11 after evaporation Cacetonitrile added and evaporated three times~ and trituration with ether,m.p.,-250(dec.).
Analysis calc'd for cl3~lsNso8s2K2 C~ 30-51; H~ 2-gS;
N, 13.6~; S, 12.53; R, 15.28 Found: C, 29.63; H, 3.2Q; N, 12.96; S, 11.~4;
X, 12.78 NMR(D20) 1.46(S, 6H), 1.58(1H, d, J=7) 4.28(1H, d of q, J=7~ 2.5), 4.67 (lH, d, J=2), 6.95ppm(S, lH).
The remaining 1.22 g of I3S-I3a(Z~,4~ 3-~l(2-amino-4 -~h i ~ zolyl 2 I Cl-diphenylmethoxycarbonyl-l-methylethoxy~imino]acetyl]amino-4-methyl-2-oxo-l-azeti~inesulfonic acid, potassium salt ~crops 1 and 2) are treated as above (4.2 ml anisole, 16 ml of trifluoroacetic acid, 13 minutes at 23 -15 C). Chromatography on a 300 ml HP-20AG
column gives 694 mg of the title compound in fractions (60 ml) 6-9 after treatment as above.

Examples 104-133 Following the procedure of Example 11, but substituting the acid listed in column I for (Z)-2-amino--[ I lhydroxy(phenylmethoxy)phosphinyl~-methoxy]imino]-4-thiazoleacetic acid, yields the compound listed in column II.

GC155f -- -146- '1 338670, -- C
~; U , - ~ o 8 ~
.~ ~ , C ~ C . `, ~
~ S ~ ~ q-l 1~ ~ - rA ~ rn ~ ~ ~ S ~ -I ~
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. ~ C~ I . C ~ ,~ _ I ~ ' U C~ I C U
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~ ~ 0 Column I Column II
119. a-1[[3-[(2-furanylmethylene)aminol-2-oxo-1- (3s)-3-tl[[t3-1(2-furanylmethylene)amino]-2-imidazolidinyl]carbonyl]amino]-4-hydLo~y- oxo-l-imidazolldinyl]carbonyl]amino](4-benzeneacetic acid l,~d.vA~hel.yl)acetyl]amino]-2-oxo-l-azet~Ain~ulfonicOacid, potassium saltt meltlng polnt 244 C, dec.
120. (R)-a-[[[3-[[3-(2-furanyl)-2-propenylidene]- ¦3S(R*)]-3-[[[[t3-[[3-(2-furanyl)-2-aminol-2-oxo-1-i ;AA7olidinyl]carbonyl]- ~ ope,.ylidene]amino]-2-oxo-1-i ~AA7olidinyl]-amino]benzeneacetic acid carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetiA;no~ulfonoc acid, potassium 8altt melting point 195 C, dec.
121. (Z)-2-amino-a-[[[(ethylamino)carbonyl]oxy]- [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[[(ethyl-imino]-4-thiazoleacetic acid amino)carbonyl]oxy]imino]acetyl]amino]-2-oxo-l-azet~di n~ l fonic acid, potassium saltt melting point 230 C, dec.
122. (R)-a-[[[2,3-dloxo-4-(phenylmethyl)-1- 13S(R*)]_3{[1[ t 2~3-dioxo-4-(phenylmethyl)-piperazinyl]carbonyl]amino]benzeneacetic acid l-piperazinyl]carbonyl]amino]phenylacetyl]-amino]-2-oxo-1-azetiAin~sulfonic acid potassium saltS melting point 158-159C, dec.
123. (R)-a-[[[4-(1-methylethyl)-2,3-dioxo-1- t3S(R*)]-3-[[[[[4-(1-methylethyl)-2,3-piperazinyl]carbonyl]amino]benzeneacetic acid dioxo-l-piperazinyl]carbonYl]aminO]phenyl- CO ~
acetyl]amino]-2-oxo-1-azetidinesulfonic C~ ~n acid, pOtassium salt~ melting point ~~

GC155f , 1 338670 - U
I ~ N l C
~ C O ~r C C r~
_~ O--I ; r~

H ~ AUJ V N _ ~ 0 H ~ ~ r_A ~A
~ U U~
C O ~ ~O r-~ I ~ N m 0 . ~ c ~ I ~ ~ 8 0 8 . ¦ 1~ , u A , e ~ .~ ,AJ _ ~AJO
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~ N ~ ~
82~ 0 0 25 ~ O
I C C I . I C
6 6 î~ 6 ~ ui ~D t--Column I Column II
128. (R)-a-1[[2-oxo-3-[[(phenylmethoxy)carbonyll- [3S(R*)]-2-oxo-3-t[[[[2-oxo-3-[[(phenyl-aminol-l-imidazolidinyl]carbonyllaminol- methoxy)carbonyllamino]-l-~ l~A ~olidinyl]-benzeneacetic acid carbonyl]amino]phenylacetyl]amino]-l-azet~ne~ulfonic acid, potassium salt;
meltlng polnt 175 & , dec.
129. (Z)-2-amino-a-[(2-amino-1,1-dimethyl-2- [3S(Z)]-3-[[[(2-amino-1,1-dimethyl-2-oxo-oxoethoxy)iminol-4-thiazoleacetic acid ethoxy)-imino]~2-amino-4-thiazolyl)acetyl]-amino]-2-oxo-1-azet1d~n~sulfonic ocid, potassium salt~ melting point 210 C, dec.
130. (R)-a-[[[4-(1-methylethyl)-2,3-dioxo-1- [3S~R*)]-3-[1[[[4-(1-methylethyl)-2,3-piperazinyllcarbonyllamino]-4-hydroxybenzene- dioxo-1-piperazinyl]carbonyl]amino](4-acetic acid l,~dLoAy~henyl)acetyl]amino]-2-oxo-1- I Co azet;~ne~ulfonic aciO, potassium salt; C~
melting point 201-203 C, dec. -J
o 131. (R)-a-[[[3-(1-methylethyl)-2-oxo-1- [3S(R*)]-3-[[[[[3-(1-methylethyl)-2-oxo-1-imidazolidinyl]carbonyllamino]benzeneacetic acid t ~A 7Olidinyl]C~L~ 11amino]phenyl-acetyl]amino]-2-oxo-1-azet~nesulfonic acid, poOtassium salt; melting point 195-200 C, dec.
132. (Z)-2-amino-~-[[2-(diphenylmethoxy)-1-methyl- [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[2- g - 2-oxoethoxy]imino]-4-thiazoleacetic acid (diphenylmethoxy)-1-methyl-2-oxoethoxy]-imino]acetyl]amino]-2-oxo-1-azetidine-sulfonic acidO potassium salt; melting point 145-150 C

$C155f --`152-I CJ
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rl Ul _1 1 338670 GC155f ~153~

Example 134-135 Following the procedure of Example 70, but substituting the compound listed in column I for [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[2-(diphenyl-methoxy)-2-oxoethoxy]imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt, yields the compound listed in column II.

1 3 3 8 6 7 0 ~C155f ~154~

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GC155f Example 136 [3a(Z),4a]-3-[t(~-~mino-4-thiazolyl)(methoxy-imino-)acetyl]amino]-4--methyl--2-oxo-1-azetidine-sulfonic acid, potassium salt A solution of 51.8 mg of (cis)-4-methyl-2-oxo-3-[[(phenylmethoxy)carbonyl]aminol-l-azetidinesulfonic acid, potassium salt and 51 mg of tetra-n-butyl-ammonium bisulfate in 5 ml of water is extracted with methylene chloride (four 10 ml portions) to give 81 mg of oil.
This is stirred in an atmosphere of hyd~o~en for 2 hours with 40 mg of 10% palladium on charcoal in 4 ml of dimethylformamide. The catalyst is filtered and washed with 1 ml of dimethylformamide. The filtrate and washings are combined and stirred for about 16 hours with 31 mg of (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid, 27 mg of N-hydroxybenzo-triazole, and 31.5 mg of dicyclohexyl-carbodiimide. The solution is evaporated in vacuo and the residue triturated with 3 ml of-acetone. The resulting slurry is centrifuged and the liquid treated with 51 mg of potassium perfluorobutanesulfonate. Dilution with 5 ml of ether and filtration gives a solid. Chroma-tography on HP-20 AG (40 ml) gives Rydon positive material in fractions (20 ml) 3-5 (elution with water). Evaporation and ether trituration gives 23 mg of product, as a hygroscopic solid.
Analysis calc'd for CloHl2N5o6s2K
C,29.91; H,3.01; N,17.44 Found: C,29.30; H,3.31; H,16.66 NMR(D20) 1.40(3H, d, )=7), 3.97 (3H,S), 4.46 (lH, apparent pentet, )=7), 5.37(1H, d, )=7), 6.97 ppm(lH,S).

GC155f Exampl-e 137 (3S-cis)-3-Amino-4-methyl-2-oxo-1-azetidinesulfonic acid A) t-Boc-1-allothreonine A suspension of 6.72 g of l-allothr~o~
in 70 ml of 50% aqueous ~ioY~e is treated with 9.45 ml of triet~ylamine and 18.1 g of t-~utyl-pyrocarbonate. The resul~ing mixture isstirred at room temperature for 4 hours, and then diluted with 70 ml of water and 14Q ml of ethyl acetate. After thoroug~ ~h~ ki n~, the layers are separated and the organic layer is washed with 30 ml of 2:1 water:brine. Com~ined aqueous layers are then back-extracted with 70 ml of ethyl acetate. The aqueous layer is cooled in an ice bath and 10% potassium bisulfi~e solution is added to pH 2 3. The acidified 2~ solution is èxtracted with ethyl acetate ~four 1~0 ml portions). Combined organic layers are dried over anhydrous sodium sulfate and stripped of solvent to give 9.13 g of the title compound.

B) N-Methoxy-t-boc-l-allothreonine amide t-Boc-l-allothreonine (9.13 g) is dissolved in 85 ml of water and 41 ml of lN potassium hydroxide solution. Methoxyamine hydrochloride (5.22 g) and 8.67 g of 1-ethyl-3,3-(dimethyl-aminopropyl)carbodiimide HCl are added. The GC155f mixture i5 stirred at room temperature for 4 hours and then saturated with sodium potassium tartarate.
The resulting mixture is extracted with ethyl acetate (four 150 ml portions) and the organic layer is dried over anhydrous sodium sulfate and stripped of solvent to give 7.38 g of the title compound as a solid.

C) -C-Methanesulfonyl-N-methoxy-t-boc-l-allothreonine amide N-Methoxy-t-boc-l-allothreonine amide ~7.32 g~
is dissolved in 4Q ml of pyridine and cooled to -20 C under nitrogen. MethAneculfonyl chloride (3 ml~ is added dropwise by syringe over a 5-minute period. The resulting mixture is slowly warmed to 0C and stirred at that temperature for 3 hours. Ethyl acetate (500 ml) is added and the solution washed with 250 ml of ice-cold 3N HCl solution, then 100 ml of 5% NaHCO3 solution.
The ethyl acetate layer was dried over anhydrous sodium sulfate and stripped of solvent to give -8.64 g of the title compound as a white solld.

D) (3S-cis)-3-t-R~ltoYycarbony1~;no-1-meth~xy-4-methylazetidinone O-Methanesulfonyl-N-methoxy-t-boc-l-allo-threonine amide (8.64 g) is dissolved in 530 ml of acetone and 11 g of solid potassium carbonate is added. The mixture is slowly heated to 65C
under nitrogen and stirred at this temperature for one hour. The reaction mixture is then GCl55f -158- l 3 3 8 6 7 0 filtered through Celite and the filter cake is washed with ethyl acetate. The filtrate i5 concentrated and the residue is taken up in 25Q ml of ethyl acetate. The ethyl acetate solution is washed with lO0 ml of lN hydrochloric acid solution and lO0 ml of 5% sodium bicarbonate solution. The ethyl acetate layer is dried over anhydrous sodium sulfate and stripped of solvent to give 6.63 g of crude product.

E~ (3S-cis)-3-t-Butox~carbonylamino-4~m~thyl-azetidi none Sodium (1.35 g2 is dissolved in about 3QQ ml of liquid ~luuonia at -50C and 5.87 g of (~S-cis)-3-t-Butoxycarbo~ylamino-l-metihoxy-4-methylazetidinone in 35 ml tetrahydrofuran is added dropwise via syringe. An additional 10 ml of tetrahydrofuran is used for rinsing. Near the end of the addition about 100 mg of additional sodium is added. The mixture is stirred for five more minutes, then qllenchP~ by adding 3.35 g of solid ammonium chloride in one portion. Ammonia is ~lown off with a nitrogen stream and 250 ml of ethyl acetate is added to the residue. After filtration and washing the solid with ethyl acetate, the combined filtrate is stripped of solvent to give 4.82 g of the title compound.

- -GC155f -F~ (3~-cis~-3-t-Butoxycarbonylamino-4-methyl-2-oxo-1-azetidinesulfonic acid,- tetrabutyl ammonium salt ~3S,4R]-3-t-8utoxycarbonylamino-4-methyl-azetidinone (4.98 g~ is dissolved in 30 ml of dimethylformamide. Pyridine-sulfur trioxide complex 11.9 g is added and the mixture is stirred at room temperature under nitrogen.
After 14 hours ~tirring, an additional 1.8 g of pyridine-sulfur trioxide complex is added and stirring is continued for 8Q hours. The reaction mixture is poured into 70Q ml of Q.5 M
monobasic potassium phosphate solution and washed with methylene chloride (three 30Q ml portions). tetra-n-Butylammonium bisulfate (8.45 g) is added to the aqueous solution and the mixture is extracted with methylene chloride (four 300 ml portions). The combined methylene chloride layers are dried over anhydrous sodium sulfate and stripped of solvent to give 10.76 g of the title compound as a gum.

G) (3S-cis)-3-Amino-4-methyl-2-oxo-1-azetidine-sulfonic acid (3~-cis)-3-t-ButoxycarbonylAmino-4-methyl-2-oxo-1-azetidinesulfonic acid, tetrabutyl ammonium salt (10.76 g) is dissolved in 50 ml of 95-97% formic acid and stirred for 4 hours under nitrogen. A small amount of product from a previous reaction is added as seed and the mixture is stirred for one more hour. The GC155f -l6a-mixture is stored in the freezer for about 16 hDursand the frozen mixture i8 warmed to room temperature and stirred for an additional hour. The solid formed is filtered and washed with methylene chloride to yield ~82 mg of the title compound.
The filtrate is diluted with 1 liter of methylene chloride and kept at -20C for 4 hours. The precipitate that forms is recrystallized from water-methanol-acetone to give an additional 167 mg of title compound.
NMR(D20) 1.63 (3H, d, J=6.5 cps), lR(nujol)1775 cm Example 138 I3s- I~.(Z) ,4~J]-3-Ir2-Amiho-4-thiazolYl)-methoxyimino~acetyl]-amino-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt A solution of 201 mg of (Z)-2-amino-a-~methoxyimino)-4-thiazoleacetic acid and 153 mg of N-hydroxy-benzotriazole monohydrate in 3 ml of dimethylformamide is treated with 206 mg of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 20 minutes under nitrogen and a solution of 180 mg of (3S-cis)-3-amino-4-methyl-2-oxo-1-azetidinesulfonic acid and 0.14 ml of triethylamine in 2 ml of dimethyl-formamide is added (.an additional 1 ml of dimethylformamide is used for rinsing) and the mixture is stirred for about 16 hours. The slurry is evaporated in vacuo, triturated with 12 ml of acetone, centrifuged and the liquid treated with 338 mg of potassium perfluorobutane-sulfonate. Dilution with 10 ml of ether andfiltration gives a solid product which is chroma-tographed on 200 ml of HP-20 resin eluting with 1 338670 GC155f water. Fractions (20 ml each) 18-30 are combined and lyophilized to give 274 mg of the title compound as a hygroscopic solid.
Anal. calc'd. for CloH12N5O6S2 N,17.44 Found: C,30.03; H,3.21; N,17.06 NMR(D20) 1.40 (3H, d, J=6.5)~ 3.98(3H,S), 4.48 lH, d of t, )=6.4~ 5.5), 5.36(1H, d, J=5.5) 6.97 (lH,S).

Example 139 (3S-trans)-3-Amino-4-methyl-2-oxo-1-azetidine-sulfonic a _ A) Threonine, methyl ester, hydrochloride Under an atmosphere of nitrogen, a flask containing 500 ml of methanol is cooled to -5C (ice/brine) and 130 ml (excess) of thionyI chloride is added at such a rate as to maintain the reaction temperature between 0 and 10C. After recooling to -5C, 59.5 g of 1-threonine is added and the mixture is allowed to reach room temperature and stirred for 16 hours. The mixture is concentrated and evacuated at 10 1 torr for 2 hours to yield a vis-cous oil. This material is used directly in the following step.

~ GC~55f ~ -162- 1 338670 B) Threonine amide The crude product from part A is dissolved - in 2.5 1 of methanol and cooled to -5C Cice/brine).
The solution is saturated with ammonia gas, the cooling bath is removed and the sealed vessel is allowed to stand for 3 days. After removing the bulk of the unreacted ammonia via aspirator, 100 g of sodium ~icar~onate and 50 ml of water is added and t~e mixture is stripped to a viscous oil.

C2 Benzyloxycar~onylthréonine amide The crude product from part B (already cont~i n i ng the requisite amount of sodium bicarbonate is diluted to a volume of 1 liter with water. To this rapidly stirring solution, 94 g (88 ml of 90% pure material) of benzyloxy-carbonyl chloride is added as a solution in 80 ml of tetrahydrofuran over a 1 hour period. The reaction mixture is then stirred for an additional 16 hours and extracted with ethyl acetate (one 500 ml portion, two 250 ml portions). The combined extracts are dried over magnesium sulfate and concentrated. The crystalline residue is then dissolved in 250 ml of hot ethyl acetate and 300 ml of hexane is added followed by boiling until a clear solution is reached. Cooling and filtration of the crystalline mass give, after drying, 104 g of the title compound.

GC155f ~ -163- 1 338670 Dl Benzyloxycarbon~lthreonine amide~ mesylate Under an atmosphere of argon, 100 g of benzyloxycarbonylthreonine amide is dissolved in 400 ml of anhydrous pyridine and cooled in an ice/salt bath. To this stirring solution, 36.8 ml (54.5 gl of meth~n~sulfonyl chloride is added over a 15 minute period. After 2 hours of stirring an additional 0.3 equivalents of me~h~nesulfon~l c~Ioride is added. The reaction is then stirred for 1 ~our and poured into a mixture of 1.5 1 of ice and 2 1 of water. The resulting slurry is stirred for about 3Q minutes and filtered. Drying of the crude product at 60C for about 16 hours in a vacuum oven gives 109 g of the title compound.

E) N-Sulfonyl benzyloxycarbonylthreonine amide, o-mesylate, tetrabutylammonium salt A solution of 2-picoline (17.8 ml) in 90 ml of methylene chloride is cooled to -5C
(ice-brine) and chlorosulfonic acid 5.97 ml is added at such a rate as to maintain the internal reaction temperature below 5C. The resulting solution is added via canula, to a suspension of 7.56 g of benzyloxycarbonylthreonine amide, ~-mesylate in 120 ml of methylene chloride. The resulting heterogeneous mixture is refluxed for about 16 hours yielding a clear solution.
The solution is poured into 500 ml of pH 4.5 phosphate buffer (0.5 M) and further diluted with 120 ml of methylene chloride. The separated GC155f ~164~

organic layer is then washed once with lOQ ml of buffer solution and the combined aqueous phases are treated with 10.2 g of tetra-n-butyl-ammonium hydrogensulfate and extracted with methylene chloride ~one 3QQ ml portion and two 150 ml portionsl. After drying the combined organic extracts over sodium sulfate, the solution is concentrated to yield 12.7 g of a foam.

F~ (3S-trans-~-3-Ami;no 4~meth~1-2-oxo--1-azeti~;~e-sulfonic acid A mixture consisting of 5.52 g of potassium carbonate in 20 ml of water and 160 ml of 1,2-dichloroethane is brought to reflux and 15.5 mmole of N-sulfonyl benzyloxycarbonylthreonine amide, O-mesylate, tetrabutylammonium salt is added in 20 ml of 1,2-dichloroethane (20 ml used as a rinse). After refluxing for 30 minutes, the mixture is poured into a separatory funnel, diluted with 50 ml of water and 100 ml of methylene chloride and the phases split. The resulting organic ph.ase is dried over sodium su-lfate and co~c~ntrated to yield crude ~3S-trans)-3-benzyloxycarbonylamino-4-methyl-2-oxo-1-azetidine-sulfonic acid, tetrabutylammonium salt. The crude azetidinone is treated in 250 ml of ethanol with 0.8 g of 5~ palladium on charcoal catalyst and hydrogen is bubbled through the solution.
After 90 minutes the mixture is filtered through Celite with 50 ml of ethanol used as a rinse.

GC155f -165- 1 3 3 8 6 7 o The addition of 1.2 ml of formic acid to this solution causes an immediate precipitation of the title zwitterion which is filtered after stirring for 1 hour to yield, after drying at 10 1 torr for 1 hour, 1.1 g of product. A
second crop of product is obtained upon concentration of the filtrate and addition of more formic acid to give 1.3 g of the title zwitterion.
m.p. ~218dec., [a]D = -41.1(C=l, H2O).
NMR(D20) 1.58(3H, d )=7), 4.80(2H,M).

Examples 140-143 Following the procedure of Example 138, but substituting (3S-trans)-3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid for (3S-cis)-3-amino-4-methyl-2-oxo-1-azetidinesulfonic acid and the acid listed in column I for (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II.

140. (R)-a-[[[3-[(2-furanylmethylenelamino]- [3S-t3d(R*),4~]]-3-[[t[t3-[(2-furanyl-2-oxo-1-imidazolidinyl]-carbonyl~amino]- ~ methylene)amino~-2-oYo-l-~ ;dA7olidinylJ-benzeneacetic acid carbonyl]amin~phenylacetyl~amino~-4-methyl-2-oxo-1-azeti~lnPsul~onOc acid, potassium salt; melting point 213 C, dec.
141. (R)--[[(4-ethyl-2,3-dioxo-1-piperazinyl)- [3S-[3a(R*),4~]~-3-[[[[(4-ethyl-2,3-dioxo-carbonyl]amino]benzeneacetic acid l-piperazinyl)carbonylJaminoJphenylacetyl]-amino]-4-methyl-2-oxo-1-aze~i~ine~ulfonic acid, potassium salt; melting point 177 C,dec.
142. (Z)-2-amino--(hydroxyimino)-4-thiazole- [3S~[3a(Z),4~]-3-[[(2-amino-4-thiazolyl)-acetic acid (hydroxyimino)acetyl]amino]-4-methyl-2-oxo- 1 ~
l-azetidinesulfonic acid, potassium salt; W
melting point 230C. Co 143. (+)-~-[(amïnooxoacetyl)amino]-2-thio- 13S-[3at+)~4~]-3-ltt~aminQnyo7-eLyllamino]-2- ~J
pheneacetic acid thienylacetyl~emino]-4-methyl-2-oxo-1-azetidine-sulfgniC acid, pota~sium salt; melting paint 135 C, dec.

1 33s6~o GC155f Example 144 [3S-[3~(Z),4~]]-3-[[(2-Amino-4-thiazolyl)[tl,l-dimethyl-2-[(4-nitrophenyl)methoxy]-2-oxoethoxy]-imino]acetyl]amino]-4-methyl-2-oxo-1-azetidine-sulfonic acid, potassium salt To a slurry of (3S-trans)-3-amino-4-methyl-2-oxo-1-azeti~;ne-sulfonic acid (0.36 g; see example 139) in dry dimethylformamide (30 ml) under nitrogen at 26C is added triethyl-amine (309 ~1). After about 5 minutes a clear solution is obt~ine~ and (~)-2-amino-a--[~
~ethyl-2-I(4-nitrophenyl)methoxy~-2-oxoethQxy~-imino]-4-thiazoleacetic-acid (Q.816 g~ is added followed by N-hydroxybenzotriazole (0.334 g) and dicyclohexylcarbodiimide (0.453 g). The mixture is stirred for twelve hours at 26C, whereupon the solvent is removed in vacuo, and the residue is triturated with acetone (30 ml).
After stirring five minutes, the solids are removed and the filtrate is treated with potassium perfluorobutane sulfonate (3.680 gj in acetone, (5 ml). Addition of ether (approximately 40 ml) affords a precipitate which is collected and dried in vacuo (1.073 g; second crop 0.066 g;
total of 1.14 g).
Anal. calc'd. for C20H21N6Olo 2 2 H, 3.70; N, 13.41; S, 10.23; K, 6.24 Found: C, 38.30; H, 3.63; N, 13.41; S, 9.88;
K, 5.98 1 3 3 8 6 7 0 GC155f .

Example 145 [ 3a ( Z ), 4~]- 3- [1(2-Amino-4-thiazolyl)[(l-carboxy-l-methylethoxy)iminolacetyl]amino~-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt (1:2) s A) [3a (Z) ,4a]-3-[~2-Amino-4-thiazolyl)[(l-~ nylmethoxycarbonyl-l-methylethoxy)imino]-acetyl~amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt A solu~ion of (cis)-4-methyl-2-oxo-3-[l(phenyl-methoxy)carbonyl~amino]-l-azetidinesulfonic acid, tetrabutylammonium salt (201 mg.; prepared from the correspo~ potassium salt of example 98 as described $n example 136) in 5 ml of dimethyl-form~m;~e is stirred with 90 mg of 10% palladium on calcium carbonate in an atmosphere of hydrogen for 2 hours. The slurry is filtered and the filtrate is stirred for about 16 hours with 146 mg of ~Z)-2-ami~-[1-dipheny~-~PUluxy~L~lJyl~ ~UIyletho~y3imino~-4-thiazoleacetic acid, 73 mg of dicyclohexy~ rl~l;;m;~P

and 51 mg of N-hydroxybenzotriazole under nitrogen.

The slurry is evaporated in vacuo and triturated with 4 ml of acetone. The slurry is filtered and the solid washed twice with 2 ml portions acetone. The filtrate and washings are combined and treated with 113 mg of potassium perfluoro-butanesulfonate. Dilution with 24 ml of ether gives a solid that is isolated by centrifugation and washed three times with ether yielding 186 mg of the title compound.

1 338670 GC155f -B) 13~(Z),4~]-3-[~(2-Amino-4-thiazolyl)~l-carboxy -l-methylethoxy]imino]acetyl]amino]-2-methyl-4-oxo-l-azet;~;nesulfonic acid, potassium salt (1:2) A slurry of 186 mg of [3(Z),4a]-3-1[2-amino-4-thiazolyl)[(l-diphenylmethoxycarbonyl-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt in O.6 ml of distilled anisole is cooled to -12C
and 3.0 ml of distilled trifluoroacetic acid io (at -10C) is added. The solution is stirred for 10 minutes and 12 ml of ether followed by 6 ml of hexane are added. After 5 minutes at -10C and stirring for 15 minutes at ambient temperature, the solid is isolated by centrifugation and washed four times with ether to give 141 mg of material. This is dried in vacuo, powdered, dissolved in 5 ml of cold water and immediately adjusted to pH 5.6 with 0.4 N potassium hydroxide.
The solution is applied to a 100 ml HP-20AG column and eluted with water. Fractions (10 ml) 8-12 are combined and evaporated in vacuo (acetonitrile is added three times and evaporated. The residue is triturated with ether to give 101.7 mg of ~ G~u~t~ as a hygroscopic solid.
Anal. Calc'd for C13N15N5O8S2 N, 13.69; S, 12.53; K, 15.28 Found: C, 30.11; H, 3.26; N, 13.35; S, 12.12;
K, 15.02 GC155f Example 146 [3S-[3a(Z),43]]-3- [[(2-Amino-4-thiazolyl)-~(1-carboxy-l--methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid [3S-[3~(Z),4B]]-3-[[(2-Amino-4-thiazolyl)-[(l-carboxy-l-methylethoxy) imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, dipotassium salt (87.3 mg; see example 103) is dissolved in 1.38 ml of water, cooled to 0C, treated with 0.34 ml of lN hydrochloric acid and the resulting crystals separated by centrifugation.
The wet solid is dissolved in methanol, filtered, concentrated to about 0.5 ml and mi ~re~ with 1 ml of water, giving 55.9 mg of the title compound.

Example 147 ~3S-[3(Z),4~]]-3-[~(2-Amino-4-thiazolyl) ~(1-carboxy-l-methylethoxy)imino]-4-methyl-2-oxo-1-azetidinesulfonic acid, sodium salt A 99.7 mg sample of ~3S-[3~(Z),4~-3-[[(2-amino-4-thiazolyl) [(l-carboxy-l-methyl-ethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid is mixed with 0.207 ml of lN sodium hydroxide and the resulting mixture is gently wanned to dissolve the r~mA;ning solid. Water is removed azeotropically with acetonitrile and the residue is crystallized from a mixture of 0.5 ml of methanol (to dissolve the residue) and 1 ml of acetonitrile, giving 81.8 mg of solid. A second recrystallization from 0.8 ml of methanol gives 47.9 mg, a third from 0.24 ml of methanol and 0.24 ml of absolute ethanol gives 44.8 mg, and - 1 3 3 8 6 7~,~
GC155f a fourth from 0.225 ml of methanol and 0.225 ml of absolute ethanol gives 38.8 mg. The solid is dried at 20C and 0.01 mm of Hg for 18 hours and then equilibrated with atmospheric moisture for 24 hours, giving 40.9 mg of the title compound.

Example 148 [3S-[3a(Z),4B]]-3-[[(2-Amino-4-thiazolyl)~(1-carboxy-l-methylethoxy)imino]-4-methyl-2-oxo-1-azetidinesulfonic acid, disodium salt A 3.00 g sample of ~3S-[3(Z),4~]]-3-[L(2-Amino-4-th;~zolyl)-[(l-carboxy-l-meth ethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, (see exam~le 146), is suspended in 30 ml of water and titrated with lN sodium hydroxide requiring 12.0 ml to give the title disodium salt. The pH is reduced to 6.5 by the addition of a little Dowex 50W-X2 (H+).
The mixture is filtered and the filtrate diluted to 66.3 g with water. A 6.63-g portion is removed for other purposes. The remaining filtrate is lyophilized, giving 2.38 g of solid. Partial equilibration (24 hours) with atmosphereic moisture gives 2.54 g of the title cG.~ul-d.

~Y~rles 149-151 Following the procedure of Example 138, but substituting the compound listed in column I
for (Z)-2-amino--(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II.

Column I Cblumn II

149. (R)-a-[[[2,3-dioxo-4-1[(phenylmethoxy)- ~3S-~3a(R*),4a]~4-[1l2-l(4-methyl-2-oxo-1-carbonyl]amino]-l-piperazinyl]carbonyl]- sulfo-3-azetidinyl)amino]-2-oxo-1-amino]benzeneacetic acid phenylethyl]amino]carbonyll-2,3-dioxo-l-piperazinyl]carbamic acid, phenylmethyO
` ester, potassium 6alt, melting point 191 C,dec.
150. (R)--111[(2-furanylmethylene)amino]-2-oxo- [3S-¦3a(R*),4]~-3-[1[¦[3-¦(2-furanylmethylene)-imidazolidinyl]carbonyl]amino]benzeneacetic amino]-2-oxo-1-~ olidinyl]carbonyl]-acid amino]phenylacetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt 151. (R)-a-[[(4-ethyl-2,3-aioxo-1-piperazinyl)- [3S-[3a(R*),4a~]-3-~[~[l4-ethyl-2,3-dioxo-carbonyl]amino]benzeneacetic acid l-piperazinyl)carbonyl~amino]phenylacetyl~- ~
amino]-4-methyl-2-oxo-1-azeti~ine~ulfonic 1 _, acid, potassium salt ' - - 1 338670 GC155f Example 152 ~3S-13~(Z),4]]-3-I~(2-Amino-4-thiazolyl)~
carboxy-l-methylethoxy)imino]acetyl]amino~-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt (1:2) s A) [3S-13a(Z),4~]-3-~1(2-Amino-4-thiazolyl)-I(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]-acetyl]amino]-4-methyl-2-oxo-1-azet;~;nesulfonic acid, potassium salt A solution of 440 mg of (Z)-2-amino--[(l-c~rhoYy-l-methylethoxy)imino]-4-thiazoleacetic acid and 153 mg of N-hydroxybenzotriazole mono-hydrate in 3 ml of dimethylform~m;~e is treated with 206 mg of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 30 minutes under nitrogen and a solution of 180 mg of (3S-c )-3-amino-4-methyl-2-oxo-1-azetidine-sulfonic acid, (see example 137) and 0.14 ml of triethylamine in 2 ml of dimethylformamide is added (an additional 1 ml of dimethylfor~-m;~e is used for rinsing) and the mixture is stirred for about 16 hours. The slurry is evaporated in vacuo and triturated with 12 ml of acetone.
The slurry is filtered and the solid washed with acetone (two 3 ml portions). The combined filtrate and washings are treated with 338 mg of potassium perfluorobutanesulfonate. Dilution with 30 ml of ether gives a gummy solid, which slowly solidifies. The solid is filtered and washed with ether to give 656 mg of the title compound.

1 338670 GC155f B) [3S- [3~ (Z), 4a] ] -3~ t~(2-Amino-4-thiazolyl)-[(l-carboxy-l-methylethoxy) imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt (1:2) A slurry of 656 mg of [3S-[3~(Z),4]]-3-[[(2-Amino-4-thiazolyl) ~(l-diphenylmethoxycarbonyl-l-methylethoxy) imino] acetyl]aminol-4-methyl-2-oxo-l-azet;~linesulfonic acid, potassium salt in 2.3 ml of distilled anisole is cooled to -12C
and 11.5 ml of trifluoroacetic acid (precooled to -10C) is added. The solution is stirred for 15 minutes and 46 ml of ether followed by 23 ml of hPYAn~ are added. After 5 minutes at -10 C
and stirring for 15 minutes at room temperature, the solid is filtered and washed with ether to give 457 mg of a very hydroscopic gum. This is dissolved in 6 ml of cold water and i~nediately adjusted to pH 5.6 with 0.4N potassium hydroxide solution. The solution is applied to a 200 ml HP-20 resin and eluted with water. Fractions (50 ml each) 7-11 are co~ined and lyophilized to give 239 mg of t~e title compound as a solid.
Anal- Calc'd. for C13H15O8N5S2R2-1/2 H2O C~ 29-99;
H, 3.10; N, 13.45; S, 12.32 Found: C, 29.94; H, 3.30; N, 13.30; S, 11.93 NMR(D20) 1.44 (3H d, )=75), 1.46(6H,S), 4.48(1H,d of t )=75, 5.5), 5.34(1H, d, )=5.5) 6.96ppm(1H,S).

1 33 8 6 70 GC155f Example 153 (i)-3-Amino-4,4-dimethyl-2-oxo-1-azetidinesulfonic acid A) (+)-4,4-Dimethyl-2-oxo-1-azetidine-tertbutyl-diphenylsilane A solution of 40.5 ml of t-butylchlorodiphenyl-silane in 112 ml of dimethylformamide is cooled to 0C. To this is added 22 ml of triethylamine.
A solution of 12.87 g of 4,4-dimethyl-2-azeti~ino~e in 25 ml of dimethylformamide is added dropwise over 10 minutes to the cooled triethylamine solution.
The resulting cloudy solution is stirred for 18 hours at 5C under argcn. This mixture is poured into 400 ml of ice water and extracted with three 150 ml portions of 2:1 ether:ethyl acetate. The combined extracts are washed with four 100 ml portions of 0.5M monobasic potassium phosphate buffer, one 150 ml portion of sodium bicarbonate solution, two 150 ml portions of water and one 150 ml portion of saturated sodium chl~ride solution. The solution is dried over sodium sulfate and concentrated in vacuo to yield 33.03 g of the title compound as a solid.

B) (i)-3-azido-4~4-dimethyl-2-oxo-l-azetidine tertbutyldiphenylsilane A solution of 4.25 ml of 1.6M (in hexane) n-butyllithium and llml of dry tetrahydrofuran is prepared at -50C under argon in a 100 ml three-necked flask. A solution of 0.083 g of triphenylmethane in 1 ml of tetrahydrofuran is 1 33~670 _ ~C155 ~176~

added. The resulting solution is cooled to -60 & , and 1.0 ml of diisopropylamine is added dropwise ~
by syringe. This is stirred for 15 minutes and then cooled to -78C. A solution of 2.3 g of (I)-4,4-dimethyl-2-oxo-1-azetidine-tertbutyl-diphenylsilane in 8 ml of tetrahydrofuran is added slowly by syringe. The resulting solution is stirred for 20 minutes at -78C, during which time heavy precipitation occurs and uniform stirring becomes difficult. A solution of 1.33 g of p-toluenesulfonyl azide in 5 ml of tetrahydrofuran is added dropwise. The resulting mixture is allowed to stir at -78C for 20 minutes, and 2 ml of trimethylsilyl chloride is added dropwise.
The reaction mixture is warmed to ambient temperature and stirred for 1 hour. Then the mixture is cooled to 0C and poured into 150 ml of 0C ethyl acetate.
Enough 0.5 M monobasic potassium phosphate buffer is added to make both the aqueous and organic layers clear. The two layers are separated and the organic layer is washed with three 150 ml portions of 0.5 M monobasic potassium phosphate solution, one 150 ml portion of sodium chloride solution, one 150 ml portion of saturated sodium chloride solution and dried over sodium sulfate.
The solution is concentrated in vacuo to 2.83 g of oil, which upon trituration with hexane yields 1.67 g of the title compound as a solid.

1 3 3 8 6 7 0 GCli5f C) (+)-3-Azido-4,4-dimethyl-2-oxo-1-azetidine In a 50 ml three-necked flask, 1.52 g of (+)-3-azido-4,4-dimethyl-2-oxo-1-azetidine-tertbutyldiphenylsilane is dissolved in 25 ml of acetonitrile. To this stirred solution is added 0.25 ml of 48% hydrofluoric acid. This is stirred at ambient temperature, and 0.5 ml portions of 48% hydrofluoric acid are added every 60 minutec until, after 6.5 hours, a total of 3.25 ml of 48% hydrofluoric acid has been added.
The reaction mixture is then cooled to 0C, neutralized with saturated sodium bicarbonate, and extracted with 120 ml of ethyl acetate. The organic layer is then washed with 100 ml of water, 100 ml of saturated sodium chloride solution, and dried over sodium sulfate. The dry solution is concentrated in vacuo to yield 1.34 g of oil.
This impure oil is chromatographed on 27 g of silica gel with hexane, followed by 33% ethyl acetate in hexane, to yield 0.358 g of the title compound as a solid.

D) (+)-3-Azido-4,4-dimethyl-2-oxo-1-azetidine-sulfonic acid, tetrabutylammonium salt To 0.100 g of (+)-3-azido-4,4-dimethyl-2-oxo-l-azetidine at 0C is added under argon 2.8 ml of 0.5M dimethylformamide-sulfur trioxide complex. This mixture is allowed to warm to ambient temperature and stir for 45 minutes.
The solution is then poured into 20 ml of 0.5 M
pH 5.5 monobasic potassium phosphate buffer.

GC155f This is washed with three 20 ml portions of methylene chloride (~discarded~ and Q.237 g of tetrabutylammonium hydrogen sulfate is added to the aqueous solution. This was extracted with four 20 ml portions of methylene chloride and the combined organic extracts are washed with 20 ml of 8% sodium chloride solution. The methylene chloride solution is dried Csodium sulfate) and co~centrated in vacuo to yield 0.31 g of oil, which appeared by nmr to be 50~ dimethyl-formamide and 50% of the title compound.

E2 (+)-3-Amino-4,4-dimethyl-2-oxo-1-azetidi-ne-sulfonic acid A solution of 0.155 g of (+~-3-azido-4,4-dimethyl-2-oxo-1-azetidinesulfonic acid, tetra-butylammonium in 0.6 m 1 of methanol is hydrogenated over 10% palladium on charcoal for 20 minutes at 1 atmosphere. The catalyst is filtered off and rinsed with methylene chloride which is combined with the methanol solution. This clear solution is treated with 0.123 ml 97% formic acid. Upon addition of the acid the solution immediately becomes cloudy. After s~An~i~g for 1 hour at 5C, the solid is filtered off to yield 0.0664 g of the title compound, m.p. 200-202C(dec.).
NMR(D20) 1.64(3H,S), 1.68(3H,S), 4.42(1H,S), IR(KBr) 1765cm 1 338670 GC155f Example 154 [3+(Z)3-3-1[(2-Amino-4-thiazolyl)(methoxyimino)-acetyl]aminol-4,4-dimethyl-2-oxo-1-azeti~;neculfonic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate (~0 mg) and (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid (0.323 mmole) in 0.5 ml of dimethylformamide is treated with 67 mg of dicyclohexylcarbodiimide under argon at ambient temperature. The resulting mixture is stirred for 1 hour at which time (i)-3-amino-4,4-dLmethyl-2-oxo-1-azetidinesulfonic acid (57 mg; see exam~le 153) is added as a solid followed by triethy~ e dropwise (0.05 ml).
The reaction is stirred at ambient t~mperature for 16 hours. The dimethylformamide is removed under high vacuum at 30C, and the residue is slurried in 4 ml of acetone and filtered. The filter cake is washed with an additional 4 ml of acetone, and potassium perfluorobutanesulfonate (85 mg) is added to the filtrate, followed by ether. Trituration of the resulting gum with ether gives 40 mg of a tan solid which is chromato-graphed on a 70 ml HP-2QAG column. Elution with 2S water gives 20 mg of the title compound in fractions (5 ml) 16-40 after evaporation, trituration with 1:1 acetone-hexane and drying.
m.p. 225(dec).
Anal. Calc'd. for CllH14N5O6S2 K
N, 16.86; S, 15.43 Found: C, 29.47; H, 3.48: N 14.98 - 1 338670 GC155f Example 155 (+)-4,4-Dimethyl-2-oxo-3-[(phenylacetyl)amino]-l-azetidinesulfonic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate (45 mg) and phenylacetic acid (40 mg) in 0.5 ml of dimethylforr-m;~ is treated with dicyclohexyl-carbodiimide (61 mg) under argon at ambient temperature. The resulting mixture is stirred for 1 hour and (+)-3-amino-4,4-dimethyl-2-oxo-l-aze~i~ineculfonic acid, (52 mg; see example 153) is added as a solid, followed by triethylA~ine dropwise (0.04 ml). The reaction is stirred at ambient temperature for 24 hours.
The dimethylformamide is removed under high vacuum at 30C and the residue is slurried in acetone and filtered. Potassium perfluorobutane-sulfonate is added to the filtrate, ether~ is added and the mixture is cooled. The resulting solid is washed with acetone, hexane, and dried yielding the title compound as a powder.
Anal. calc d. for C13H15N205SK: C, 44.55; H, 4.32;
N, 8.00; S, 9.15; K, 11.16 Found: C, 43.83; H, 4.16; N, 7.96; S, 8.76;
K, 11.43 NMR(D20) 1.33(S,3H), 1.58(S,3H), 3.68(S,3H), 4.70 (S,lH), 7.56ppm (broad S, 5H).
EXample 156 (3S-trans)-3-~[(2-Amino-4-thiazolyl)oxoacetyl]-amino~-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt To a solution of diphenylphosphinyl chloride (1.85 g) in dry dimethylformamide (15 ml) cooled in an ice-methanol bath (-15 ~o -20C) is added (2-amino-4-thiazolyl)glyoxylic acid, triethylamine GC155f salt (2.14 g). After stirring for 0.5 hour a solution of (3S-trans)-3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid (1.08 g;
see example 139) and triethylamine (1.92 ml) in dry dimethylformamide (5 ml) is added to the cold mixed anhydride solution and the reaction mixture is stirred at 5C for 24 hours. Solvent is removed in vacuo, the residual dark oil is dissolved in water, and chromatographed on Dowex 50 X 2-400 resin (~ form, 200 ml). Upon elution with water ~15 ml fractions~ the crude product is collected in fractions 13-27 (3.37 g).
Chromatography on EP-20 resin (200 ml~, eluting with water (15 ml fractions), gives the desired product in fractions 18-26. Removal of water in vacuo gi~es the title compound as an amorphous powder.
Anal. Calc'd for CgHgN4O6S2K (372.42): C, 29.02;
H, 2.44; N, 15.04; S, 17.22; K, 10.50 Found: C, 28.87; H, 2.62, N, 14.85; S, 15.09;
K, 10.81 Example 157 [3S(R*)]-3-[1[(Aminoacetyl)amino]phenylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt, trifluoroacetate (1:1) salt The deprotection of r3s(R*)~-3-I~ L(4-methoxyphenyl)methoxy]carbonyl]amino]acetyl]amino]-phenylacetyl]amino]-2-oxo-1-azetidinesulfonic acid potassium salt (see example 127) using trifluoroacetic acid and anisole yields the title compound, melting point 165C, dec.

1 338670 GC155f Example lg8 (3S-tFans)-3-Methoxy-4-methyl-2-oxo-3-'[1(phenyl-methoxy)carbonyl~amino-] l-azetidinesulfonic acid, potassium salt A) (3~-trans)-4-Methyl-3-methoxy-2-oxo-4-[[(phenylmethoxy)carbonyl-~amino~aze~i~ine A solution of 2.5 g (0.0106 mole) of (3R-trans)-4-methyl-2-oxo-3[~(phenylmethoxy)-carbonyl~amino~azeti~ine (prepared from d-threo~in~ in 12.6% yield essentially asdescribed for the racemic cis isomer in Example 98C) in 112 ml of 4% borax in methanol is cooled to 0C and 3.5 ml of t-butyl hypo-chlorite is added. After 20 minutes the solution is poured into 1 liter of cold water and extracted with two 750 ml portions of cold ethyl acetate. The organic layer is washed with cold water (two 750 ml portions), saturated salt, dried and evaporated to give 3.05 g of crude N-,N'-dichloroamide.
A solution of 426 mg of lithium methoxide in 20 ml of dry methanol is cooled to -78C
and diluted with 40 ml of dry tetrahydrofuran.
Over 30 seconds a solution of the above chloro-amide in 20 ml of tetrahydrofuran (-78C) are added via syringe. After 20 minutes at -78C, 2 ml each of acetic acid and trimethyl phosphite are added. After 40 minutes at room temperature the solution is poured into 500 ml of water and extracted with ethyl acetate (two 300 ml portions). The organic layer is washed with GC155f water, dried, and evaporated to give an oil.
Chromatography on a 200 ml silica gel column eluting with 3:1 chloroform-ethyl acetate gives a total of 1.25 g of the title compound.
B) (3S-trans)-3-Methoxy-4-methyl-2-oxo-3-~l(phenylmethoxy)carbonyl]amino-1-azetidinesulfonic acid, potassium salt A solution of sao mg (0.00303 mole) of (3S-trans)-4-methyl-3-methoxy-2-oxo-4- I [ (phenyl-methoxy)carbonyl]amino]azetidine in 2 ml of dimethyl-formamide is cooled to 0C and 4 ml of dimethyl-formamide-sulfur trioxide complex is added. After 1 hour at 0C and 4 hours at room temperature the solution is poured into 80 ml of 0.5 M monobasic potassium phosphate (adjusted to pH 5.5) and extracted with methylene chloride (two 50 ml portions, discard). The aqueous layer is treated with 1.04 g of tetrabutyl ammonium sulfate and extracted with dichloromethane to give 1.42 g of oil. This is dissolved in acetone and treated with 1.04 g of potassium perfluorobutanesulfonate in 10 ml of acetone. Dilution with 250 ml of ether and extensive trituration of the oily solid gives 584 mg of crude product. Chromatography on HP-20 AG
(200 ml) gives 418 mg of purified product in fractions (100 ml) 13-16 (elution with 1 liter of water and then 9:1 water-acetone). Trituration of 114 mg of this material with ether gives 104 mg of an analytical sample.
Analysis calc'd for C13H14N2O7SK H2O: C, 39.06;
H, 4.04; N, 7.01; S, 8.03; K, 9.78 Found: C, 38.91; H, 3.62; N, 6.91; S, 8.06;
K, 9.51 NMR(D20) 1.33(3H, d, )=7), 3.46(3H,S), 4.22(2H, d of d, ) =6), 5.18 (2H,S), 7.43ppm (5H,S) .

GC155f Exa~ple l59 (3S-trans)-3 ~clhoxy-4-methyl-2-oxo-3-~(phenylacetyl)-amino]-l-azetidines~lfonic acid, potassium sa-lt (3S-trans)-3-am~no-3 ~U~y 4 1~Ll~yl-2-X~l~
5 azetidinesulfonic acid, tetrabutylammonium salt is prepared by the catalytic hydrogenation of (3S-trans)-3-methoxy-4-methyl-2-oxo-3-I[(phenyl-methoxy)carbonyU amino~-l-azeti~ineæulfonic acid, potassium salt (see example 158) after conYersion -to lO the tetrabutyla~onium salt. Following the procedure of example 88,hlt u~ ;n~ (3s-trans)-3-arrllno-3 4-methyl-2-oxo-l-azetidinesulfonic acid, tetra-butyla~onium salt and phenylacetyl chloride yields the title co...yound.
Anal. calc'd for Cl3Hl5N2O6SX: C,42.61; H,4.31; N,7.65 Found: C,39.67; H,4.09; N,7.30 r~R (D20) 1.29(BH, d, )=7) 3.45(3H,S), 3.73(2H,S), 4.36 (2H, d of d, )=6), 7.38ppm (5H,S).

Example 160 [3S-13a(Z),4~]]-3- [I(2-Amino-4-thiazolyl) 1[2-(diphenylmethoxy)-2-oxoethoxy]imino]acetyl]-amino]-2-methyl-4-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of example 138, but substituting (Z)-2-amino-a- [12-(diphenylmethoxy)-2-oxoethoxy]imino]-4-thiazoleacetic acid for (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid and first treating the (3S-cis)-3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid, with triethylamine, yields the title compound, melting point 155-160C, dec.

GC15-5f Example 161 13S_~3~(Z),4~]] -3-I[~(Carboxymethoxy) imino] (2-amino-4-"hiazolyl)acetyl-]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, dipotassium salt The deprotection of l3S-~3c~(Z1~4 ~ ~ -3-Il(2-amino-4-thiazolyl)ll2-(diphenylmethoxy)-2-oxoethoxy]-imino]acetyl]amino]-2-methyl-4-oxo-1-azetidine-sulfonic acid, potassium salt (see example 160) using trifluoroacetic acid and anisole yields the title compound, which decomposes a.~.>250C.

Example 162 (S)-3-1[~(2,6-Dichloro-4-pyridinyl) thio~ acetyl]-amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Acylation of (S)-3-amino-2-oxo-1-azetidine-sulfonic acid, tetrabutylammonium salt (see example 6A) with ~(2,6-dichloro-4-pyridinyl)thio]acetic acid, 4-nitrophenyl ester, followed by treatment with potassium perfluorobutane sulfonate, yields the title compound, melting point 212-214C.

Example 163 I3S (R*)] -3-I I ~ I ( 4-~mino-2,3-dioxo-1-piperazinyl~-carbonyl] ami-no]phenylacetyl~amino]-4-methyl-2-25 oxo-l-azetidinesulfonic acid, potassium salt [3S(R*)]-I4-~I[2-~(4-methyl-2-oxo-1-sulfo-3-azetidinyl)amino]-2-oxo-1-phenylethyl]amino] -carbonyl]-2,3-dioxo-1-piperazinyl]carbamic acid, phenylmethyl ester, potassium salt (see example 149) 30 is hydrogenated using gaseous hydrogen and 10%
palladium on charcoal as catalyst, yielding the title compound, melting point 165C, dec.

GC155f _ -186~

Example 164 [3S(Z)]-3-[f(2-Amino-4-thiazolyl)~[(l-carboxy-1-methylethoxy)imino]acetyl]amino]-2-oxo-1-azetidine-sulfonic acid, sodium salt ~1:2) [3S(Z)]-3-~1(2-Amino-4-thiazolyl~[~2-(diphenyl-methoxy)-l,l-dimethyl-2-oxoethoxy]imino~acetyl]amino]-2-oxo-1-azeti~ sulfonic acid, tetrabutylammonium salt (see example 28) is deprotected using trifluoroacetic acid and anisole to yield the title compound, melting point 185C, dec~ after conversion to the disodium salt with aqueous sodium hydroxide and purification on HP-20.
Examples 165-168 Following the procedure of Example 11, but substituting the acid listed in column I for ~Z~-2-amino--~lhydroxy(phenylmethoxy)phosphinyl]-methoxy]imino~-4-thiazoleacetic acid, yields th compound li~ted in column II.

Column I Column II

165. 2,6-dimethoxybenzoic acid (S)-3-[(2,6-dimethoxybenzoyl)amino]-2-oxo-l-azetidinesulfon~c acid, potassium salt;
melting point 180 C, dec.
166. (Z)-2-amino-a-[~4-(diphenyl- [3S(Z)1-2-[1(2-amino-4-thiazolyl)~[4-methoxy-)-4-oxobutoxy]imino]-4- (diphenylmethoxy)-4-oxobutoxy]imino]-thiazoleacetic acid acetyllamino]-2-oxo-1-azetidinesulfonic aciOd, potassium salt; melting point 125-130 C, dec.
167. 2-t~lphenylmethoxy)carbonyl]- (S)-2-oxo-3-~[2-t[~(phenylmethoxy)carbonyl]-amino]methyl]benzoic acid amino]methyl]benzoyl~amino~-l-azetidine-sulfonic acOd, potassium salt; melting point 234.5 C, dec.
168. ~-~[l5-hYdroxy-2-(4-formyl-1- (3S)-3-[~[~5-hydroxy-2-(4-formyl-1-piperazinyl)pyrido[2,3-d]- piperazinyl~pyrido~2,3-d~pyrimidin- 6-pyrimidin-6-yl]carbonyl~amino~- yl]carbonyl]amino]phenylacetyl]amino]-2-benzeneacetic acid oxo-l-azetidinesulfonic aci~, potassium salt; melting point 265-270 C, dec.

oo ~
`J ~n GC155f -188-.

Example 169 (trans~-3-A~ino-4-eth~l-2:-oxo-1-azat;~in~sulfonic acid A) t-Boc-N-methoxy-B-threoethylserinamide threo-D,L-~-Ethylserine (1.33 g) is dissolved in 10 ml of 2N potassium hydroxide and 5 ml of t-butanol. After t~e addition of 2.46 g of di-t-butylpyrocar~onate the Lw3 ~hase mixture is stirred for 4 hours at am~ient temperature.
O-Methylhydroxylammonium chloride (1.25 g) is added and the pH is adjusted to 4 with lN
hydrochloric acid. l-Ethyl-3-(3-dimet~ylamino-propyl)carbodiimide, hydrochloride (1.92 g) is added and the pH is again adjusted to 4. After stirring for 1 hour the reaction mixture is saturated with sodium chloride and extracted with four 50 ml portions of ethyl acetate.
The ethyl acetate extracts are combined and dried over MgSO4. Removal of the solvent in vacuo yields 1 g of the title compound.

B) t-Boc-O-me~Anesulfonyl-N-methoxy-B-threo-propionamide t-Boc-N-methoxy-~-threoethylserinamide (10.5 g) is dissolved in 65 ml of pyridine. Methanesulfonyl chloride (4.65 ml) is added dropwise at 0C.
After stirring for 3 h.ours at ambient temperature, the reaction mixture is poured into 200 g of ~ ice and 300 ml of lN h.ydrochloric acid. The pH
is adjusted to 4 with concentrated hydrochloric acid. After extraction with three 85 ml portions GC155f of ethyl acetate the comh;ned extracts are dried over MgSO4 and concentrated in vacuo. The residue is treated with carbon tetrachloride and concentrated again. Stirring with ether followed by filtration yields 6.9 g of the title compound.

C) (trans~-3-t-Butox-ycar~onylamino-4-eth-yl-1-methoxy-2-azeti-~ino~
Anhydrous potascium car~onate ~4.15 g) and 125 ml of dry acetone are brought to reflux and 3.4 g of t-Boc-Oqmethanesulfonyl-N-methoxy-~-threo-propionamide in 25 ml of acetone is added.
After 1 hour the reaction mixture is cooled and filtered, and t~e filtrate is concentrated in vacuo. The oily residue is stirred with hexane to yield 2.2 g of the title compound.

D) (trans)-3-t= Butoxycarbonylamino-4-ethyl-2-azetidinone (transl-3-t-Butoxycarbonylamino-4-ethyl-1-methoxy-2-azetidinone (3 gl is added to 170 ml of liquid ammonia at -78C under nitrogen and 1.68 g of sodium is added in 5 portions with stirring over a S minute period. Stirring is continued for 30 minutes. Ammonium chloride is then added slowly until the blue color of the reaction mixture disappears. After removal of the ammonia under nitrogen the solid is extracted with two 100 ml portions of ethyl acetate.
Removal of the solvent followed by drying in vacuo yields 2.7 g of the title compound.

GC155f -19Q~
1 33867a E) (trans)-3-t-~uLGx~ca ~.onylam-ino-4 et~ 2--oxo-l-azetidinesulfoni-c acid, tetr-abutylammonium salt To a 2 ml of ab.solute pyridine in 20 ml of dry dichlor~methane is added trimethylsilyl-sulfonyl chloride (~3.7 ml~ in 5 ml of dry dichloromethane. The addition is acoomplished at -30C, under nitrogen, over a 10 minute period. After stirring at ambient temperature for 30 minutes, the flask is evacuated to yield a pyridine-sulfur trioxide complex. (:trans~-3-' t-Butox~rcarbonylamino-4-ethyl-2-azeti~ino~e (2.67 g) and 20 ml of dry pyridine are added to the flask which. is then placed in an oil bath preheated to 90C. After 15 minutes a clear solution is obtained and poured into 200 ml of a lM solution of dibasic potassium phosphate.
After the addition of 27 g of dibasic .potassium phosphate and lQ0 ml of water a clear solution is obtained. The solution is extracted with two 60 ml portions of ethyl acetate. Tetrabutyl-ammonium hydrogensulfate is added to the aqueous layer, and the aqueous solution is extracted with three 100 ml portions of dichloromethane and the combined organic layers are dried over MgSO4. Concentration in vacuo yields 6.9 g of the title compound.

GC155f -191- 133~

F2 (trans)-3-Amino-4-ethyl-2-oxo-l-azetidinesulfonic acid (trans)-3-t-Butoxycarbonylamino-4-ethyl-2-oxo-l-azetidinesulfonic acid, tetrabutylammonium salt (6.75 g2 in 4Q ml of 98% formic acid is stirred for 3 hours at am~ient temperature.
Dichloromethane C60 ml) is added and the mixture is refrigerated for about 16 hours. The resulting precipitate is separated ~y filtration and then dried in vacuo to yield 0..85 g of the title compound, melting point 185C, dec.

Example 170 (.trans,Z)-3-rf(2-Amino-4-thiazolyl)r(l-car~oxy-1-methylethoxy)imino]acetyl]amino]-4-ethyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt A) (trans,Z)-3-I~(2-Amino-4-thiazolyl)[(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]-acetyl]amino]-4-ethyl--2-oxo-1-azetidinesulfonic acid, dipotassium salt (trahs1-3-Amino-4-ethyl-2-oxo-l-azet;~;ne-sulfonic acid (Ø55 g2 and 335 mg of triethylamine are dissolved in 50 ml of dry dimethylformamide.
(Z)-2-Amino-~-r(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]-4-thiazoleacetic acid (1.14 g) is added with stirring at 0C followed by 450 mg of hydroxybenzotriazole and then 0.69 g of dicyclohexylcarbodiimide. After stirring for about 16 hours at 0C the flask is evacuated.
Dry acetone (25 ml) is added to the s~olid with _ GC155f stirring. The mi~ture is filtered and Q.94 g of potassium perfluoro~utanesulfonate is added to the filtrate followed ~y 100 ml of ether.
After standing for 1 hour at 0C the solid is filtered, washed with ether and dried -in vacuo yielding 1.58 g of the title c~mpound.

B) (trans,Zl-3-I-~(2-Amino-4-thiazol~ C1-carboxy-l-methyle-thoxy)imino]acetyl~amino`]-4-ethyl-2-oxo-l-azetidinesulfonic acid,-dipotàssium salt To a suspension of 1.31 g of (transl,Z)-3-[~2-amino-4-thiazolyl~](l-diphenylmethoxycarbonyl-l-methylethox~imino~acetyl~amino]-4-ethyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt in 10 ml of anisole is added 5 ml of trifluoro-acetic acid over a 10 minute period at -15C.
After stirring for 2 hDurs at -10 C a clear solution is obtained. At -30C 80 ml of dry ether is added and the resulting precipitate is filtered and then treated with 5 ml of water.
The pH is adjusted to 5.5 with lN potassium hydroxide at 0C, and the mixture is filtered to remove unconverted starting material. The filtrate is chromatographed on HP-20 with water as eluent. Lyophilization yields 185 mg of the title compound, melting point 160C, dec.

GC155f Exa~ple''171 ~3S(.Z)]-3-~['('2-Amino-4-thiazolyl')[t4-hydroxy-4-oxobutoxy)imino]ace-tyl]amino]-2-oxo-1-azetidine-sulfonic acid, potassium' salt The deprotection of I 3S(Z)]-3-~[(2-amino-4-thiazolyl)[~4-(diphenylmethoxy)-4-oxobutox~]imino]-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Csee exæmple 1661 using trifluoro-acetic acid and anisole yields t~e title compound, melting point ~2Q0C.

EXam'ple 172 ~S2-3-I [2-(Amihomethyl~benzoyl]amino]-2 azetidinesulfohic ac'id','inher salt The deprotection of (S)-2-oxo-3-[[2-~l[(phenyl-methoxy)carbonyl]amino]methyl]benzoyl]amino]-l-azetidinesulfonic acid, potassium salt using hydrogen gas, palladium on charcoal, and hydrochloric acid, yields the title compound, melting point 162-165C.

Example 173 (S)-3-[~2-(4-Formyl-l-piperazinyl)-5-hydroxy-pyrido[2,3-d~pyrimidin-6-yl]carbonyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (see example 6A) is coupled with 2-(4-formyl-1-piperazinyl)-5-hydroxy-6-L(4-nitrophenoxy)carbonyl]pyrido[2,3-d]pyrimidine and treated with potassium perfluorobutanesulfonate in acetone to yield the title compound, melting point 290 C, dec.

GC155f F~ le 174 (3S-trans~ l I (4-Methyl-2-oxo-1-~ul~o-3-azetidiny-1)-amino]carbonyl]benzeneacetic acid', d'ipo'tassium salt (3S-trans)-3-Amino-4-methyl-2-oxo-1-azetidine-sulfonic acid (see example 139) is coupled with ~-(carboxyllbenz~ne~cPtyl chloride and treated with triethyl~mine and potassium perfl~or~utane-sulfonate to yield the title compound, melting point 147 C, dec.
Ex'ampl'e''1'75 ~3S-trans~-3-hmin~-4-~y~lohexyl-2-oxo-1-azetidine-sulfonic acid A) -(t-Butoxycar~onylamino)-~-cyclohexyl--~-hydroxy-threo-propionic acid B-Cyclohexyl-~-amino-B-hydroxy-threo-propionic acid (15 g) is suspended in 150 ml of acetonitrile and 70 ml of water. Triethylamine (17.8 g) is added and the mixture is heated with stirring to 60C. At this temperature a clear solution is ob~ P~ and 21.0 g of di-t-butylpyrocarbonate is added and stirring at 60C is continued for 1.5 hours. The solvent is ~ oved in vacuo and 50 ml of water is added. The aqueous layer is extracted with ethyl acetate at a pH of 2, which is adjusted by addition of 3N HCl. The organic layer is separated, dried over Na2SO4 and evaporated to dryness. The remaining crystalline material is filtered with petrol ether, yielding 20.4 g of the title compound, melting point 113-115C.

_ GC155f B~ -(t-Butoxycarhony-lamino)-B-cyclohexyl-~-hydroxy-N-methoxy--threo-propionamide ~ -(t-Butoxycarbon~l A~; no) -B-CyClohexyl-B-hydroxy-threo-propionic acid (20.2 g) and 7.6 g of O-methylhydroxylamine hydroch.loride are suspended in 350 ml of water and 175 ml of t-butanol. Th~ pH of the mixture is adjusted with potassium carbonate to 4.1-Ethyl-3-~.3-dimethyl-aminoprop~l)carbodiimide C16 .4 gl is added and the pH is maintained at 4 with stirring for 1.5 hours. t-Butanol is removed in vacuo and the remaining aqueous solution is saturated with sodium chloride and extracted twice with. 100 ml portions of ethyl acetate. The organic layers are combined, dried with Na2SO4 and evaporated to dryness. The remaining crystals are filtered off with petrol ether yielding 18.6 g of the title compound, melting point 125-127C.

C) a-(t-Butoxycarbonylamino)-~-cyclohexyl-~-(methanesulfonyloxy)-N-methoxy-threo-propionamide a- (t-Butoxycarbonyl ~m; no) -B-cyclohexyl-B
hydroxy-N-methoxy-threo-propionamide (18.3 g) is dissolved with stirring in 100 ml of dry pyridine. The solution is cooled with stirring to 0 C and 9.3 g of methanesulfonyl chloride is dropped in. After one hour at 0C an additional 3.3 g of methanesulfonyl chloride is added and stirring is continued for one more hour. The solution is poured into 300 ml of ice water, 1 33867~
-GC155f 2Q0 ml of ethyl acetate is added and the pH is adjusted to 3 with dilute sulfuric acid. The organic layer is separated, dried with Na2SO4 and the solvent is removed ln vacuo. The - 5 remaining solid is collected with petrol ether yielding 1~.0 g of the title compound, melting point 150-152C.

D) [3S-trans]-3~ Bu-toxycarbonyl~minol-4-cyclohexyl~ methoxy-'2-azetid'inone ~-(t-Butoxycarbonylamino)-~-cyclohexyl-B-(methanesulfonyloxy)-N-methoxy-threo-propionamide (18.7 g) is dissolved in 50Q ml of dry acetone.
Potassium carbonate (9.8 g) is added and the suspension is heated to reflux temperature with stirring for 5 hours. The insoluble inorganic material is filtered off and the solvent removed _ vacuo and the remaining oil is dissolved in 30 ml of ethyl acetate. Upon the addition of petrol ether, the title compound precipitates and is filtered off (12.9 g), melting point 110-112C.

E) ~3S-trans]-3-('t-Butoxycarbonylamino)-4-cyclohexyl-2-azetidinone ~3S-trans]-3-(t-Butoxycarbonylamino)-4-cyclohexyl-l-methoxy-2-azetidinone (1 g) is added to 50 ml of liquid ammonia with stirring.
Sodium (0.154 g) is added in 5 to 6 portions within 5 minutes. After this time an additional _ GC155f amount of 0.025 g sodium is added and stirring - is continued for 5 minutes. Ammonium chloride (Q.89 ~ is added and t~ ammonia i5 removed.
The residue is extracted with warm ethyl acetate.
The organic extract is evaporated to dr~ness and the remaining crystals of the title compound are filtered with petrol et~er, yielding Q.5 g, melting Point 13Q-132C.

F) ~3S-transl-3-(t-Bu'toXyc'ar~oh'yl'aminO'1-4-cyclohexyl-2-oxo-1-azetidinesulfohic ac'id, pyridine salt ~ 3S-trans]-3-(_-Butoxycarbonyl ~mi no~ -4-cyclohexyl-2-azetidinone (5.3) is dissolved in 20 ml of methylene chloride and 80 ml of dimethylformamide. After the addition of 60 mmole of pyridine-sulfur trioxide complex the solution is stirred for ~ hours at room temperature. Removal of the solvent in vacuo yield 11.3 g of the title compound as an oil.

G) [3S-trans]-3-(t-Butoxycarbonyl-amino~-4-cyclohexyl--2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt L3s-trans]-3-(t-Butoxycarbonylamino)-4 cyclohexyl-2-oxo-1-azetidinesulfonic acid, pyridine salt (11.3 g) is dissolved in 250 ml of water. Tetrabutylammonium hydrogensulfate (9.0 g) is added with stirring and the pH is adjusted to 6.5 with lN potassium hydroxide.
The aqueous solution is extracted twice with GC155f 2Q0 ml portions of methylene c~loride. The organic portions are dried with Na2SO4, filtered and the solvent is distilled off, yielding 8 g of the title c~.~o~l.d, melting point 135-138C.

H) ~3S-trans~-3-Amino-4-cyclohexyl-2-oxo~1-azetidinesulfonic acid 13S-trans]-3-(t-Butoxycarbonylaminol-4-cyclohexyl-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (3.8 g) is stirred in 20 ml of formic acid for 3 hours, followed ~y the addition of 2~ ml of methylene chloride.
The precipitated title compound (1.0 g) is filtered off, melting point 217-219C.

Exam~le 176 [3S-~3(Z),4B]]-3-[[(2-Amino-4-thiazolyl)-(methoxyimino)acetyl]amino]-4-cyclohexyl-2-oxo-l-azetidinesulfonic acid, potassium salt ~3S-transJ-3-Amino-4-cyclohexyl-2-oxo-l-azetidinesulfonic acid (0.25 g) is dissolved in 30 ml of dry dimethylformamide and 0.12 g of triethylamine with stirring. When a clear solution is obtained, (Z)-2-amino-~-(methoxy-imino)-4-thiazoleacetic acid (0.2 g), 0.16 g of hydroxybenzotriazole and 0.42 g of dicyclo-hexylcarbodiimide are added. Stirring is continued for 48 hours at ambient temperature.
The precipitated urea is filtered off and the solvent is removed in vacuo. The residue is _ GC155f dissolved in 10 ml of acetone and Q.41 g of potassium perfluoro~utanesulfonate is added.
After the addition of 50 ml of ether, the title compound precipitates and is filtered.
Column chromatograph~ using HP-20 and water~
acetone (9:11 as eluent, yields ~.36 g of product, melting point 200-2Q5C Cafter lyophilization~.

EXample' 177 [3S-[3~(Z)~,4B~-3-[I~2-Amino-4-thiazol~l~ I (l-carboxy-l-methylethoxyJimino]acetyl~amino~-4-cyclohexyl-2-oxo-1-a'zetidinesulfonic'acid, dipotassium salt A) [3S-~3~(Z),4~]]-3-~[(2-Amino-4-thiazolyl)[(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]-acetyl]amino]-4-cyclohexyl-2-oxo-1-azetidinesulfonic acid, potassium salt [3S-trans]-3-Amino-4-cyclohexyl-2-oxo-1-azetidinesulfonic acid (0.2 g; see example 175) is dissolved in 30 ml of dimethylformamide and 0.09 g of triethylamine with stirring. Hydroxy-benzotriazole (0.12 g), 0.30 g of (Z)-2-amino-~-l(l-diphenylmethoxycarbonyl-l-methylethoxy)imino]-4-thiazoleacetic acid and 0.33 g of dicyclohexyl-carbodiimide are added and stirring at ambient temperature is continued for 12 hours. The precipitated urea is filtered off and the mother liquor is evaporated to dryness. The remaining oil is dissolved in 5 ml acetone, treated with 0.3 g of potassium perfluorobutanesulfonate and - ' GC155f poured into lQ0 ml of ether wit~ stirring.
~3S-~3CZ~,4~]]-3-~(2-Amino-4-thiazolyl~-~(l-diphenylmethoxycar~onyl-l-methylethoxy2-imino]acetyl]amino]-4-cyclohexyl-2-oxo-1-azetidine-sulfonic acid, potassium salt ~0.61 g~ precipitatesand is filtered off.

B) l3S-[3~(.Z~,4~-3-~C2-Amino-4-thiaZolyl)t(l-carboxy-l-meth.yleth~xy~i~ino~acetylJamino~-4-cyclohexyl-2-oxo-1-azetidine'sul'fo'nic''acid, dipotassium salt [3S-13a~Z),4B]]-3-[ I (2-Amino-4-thiazolyl)-[(l-diphenylmethoxycar~onyl-l-methylethoxy~imino]-acetyl]amino]-4-cyclohexyl-2-oxo-1-azetidinesulfonic acid, potassium salt (0.61 g) is suspended in 6.ml of anisole, cooled to -15C and 5 ml of trifluoroacetic acid is dropped in with stirring.
The temperature is maintained for one hour and then lowered' to -30C. About 100 ml of dry ether is added at such a rate, that the temperature does not exceed -10C. The precipitated compound is filtered off and chromatographed using HP-20 resin and water~acetone (9:lJ as eluent, yielding 0.3 g of the title compound, melting point 11-12QC, dec. (after lyophilization).

-- GCl55f .. -201-Example 178 13S-~3~,4~]-4-Cyclohexyl-3-I[I I 13- ~ C'2-furanyl-methylene~amino]-2-oxo-l-imidazolidinyl'~car~onyl~-amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfohic acid, potassium salt 3S-trans~-3-Amino-4-cyclohexyl-2-o azetidinesulfonic acid (O.l g; see example 175 is dissolved in a mîxture of 3~ ml of dry dimethylformamide and 0.~5 g of triethylamine with stirring. I 1 I ~C2-Furanylmethylene)amino]-2-oxo-l-imidazolidinyl]carbonyl~amino~phenylacetic acid (.Q.14 g), 0.06 g hydroxybenzotriazole and 0.17 g of dicyclohexylcarbodiimide are added and the solution is stirred for 5 days at room temperature. The solvent is removed in vacuo, the residue is dissolved in lO ml of acetone and the precipitated urea is filtered off.
The mother liquor is agitated with 0.15 g of potassium perfluorobutanesulfonate and diluted with 50 ml of ether. The precipitate is filtered, and chromatographed with HP-20 resin using water/acetone (9:l) as eluent yielding 0.14 g of product, melting point 195-200C, dec.
(after lyophiliaztion).

GC155f EXample 17~
. ~ 3S - 13 a. (R* 2 34.~ ] ] -4 ~ Q~ P~yl -3~ 3 -. ~4.-.e.thyl.-2., .3 -dioxo-l-piperaz`inyl'~-1.'3'-dioxo-2-phenylpropyl]-amino]--2-oxo--1-azet;dinesulfonic 'ac'id','potassium salt ~3S-trans]-3-Amino-4-cyclohexyl-2-oxo-1-- azeti~inesulfonic acid (0.1 g; see example 175) is dissol~ed in 30 ml of dimethylformamide and O.5 g of triethylamLne wit~ stirring. (R~--[t(4-ethyl-2~3-dioxo-l-piperazinyl~car~onyl]
amino]~enzeneacetic acid, Q.06 g of hydroxy-benzotriazole and n. 17 g of dicy~lohexylcar~odiimide are added, and the mixture is stirred for a~out 16 hours at room temperature. The solvent is distilled off in vacuo and the remaining oil is dissolved in 10 ml of acetone. The precipitated urea is filtered off and the mother liquor is agitated with 0.15 g of potassium perfluoro-butanesulfonate and diluted with 50 ml of ether.
.The precipitate is filtered off and chromatographed with HP-20 resin, using water/acetone (9:1) as - eluent, yielding 0.15 g of the title compound, melting point 17~180C (after lyophilization~.

EXample 180 (.trans,Z)-3-~(2-Amino-4-thiazolyl)(me-thoxyimino)-acetyl]amino2-4-ethy1-2-oxo-1-azetidinesulf~nic acid, potassium salt Following the procedure of example 170, part A, but substituting (Z)-2-amino-~-(methoxy-imino)-4-thiazoleacetic acid for (z)-2-amino-~-[(l-diphenylmethoxycarbonyl-l-methylethoxy)imino~-4-thiazoleacetic acid, yields the title compound, melting point 190C, dec.

-- GC155f Example 181 (i)-(trans2-3-Amino-2-oxo-4-phenyl-l-azeti~;~e sulfonic acid S A~ (~)-(trans)-2-oxo-4-p~enyl-1-azet;~i~e LerL-- butyldiphenylsilane A solution of tert-~utylchlorodip~enylsilane (20.56 gl in dimethylformamide C45 ml~ is cooled to 0C under argon and treated with triethylamine (10.4 ml2 and then (~-2-oxo-4-phenyl-l-azet;~ine.
After several h~urs at 0C, the resulting mixture is treated with additional trieth~lamine (1 mll and tert-butylchlorodiphenylsilane (2.11 g), and allowed to stir for 65 hours at 5C. The reaction mixture is poured into ice water (300 ml~
and extracted with 3:1 ether-ethyl acetate (three 125 ml portions). The organic extracts are washed with pH 4.5 phosphate buffer (three 50 ml portions), saturated sodium bicar~onate solution (50 ml), water (two 50 ml portions) saturated sodium chloride solution and dried (Na2SO4). Filtration, and concentration in vacuo yields a~solid which is washed with hexane to give after drying (high vacuum) 15 g of the title compound as a solid.

~~ GC155f B) (~ (transl-3-azido-2-oxo-4-phsnyl-1-azetidine-tert-~u*yldiphenylsilane A 50 ml flask equipped with stirring bar, gas inlet, and septum is flame dried under argon and charged with n-butyl lithium ~0.65 ml of a 1.6 N solution in heYAn~) whic~ is cooled to -4QC and dissolved in tetrahydrofuran ~2 ml).
Diisopropylamine ~0.16 mll is added dropwise, the resulting mixture is stirred for 30 minutes and cooled to -78C. A solution of C~-(trans)-2-oxo-4-phenyl-1-azetidine-tert-~utyldiphenylsilane (40Q mg1 in tetrahydrofuran n.5 ml) is added dropwise over a~out 5 minutes. After stirring an additional 20 minutes the solution is treated with p-toluenesulfonyl azide (204 mg) in tetrahydrofuran (0.5 ml). The resulting mixture is stirred 10 minutes at -78C and treated dropwise with chlorotrimethylsilane (0.4 ml). After an additional 10 minutes of stirring, the cooling bath is removed and the reaction mixture is stirred at ambient temperature for 2.5 hours.
Then, while cooling at 0C, ethyl acetate (20 ml) is added followed by pH 4.5 phosphate buffer (8 ml). The organic layer is washed with additional buffer (two 8 ml portions), 5% sodium bicarbonate solution (three 10 ml portions), 50% sodium chloride solution (10 ml)l saturated sodium chloride solution (10 ml) and dried (~a2SO4).
Filtration and concentration ln vacuo yields 500 mg of oil which is flash chromatographed with 5% ethyl acetate-hexane, yielding the title compound (253 m~).

GC155f -205- ~ 338670 Cl L+~-(trans)~3-azi.do-2-oxo-4-phen~l-1-aze*idine A solution of 17 g of crude C+~-(trans)-3-- azido-2-oxo-4-ph.enyl-1-azetidine-tert butyl-diphenylsilane is dissolved in methanol (240 ml) and treated dropwise at ~C with concentrated - HCl ~35 ml~. The cooling bath is removed, the reaction stirred at ~m~ient temperature for 1 hour, and recooled to OC whereupon saturated - sodium bicar~onate solution is added to neutrality.
The resulting mixture i5 extracted Wit~ et~l acetate (one 300 ml portion and four lOQ ml portions) and the organic extracts are washed with 1:1 5% sodium ~icar~onate 50% sodium chloride solution, saturated sodium chloride lS solution, and dried (Na2SO4). Filtration and concentration in vacuo yields;15 g of a heavy oil which is chromatographed on 100 g of silica gel with 20~ ethyl acetate-hexane, yielding 460 mg of the title compound.
D) (.+)-(trans)-3-azido-4-phenyl~ azetidine-sulfonic acid, tetrabutylammonium salt A solution of (+)-(transl-3-azido-2-oxo-4-phenyl-1-azetidine (300 mg) in dimethyl-formamide (3 ml) is cooled to 0C under argon and treated dropwise with a complex of dimethyl-formamide and sulfur trioxide (4.78 ml of a 0.5 M solution in dimethylformamide). The cooling bath is removed, the reaction mixture is stirred at ambient temperature for 2 hours GC155f and poured'into 80 ml of a.5 M monobasic potassium phosphate (pH 5.5~. The 'solution is extracted ' with dichloromethane '(:discarded2 and 541 mg of tetrabutylAm~non;um bisulfate is added. The 5 resulting mixture is extracted with dicElloromethane and the organic extracts are washed with 10% sodium chloride solution and dried CNa2SO41. Filtration and concentration in vacuo affords 8Q0 mg of oil; approximately 4Q% the desired product, 10 the remainder dimethylformamide. This mixture is used without purification in the next step.

E) (~)-(trans)-3--amino-4-phenyl-1-azetidine-sulfonic acid A solution of (+)-(trahs)-3-amino-4-phenyl-l-azetidinesulfonic acid, tetrabutylammonium salt in 4 ml of methanol is hydrogenated over 30 mg of platinum oxide at 1 atmosphere and room temperature. After 15 minutes, the system is 20 evacuated and fresh hydrogen is introduced.
After an additional 45 minutes the reaction is complete, and the system is flashed with nitrogen. After several days at room temperature in dichloromethane-methanol (4:1, 200 ml) 25 catalyst aggregation is complete and filtration is accomplished. The filtrate is concentrated _ vacuo to 18 ml and 0.2 ml of 97~ formic acid is added. /After cooling at 5C for several hours the resulting solid is filtered and 30 washed with dichloromethane to afford, after drying, 150 mg of the title compound as a solid.
Analysis calctd for CgHlgN2O4S: C, 44.62; H, 4.17;
N, 11.57; S, 13.23 Found: C, 43.36; H, 4.31; N, 11.09; S, 13.02 _ ' GC155f Example 182 ~ (trans)-2-~xo-4-phenyl-3-E(:phenylacetyl)amino~-l-azetidihesulfonic' acid, potassium salt Al (+)-trans-2-ox~-4-p~.enyl-3-I(phenylacetyl~amino]-~aze~i~inesulfonic acid tetra~ut~lammonium salt A solution of N-hydroxy~enzotriazole mono-hydrate (52 mg) and phenylacetic acid C46 mg) in dimethylformamide (0.3 ml) is treated with solid dicyclohexylcar~odiimide (~7Q mg~ under argon at 0C. T~e cooling bath. is removed and the resulting mixture stirred at ambient temperature for 1 hour. After dilution~with additional dimethyl-formamide (0.3 ml), (+)-(trans)-3-amino-2-oxo-4-phenyl-l-azetidinesulfonic acid is added as a solid (75 mg, see example 181) followed by triethylamine (0.05 ml) dropwise. The reaction is stirred at ambient temperature for 23 hours, filtered and the filter cake washed with dimethyl-formamide. The filtrate was added to 20 ml0.5 monobasic potassium phosphate (pH 4.5), the mixture washed with three 8 ml portions of ethyl acetate (discard), and 105 mg of tetrabutyl-ammonium bisulfate (0.31 mmole) is added. The resulting mixture is extracted with dichloromethane (three 15 ml portions). The extracts are washed with 10% sodium chloride solution (two 15 ml portions), saturated sodium chloride solution (10 ml) and dried (Na2SO4). Filtration and concentration in vacuo yields (after heating at 32C under high vacuum) 165 mg of oil; approximately 40~ is the desired compound, and 60% dimethylformamide.

GC155f B2 (+)- -trans-2-~xo-4-p~enyl-3-I (~11~ nylacetyl~
amino~-l-azetidinesulfohic acid', p'otassium'salt A solution of ~)-*rans-2-oxo-4-phenyl-3-~(phenylacetyl~amino]-l-azetidinesulfonic acid tetrabutylammonium salt in 1.5 ml acetone is treated with 41 mg (a.121 mmole~ of potassium perfluorobutanesulfonate.~ Dilution with 12 ml of ether affords a glass, whic~ upon trituration with ether affords 43 mg of solid which contains about 20% of an Lmpurity contAining a tetrabutyl-ammonium moiety. The solid is dissol~ed in 5Q% aqueous acetone and passed through a Dowex 50W-X2 ~ ion-~h~nge resin (1 ml).
Removal of solvent yields a solid whïch is washed with acetone, hexane and dried (60C, high vacuum). The yield of the title compound is ]5 mg.
Analysis calc'd for C17H15N2O5S K: C, 51.23; H, 3.80;
N, 7.03; S, 8.05; K, 9.81 Found: C, 50.44; H, 4.20; N, 7.01; S, 7.59;
K, 9.40 Example 183 (i)-(trans,Z)-3-~12-Amino-4-thiazolyl)(methoxyimino)-acetyl]amino]-2-oxo-4-phenyl-1-azetidinesulfonic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate (52 mg) and (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid (69 mg) in dimethylformamide (Q.3 ml) is treated with solid dicyclohexyl-carbodiimide (7Q mg) under argon, at ambient GC155f temperature. The resulting mixture is stirred for 1 hour, ~ (trans)-3-amino-2-oxo-4-phenyl-l-azetidinesulfonic acid (75 mg; see example 1812 is added as a solid, followed by triethyl~mi ne dropwise (0.05 ml~. The reaction is stirred at ambient temperature for 23 hour5 . The dimethyl-formamide is removed under high vacuum at 30C, and the residue triturated wlt~ 2 ml of acetone and filtered. The filter cake is was~ed with additional acetone (two 3 ml portions~ and potassium perfluorobutanesulfonate (86 mg) is added to the filtrate. Dilution with 10 ml of ether produces a gummy solid which is triturated, washed with acetone and hexane to yield, after drying, 82 mg of the title compound as a solid.
Analysis for C15H14N5O6S2 Calc'd: C, 40.26; H, 3.16; N, 15.65; S, 14.33;
K, 8.74 Found: C, 38.60; H, 3.19; N, 15.07; S, 13.87;
K, 7.5 Example 184 (cis)-2-Oxo-4-phenyl-3-l(phenylacetyl)amino]-1-azetidinesulfonic acid, potassium salt A) N-Benzylidene-2,4-dimethoxybenzylamine 12.0 g of 2,4-dimethoxybenzylamine hydrochloride is added to 100 ml of lN sodium hydroxide solution and the mixture is extracted with 125 ml of ethyl acetate. The organic layer is dried over anhydrous sodium sulfate and stripped of solvent to give GCl55f -10.2 g of 2~4-dimethDxybenzy~ `n~ as an oil.
~his amine is dissolved in 150 ml of ~enzene;
6.47 g of benzaldehyde and 0.6 g of ~-toluene-sulfonic acid monohydrate are added. The mixture is heated under reflux removing water wit~ a Dean-Stark 5eparator and in two hours the calculated amount of water Cl .1 ml~ separates out. The mixture is cooled to ro~m temperature.
Upon further cooling thB ~enzene solutîon deposits some precipitate. Benzene is lemoved under reduced pressure and 60 ml of petroleum ether is added to the residue. An oily- layer separates out with more precipitate. Benzene (lO ml) is added to make the layers homogeneous and the remaining precipitate is filtered. The filtrate is stripped of solvent to give 14.2 g of the title compound as an oil.

B) (+)-(cis)-4-Phenyl-1-(2,4-dimethoxybenzyl)-2-oxo-3-azidoazetidine ~ -Azidoacetic acid (1.62 g) is dissolved in 25 ml of methylene chloride under nitrogen.
To this solution are added 3.24 g of triethyl-amine and 1.02 g (4.0 mmole) of the imine N-benzylidene-2,4-dimethoxybenzylamine dissolved in 10 ml of methylene chloride. The mixture is cooled in an ice-bath and 3.36 g o~ trifluoro-acetic anhydride is added slowly; the solution becomes dark colored. After stirring for l hour - 30 in an ice-bath, the mixture is warmed to room temperature and stirred an additional 15 minutes.

- GC155f The solution is then was~ed with ~ater ~60 ml), 5% NaHCO3 solution Ctwo 50 ml portionsl, and lN
HCl solution ~60 ml~. The organic layer is dried over anhydrous sodium ~ulfate and stripped of solvent to give 1.72 g of crude product as dark gum. The gum i5 treated with charcoal several times and the resulting ~rown mixture is chromato-graphed on 40 g silica gel eluting with 1:1 petroleum ether:ethyl acetate. T~e com6ined fractions yield crystal upon quick-freezing in a dry ice-acetone ~ath. Using this as a seed the product i5 recrystallized'from petroleum ether-ethyl acetate to give 817 mg of the title compound as needles which melt upon warming to room temperature.

C) (+7-(cis)-4-Phenyl-2-oxo-3-azidoazetidine (+)-(cls)-4-Phenyl-1-(2,4-dimethoxybenzyl)-2-oxo-3-azidoazetidine (737 mg) is dissolved in 25 ml of acetonitrile and heated to 80 -83C
under nitrogen. To the resulting solution are added over a 1 hour period 943 mg of potassium persulfate,and 570 mg of potassium monohydrogen phosphate, both dissolved in 25 ml of water.
After the addition, the mixture is further heated at 80-83C for 7 hours. The mixutre is cooled and the pH is adjusted to 6-7 by adding solid potassium monohydrogen phosphate. Most of the acetonitrile is removed under reduced pressure and the resulting mixture is extracted with GC155f ~212~

60 ml of chloroform. The chlorofor.m layer is washed with water (60 mll, dried over anhydrous sodium sulfate and stripped of solvent to gi~e a crude product as an oil. Th.e crude product is chromatogrpahed on 40 g of silica gel eluting with 1:1 petroleum ether-ethyl acetate. The combined fractions y-ield crystals and the product is recrystallized from petroleum ether-ethyl acetate to give 267 mg of t~e title compound.
D) (+)-(cis~-4-Pheny1--2-ox~-3-azido l-azetid-ine-sulfonic acid, tetra~utyl ~ onium salt (+)-(cis)-4-Phenyl-2-oxo-3-azidoazetidine (162 mg) is cooled to 0C under nitrogen and 3.5 ml of caD . SM dimethylformamide-sulfur trioxide complex solution in dimethylformamide is added dropwise via syringe. The resulting clear solution is stirred at 0C for 15 minutes. The mixture is then poured into 50 ml of 0.5M
monobasic potassium phosphate solution and washed with methylene chloride (three 50 ml)portions).
tetra-n-Butylammonium bisulfate (292 mg) is added to the aqueous solution and the mixture is extracted with methylene chloride (six 5Q ml portions). The combined methylene chloride layers are dried over anhydrous sodium sulfate and stripped of solvent to give 272 mg of the title compound as a gum.

_ GC155f E) (~-(cis)-2-oxo-4-phenyl-3- I Cphenylacetyl)ami-no~-l-azetidinesulfonic acid, potassium ~alt C~ - (Ci5) -4-Phenyl-2-oxo-3-azido-1-azetidine-sulfonic acid, tetra~utyl ammonium salt ~2~3 mg) is disaolved in 4 ml of ethanol and hydrogenated with 8Q mg of platinum oxide catalyst at one abmosphere. After 1 ~our stirring t~P catalyst iS filtered through a Millipore filter with Celite; some catalyst particles pass throug~ the 1 filter to give a ~lack filtrate. Et~anol is removed under reduced pressure and the residue is dissolved in 4 ml of dLmethylformamide.
N-Hydroxybenzotriazole -m~nohydrate (81 mg~, 78 mg of phenylacetic acid, and 117 mg of dicyclohexylcarbodiimide are added and the mixture is stirred for about 16 hours under nitrogen. The slurry is evaporated in vacuo and triturated with 10 ml of acetone. The resulting slurry is filtered through a Millipore filter with a Celite top and the brown filtrate is treated with 193 mg of potassium perfluoro-butane sulfonate. Upon adding 20 ml of ether a gum separates out. Liquid is removed and the gum is washed with ether. The gum is dissolved in 10 ml of methanol. Upon adding ether, a small amount of precipitate is formed. The mixture is filtered and the colored filtrate is treated with more ether. The precipitate formed is filtered and recrystallized twice from ether-methanol to give 26 mg of the title compound.
Analysis calc'd for C17H1505N2 2 C, 46.99; H, 4.41; N, 6.45 Found: C, 47.24; H, 4.19; N, 6.34 GC155f Example 185 (cis,ZI-3-~I-(-2-Amino-4-t~iazolyl) ~methoxyimino)-acetyl] amino] -2-oxo-4-phenyl-1-a'zetidinesul'fohic acid, potassium salt (cis~-2-Oxo-4-phenyl-3-I(phenylacetyllamino]-l-azetidinesulfonic acid, potassium salt ~560 mg;
see example 184, part DI is d;ssol~ted in 5 ml of etlianol and hydrogenated wi~h llQ mg of platinum oxide catalyst a~ one atmosphere. After one hour stirring the catalyst is filtered through a Millipore filter with Celite; catal~st particles pass through the filter to give a black filtrate.
Ethanol is removed under reduced pressure and the residue is dissolved in 4 ml of dimethyl-formamide. N-Hydroxybenzotriazole monohydrate (168 mg), 221 mg of the (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid and 227 mg of dicyclohexyl-carbodiimide are added and the mixture is stirred for about 16 hours under nitrogen. The slurry is evaporated in vacuo and triturated with 15 ml of acetone. The resulting slurry is filtered through a Millipore with Celite and the filtrate is treated with 372 mg of potassium perfluoro-butane sulfonate. Upon adding 15 ml of ether a gum separates out. Liquid is removed and the gum is washed with ether. The gum is dissolved in 5 ml of water and applied on 150 ml of HP-20 resin eluting with water. Fractions (30 ml each) 16-34 are combined and lyophilized to give 201 mg 30 of the title compound as a solid.
Analysis calc'd for C15H14O6N5S2 / 2 C, 36.73; H, 3.49; N, 14.28; S, 13.07;
K, 7.97 Found: C, 36.65; H, 3.00; N, 13.~9; S, 13.48;
K, 8.30 , GC155f Example '186 (cis)-3-Amino-2--oxo-4-(2-phen~letfienyll-1-azetidinesulfonic acid A~ N-(,3-phenyl-2-prope-nylidene~'-4-me'thoxyaniline ~ -Anisidine a2.32 g) is dissolved in 160 ml of methylene c~loride and 2Q g of anhydrous magnesium sulfate is- added. The mixture is cooled in an ice bat~ and 13.22 g of' trans-cinnamaldehyde is added. The mixture is stirredunder nitrogen for 2 hours and then filtered.
The filtrate is e~aporated to give a solid.
- The crude product is recrystallized from met~ylene chloride-petroleum ether to give 20.96 y of the title compound as a solid.

B) (+)-~cls)-3-Azido-1-(4-Methoxyphenyl)-2-oxo-4-(2-phenylethenyl)azetidine 2-Azi~oacetic acid (24.26 g) is dissolved 20 ih 100 ml of methylene chloride and cooled in an ice bath. To this solution is added 48.57 g of triethylamine and 14.24 g of N-(3-phenyl-2-propenylidene)-4-methoxyaniline dissolved in 250 ml of methylene chloride. To the resulting solution is added 50.41 g of trifluoroacetic anhydride dropwise over a one hour period. After stirring for one hour in an ice bath, tne mixture is warmed to room temperature and stirred for about 16 hours. The mixture is then diluted with 250 ml of methylene chloride and washed _ GC155f with water (750 mll, 5% sodium bicar~onate solution (two 75Q ml portions), and lN HCl solution (75Q ml~. The organic layer is dried over anhydrous sodium sulfate and stripped of solvent to give a solid. The crude product is recrystallized from ethyl acetate to give 11.39 g of the title compound a5 a solia.

C~ (+)--(C1S~-3 - AZidO-2 OAO ~-~2-p~enylet~en~l) azetidine To a solution of 10.22 g of ceric ammonium nitrate in 13 ml of water at 0C is added 1.99 g of (+)-(cis~-3-azido-1-(4-methoxyphenyl)-2-oxo-4-(2-phenylethenyl)azetidine is dissolved in 65 ml of acetonitrile during a 15 minute period (additional 10 ml of acetonitrile is used for rinse). The mixture is stirred for an additional 15 minutes at 0 C, diluted with 750 ml of ethyl acetate, washed with water (six 600 ml portions), dried over anhydrous sodium sulfate, and stripped of solvent to give an oil. The crude product is chromatographed on 90 g of silica gel, eluting first with 250 ml of 30% ethyl acetate/petroleum ether, then 50% ethyl acetate/petroleum ether.
Fractions (50 ml each) 11-16 are combined and evaporated to give 802 mg of the title compound as an oil.

GCl55f -217- ~ 338670 D) (+)-(cis)-3-Azido-2 o~o q-~2-phenylet~enyl)-l-azetidinesulfonic ac:id, tetra-n ~.uLylammonium salt (~)-(cis~-3-Azido-2-oxo-4-(2-phenylethenyl)-azetidine (334 mgl is dissolved in 3 ml of dimethylformamide and 868 mg of pyridine-sulfur trioxide is added. The mixture is stirred at room temperature for 40 hours under nitrogen and then poured into 20~ ml of n.5 M monobasic potassium phosphate solution and was~ed wit~ 30 ml of methylene chloride. tetra-n-Butyl ammonium bisulfate (530 mg~ is added to the aqueous solution and the mixture is extracted with methylene chloride (four 50 ml portions). The combined organic layers are back-washed with water (two lO0 ml portions), dried over anhydrous magnesium sulfate and stripped of solvent to give 824 ma of the title c~mpound as a gum.

E) (+)-(cis)-3-Azido-2-oxo-4-(2-phenylethenyl)-1-azetidinesulfonic acid (+)-(cis)-3-Azido-2-oxo-4-(2-phenylethenyl)-l-azetidinesulfonic acid, tetra-n-butylammonium salt (300 mg) is dissolved in 4 ml of tetrahydrofuran and stirred rapidly. To the mixture is added 600 mg of zinc dust followed by 0.8 ml of lN
monobasic potassium phosphate solution. The mixture is heated to 45C and stirred at this temperature for 3 hours. The mixture is then ~ filtered and the filtrate is taken in 4n ml of methylene chloride and 10 ml of water. The _ GC155f 1 33867~

aqueous layer is further extracted with methylene chloride (three 40 ml portions2 and t~e com~ined methylene chloride layers are stripped of solvent to give 256 mg of a foam. This crude product is dissolved in a small amount of ca. 3~%
acetone~water and applied on 7.5 ml of Dowex CR 2 resin (0.7 meq./vnl~ eluting wlt~ 4a ml of water. The eluent is evaporated to give 151 mg of foam, which is dissolved in 2 ml of water and acîdified to p~ 2 with lN HCl solution.
A small amount of acetonitrile is added to dissolve the precïpitate and the resulting solution is applied on 15 ml of HP-20 resin, eluting with 150 ml of water, then 10% acetone/water. Fractions (15 ml each) 2-13 are combined and evaporated to give 101 mg of the title compound as a foam.

Example 187 (+)-tcis,Z)-3-[[(2-Amino-4-thiazolyl)(methoxyimino)-acetyl]amino]-2-oxo-4-(2-phenylethenyl)-1-azetidine-sulfonic acid, potassium salt A solution of 68 mg of (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid and 51 mg of N-hydroxybenzotriazole monohydrate in 2 ml of dimethylformamide is treated with 69 mg of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 30 minutes under nitrogen.
(cls)-3-Amino-2-oxo-4-(2-phenylethenyl)-1-azetidinesulfonic acid (90 mg; see example 186) and 34 mg of triethylamine are added and the mixture is stirred for 20 hours under nitrogen.

GC155f The slurry is evaporated in' vac~o and triturated with lQ ml of acetone. TEle slurry îs filtered and the filtrate is; treated w~th 113 mg of potassium perfluorobutanesulfonate. Dilution with 5 30 ml of ether and filtration gives 169 mg of a solid product, which is dissolved in a small amount of ca. 109~ acetonitrile/water and applied on 34 ml of ~P-20 resin, eluting with 15Q ml of water, thsn lQ~ acetone~water. Fractions (15 ml each) 16-1~ are cam~iined and stripped of solvent to gi~re llQ mg of the title cG...~ound as a solid.
Analysis calc'd for C17H16O6N5S2K H2O: C, 40.23;
H, 3.57; N, 13.80; S, 12.63; K, 7.70 Found: C, 40.03; H, 3.05; N, 13.61;
S, 12.31; K, 7.56 Example 188 (cis)-3-Amino-4- (methoxycarbonyl)-2-oxo-1-azetidinesulfonic acid A) ~(4-Methoxyphenyl)imino]acetic acid, me'hyl ester A dry 3-necked, 1 liter flask equipped with a nitrogen inlet and stirring bar is charged with 56.88 g of MgSO4 followed by a solution of recrystallized anisidine ('19.43 g) in dichloromethane (250 ml). After cooling to 0 C a solution of methyl glyoxylate hemiacetal (19.92 g) in dichloromethane (250 ml) is added over 1.5 hours.
30 After stirring an additional 20 minutes at 0 C, GC155f , the reaction mixture is suction filtered, dried over sodium sulfate, filtered and concentrated in vacuo to one-quarter volume. ~Y~ne C30~ ml) is added, and t~e solution is concentrated to an oil which semi-solidifies on stAn~ing under high vacuu~ at 5C.

B) (cisl-3-(-1,3-~ihydro-1,3-dioxo-2H-isoind~l-2-yl)-4-methoxycar~onyl-2-oxo-1-t:4-methoxyph.enyl) azetidine A dry 3-necked 500 ml flask equipped with stirring bar, addition funnel, septum and nitrogen inlet is charged with a solution of [(4-Metho~y-phenyl)imino]acetic acid, methyl ester (21.09 g) in dichloromethane (150 ml) and cooled to 0 C.
Triethylamine (19.2 ml) 0.14 mole is added dropwise followed by a solution of (N-phthalimido)-acetyl acid chloride (28.4 g) in dichloromethane (150 ml) over 1 hour. The resulting mixture is stirred for 1.5 hours at 0C, and diluted with 2.5 1 of dichloromethane. The organic solution is washed with pH 4.5 monobasic potassium phosphate (two 500 ml portions), 5~ sodium bicarbonate (two 500 ml portions), saturated sodium chloride solution (500 ml), and dried over sodium sulfate.
Filtration and concentration in vacuo yields a solid which is washed with ethyl acetate, cold acetone and hexane to yield 18.65 g of product.

-. GC155f C) (cisl-4-~Methoxycarbon~1)~ 4-methoxyphenyl)-2-oxo-3-~l(pheny-lmethoxylcarbonyl~amino~azetidine A dry 50Q ml flask equipped with. nitrogen inlet, stirring bar, and septum was charged with 18.65 g of (.cis1-3-(1,3-dioxo-2H-isoindol-2-yl)-4-methoxycar~onyl-2-oxo-1-~4-methoxyphenyl)azet;~;ne and 325 ml of dichloromethane. Th.e res~lting sl-cpension i8 cooled to -3aC, and metAyl hydrazine C3.52 mlI is added dropwise. The reaction is warmed to ac and stirred for l hour.
An additional Q.4 ml of methy-l h~ydrazine is added and the mixture is stirred for 10. minutes. Th.is sequence is repeated with a total of 2.9 equivalents of methyl hydrazine (7.7 ml) has been added.
The solvent was removed in vacuo; 200 ml of fresh dichloromethane is added, and the mixture is again concentrated. This sequence was repeated t~o additional times, the resulting foam . was dried under high volume for 20 minutes, redissolved in 225 ml dichloromethane, and allowed to stand at ambient temperature for about 16 hours during which time a considerable amount of solid precipitates. The mixture is filtered under nitrogen, the filtrate cooled to 0C (nitrogen atmosphere) and treated with diisopropylethyl amine (17 ml) followed by benzyl chloroformate - (7 ml) dropwise. The reaction is stirred at 0C for 30 minutes, then at ambient temperature for 1.5 hours. The mixture is washed with two 300 ml portions of pH 4.5 monobasic potassium phosphate buffer, 5% sodium bicarbonate (two 300 ml portions), saturated sodium chloride (300 ml), ~ GC155f dried (sodium sulfatel, and filtered. Concentration in ~acuo, yields a foam which on tr;turation with ether yields 2.~ g of the title compound as a solid.

D) (cis)-4-(IIe~lG~carbonyl)-2-oxo-3~ henyl-methoxy)carb~nyl]amino~ azetidine A solution of ceric ~wnium nitrate (8.59 g) in 6Q ml of 1:1 acetonitrile-water is treated with a slurry of 2 g (cls~-4-(methoxycarbonyll-1-C4-methoxyphenyll-2-oxo-3-I[(Phenylmethoxy2car~onyl~amino]azetidine in 50 ml acetonitrile over 10 minutes. The reaction mixture is stirred an additional 10 minutes at ambient temperature and diluted with ethyl acetate (100 ml). The separated aqueous layer is washed with ethyl acetate (three 40 ml portions) and the combined organic extracts are washed with 50% sodium bicarbonate (three 70 ml portions).
The basic washings are back-washed with ethyl acetate (50 ml) and the combined organic extracts are washed with aqueous sodium sulfite, 5%
aqueous sodium car~onate (100 ml), 5% sodium chloride solution (two 100 ml portions), saturated sodium chloride (two 50 ml portions), and stirred over Darco G-60 charcoal for 30 minutes. Sodium sulfate is added, and the mixture is filtered and concentrated ln vacuo to yield an oil which on trituration with ether yields 685 mg of the title compound as a solid.

GC155f E) (cis)-4-(Met-hoxyca~ yl~ -2-oXD-3-~ (phenyl-methoxy)car~onyl]amino~ azetidinesulfonic acid, tetrabutylammonium salt A mixture of (cis)-4-(methoxycarbonyl)-2-oxo-3-l~(phenylmethoxy~carbonyl]amino]-1-azetidine (100 mg) and 172 mg of a pyridine-sulfur trioxide complex in 1 ml of pyridine is stirred under argon for 3 hours at 80C. The reaction mixture is poured into 7~ ml of Q.5 M monobasic potassium phosphate (pH 5.51 and extracted wit~ four 30 ml portions of dichloromethane (discard~. Tetrabutyl-ammonium hydrogen sulfate ~122 mg) is added to the aqueous layer which is then extracted with dichloromethane (four 30 ml portions). The organic lS extracts are washed with 8% sodium chloride solution, dried over sodium sulfate and filtered. Concentration in vacuo yields 186 mg of the title compound as a viscous oil.

F) (cis)-3-Amino-4-~methoxycarbonyl)-2-oxo-1-azetidinesulfonic acid A solution of 186 mg of (cis)-4-(methoxy-carbonyl)-2-oxo-3- I ~ (:phenylmethoxy~carbonyl]amino]-l-azetidinesulfonic acid, tetrabutylammonium salt in 2 ml of methanol is hydrogenated over 10%
palladium on charcoal (95 mg) for 1.5 hours at 1 atmosphere. The catalyst is filtered off and rinsed with dichloromethane-and the filtrate is treated with 97% formic acid and cooled to -50 C (the presence of a seed crystal at this staqe is necessary to induce crystallization).
After crystallization commences, the mixture is allowed to stand for about 16 hours at 10C.

- GC155f -224- 1 3 3 ~ 6 7 0 The resulting solid is ~ashed with dichloromethane, hexane, and dried in wacuo, ~ielding 50 mg of the title c~ d.

Example -182 (cis)-3-~2-Amino-4-thiazol~ (diphenyl-methoxycarbo-nyl)-l-methylethoxyJimino]acetyl]-amino]-4-Cmethoxyca-r~onyl)-2-oxo-1-azetidinesulfonic acid, potassium salt A solution of N-hyd~o~y~enzotriazole hydrate (34 mg) and lQl mg of 2-amino-a-IIl-(dip~enyl-methoxycar~onyll-l-methylethoxy~imino~-4-thiazoleacetic acid in 0.5 ml of dimethylform~m;~e is treated with solid dicyclohe~ylcarbodiimide (45 mg) and the mixture is stirred under argon for 45 minutes (ambient temperature). (cls)-3-Amino-4-(methoxycarbonyl)-2-oxo-1-azetidinesulfonic acid (45 mg; see example 188) is then added as a solid followed by triethylamine (0.03 ml) dropwise. The reaction is stirred at ambient temperature for about 16 hours. The dimethyl-formamide is removed under high vacuum at 30C
and the residue is triturated with acetone.
The supernatant is treated with potassium perfluorobutanesulfonate (67 mg). Dilution with ether produces a solid which is washed with ether and dried in vacuo to yield 93 mg of the title compound.

GC155f -225- 13386 a Example l~Q
(cis ) -3- I-I (2-P~nino-4-thi,azo-lyl ~ c~ r~y-l-methylethoxy)imino]acetyl]amino]-4-~methoxy-carbonyl)-2-oxo-1-azetidinesulfonic acid, dipotassium salt A slurry of (cis~-3-~12-Amino-4-thiazolyl)-~ (diphenylmethoxycar~onyl~-l-methylethoxy)-- imino~acetyl]amino~-4-~methoxycar~onyl~-2-oxo-1-azetidinesulfonic acid, potassium salt in n . 4 ml of anisole is stirred at -12C under argon, and 0.9 ml of cold (-10C) trifluoroacetic acid is added. After 1.5 hours, 4 ml of ether and 2 ml of hexane are added and the resulting slurry is stirred for 15 minutes at -10C, then 15 minutes at ambient temperature. The solid is isolated by centrifugation and washed with ether. The pH of a suspension of this material in O.S ml of cold water is adjusted to 6 with lN potassium hydroxide and then applied to a 30 ml HP-20AG column. Elution with water yields 30 mg of the title compound after evaporation (acetonitrile added and evaporated twice).
AnalysiS calc'd for C14H15K2N5O9S2 C~ 31-15;
H, 2.81; N, 12.98 Found: C, 29.08; H, 3.03; N, 12.19 f Example 191 (S)-(trans)-3-Amino-4-ethynyl-2-oxo-1-azetidine-sulfonic acid A) 2-(Trimethylsilyl)ethynylmagnesium bromide To a flame-dried 50 ml flask maintained under positive nitrogen pressure is added 20 ml of dry tetrahydrofuran, 2.20 ml of trimethylsilyl acetylene and 5.05 ml of 3.06 M solution of methylmagnesium bromide in ether. The mixture is stirred for 140 minutes yielding the title compound.

B) (S)-(trans)-4-[2-(trimethylsilyl)ethynyl]-2-oxo-3-[(triphenylmethyl)amino]azetidine To a flame dried 250 ml 3-necked flask is added 6.00 g of (S)-(cls)-4-(methylsulfonyl)-2-oxo-3-[(triphenylmethyl)amino]azetidine. The flask is flushed with nitrogen, and then maintained under positive nitrogen pressure.
After the reaction mixture is cooled in a dry ice/isopropanol bath, 4.65 ml of a 3.06 M
solution of methylmagnesium bromide in ether is added dropwise via syringe with rapid stirring. The solution of 2-(trimethylsilyl)-ethynylmagnesium bromide prepared in part A
is added via a Teflon tube under positive nitrogen pressure (the flask containing the reactant is rinsed with 7 ml of tetrahydrofuran).
When the addition is complete the-cold bath is removed. After 45 minutes, a solution of 3.5 g -- ~-C1~5f -226a-.
of potassium bisulfate in 20 ml of water is added. Most of the tetrahydrofuran is removed on the rotary evaporator. The residue is transferred to a separatory funnel with ether and water. The water layer is separated and extracted twice with ether. The combined ether layers are washed once with saturated aqueous sodium chloride, dried over sodium sulfate, and filtered. Removal of the solvent gives a foam which is chromatographed on a silica column. Elution with 2 liters of dichloromethane, 1 liter of 1% ether/dichloromethane, 2 liters of 2% ether/dichloromethane and 1.5 liters of 10% ether/dichloromethane (fraction 1=1000 ml; fraction 2,3=500 ml; fraction 4-end=250 ml) gives 1.30 g of the title compound in fractions 2-8 and 1.80 g of the corresponding trans-isomer in fractions 12-19. Fractions 9-11 contain 1.19 g of a mixture of cls and trans isomers.
C) (S)-(trans)-4-ethynyl-2-oxo-3-[(triphenyl-meth~yl)amino]azetidine (S)-(trans)-4-[2-(trimethylsilyl)ethynyl]-2-oxo-3-[(triphenylmethyl)amino]azetidine (2.97 g) is dissolved in 30 ml of dichloromethane and 330 mg of tetrabutylammonium fluoride (containing 20-25% water) is added. After 20 minutes, the solvent is removed _ vacuo. The residue is taken up in ethyl acetate and water. The organic layer is separated, washed once with water and once with saturated aqueous sodium chloride, r l _ 5 f -226b-dried over sodium sulfate, and filtered.
Removal of the solvent gives an oil which is stirred for 15 minutes with 60 ml of pentane to afford 2.35 g of the title compound as a powder (after drying in vacuo).

D) (S)-(trans)-3-Amino-4-ethynyl-2-oxo-1-azetidinesulfonic acid (S)-(trans)-4-ethynyl-2-oxo-3-[(triphenyl-methyl)amino]azetidine (404 mg) and 560 mg of a complex of pyridine and sulfur trioxide are added to a 25 ml flask. After the flask is flushed with nitrogen, 4.0 ml of dry pyridine is added and the mixture is heated at 80-85C
for 3 hours. The mixture is added to a rapidly stirred mixture of 4.0 ml of concentrated hydrochloric acid, 50 ml of water, and 50 ml of ethyl acetate. The pH is adjusted to 3.15 with sodium carbonate. The water layer is separated and extracted once with ethyl acetate.
The combined organic layer is washed once with saturated aqueous sodium chloride, dried over sodium sulfate and filtered. Solvent removal ln vacuo gives a foam which is taken up in 10 ml of dichloromethane. Formic acid (98%, 8 ml) is added, and after 15 minutes the mixture is concentrated to 4 ml and 10 ml of dichloromethane is added to give a solid suspended in solution.
Filtration gives 100 ml of the title compound as a solid (obvious discoloration with melting >180C).

GC155f -226c-Example 192 I3S- [3~(Z),4B]]-3-~[(2-~mino-4-thiazolyl)(methoxy-imino)acetyl]amino]-4-ethynyl-2-oxo-1-azetidine-sulfonic acid, potassium salt (Z)-2-Amino-a-tmethoxyimin~o)-4-thiazole-acetic acid (100 mg), 85 mg of N-hydroxybenzo-triazole monohydrate, and 113 mg of dicyclo-hexylcarbodiimide are weighed into a 10 ml flask. The flask is flushed with nitrogen and cooled in an ice-water bath. Then 0.6 ml of dimethylformamide is added and the mixture is stirred for 10 minutes, at which point an additional 0.6 ml of dimethylformamide is added. (S)-(trans)-3-Amino-4-ethynyl-2-oxo-1-azetidinesulfonic acid (95 mg; see example 191) is added as a solid with 1.0 ml of dimethyl-formamide and 56 ~1 of triethylamine. The cold bath is removed and the mixture is stirred for 22 hours. Acetone (3 ml) is added and the solids present are removed by filtration and washed with an additional 4 ml of acetone.
All solvents are removed in vacuo and the residue is taken up in 5 ml of methanol and 162 mg of potassium perfluorobutanesulfonate is added and dissolved. After standing/ a solid is deposited and then isolated by centrifugation to afford 68 mg of the title compound, melting point >230C.

GC155 f -- 26u ~ 338670 Exam le 193 (S)-3-[[[(2,5-Dichlorophenyl)thio]acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt 3-Amino-2-oxo-1-azetidinesulfonic acid (100 mg) is dissolved in dry dimethylformamide (2 ml) with triethylamine (0.083 ml). 2,5-Dichlorophenylthioacetic acid (123 mg) 0.602 mmol, N-hydroxybenzotriazole hydrate (81 mg) and dicyclohexylcarbodiimide (124 mg) are added, the mixture is stirred for 2 hours at room temperature, and then 2 days at 5 C. Solvent is removed in vacuo, the residue is taken up in water, filtered through Celite, and the filtrate is washed with ethyl acetate. The aqueous layer is combined with dichloromethane, tetrabutylammonium bisulfate (612 mg) is added, the pH was raised to 3 with lN potassium hydroxide solution, and after extracting a total of three times with dichloromethane, the combined extracts are dried (Na2SO4), and solvent is removed _ vacuo yielding an oil. A solution of the oil in acetone is added to a solution of potassium perfluorobutanesulfonate (612 mg) in acetone causing precipitation of the product.
After addition of a small amount of ether the solid is collected by filtration, washed several times with acetone, and dried to give a powder (206 mg).
Anal. Calc'd for CllHgN2O5S2C12K:
C, 31.21; H, 2.14; N, 6.62; Cl. 16.75 Found: C, 27.90; H, 2.11; N, 5.84; Cl, 18.04 _ ~ 5C155f ~ 226~-Example 194 (3S-trans)-3-[[[(2,5-Dichlorophenyl)thio]acetyl]-amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt (3S-trans~-3-Amino-4-methyl-2-oxo-1-azetidinesulfonic acid (250 mg; see example 139) is dissolved in dimethylformamide (2 ml) with triethylamine (193 ~1). 2,5-Dichlorophenyl-thioacetic acid (285 mg), N-hydroxybenzotriazole hydrate (213 mg) and dicyclohexylcarbodiimide (287 mg) are added. After stirring for about 16 hours at room temperature, the mixture is filtered and solvent is removed in vacuo. The residue is taken up in water and filtered.
The filtrate is washed with ethyl acetate, layered with dichloromethane and tetrabutyl-ammonium bisulfate (4.2 mmol) is added. After a total of three extractions with dichloromethane, the combined extracts are dried (Na25O4) and solvent is removed ln vacuo yielding an oil (920 mg). To a solution of the oil in acetone is added potassium perfluorobutanesulfonate (946 mg) dissolved in acetone. A solid slowly precipitates, is collected, washed twice with ether, and dried to give a powder (306 mg).
Chromatography on HP-20 resin (100 ml column), eluting with 20% acetonitrile: 80% water, yields the desired product, which crystallizes upon evaporation of a ~Jater:methanol mixture.
Trituration of the residue with acétone gives a powder (233 mg); melting point 212-213(dec.).

C, 32.95; H, 2.54; N, 6.41; Cl, 16.21; S, 14.66 Found: C, 32.91; H, 2.60; N, 6.42; Cl, 16.50;
S, 13.77 - rc~
-226f-1 3386,~o Examples 195-196 Following the procedure of example 138, but substituting (3S-trans)-3-amino-4-methyl-2-oxo-1-azetidinesulfonic acid for (3S-cls)-3-amino-4-methyl-2-oxo-1-azetidinesulfonic acid and the acid listed in column I for (Z)-2-amino-~-(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II.

195. ~RJ-[(aminooxoacetyl)amino](4- [3S-[3~(R*),4~]]-3-~[[
hydroxyphenyl)acetic acid (aminooxoacetyl)amino]-(4-hydroxyphenyl)acetyl]-amino]-4-methyl-2-oxo-1-azetidines~lfonic acid, potassium salt 196. (R)-[(aminooxoacetyl)amino]- [3S-[3~(R*),4B]]-3-[[[-phenylacetic acid (aminooxoacetyl)amino]-phenylacetyl]amino]-4-methyl-2-oxo-1-azetidine-sulfonic acid, potassium salt; melting point 187 C, dec.

f -226g-Example 197 ~3S(R*)~-3-[ L [ (Aminooxoacetyl)amino](4-hydroxy-phenyl)acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure described in Example 28, but substituting (R)-L(aminooxoacetyl)-amino](4-hydroxyphenyl)acetic acid for (Z)-2-amino--~[2-(diphenylmethoxy)-1,1-dimethyl-2-oxoethoxy]-imino]-4-thiazoleacetic acid, yields the title compound melting point 128C, dec.

Example 193 [3S(R*)]-3-~[(2-Amino-4-thiazolyl)[[[3-~(2-furanyl-methylene)amino]-2-oxo-1-imidazolidinyl]carbonyl~-amino]acetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of example 6, but substituting (R)-2-amino-~-~[[3-[(2-furanyl-methylene)amino]-2-oxo-1-imidazolidinyl]carbonyl]-amino]-4-thiazoleacetic acid for aminothiazoleacetic acid yields the title compound, melting point >250C.

GC155f -Example 195 Biological Production of EM5117 9 Liter Fermentation Chromobacterium violaceum SC 11,378 A.T.C.C.
S No. 31532 is maintained on the following sterilized agar medium (A):
Grams Yeast Extract Beef Extract NZ Amine A 2 Glucose 10 Agar 15 Distilled H2O to 1 liter The pH is adjusted to 7.3 before sterilization lS at 121C for 30 minutes.
A loopful of surface growth of the micro-organism is used to inoculate each of three 500 ml Erlenmeyer flasks, each contA;ning 100 ml of the following sterilized medium (B):
Grams Oatmeal 20 Tomato Paste 20 Tap H2O to 1 liter Adjust pH to 7.0 before sterilization at 121 C
25 for 15 minutes.
The flasks are then incubated at 25 C on a rotary shAker (300 rpm; 2 inch stroke~ for approximately 24 hours.
After the appropriate incubation as 30 described above, 1% (vol/vol) transfers are made from the growth culture flasks to one hundred 500 ml Erlenmeyer flasks each containing 100 ml of GC155f the following sterilized medium (C):
Grams Oatmeal 20 Tomato Paste 20 S Glucose 30 Tap H2O to 1 liter me pH is adjusted to 7.0 before sterilization of 121C for 15 minutes.
After inoculation, the flasks are incubated at 25C on a rotary shaker (300 rpm; 2 inch stroke~
for approximately 18-24 hours. At this time the contents of the flasks are pooled and the broth is centrifuged yielding approximately 9 liters of supernatant broth.
250 Liter Fermentation A loopful of surface growth from an agar slant (medium A) of Chromobacterium violaceum SC 11,378 A.T.C.C. No. 31S32 is used to inoculate each of five--500 ml Erlenmeyer flasks each cont~; n; ng 100 ml of sterilized medium (B).
The flasks are then incubated at 25C on a rotary sh~ker (300 rpm; 2 inch stroke) for approximately 24 hours. After the appropriate incubation, as described above, 1% (vol/vol) transfers are made from the grown culture flasks to five 4 liter Erlenmeyer flasks each containing 1.5 liters of sterilized medium B.
After inoculation the flasks are then incubated at 25 C on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After the GC155f -229- 1 3 3 8 6 7~

appropriate incubation as described above, a 1% transfer (vol/vol) is made to an agitator equipped fermentation tank containing 250 liters of sterilized medium (C). After inoculation the fermentation is continued under the following conditions: temperature - 25 C; pressure - 10 psig;
aeration - 10 cfm; agitation - 155 rpm. Ucon is added as needed as antifoam agent. After approximately 18-24 hours the fermentation is completed. The fermentation broth is then adjusted to pH 5.0 using HCl and the broth contents of the tank is centrifuged yielding approximately 230 liters of supernatant broth.

Isolation and Purification The broth supernatant from the 250 liter fermentation is adjusted to pH 5 using sulfuric acid and filtered using 3-5% diatomaceous earth (Celite). The broth filtrate is extracted 20 with two 30 liter portions of O.OOS M cetyl-dimethylbenzylammonium chloride in methylene chloride.
The combined lower phase is extracted with 6 liters of 0.05 M sodium iodide which has been 25 adjusted to pH 5 with acetic acid. The lower phase is discarded and the upper phase is concentrated ln vacuo to 500 ml.
The concentrated material is extracted with 400 ml of n-butanol. The upper phase is GC155f discarded and the lower phase is concentrated to dryness in vacuo. The residue is dissolved (to the extenl possible) in 150 ml of methanol.
The insoluble material is discarded and the methanol solution is concentrated to dryness in vacuo, yielding 38.6 g of crude antibiotic.
The crude product is dissolved in lQ ml of methanol-water (1:1) and chromatographed on a 500 ml ~olumn of cross-linked dextran gel (Sephadex ~-lQ~ in the same solvent mixture, eluting at 2 ml/minute and collecting 20 ml fractions. Active fractions (19-26 are combined and concentrated in vacuo. The residue (5.23 g) is mixed with 50 ml of methanol. Insoluble material is filtered out and discarded.- The filtrate is concentrated in vacuo.
The residue, 5.0 g of material,is dissolved in 10 ml of pH 5 sodium 0.01 M phosphate buffer and applied to a column of DEAE cellulose (Whatman DE52 cellulose) packed and equilibrated in the same buffer. The column is eluted at 5 ml/minute with a linear gradient prepared from 4 liters of pH 5 sodium 0.01 M phosphate buffer and 4 liters of pH 5 sodium 0.1 M phosphate buffer, collecting 20 ml fractions. Active fractions (192-222) are 25 combined and concentrated in vacuo, and methanol-insoluble material is removed, washing well with methanol. Removal of solvent leaves 576 mg of material.

GC155f -231- 1 3386 7~

The 576 mg of residue is dissolved in 4 ml of water and the pH adjusted to S with about 1 ml of 0.1 N sodium hydroxide. The solution is chromatographed on a column Df alkylated cross-linke~ dextran gel (Sephadex LH-20) in water, eluting at l ml/minute and collecting 10 ml fractions.
Active fractions (38-44) are combined and concen-trated, giving 459 mg of residue.
Three hundred and forty eight ~348)mg of the above residue is dissolved in water and the solution placed on a column of macroreticular styrene-divinylbenzene copolymer resin (Diaion HP20AG~;
the column has been first prepared by washing with -methanolic potassium hydroxide, methanol, methanolic 15 hydrogen chloride, methanol and water, and then packed in water. The column is eluted with water at 1 ml/minute, collecting 10 ml fractions.
Active fractions (36-43) are combined and concen-trated giving 186.4 mg of material. Chromatography (in the same way) of another 100 mg of the 459 mg of residue from the previous step yields 51.5 mg of material. At this stage the 186.4 mg of material and 51.5 mg of material are nearly pure EM5117 as shown by thin-layer chromatography and nuclear 25 magnetic resonance spectra.
The 186.4 mg portion of EM5117 from above is dissol~ed in water and passed through a column of ion-exchange resin ~Dowex 50W-X2, 100-200 mesh, potassium form), washing with two 30 bed volumes of water. Concentration of the effluent .

-- GC155f yields 189.0 mg of crystalline solid. Recrystal-lization of this material is acco.nplished by dissolving it in 0.38 ml of water and adding 3.42 ml of methanol. The resulting mixture is cooled on 5 ice and filtered, yielding 145 mg of crystals.
Two further recrystallizations in this n~-nn~r from water-methanol, 1:9, yields 95.9 mg of EM5117, potassium salt, m.p. 194(dec.).
Optical rotation in water at 21C (c=l) ~ (mn) ~]

- - 589 +94.3 579 +98.6 546 +113.1 - 15 436 +203 365 +348 The following fermentation media are effective for the production of EM5117, and may be substituted for medium (B) and (C) in the above example.
- MEDIUM D
Grams Nutrisoy Flour 30 Glucose 50 Yeastamine 2.5 CaCO3 7 Distilled H2O to 1 liter MEDII~M E
Grams Yeast Extract 4 Malt Extract 10 Glucose 30 Glycerol 2 Distilled H2O to 1 liter Adjust pH to 7.3 before sterilization GC155f ~~33~1 3 3 8 6 7 0 MEDI~ F
Grams Gl~cerol 10 L-asparagine S

Na2HPO4 2 Glucose 50 gSO4 7H2O 0.2 Yeast Extract 2.5 Tap H2O to 1 liter MEDIUM G
Grams (NH4)2S04 2 L-asparagine 5 lS Glucose S0 Glycerol 10 MgSO4.7H2O 0.2 Yeast Extract 2.5 Distilled H2O to 1 liter Ad~ust pH to 7.0 MEDIUM H
Grams KH2PO4 . 3 Na Citrate 0.5 MgS04 0 . 1 (NH4)2SO4 Glucose 30 Yeast Extract 2.5 Distilled H2O to 1 liter _ GC155 ~234~

MEDIUM I
- Grams Nutrisoy Flour 10 ~NH4)2S4 5 S Glucose 50 Yeast Extract 2.5 CaC03 5 Distilled H2O to 1 liter MEDIUM J
- Grams Gerber's Baby Oatmeal 20 Cont~i n~ Tomato Paste .S
Glucose 20 Tap H2O to 1 liter lS Adjust pH 7.0 ~EDIUM K
Grams Gerber's Baby Oatmeal 5 Contadina Tomato Paste 20 Glucose 20 Tap H2O to 1 liter Adj~st pH 7.0 ~DIU~ L
Grams Yeast Extract 4 Malt Extract 10 Glucose 34 MEDIU~ M
Grams Amberex (1003) 5 Glucose 30 Tap H2O to 1 liter GC155f MEDIUM N
Grams Amberex 5 Cerelose 33 Tap H20 to 1 liter - l~S155f, -~236- 1 3 3 8 6 7 0 Example 196 Biological Production of EM5210 Gluconobacter species SC11,435 A.T.C.C.
No. 31581 is maintained on the following 5 sterilized agar medium (A):
Grams Yeast Extract Beef Extract NZ amine A 2 Glucose 10 Agar 15 Distilled H2O to 1 liter ~he pH is adjusted to 7.3 before sterilization at 121C for 30 minutes.
A loopful of surface growth from the agar slant (medium A) of Gluconobacter species SC11,435 is used to inoculate each of three 500 ml Erlenmeyer flasks each containing 100 ml of the following sterilized medium (B):
Grams Yeast Extract 4 Malt Extract 10 Dextrose 4 Distilled H2O1 liter 25 The pH is adjusted to 7.3 before sterilization at 121C for 15 minutes.
After inoculation, the flasks are incubated at 25C on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After the 30 appropriate incubation, as described above, 1% (vol/vol) transfers are made from the grown culture flasks to one hundred 500 ml Erlenmeyer flasks each containing 100 ml of the following . GC155f -sterilized medium (C):
Grams Yeast Extract 5 Glucose 10 Distilled H2O 1 liter The.medium is sterilized at 121C for lS minutes.
After inoculation, the flasks are incubated at 25C on a rotary shaker (300 rpm; 2 inch stroke) for 18 hours. At this time the contents of the fiasks are pooled and the broth is centrifuged yielding approxLmately 9 liters of supernatant broth.

Isolation and Purification (small scale) Activity from the broth supernatant (1~0 liters) is absorbed on a 500 g column of strongly basic anion exchange resin with quaternary ammonium groups attached to a styrene-divinylbenzene copolymer lattice (Dowex AGl-X2(Cl-)),washed with water and eluted with 5% sodium chloride in aqueous 0.01 M NaH2PO4. The eluate is concentrated in vacuo.
The residue is ~dsorbed on 250 g of charcoal which is washed with water. EM5210 is eluted with methanol-water (1:1). The active fractions are combined and concentrated in vacuo yielding crude EM5210.
The crude EM5210 is chromatographed on . a 280 ml column of strong base anion exchange resin (Bio.Rad AG l-X2 (Cl )) using a linear gradient prepared from 1 liter of water and *Trade Mark ., .~
~. ,~

GC155f _ . .

1 liter of 2M pyridinium acetate (pH 4.5). The active fractions are combined and concentrated in vacuo.
The partially purified EM5210 is further purified by gel filtration of the residue on a 500 ml column of cross-linked dextran gel (Sephadex G-10) eluting with water. The active fractions are combined and concentrated yielding 26 mg of EM5210. The potassium salt of EM5210 is prepared by passing EM5210 through a column of cation-exchange resin (Dowex 50-X2) in the potassium form.

Isolation and Purification (large scale) The broth filtrate of a 250 liter fermentation (pH 3.7) of Gluconobacter species SC11,435 is absorbed on 10.8 kg of strong base anion exchange resin (Dowex l-X8(Cl-)). The resin is washed with water and eluted with 5% sodium chloride in 0.01 M sodium dihydrogen phosphate. The active fractions are combined and concentrated to a small volume. The precipitated salt is removed and the filtrate is desalted by passing it through a column of charcoal (1.1 kg of 20-40 mesh Darco) in water. The column is washed with water and the EM5210 is elute~d with methanol-water,l:l. Active fractions are combined and concentrated. The residue (16 g) is dissolved in water and chromatographed on a column (600 ml) of strong base ion exchange resin (Bio.Rad AG 1-X2(Cl-), 200-400 mesh), eluting with a linear GC155f gradient prepared from 1 liter of water and 1 liter of 10% sodium chloride in 0.01 M sodium dihydrogen phosphate. Active fractions are combined !
concentrated in vacuo to a small volume, and precipitated salts are removed by filtration.
The filtrate is applied to a column of macroreticular styrene-divinylbenzene copolymer resin. The column is eluted with water. The active fractions are combined, concentrated to a small volume and freeze-dried, yielding 120 mg of the sodium salt of EM5210.
To transfonm the sodium salt to the lithium -salt an ion-~YchA~ge resin (Dowex 50 W-X2, lithium form) column is used. The sodium salt (100 mg) is dissolved in 0.5 ml of water, applied to the column and eluted with water. The active fracti~ns are com~
bined and directly freeze-dried, yi~l ~;ng 95 mg of the lithium ~lt of EMs~Io or as an ~I~L~ S solid.
The free acid (inner salt) of EM5210 is prepared by passing a base salt of EM5210 through a column of weak acid ion-exchange resin in the H+ form. For example, about 2.5 mg of the lithium salt can be applied to a column of Bio.Rad Bio.Rex 70 (H+) and eluted with water to give 1.45mg of the free acid (inner salt).

Chemical properties of EM5210 1) Ninhydrin positive.
2) Acid hydrolysis (6N-HCl at 115 C for 16 hours) gives two major ninhydrin positive spots by ~ paper chromatography (.~atman No. 1, ~utanol-acetic GC155f -1 33867~

acid-water (5:1:4), and one weak ninhydrin positive spot that quenches W-excited fluorescence. The two major ninhydrin pasitive spots are D-glutamic acid and D-alanine.
Physical characteristics of EM5210 1) W spectrum of the sodium salt in water:
end absorption 2) IR - Major peaks of the lithium salt in KBr:
1770, 1640, 1530, 1384, 1242, and 1051 cm~l.
3) PMR - Chemical shifts of the lithium salt in deuterated water, ppm down field from TSP:
1.40 (d,J=7Hz), ca. 2.14 (m), ca. 2.44 (m), 3.49 (5) 3.73 (t,J=6Hz), 3.94 (s), 4.28 (m) Optical rotation of the free acid (inner salt) in water at 24 C (C=0.15%) (pH 2.7):

~ (nm) [~]
- 589 +73 578 +79 54 6 - +91 4 36 +159 365 +263 - The following fermentation media have been found effective for the production of EM5210 and may be substituted for medium (B) and (C) in the text.

MEDIUM D
Grams Oatmeal 20 Tomato Paste 20 GC155f MEDIUM D (continuçd) Grams Tap H2O to 1 liter Adjust pH to 7.0 before sterilization of 121C for 15 minutes.

MEDIUM E ..
- ~rams Yeastamine 5 Cerelose 11 Tap H2O to1 liter - Sterilization at 121C for 15 minutes.

MEDIUM F
Grams Glucose 5 Tartaric Acid 2 Yeast Extract0.5 (NH4)2PO4 (NH4)2SO4 2 K2HPO4 0.5 NaH2PO4 0.5 - g 4 2 0.2 CaC03 Distilled H2O1 liter Adjust pH to 6.0 before sterilization of 121C for 15 minutes.

MEDIUM G
Grams Nutrisoy flour~ 30 Glucose 50 Yeastamine 2.5 CaCO3 7 Distilled ~I2O to 1 liter .

GC155f MEDIUM G (continued) Sterilization at 121C for 30 minutes.

MEDIUM H
Grams NZ Amine A 10 Cerelose 33 .
YeastAr; n~ 2 . 5 Tap H2O to 1 liter Sterilization at 121C for 15 minutes.

MEDIUM I
Grams Nutrisoy flour 15 Soluble starch 15 Glucose 50 CoC12.6H2O 0.005 CaCo3 10 Distilled H2O to 1 liter Sterilization at 121C for 30 minutes.

GC155f Biological Activity The following methDdology is used to deterrine the m; n;mum inhibitory concentration (hereinafter referred to as MIC) of the B-lactams of this invention.
The test organisms are grown in approximately 15-20 ml of Antibiotic Assay broth (Difco) by inoculating (in tubes) the broth with a loopful of the organism from a BHI (Difco).agar slant.
The inoculated tubes are incubated at 37C for 18 to 20 hours. These cultures are assumed to contain 109 colony forming units (hereinafter CFU) per milliliter. The cultures are diluted 1:100 to give a final inoculum level of 104 CFU; dilutions are made with K-10 broth*.
The compounds are dissolved in the appropriate diluent at a concentration of 1000 ~g/ml. Two-fold dilutions are made in K-10 broth resulting in a range from 1000 ~g/ml to 0.5 ~g/ml. 1.5 ml of each dilution is placed into individual square petri dishes to which 13.5 ml of K-10 agar** is added. The final drug concentration in the agar ranges from 100 ~g/ml to 0.05 ~g/ml.
Organism growth control plates containing agar only are prepared and inoculated before and after the test plates. The organisms are applied to the `agar surface of each plate with the Denley Multipoint Inoculator (which delivers approximately 0.001 ml of each organism) resulting in a final - inoculum level of 10 CFU on the agar surface.

.

GC155f The plates are incubated at 37 C for 18 hours and the MIC's are deterrined. The MIC is the lowest concentration of compound inhibiting growth of the organism.
*K-10 broth is a yeast beef broth containing:
Beef extract 1.5 g Yeast extract 3.0 g Peptone 6.0 g Dextrose 1.~ g Distilled water q.s. 1 liter **K-10 agar Beef extract 1.5 g Yeast extract3.0 g Peptone 6.0 g Dextrose 1.0 g Agar 15.0 g Distilled water q.s. 1 liter The tables that follow are tabulated results obtained when the ~-lactams of this invention are tested against various organisms. The number following each organism refers to the number of the organism in the collection of E. R. Squibb &
Sons, Inc., Princeton, New Jersey. A dash (-) in the tables means that the compound tested did not show activity against the particular organism at 100 ~g~ml. The symbol "N.T." means not tested.

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Claims (5)

1. The .beta.-lactam which is 3-[[(2-amino-4-thiazol-yl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-4-me-thyl-2-oxo-1-azetidinesulfonic acid of the formula:

or an isomer thereof or a pharmaceutically acceptable salt thereof.
2. The .beta.-lactam as claimed in claim 1 which is [3S-[3.alpha.(Z),4.beta.]]-3-[[(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azeti-dinesulfonic acid.
3. The .beta.-lactam as claimed in claim 1 which is a salt of [3S-[3.alpha.(Z),4.alpha.]]-3-[[(2-amino-4-thiazolyl)[(1-car-boxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid.
4. The .beta.-lactam as claimed in claim 1 which is a salt of [3S-[3.alpha.(Z),4.beta.]]-3-[[(2-amino-4-thiazolyl)[(1-car-boxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid.
5. The .beta.-lactam as claimed in claim 4 which is [3S-[3.alpha.(Z),4.beta.]]-3-[[(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-1-azeti-dinesulfonic acid, dipotassium salt.
CA000370320A 1980-02-07 1981-02-06 .beta.-lactam antibiotics Expired - Fee Related CA1338670C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA000617057A CA1340253C (en) 1980-02-07 1996-08-28 B-lactam antibiotics

Applications Claiming Priority (4)

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US11927680A 1980-02-07 1980-02-07
US119,276 1980-02-07
US18889380A 1980-09-29 1980-09-29
US188,893 1980-09-29

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CA000617057A Division CA1340253C (en) 1980-02-07 1996-08-28 B-lactam antibiotics

Publications (1)

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CA1338670C true CA1338670C (en) 1996-10-22

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GB2139618B (en) 1985-05-01
PT72465A (en) 1981-03-01
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KR860001289B1 (en) 1986-09-10
FR2509299A1 (en) 1983-01-14
CH651020A5 (en) 1985-08-30
DE3104145C2 (en) 1999-05-12
FI80271B (en) 1990-01-31
AU4574885A (en) 1985-11-07
GB2071650B (en) 1984-12-05
NL192924B (en) 1998-01-05
NO810410L (en) 1981-08-10
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SE8602193L (en) 1986-05-14
ES499171A0 (en) 1982-06-01
KR830005131A (en) 1983-08-03
IE862297L (en) 1981-08-07
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CH653993A5 (en) 1986-01-31
NO170015B (en) 1992-05-25
GB2139618A (en) 1984-11-14
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