NZ205240A - Sulphonamide derivatives,being starting materials for producing beta-lactams - Google Patents

Sulphonamide derivatives,being starting materials for producing beta-lactams

Info

Publication number
NZ205240A
NZ205240A NZ205240A NZ20524081A NZ205240A NZ 205240 A NZ205240 A NZ 205240A NZ 205240 A NZ205240 A NZ 205240A NZ 20524081 A NZ20524081 A NZ 20524081A NZ 205240 A NZ205240 A NZ 205240A
Authority
NZ
New Zealand
Prior art keywords
amino
oxo
acid
salt
solution
Prior art date
Application number
NZ205240A
Inventor
Richard B Sykes
William L Parker
Christopher M Cimarusti
William H Koster
William A Slusarchyk
Alan W Fritz
David M Floyd
Original Assignee
Squibb & Sons Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Squibb & Sons Inc filed Critical Squibb & Sons Inc
Publication of NZ205240A publication Critical patent/NZ205240A/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • 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
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • 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
    • C07F7/10—Compounds having one or more C—Si linkages containing nitrogen having a Si-N linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Communicable Diseases (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Cephalosporin Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Pyridine Compounds (AREA)

Description

New Zealand Paient Spedficaiion for Paient Number £05240 20524-0 Priority Date{ s): .1. 2, % P.). ^~fi .' 9. ~&Q Complete Specification Filed: T. :V^M. Coici^r> ^ I t r*a I i i I I I t rM lit ««>*«• ••••■■ ^ 01 JUL 1984 Publication Date: . .t7.
P.O. Journal, No: .VrM1? L Under the provisions of ftegtft Ittton 23 jfl) the J J Specification has been ante-datedj to l9' -LACTAM ANTIBIOTICS" K/We, E. R. SQUIBB & SONS, INC., a corporation of Delaware, the United States of America, having its offices at Lawrencevilie, Princeton, New Jersey, United States of America, hereby declare the invention for which ix/ we pray that a patent may be granted to>«Mfex/u.s, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 (followed by pa It had! been discovered that the 8-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 salt (including "inner salt") substituent in the 1-position and an acylamino substituent in the 3-position exhibit activity against a range of gram-negative and gram-positive bacteria.
The present invention relates to novel starting materials for the production of members of the novel family of B-lactam antibiotics of our New Zealand Patent Specification No. 196202 and accordingly the full content of that specification is herein incorporated by way of reference.
In one aspect the persent invention consists in A compound of the formula v R1~NH CH- 'R4 R3 C NH-SO-M ^ a 3 ^ .0 J. P. & 5. wherein R]_ is hydrogen or acyl or the group R^-NH- is protected amino; H© k Hor <\ ccdio^ • V is a leaving groupand R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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. a 205240 In a further aspect tJie Invention consists in A process for preparing a compound having the structure V .R ANH - CH // 0 -nhso^m® wherein A is a protecting group; V is a leaving group; R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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; and M® is hydrogen or a cation character!'sed by sulfonating a compound having the formula anh - ch c // o v I • c nh.
■R, wherein A, V, r3 and r4 are as herein defined.
In yet a further aspect the invention consists in A compound having the structure anh - ch // 0 V I c ■ R, •nhso^m® 3 2 0 52A0 wherein A is a protecting group; V is a leaving group; R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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; and M® is hydrogen or a cation.
The preferred members of the novel family of B-lactam antibiotics of that invention are those encompassed by the formula §2 §4 R-^-NH-C C-R3 C N-SO®M® In addition to the above described S-lactams having a sulfonic acid salt substituent in the 1-position and an. acyiamino substituent in the 3-position, that invention also encompasses B-lactams having a sulfonic acid salt (including "inner salt") substituent in the 1-position and an amino substituent in the 3-position.
-Get ,205240 The preferred compounds of this type have the formula la R2 R4 nh2-c -C-R.
.C N-SO-.M As used in formulas I and la, and throughout the specification, the symbols are as defined below.
R-^ is acyl; R^ is hydrogen or alkoxy of 1 to 4 carbons; R-j and R^ are the same or different and each is hydrogen, alkyl, cycloalkyl, phenyl or substituted phenyl, or one of R^ and R^ is hydrogen and the other is alkoxycarbonyl, alken-l-yl, alkyn-l-yl, 2-phenylethenyl or 2-phenylethynyl.
© M is hydrogen or a cation. 205240 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 "cycloalkyl" and "cycloalkenyl" refer to cycloalkyl and cycloalkenyl groups having 3,4,5,6 or 7 carbon atoms.
The term 'klkenyl" 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 tern "substituted phenyl" refqrs, to a phenyl group substituted with 1,2 or 3 substituents selected from, amino, halogen, . hydroxyl, trifluoromethyl and lower alkyl and alkoxy groups.
The term "protected carboxyl" refers to a carboxyl group which has beefT^s±erified~ with a conventional -ester protecting group.
These groups are well known in the art; see, for example, United States patent 4,144,333/ issued March 13, 1979. The preferred protected carboxyl qroups ire 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, preferred, but this preference should not be viewed as a limitation of the scope of this invention. Exemplary acyl groups are those acyl groups which have been used in the past to acylate 8-lactam antibiotics including 6-aminopenicillanic acid and derivatives and 7-aminocephalosporanic acid and derivatives; see, for example, Cephalosporins and Penicillins, edited by Flynn, Academic Press (1972), United States patent 4,152,432, issued 2cQ?3 ^ ^ 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 portions of these references describing various acyl groups are incorporated herein by reference. 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 li R5'C~ wherein is alkyl; cycloalkyl; alkoxy; alkenyl; cycloalkenyl; cyclohexadienyl; or alkyl or alkenyl substituted with one or more halogen> cyano, nitiro, amino, mercapto, alkylthio, or cyano-methylthio groups. (b) Carbocyclic aromatic groups having the formula l A o R.
R5vrf o II 0-CH2"C-; or R_ 'O^-sX R,., R_,, and R_ each o / o wherein n is 0, 1, 2 or 3; 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, 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' o ii CH2-C- o II CH2-C- h2nh2 -I- 2 4 9 \\ ?t HO— Preferred heteroaromatic acyl groups include those groups of the above formulas wherein R^gis 2-amino-4-thiazolyl, 2-amino-5-halo-10 4-thiazolyl, 4-aminopyrimidin-2-yl, 5-amino-l,2,4- thiadiazol-5-yl, 2-thienyl or 2-furanyl. (d) [[(4-Substituted-2,3-dioxo-l-piperazinyl) carbonyl]amino]arylacetyl groups having the formula O II -C-CH-NH-C-N N-R12 cf\ wherein R^ is an aromatic group (including. carbocyclic aromatics such as those of the formula R- R, and heteroaromatics as included within the definition of R]_o); an<3 Rj.2 "*"s alkY1' substituted alkyl (wherein the alkyl group is substituted with one or more halogen, cyano, nitro, amino or mercapto groups), arylmethyleneamino (i.e., -N=CH-R-^^ wherein R11 is as defined above), arylcarbonylamino 0 II (i.e. , -NH-C-Rj^ wherein R-^ is as defined above) or alkylcarbonylamino. r i - 2 0 5 2 4 0 Preferred [[(4-substituted-2,3-dioxo-l-piperazinyl)carbonylJ amino]arylacetyl groups include those wherein R-^ is ethyl, phenylmethylene-amino or 2-furylmethyleneamino. (e) (Substituted oxyimino)arylacetyl groups having the formula 0 II -C-C=N—O-R-i „ ' I -LJ- R11 wherein R-^ is as defined above and R-j^ is hydrogen, alkyl, cycloalkyl, .alkylaminocarbonyl, arylamino- 0 U carbonyl (i.e. , -C-NH-R^^ wherein R-^ is 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 , carboxyl (including salts thereof), amido, alkoxycarbonyl, phenylmethoxycarbonyl, diphenyImethoxycarbonyl, hydroxyalkoxyphosphinyl, dihydroxyphosphinyl, hydroxy (phenylmethoxy)phosphinyl, or dialkoxy-phosphinyl substituents) .
Preferred (substituted oxyimino)arylacetyl groups include those wherein Rllis 2-amino-4-thiazolyl . Also preferred are those groups wherein R^ is methyl, ethyl, carboxy-methyl, or 2-carboxyisopropyl. (f) (Acyiamino)arylacetyl groups having the formula 0 0 II II -C-CH-NH-C-R14 R11 wherein R]_^is as defined above and R^4 is -If- 4 0 • R, (CH2)n-0-, amino, alkylamino, (cyanoalkyl)amino, amido, alkylamido, (cyanoalkyl) - NH ll amido, -CH2"NH-C- D NH_ .0 I 2 If , -CH-CH_-C-NH-CH 3 ' HCk S02-N(CH -CH -OH) , _// \V_CH OH \_ / 3 N—N or / \ 0 / \ II N N -CH Preferred (acyiamino)arylacetyl groups of the above formula include those groups wherein R^ is amino, or amido. Also preferred are those groups wherein R-^is phenyl or 2-thienyl (g) [[[3-Substituted-2-oxo-l-imidazoli- dinyl]carbonyl]amino]arylacetyl groups having the formula 0 7n II 0 0 .C^ II ' II / \ -C-CH-NH-C-N N-Rn k I 1 ^.1 CH- CH. ( 2=0*5240 -M- wherein R^ is as defined above and R-^ is hydrogen, alkylsulfonyl, arylmethyleneamino (i.e., -N=CH-RjL2 wherein R^-|_is as defined above), O II -(--r16 (wherein is hydrogen, alkyl or halogen substituted alkyl), aromatic group (as defined by R-^above), alkyl or substituted alkyl (wherein the alkyl group is substituted with one or more halogen,cyano, nitro, amino or mercapto 10 groups).
Preferred [[3-substituted-2-oxo-l-imidazoli-dinyl]carbonyl]amino]arylacetyl groups of the above formula include those wherein R^]_is phenyl or 2-thienyl. Also preferred are those groups .15 wherein Rj_5 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 "-SO^M " substituent 20 on the nitrogen atom of the e-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 25 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 30 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; 35 alkaline earth metal, such as calcium and magnesium; 205 2 4 -ij- pyridinium; dicyclohexylammonium; hydrabaminium; benzathinium; N-me thy 1-D-gl uca minium.
As set forth in formula I, and in the definitions following formula I, M® can be hydrogen As is described hereinafter, the B-lactams of that invention can be prepared by synthetic means .... The non-alkoxylated'4-unsubstituted 6-lactams of fonnula I, i.e., those compounds of formula I wherein R2 , R3 and are hydrogen -can be prepared using 6-amino-penicillanic acid or a 6-acylaminopenicillanic acid as a starting material. The 6-lactams of formula I wherein R2 is alkoxy can be prepared from the corresponding non-alkoxylated 6-lactam.
Some of the compounds of the", invention may be crystallized or recrystallized from solvents containing water . In these cases water of hydration may be formed. The invention contemplates stoichiometric hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilization.
Some of the 6-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-l-azetidinesulfonic acid. Cultivation of various acetic acid bacteria e.g., Gluconobacter species SC 11,435, yields a salt of (R)-3-[ IN-(D-y-glutamyl)-' D-alanyl ] amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid ( -X \- 2052 B-Lactams having a sulfonic acid salt substituent in the 1-position and an amino or acyiamino substituent in the 3-position contain (in the 3-position of the 6-lactam nucleus) to which the amino or acyiamino substituent is attached. The invention is directed to those 6-lactams which have been described above, wherein the stereochemistry at the chiral center in the 3-position of the 8-lactam nucleus is the same as the configuration at the carbon atom in the 6-position of naturally occurring penicillin (e.g., 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 la, 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 la wherein hydrogen have the S configuration and those compounds of formulas I and la wherein is alkoxy have the R configuration.
Also included within the scope of that invention are racemic mixtures which contain the above-described 8-lactams. at least one chiral center the carbon atom ( ( 205240 -yr- 3-Lactams having a sulfonic acid salt substituent in the 1-position of the 0-lactam nucleus and an amino or acyiamino substituent in the 3-position of the B-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 the invention. The compounds wherein and/or R4 are hydrogen or alkyl, especially methyl, exhibit especially useful activity.
The compounds of . the invention can be used as agents to combat bacterial infections (including urinary tract infections and respiratory infections) in mammalian 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 f3-lactams Qf this invention. Such methods of administration include oral, intravenous, intramuscular and as a suppository. f ( - . 205240 —1 &— The g-lactam products of the invention are generally prepared by the introduction of a sulfonic acid substituent (a sulfo group -SO^-) onto the nitrogen atom in the 1-position of the 5 B-lactam nucleus. This sulfonation reaction is readily effected by treating the B-lactam with a sulfur trioxide complex or with an equivalent sulfonating reagent such as a chlorosulfonate.
The sulfur trioxide -complexes most commonly used 10 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. 15 Alternatively, sulfonation can be effected by way of an intermediate compound as for example first silylating the nitrogen atom of the B-lactam nucleus and then subjecting the silated compound to a silyl interchange reaction with trimethylsilylchlorosulfonate or a similar 20 reagent. Exemplary silylating agents are monosilyltri-f luoroacetamide, trimethylsilylchloride/trieth.ylamine and bis-trimethylsilyl trifluoroacetamide.
Generally, the sulfonation reaction is carried out in the presence of an organic solvent such as pyridine or 25 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 B-lactam.
When pyridine-sulfur trioxide is the sulfonating complex the product initially formed is the B-lactarn~"sulfonated pyridinium salt of the sulfonated B-lactam wherein M+ in the formula below is the pyridinium ion: O n-s03 m+ ( 2 4 0 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 5 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 10 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-15 tion prior to the formation of a g-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 20 preferably conducted with the amino function protected.
Using a benzyloxycarbonyl protecting group as an example, the sulfonation reaction can be visualized as follows: C^HcCHn OCO—NH o 5 2 r, -r- h2n- 0.
II J$H 1) sulfonation 2) deprotection ^ N-S03 M+ III Other protecting groups can be used to protect the amine function, for example, a t-butyloxycarbonyl group, a simple acyl group 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 (R-^) can then be affixed by a conventional acylation reaction. f -1M- TQr-5 ? a o Exemplary acylation techniques for converting a compound of formula III to a product of formula I include reaction with a carboxylic acid (R-^-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 (R^) 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., R, R1-NH- 0 _ 4 t-R- ■NH sulfonation' RX-NH- /? 0 -R, tf-S03M+ 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 ( ( -17- ci h I acyl-N- r 4 (R^)alkoxide R2 -R^ .. acyl-NH- n-so3 m+ _4 • f R- 0 ^j-s03 m+ vi The acyl groups in the above reaction also include 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 B-lactam ring can be formed via a cyclization reaction and the sulfonation reaction can be effected prior to cyclization as well as subsequent thereto, i.e., acyl-NH- V , R4 -NH-O-Y ( . f ^52 4 C -treat least one of and R^ 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 fully protected compound having the formula ; ■ V " "I/*4 BOC-NH-CH cC XV I ^R. o- 3 -C— NH-O-Y (at least one of R^ and R^ 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 R, BOC-NH- XVI 4 ■R. 3 N-O-Y (at least one of R^ and R^ 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 R^ and R^ is hydrogen. -7#- 2^- 7 A 0 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 boc-nh- ?4 XVII 0' -NH (at least one of R^'and R^ 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 stereochemical configuration of the R3 and R4 substituents.
As previously mentioned, the above azetidinone can be sulfonated to form a comgound having the formula XVIII boc-nh- 0* n-so3~m+ (at least one of R^ and R^ is hydrogen).
An alternative procedure for preparing a compound of formula I wherein R2 is hydrogen and at least one of R^ and R^ is hydrogen, utilizes as a starting material an amino acid amide having the formula oh xix nh2-ch- (y -nh. (at least one of R^ and R^ is hydrogen). 2 0 5 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 ^ R XX ANH——CH- C I R JZ— NH. 2 (at least one or R^-"and R^ is hydrogen), wherein A is a protecting group.
Sulfonation of a compound of formula XX yields a compound having the formula )Ms r , R4 ANH CH C"T XXI | ^R_ ^-rC NHS03 M (at least one of and R^ 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 R, ANH- XXII R3 N-S0-, M+ CP5 " (at least one of R^ and R^ 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~ alkoxy group by catalytic hydrogenation yields a compound having the formula -2jr ^s£2=ErS€ 52 4 0 R, h2n- xxiii 0 R, n-so3 m+ (at least one of R^ and R^ is hydrogen), wherein R2 is hydrogen or alkoxy which can be converted to the corresponding zwitterion having the formula XXIV nh- y R4 -N-SO. (at least one of 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 XIV.
An excellent source for the 3-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 rx-nh- XXVa 0^ c /CH T"^"CH3 ■n c-cooh and XXVb R. r1-nh" 0 & .N ch- COOH "2T0524O •21" respectively wherein is hydrogen or alkoxy and R^ 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-aminocephalosporanic 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 R2 R -NH- XXVI 0' -N-CH-CH(CH )_ I 3 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. I wherein R^ is hydrogen: or acyl, R^ is hydrogen or lower alkoxy and R^ and are hydrogen. Treatment. of a compound of formula XXVI with supric acetate and lead tetraacetate in an organic solvent (e.g., acetonitrile) replaces the carboxyl group with an acetate group.
Hydrolysis of the resulting compound can be accomplished using potassium carbonate in the presence of sodium borohydride.
Introduction of a sulfo group in the 1-position of above 3-amino-3-alkoxy-2-azetid±none cctnpound can be acccmplished by reacting the intermediate with a complex of dimethyl-formamide and sulfur trioxide. -2*- 2 0 S 2 -4- 0 A 3-azido-2-azetidinone starting material can be prepared by first reacting an olefin having the formula i4 XXVII CH2=C—R3 with a halosulfonylisocyanate (preferably chloro-sulfonylisocyanate) having the formula 1Q XXVIII 0=C=N—S02-halogen to yield an azetidinone having the formula R4 XXIX * " "~2 o ^ •R3 N-SO^-halogen Reductive hydrolysis of an azetidinone of formula xxix yields an N-unsubstituted ^-lactam having the 2 0 formula R4 -R- -NH XXX C 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 3-30 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 -2^- 205240 r4 n 3 XXXI nh The reaction proceeds best by first protecting the 5 azetidinone 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 10 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 with an aldehyde of the formula R3CH=Q to yield the corresponding Schiff base. A [2+2]cycloaddition with an activated form of -azido acetic acid yields a 3-25 azido-2-azetidinone having the fonnula or O-alkyl r4 N 3 R 3 n—q ( wherein Q is 205240 O-alkyl _J/ S\_0-alkyl or \=J 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 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 T R, -N- XXXII ?4 -R.
N-SO-.~M+ CT 3 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, -2?- 2trs2.+o 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 R^ is alkoxy comprises first alkoxylating an intermediate of formula VI wherein TR^NH ts a carbamate (e.g., R^ is benzyloxycarbonyl) and R^ is hydrogen and then introducing a sulfo group in the 1-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 CI R, XXXIII c6h5-ch2-o-co-n N—CI Using the alkoxylation procedure described above 25 (for converting a compound of formula XXXII to a product of formula I), and subsequently adding a reducing agent such as trimethylphosphite, the compound of formula XXXIII can be converted to an intermediate having the formula 30 O-alkyl R XXXIV c _h._0—co—nh-6 b ✓ -NH in a combination with its enantiomer.
To 52 ->8- The above procedures yield those products of formula I wherein 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.
Biological Production of the Antibiotic EM5117 Salts of (R) -3- (acetylamino) -3-methoxy-2-oxo-=-1-azetidinesulfonic acid (formula I, R^ is acetyl and R2 is methoxy; referred to hereinafter as EM5117) can also be prepared by cultivation of a strain of the microorganism Chromobacterium violaceum SC 11,378, which has been deposited in the American Type Culture Collection as A.T.C.C. No. 315T2.
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, Maryland. 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 EM5117.
'Chromobac terium violaceum SC 11,378, A.T.C.C. No. 31532 can be isolated from a moist soil sample containing the microorganism by first stamping 2052 -QCTS5T~ 33. -3-0"- the soil sample on a medium containing: Soil Extract - 4 00 ml Distilled Water- 600 ml Yeast Extract - 5.0 g Glucose -10.0 g Agar ~ - 17.5 g The medium is adjusted to pH 6.0 and sterilized in an autoclave at'121°C for 20 minutes. After 24 to 72 hours incubation at 25°C 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 121°C 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 (' 2052 . '---GClSS-f—'" -23-- 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 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 15-37°C; no growth occurs at 4°C or above'37°C. Casein is hydrolyzed strongly by the microorganism, which is oxidase positive . In the presence of Chromobacterium violaceum 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 the microorganism and aesculin hydrolysis is negative.
The above described key characteristics provide the basis for the identification of the microorganism as Chromobac terium violaceum as distinguished from Chromobac terium lividum, the only other species of the genus Chromobacterium recognized in the 8th edition of Bergey's Manual of Determinative Bacteriology.
Additional .strains of Chromobacterium violaceum can also be cultured to produce EM5117. ( ( -■***%0 5 2^ "3f" The Antibiotic To obtain the antibiotic EM5117, Chromobacterium violaceum SC 11,378 A.T.C.C. No. 31532 is grown at, or about, 25°C 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 mediura and purified. After the fermentation has been completed the broth can be centrifuged to remove the mycelium 15 .or, alternatively, filtration can be used to remove the mycelium from the broth. After removing the mycelium from the broth EM5117 can be extracted from the broth. Preferably the broth is extracted at pH 5 by ion-pair.extraction with cetyldimethyl-20 ■ benzyl ammo niuiu 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 inrvacuo, an aqueous solution of the residue can be washed with 25 butanol. Concentration of the resulting aqueous solution to dryness yields a residue which is dissolved, to the extent possible, in methanol. Centrifugation can be used to separate insolubles which are washed with methanol and then discarded. r ( 20524 *GC155f - -aX- 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.g., 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 0.0. 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, and eluting the column with water. Active fractions are combined and concentrated. Further purification of EM5117 is then accomplished 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 Dowex 50W-X2 potassium form, washing with water. The effluent can be concentrated to yield a crystalline material 4 0 -3 £- 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 Production of the Antibiotic EM5210 Salts of (R)^.3-[ [N- (D-y-glutamyl)-D-alanyl] -amino ] -3-methoxy-2-oxo-l-azetidinesulf o'nic ac id (formula I, is D-y-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 SCll,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, 25 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. f 2052 —GC155f - Gluconobacter species SC11,435, A.T.C.C. No.31581 can be isolated from ground moss containing the microorganism 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 by plating on a mediurn containing: *Extract of -Spartina patens Grass 400 ml Distilled Water 60 0 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 pH 6.0 and sterilized in an autoclave at 121°C for 20 minutes. After about 18 hours incubation at 25°C, colonies of the Gluconobacter species SC11,435 are isolated from th plated pectin enrichment solution. 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 pEI 7.3 and autoclaved at 121°C for 30 minutes.
Gluconobacter'species SC11,435 is a pleiomorphic gram negative rod motile by means f 2Q5 2- ■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 SC11,4 35 grows . as 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 SC11,4 35 grows luxuriantly as heaped up mucoid slimy colonies. Similar growth is obtained on malt-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 20524 lactate agar plates, (ii) acid is produced 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 Hugh and Leifson's medium.
The biological production of EMS210 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 EM5210: Acetobacter pasteurianus subsp. pasteurianum A.T.C.C. 6 0 33 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 subsp. liguefaciens 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 205? L^D -2i- The Antibiotic To obtain the antibiotic EM5210, Gluconobacter species SC11,435 A.T.C.C. No. 31581 or one of the microorganisms listed above, can be grown at, or about, 25°C under submerged aerobic conditions 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 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.g., Dowex 1-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,1:1.
The active fractions are collected and dried, and then placed on an anion exchange column, e.g., Dowex 1-X2, and eluted with-a gradient of sodium Chloride buffered at pll 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. 205? —eets-sf-— 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.
I -V5- r • ( 2tf57 Exampl e 1 (S) — N— (2—Oxo—1 — sulfo— 3 — azetidinyl) —2-phenylacetamide , potassium salt Method I: ■ A) 1-((1R)-carboxy-2-methyl(propyl)]-2-oxo-(3S)- [phenyl [acetyl (amino)]] azetidine Raney nickeil is washed with water by decantation for several hours until the pH of the water (5-6 times volume of that of the Raney 10 nickel)is 7.6.
To a solution.of 9.0 g of penicillin G (Na+) in 500 ml of- water is added 54 g (9 0 ml) of Raney nickel. The flask, fitted with a reflux condenser, is immersed in a bath at 15 155°C. When refluxing begins, it is continued for 15 minutes. The flask 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 20 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) 1- [Acetyloxy-2-methyl (propyl) 3-2-oxo- (3SMphenyl-[acetyl(amino)]]azetidine Nitrogen is bubbled for 15 minutes through a stirred suspension of 608 mg (2 mrnol) of the above compound in 20 ml of dry acetonitrile.
A water bath at 4 0-45°C is used for several minutes to dissolve all of the acid. The water ( -43.- -GG±5^f— 5? 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 2 0 minutes. The "acetonitril 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) , which is used without further purification in the next reaction.
C) 2-Qxo- (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 1G0 mg (4.30 mmol) of sodium borohydride is added. The reaction is stirred at room temperature for 20 minutes. The methanol is removed in 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 bicarbonate and then a small volume of water and -Mf- 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- 1-sulf o-3-azetidinyl) -2-phenyl-acetamide, potassium' salt Pyridine-SO^ 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 diraethyl-formamide and 2 ml of dry methylene chloride under nitrogen at room temperature. The mixture 15 is stirred for 3 hours. The solvents are removed in 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) 20 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 25 solids are collected to give 49 mg of the title compound, melting point 18 9°C, dec.
Anal. Calc'd for C^H-^N O SK: C, 40-99; H, 3.44; N, 8.69; S, 9.93 Found: C, 45.96; H, 3.83; 30 N, 9.86; S, 8.99 The compound has identical spectral characteristics to the product obtained in Method II. -4f- 2 0 5 7 'f.-0 Method II: A solution of 660 mg of (S)-2-oxo-3- [I (phenyl- methoxy) carbonyl] amino] -1-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 4 0 yl portions) and 10% potassium bicarbonate solution are added--'{pH 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 TLC examination of Rydon positive fractions, followed by pooling and evaporation. Crystallization from methanol-ether gives 101 mg of the title ' o compound, meltxng point 210 C, dec.
Anal. Calc'd for C^H^N^SK-1/2 H20: C, 39.86; H, 3.65; N, 8.45; S, 9.68; K, 11.80 Found': C, 4 0.01; H, 3.37; N, 8.59; S, 9.59; K, 11.98 NMR(D2Q) 3.66 (s , 3 ) , 3 . 67 (d of d-~-, J= 6.4), 3.90 25 (t,j = j), 4.90 (d of d, . j = 6.4) , 7.36 ppm (m.5). r ■ - . ■ -GClSSt' ■"44' 20 ^ Method III: To a solution of (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (121 mg; see Example 6A) in dry methylene chloride 5 (3 ml) is added 4 0 mg of phenylacetic acid and 61 mg of dicyclohexylcarbodiimide. The mixture is stirred for 4 8 hours at room temperature, and filtered to remove dicyclohexylurea. The solvent is removed in vacuo, and "the compound residue is 10 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, 4 8 mg 15 of product is obtained, having spectral characteristics in agreement with the products of Methods I and II. Method IV: A solution of 2.8 3 g of (S)-2-oxo-3[{(phenyl-methoxy) carbonyli] amino] -i-azetidinesulfonic acid, 20 pyridine salt (see Example 2) in 36 ml of water is stirred in an atmosphere of hydrogen with 707.5 mq of 10% palladium on charcoal (175 ml of hydroqen taken up). After 2 hours the slurry is filtered, and the filtrate cooled to 0°C 25 and diluted with 4 6 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 30 maintained between 5.2 to 5.8 by the simultaneous addition of cold 10% potassium bicarbonate solution. After 4 5 minutes the slurry is 5 2-'4~0 diluted with 9 3 ml of 0 - 5M 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 5 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 1.28 5 g of the title compound in fractions 14-19 (fraction 1-15" were 200 ml, fractions 10 16-21 were 100 ml)"". - Spectral data is in agreement with that obtained in the foregoing methods. -4*- ~-SG155£ s 20524 Example 2 (,S) -2-0x0-3- I [ (phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, pyridine (1:1) salt Method I : A) 1-1 (1R) -Carhoxy-2-methyl (propyl) ] -2^oxo- (3S) - [ I (phenylmethoxy) carbonyl] amino] azetidine A slurry ofv6-aminopenicillanic acid (12.98 g, 0.06 mole) in-140 ml of water containing .18 g of sodium bicarbonate (stirred for ca. minutes without complete 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 CO.0 7 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. 1- [ (Acetyloxy) -2-methyl (propyl) ] -2-oxo- (3S) - [ [ (phenylmethoxy) carbonyl3amino]azetidine A solution of 6.83 g (0.0213 mole) of the above acid in 213 ml of acetonitrile is treated with 1.95 g (0.0107 mole) of cupric acetate monohydrate and 9.5 g (0.0213 mole) of lead Hffr- 2r05 2. tetraacetate. The slurry is immersed in a 65°C oil bath and stirred with a stream of nitrogen bubbling through the slurry until the starting material is consumed. The slurry is filtered 5 and the solids washed with ethyl acetate. The combined filtrate and washings are evaporated in 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 10 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 15 above acetate in 70 ml of methanol and 7 ml of water is cooled to -15°C and 1.33 g of potassium carbonate and 34 9 mg of sodium borohydride are added. The reaction mixture is stirred at -15°C - 0°C. After the reaction is complete 20 (ca. 2 hours), the mixture is neutralized to pH 7 with 2N HC1 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 25 in vacuo. The residue is combined with material from a similar experiment and triturated with ether to give 3.30 g of the title compound.
( ^C155f -*<■ 2 0 5 ? '4 D) (S) -2-0x0-3- { I (phenylmethoxy)carbonyl] amino] -1-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 dimethylformaraide is stirred for 2 hours under hitrogen with 350 mg (0.0022 mole) of pyridine-sulfur. trioxide complex." Th.e 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(E>2o~CD3qE>) 3.63 (1H, d of d, ;«6.4), 3.90 (1H, t, )= 6), 4.85 (1H, d of d, _,= 6.4), 5.10(2H.,S) 7.27 (5H,S) 8-0-9.oppm(m1s 5H).
Method II: Chlorosulfonyltrimethylsilyl ester (18.87 g) is added dropwise at -20°C 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 dimethylforroamide and 12 0 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.
-GClbbf*"- / -J&- r~ ' Example 3 (S) -2-0x0-3- [ I (phenylmethoxy)carbonyl] amino] -1-azetidinesulfonic acid, potassium salt Method I: (S)-2-Oxo-3-I I (phenylmethoxy) carbonyl]amino] -1-azetidinesulf onic acid, pyridine salt (135 mg; see Example 2) is dissolved in 2 ml of 0.5 M monobasic potassium phosphate (adjusted to pH 5.5 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-wa ter. Fractions (25 ml) 14-15 are highly Rydon positive. Evaporation yields 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] -1-azetidinesulfonic acid, pyridine salt (600 mg; see Example 2) is dissolved in 2 ml of water and mixed with 15 ml of pH 5.5 monobasic potassium phosphate buffer. A solid forms and the slurry is cooled to 0°C, filtered, washed with cold buffer, cold 50% ethanol, ethanol and ether to give 37 0 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) . ( 52 -GCI55T^ Fractions (50 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 C^H^I^OgSK-l/^I^O: 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, 9.12; K, 11.53 NMR(D20) 3.69(1H, d, of d, j=6.4), 3.91(1H, t, j=6), 4.76(1H, 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 50°C 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 identical to that of Method II.
! - ■ ■ , ,GClS5f .ri Example 4 (S)-2-0x0-3-I[(phenylmethoxy)carbonyl]amino] -1-azetidinesulfonic acid, tetrabutylammonnium salt (1:1) Method I: (S) -2-0x0-3- [ I (phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, pyridine salt (1:1) (34.3 g; see Example 2) is dissolved in 800 ml of 10 water. The solution is cleared with charcoal, 30.7 g of tetrabutyiammonium hydrogen sulfate in 8 0 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 15 is reached. The precipitated tetrabutyiammonium 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 20 melting point 108-110°C.
Method II: (S)-2-Oxo-3-£1 (phenylmethoxy)carbonyl] amino] -1-azetidinesulfonic acid, potassium salt (1:1) 25 (20.2 g; see Example 3) is dissolved in 500 ml of water, filtered and 20.3 g of tetrabutyiammonium hydrogen sulfate in 100 ml of water is added.. The pH is brought to 5.5 with IN potassium hydroxide. The volume is reduced iri vacuo to 30 about 100 ml and the precipitated tetrabutyiammonium salt is filtered off. The compound is i3ClS5f -**- 20 5^-^-0 dissolved in 30 ml of methylene chloride, filtered and precipitated by addition of ether, yielding 21 g of the title compound, melting point 109-111°C.
Example 5 (3S)-a- I [ (2-Oxo-l-sulf o-3-azetidinyl)amino] -carbonylJbenzeneacetic acid, phenylmethyl ester, potassium salt (1:1) A) (S)-3-Amino-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 15 absorbed, the catalyst is filtered off and the filtrate evaporated to dryness. On standing, 1.1 g of the title compound crystallizes.
B) (3S) - a- [ I ( 2-Oxo-3-a zetidinyl) amino] carbonyl j -benzeneacetic acid, phenylmethyl ester The above azetidinone (3.0 g) is dissolved in 100 ml of dimethylf ormamide. The solution is cooled to 0°C and 4.5 g of N-methylmorpholine is added followed (dropwise) by 10.8 g of 25 a-(chlorocarbonyl)benzeneacetic acid, phenylmethyl ester in 50 ml of acetonitrile with stirring. The mixture is stirred for about 16 hours at 5°C. The solvent is distilled oj 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.
I .gG-lS-5-£- "5J r- 20524 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 5 ethyl acetate and petroleum ether, yielding 8.7 g, melting point 164-166°C.
C) (3S) -a- [ I (2-OxQ-l-sulfo-3-azetidinyl) amino] -carbonyl]benzeneacetic acid, phenylmethyl ester, 10 potassium salt (1:1) The above compound (6.9 g) is suspended in 150 ml of acetonitrile. Monotrimethylsilyl-trifluoroacetamide (5.7 g)is added and the solution is heated for 30 minutes at 50°C with 15 stirring. The solution is cooled to 0°C and 3.9 g trimethylsilyl chlorosulfonate is added dropwise. When the addition is complete the mixture is heated to 50°C for 5 hours.
After cooling to 20°C, 7.6 g of potassium ethyl 20 hexanoate in 10 ml of butanol is added and stirring is continued for 30 minutes. On addition of 300 ml of ether the title compound precipitates and is filtered off. The crude product is stirred with 100 ml of dry acetonitrile 25 for 30 minutes and filtered off, yielding 4.5 g of the title compound, melting point 118-120°C. Further purification of the crude product bv HP2 0 chromatography followed by freeze-drying yields pure material having a meltina point of 188-190°C.
I •'<3CI55T -oQ- 2052 Example 6 (S) - 3- J [ (2-Amino-4 - thiazolyl) acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt A) (S) -3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (S) -2-0x0-3- [ I (phenylmethoxy) carbonyl] amino] 1-azetidinesulf onic acid, tetrabutyiammonium salt (2 g; see Example 4) is dissolved in 100 ml of diciethylformamide 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 removed leaving the title compound as an oil. NMR (CDC13) 3.82 (1H, t,} 5.5), 4.05 (d. 1H, dof d, J= 5.5, 2.5 cps) .
B) (S) -3- [ [ (2-Amino-4-thiazolyl) acetyl] amino] - 2-oxo-l-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 dimethylformamide is added dropwise. After the addition is complete, stirring is continued for 12 hours at 20°C. 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 chromatography column using water as eluent. The yield 2 0 5240 is 8 50 mg of the title compound, melting point >300°C.
Example 7 [3S (i) ] -3- I [ (Formyloxy) phenylacetyl] amino] -2- oxo-l-azetidinesulfonic acid, potassium salt (S) -3-Amino-2-oxo-l-azetidinesulf onic acid, tetrabutyl ammonium salt (1.5 g; see Example 6A) in 100.ml of dimethylformamide 10 and 2 ml of propylene oxide are cooled to 0°C.
A solution of O-formylmandelic acid chloride in 10 ml of acetonitrile is added dropwise with stirring. The temperature is maintained for 1 hour and the solvent is then distilled off 15 in vacuo. The oily residue is treated with a solution of 2 g of potassium perfluorobutane sulfonate in 1.5 ml of acetone. After adding 20 0 ml of ether, the title compound crystallizes and is filtered off yielding 1.5 g of product. The 20 product is purified by HP-20 chromatography, m.p. 180-185°C. with decomposition.
Example 8 [3S (+) ] -3- [ I (Formyloxy) phenylacetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt 25 Following the procedure of Example 7, but substituting D-O-formylmandelic acid chloride for O-f ormylmandelic acid chloride, yields the title compound, melting point 120-125°C, after freeze drying. -ecX53T 2 0 5240 Example 9 [ 3S (-)•] -3- [ I (Formyloxy)phenylacetyl] amino-2-oxo-l-azetidinesulfonic acid, potassium salt Following 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, 2Q3-20 5°C. After careful drying ■ the product melts at 228-230°C- -Example 10 (5) -2-QXO-3- I (1-oxooctyl) amino] -1-azetidine-sulfonic acid, potassium salt (S) -3-Amino-2-oxo-l-azetidinesulfonic acid, 15 tetrabutyl ammo nium salt (1.5 g; see Example 6A) in 100 ml of dimethylformamide and 2 ml of propylene oxide are cooled to 0°C. At this temperature a solution of 0.8 g of caprylic acid chloride in 2 0 ml of dry acetone is added dropwise 20 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 25 is dissolved in 5 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 :ompouni drying. compound, melting point 173-180°C, after freeze- jGGt5r5"f 2 0 5 2 4 Example 11 [3S(Z)]-3-[[(2-Amino-4-thiazolyl)[ [ [hydroxy(phenylmethoxy) phosphinyl ]methoxy] imino]acetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (0.8 g; see Example 6A) in 30 ml of dimethylformamide, 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 210-215°C,dec.' Example 12 [3S(Z)]~3—[£(2-Amino-4-thiazolyl) (ethoxyimino)-acetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-l-azetidinesulfonic acid tetrabutyiammonium salt (1.5 g; see Example 6A) in 100 ml of dimethylformamide, 0.6 g of hydroxybenzotriazole, 1 g of dicyclohexylcarbodiimide and 0.8 g of (Z)-2-amino-a-(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 of acetone. 60 ^rf' 2 0 52 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 compound precipitates, is filtered off and dried. Purification is accomplished by chromatography using an HP-20 column and water as eluent, yielding 1:1 g of the title compound, melting point 18 0-185°C,. dec.
Example 13 [35(E)3-3-[I(2-Amino-4-thiazolyl)(ethoxyimino)-acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 12, but substituting (E)-2-amino-a-(ethoxyimino)-4-thiazoleacetic acid for (Z)-2-amino-a-(ethoxyimino)-4-thiazoleacetic acid, yields the title compound, melting point 160-170°C after freeze 20 drying.
Example 14 [3S(Z)-3-[[ (2-Amino-4 -thiazolyl) ] (2,2, 2-trif luoro-ethoxy) imino] acetyl] amino] -2-oxo-l-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-a-(ethoxyimino)—4-thiazoleacetic acid, yields the title compound melting point 160-170°C after freeze-drying. . 205240 Example 15 (S)-2-0x0-3-[(1-oxopropyl)amino]-1-azetidinesulf onic acid, potassium salt Method I: (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyl ammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide and 4 ml of propylene oxide are cooled to 0°C with stirring. 10 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 vacuo and the oily residue is treated with an equivalent 15 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) -2-0x0-3- [ I (phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, tetrabutyiammonium salt" (4 g; see Example 4) is hydrogenated in 100 ml 25 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 30 added with stirring. After 30 minutes the solvent is removed in vacuo and the oily residue is treated L2 -G 'Example 16 [35 (t) ] -3- I (Hydroxyphenylacetyl)amino] -2-oxo-l-azetidinesulfonic--acid, potassium salt (5) -3-Amino~2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is stirred for about 16 hours with 1.5 g of dicyclohexylcarbodiimide, 0.5 g of hydroxybenzotriazole and 0.6 g of mandelic 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 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 138-140°C.
Example 17 (S)-3-[[[(Cyanomethyl)thio]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) and 0.72 g of [ (cyanomethyl) thio]acetic acid are dissolved in 7 0 ml of acetonitrile and a solution of 1.04 grams of dicyclohexylcarbodiimide f I (_J5C155l£ 2 0 52 4 in acetonitrile is added dropwise. The mixture is stirred for about 16 hours at 0°C 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 150-155°C.
Example 18 (S) -2-0x0-3- [ (lH-tetrazol-l-ylacetyl) amino azetidinesulfonic acid, potassium salt To a solution of 0.005 mole of (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (see Example 6A) in 70 ml of dimethylformamide 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 0.00 6 mole of a solution of potassium . perf luorobutane sulfonate in acetone, yielding 1.5 g of the title compound, melting point 170-175°C, dec.
Example 19 (S) -2-0x0-3- [ (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. ( i --iSCitlrfrf 205 2 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 lH-tetrazole-1-acetic acid, yields the title compound, melting point 180-190°C, dec.
Example 21 I3S(Z)]-3-I[(2-Amino-4-thiazolyl) (methoxyimino)-acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt (S) -3-Amino-2-oxo-1-azetidinesulf onic acid, tetrabutyiammonium salt (prepared as described in Example 6A from 7.9 g of (S)-2-oxo- 3-11 (phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, tetrabutyiammonium salt) is cooled to 0°C and 3.53 g of (Z)-2-amino-ct-(methoxyimino) - 4-thiazoleacetic acid is added, followed by a solution of 3.27 g of dicyclohexylcarbodiimide in 10 ml of dimethylformamide. The mixture is stirred for 16 hours at 5°C, 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 235°C.
^GCISSJL -«>- 7 0 5 2 4 0 Example 22 (.35) -o - I [ (2-Oxo-l-sulfo-3-azetidinyl) aminoj -carbonyl]benzeneacetic acid, dipotassium salt (3S) — ct— T I (2-Oxo-l-sulfo-3-azetidinyl) aminoj -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 evaporated in vacuo. The residue is dissolved in ml of water and the pH adjusted to 6 with IN 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 8 0-85°C.
Example 23 (S)-3-(Acetylamino)-2-oxo-l-azetidinesulfonic acid, potassium salt Method I: A) (S) -3-Amino-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-(Benzyloxycarbonylamino)-2-oxo-l-25 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 4 0 minutes. The catalyst is removed by filtration and washed with 1 ml of 50% aqueous acetone. — 6 2o^T4 Anal . for CgH O N2SK, Calc'd: C, 24 .38; H, 2.87; N, 11.37; K, 15.9 Fo und: C, 26.06; H, 3.14; N, 9.96; K, 18.04 ( ( —GCig-S-f- 2052 4 0 Method II: A solution of 2.0 g of (S)-2-oxo-3-I[(phenylmethoxy) carbonyl] amino] -1-azetidineuslf onic acid , pyridine salt (see Example 2) in 25 ml of water 5 is hydrogenated over 5 00 mg of 10% palladium on charcoal. After 2 hours the solution is filtered, cooled to 0°C and 4 0 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 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 3 00 ml HP-20 AG column 15 (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 205-210°C.
Anal.calc'd for C5H?N205SK: C,24.38; H,2.86; N, 11 . 3 8; S,13.0 2; K,15.88 Found: C,24.23; H,2.81; N,11.25; S, 12.86; K,15 .74 ( I 2 o ?? w Example 24 (S) -2-0x0-3- I (phenoxyacetyl) amino 3 -1-azetidinesulfonic acid, potassium salt (S) -3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is cooled to 0°C. 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 2 0 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 176-180°C.
Example 25 [3S(R*)]-3- [[ [ [ [ 3-[(2-Furanylmethylene)amino]-2-oxo-l-imidazolidinyl ] carbonyl ] amino] phenylacetyl] -amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S) -2-0x0-3- [ [ (phenylmethoxy) carbonyl] amino]-1-azetidinesulfonic acid, tetrabutyl ammo nium 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 dicyclohexylcarbodiimide, 2 g of (R)-a-I [ [3-1(2-furanyl-methylene) amino] -2-oxo-l-imidazolidinyl] carbonyl] - ( ( 2 0-, amino]benzeneacetic acid and 0.9 g of hydroxybenzotriazole are added. After 3 hours the reaction mixture is evaporated to dryness, dissolved in 50 ml of dry acetone and the precipitated urea removed by filtration. An equivalent amount of potassium perfluorobutane sulfonate in 20 ml of acetone1 is added and the title compound precipitates. Crystallization is completed by addition of 200 ml ether.
After filtration the title compound is recrystallized from water, yielding 2 g, melting point 220-225°C, dec. [3S (R* )■] -2-0x0-3- [ [ [ [ (2-oxo-l-imidazolidlnyl) -carbonyl ] amino] phenylacetyl ] amino] -1-azetidinesulfonic acid, potassium salt (S) -2-0x0-3- [ [ (phenylmethoxy) carbonyl]amino] -1-azetidinesulfonic acid, tetrabutyiammonium 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)-a-[ I (2-0xo-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 the residue is dissolved in 50 ml of acetone. The precipitated urea is filtered off and the mother liquor is treated with an equivalent Example 26 ( ( —Get5-5-f— Si, 2 05 2 4 0 amount of potassium perfluorobutane sulfonate in 2 0 ml of acetone. The title compound crystallizes and is filtered off after the addition of 200 ml of ether, yielding 1.8 g of product, 5 melting point 210-215°C after recrystallization from water/acetone.
Example 27 [3S (Z) 3 -3- [ [ (Methoxyimino) phenylacetyl] aminoj -2-10 oxo-l-azetidinesulfonic acid, potassium salt A) [3S (Z) ] -3- [ [ (Methoxyimino) phenylacetyl] amino] -2-azetidinone (Z) -a- (Methoxyimino)benzeneacetic acid (3.58 g) is dissolved in methylene chloride, 15 cooled to 5°C, 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 5°C and a solution of 1.72 g of 3-amino-2-azetidinone (see Example 5A) in 20 100 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. 25 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. ~7 / 2.fT5T4 0 B) [3S(Z) )]-3-[ I(Methoxyimino)phenylacetylJ -aminoj-2-oxo-l-azetidinesulfonic acid, potassium salt Pyridine (0.5 ml) is dissolved in 5 ml of 5 absolute methylene chloride, cooled to -3Q°C 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 10 to dryness. The residue is dissolved in 10 ml of dimethylformamide and treated at room temperature with a solution'of . 1.23 g of the above azetidinone in 10 ml of dimethylformamide. After stirring for two hours at room temperature, the solution is 15 evaporated to dryness to yield 2.1 g of crude [3S (Z) ] -3- [ [ (methoxyimino) phenylacetyl] amino] -2-oxo-l-azetidinesulfonic acid, pyridine salt.
Treatment of the pyridine salt with tetrabutyiammonium hydrogen sulfate yields 20 the corresponding tetrabutyiammonium salt which is extracted with methylene chloride and remains as an oil after evaporation.
Treatment of the tetrabutyiammonium salt with an equimolar amount of potassium 25 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 30 yields a product having a melting point of 220°C,dec. ( 77. -70- ( 2 0 Example 28 [3S CZ)j —3—IT(2-Amino-4-thiazolyl) f [2-(diphenyl-methoxy)-1,1-dimethyl-2-oxoethoxy3imino]acetyl] -amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (1:1) A solution of 0.005 mole of (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (see Example 6A) and 0.006 mole of (Z) -2-amino-a-112—(diphenylmethoxy) -1,1-dimethy1-2-oxoethoxy]imino]^4-thiazoleacetic acid in 60 ml of dimethylformamide is treated with 0.7 g of hydroxybenzotriazole and 1.13 g of dicyclohexylcarbodiimide. 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 perfluorobutane sulfonate in acetone. After the addition of petroleum ether the title compound precipitates and is treated with ether and filtered to yield 3.8 g of product,melting point 190°C, dec.
Example 29 [3S(Z)]-3-[[(2-Amino-4-thiazolyl) [ (1-carboxy-l-methylethoxy)imino]acetyl]amino]-2-oxo-l-azetidine-sulfonic acid, dipotassium salt (3S(Z)]-3-[[(2-Amino-4-thiazolyl)[[2-(diphenylmethoxy)-1,1-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 -73.- " GClbSt „ 20524 acid is added at -10°C. The mixture is stirred for 10 minutes at -10^C. Ether (100 ml) is added slowly at -10°C and subsequently 50 ml of petroleum ether is'added. Hie precipitate is filtered to' yield 1.6 g of the trifluoroacetic acid salt. This is suspended in 20 ml of water at 0°C, 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 225°C, dec. .
Example 30 I3S(Z)J—3—1[(2-Amino-4-thiazolyl) [12- (diphenyl-methoxy) -2-oxoethoxy"]imino] acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure described in Example 28, but substituting (Z)-2-amino-a- [ [2-(diphenylmethoxy) -2-oxoethoxy] imino] -4-thiazole-acetic acid for (Z)-2-amino-a-[ [2-(diphenylmethoxy)-1, l-dimethyl-2-oxoethoxy] imino] -4-thiazoleacetic acid, yields the title compound, melting point 180°C, dec. f -GeTSST— -7* 2052 Example 31 [3S(1)j-3-1(Azidophenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Method I: A) . (±,S)-g-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). (±)-a-Azidobenzeneacetyl chloride (5 g) dissolved in 10 ml of acetone" is added dropwise while maintaining the temperature at 0-5°C and the pH at 6.8 with sodium bicarbonate. After stirring for 1 hour, the acetone is distilled off and the remaining 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 97-100°C. ( ( j&e±5$£—- 5 2 A B) I3S(±)]-3-[(Azidiophenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt A solution of 2.4 5 g of the above azetidinone and 3 g of monosilyltrifluoroacetamide in 20 ml 5 of acetonitrile are kept for 1 hour at 40°C.
After cooling to 0°C the solution is treated with 1-88 g of trimethylsilyl chlorosulfonate 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 4 5 minutes. The solution is poured into,300 ml of ether and the precipitate is filtered off. A filtered solution of 1.2 g of precipitate in phosphate buffer 15 (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 (Rydon test on Si02). 2 5 ml 20 fractions are taken and from fractions 15 and 16 28 0 mg of the title compound are obtained. A second column chromatography of this material yields 120 mg of the title compound, melting point 14 8°C, dec.
Method II: (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (2.03 g; see Example 6A) and 0.9 g of (±)-a-azido-benzeneacetic acid are 30 dissolved in 30 ml of acetonitrile and a solution of 1.03 g of dicyclohexylcarbodiimide in 10 ml ( -7 6- 2^-0^5-2 of acetonitrile is added. The temperature is maintained for one hour at 0°C and for 10 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 149°C, dec.
Method III: a-Azidiophenylacetyl chloride (2.5 g) is added to a solution of 4.06 g of 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 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 of ether the title compound crystallizes and is filtered off, melting point 148-149°C dec.
Example 32 [3S (D) ] —3- [[J[I (4-Methoxyphenyl)methoxy]carbonyl] -amino] phenylacetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt To a solution of 2.03 g of 3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (2.03 g; see Example 6A) and 1.58 g of D-a-[[[(4-methoxyphenyl)methoxy]carbonyl]amino]benzeneacetic 205 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 5°C for one hour and for 6 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 perfluorobutane sulfonate and.ether yields 2.4 g of product, melting point 108-111°C dec.
Examples 33-37 Following the procedure of Example 32, but substituting the compound listed in column I for (D)—a—I[X(4-methoxyphenyl)methoxy]carbonylJ-amino]benzeneacetic acid, yields the compound listed in column II.
( (D)-a-[[[(4-methoxyphenyl)methoxy] carbonyl]amino]-2-thiopheneacetic acid (±)-2-amino-a-[[[(4-methoxyphenyl) methoxy]carbonyl]amino]-4 -thiazole acetic acid (+) - a- [ [ [ (4 -methoxyphenyl) methoxy] carbonyl]amino]-2-furanacetic acid (L)-a-[([(4-methoxyphenyl)methoxy] carbonyl]amino]benzeneacetic acid (L)-a-[[[(4-methoxyphenyl)methoxy] carbonyl]amino]-2-thiopheneacetic acid [3 S- (D) ]-3- [ [ I [ I (4-methoxyphenyl) methoxy] carbonyl] amino}-2-thienyl-acetyl] amino}-2-oxo-l-azetidine-sulfonic acid, potassium salt, melting point 144-146 C, dec. [3S(±)]-3-[[(2-amino-4-thiazolyl)-I [ [ (4-methoxyphenyl)methoxy]-carbonyl]amino]acetyl]amino]-2-oxo-1-azeti.dinesulfonic acid, potassium salt, melting point 232-234 C. [3S(+)]-3-[[2-fur.anyl [n^-me thoxyphenyl ) methoxy ] carbonyl ] -■ amino]acetyl]amino-2-oxo-l-azetidinesulfonic acid, potassium salt, melting point 124-126 C, dec. [3 S (L)]-3-[[[[[ (4-me thoxyphenyl) -methoxy]carbonyl]aminoj phenyl-acetyl] amino]-2-oxo-l-azetidine-sulfonic acid, potassium salt, melting point 172-175 C, dec. [3 -S (L) ] -3- [[[It (4-methoxyphenyl) -methoxy]carbonyl]amino]-2-thienyl-acetyl]amino]-2-oxo-l-azetidine-sulfonic acid, potassium salt, melting point 146-148 C.
•;Po * -77- —aw «*-• Example 38 (3S) -3- 11 [ [ (Methyl thio) thioxomethyl] thio] phenyl-acetyl] amino]-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt A solution of 1. 03 g of (±)-a-fI(methylthio)- . thioxomethyl] thio]benzeneacetic acid and 1.63 g of 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (see.Example 6A) in 30 ml of acetonitrile is treated with 0.8 g of dicyclohexylcarbodiimide dissolved in 10 ml of acetonitrile at -5°C. 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 4 00 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'-l-azetidinesulf onic acid , potassium salt (3S)-3-[I[[(Methylthio)thioxomethyl] thio]-phenylacetyl ] amino-2-oxo-l-azetidinesulf onic acid, tetrabutyiammonium 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 crystallizes the title compound, which is filtered off, yield 0.18g of product, melting point 157°C, dec. -yg- ( 20574 ^jSG±-&5f Example 4 0 [3S (D) 3 -3- [ [ I [ (4-Ethyl-2 , 3-dioxo-l-piperazinyl) -carbonyl] amino3 -2-thienylacetyl 3amino3 -2-oxo-l-azetidinesulfonic acid/ potassium salt 5 (D) —a— [ [ (4 -Eth.yl-2 , 3-dioxo-l-piperazinyl) - carbonyl]amino3-2-thiopheneacetic acid (.3.25 g) , 4.20 g of 3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt Csee 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 -10°C 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 185-187°C, dec.
Example 41 [3S (±) 3 -3- [ (Bromophenylacetyl) aminoj -2-oxo-l-azetidinesulfonic acid, potassium salt a-Bromophenylacetyl chloride (1.4 g) in 25 10 ml of acetonitrile is added dropwise to a solution of 5mM 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyl ammonium salt, and 3 g of propylene oxide in acetonitrile at 0°C. After three hours stirring, the solvent is distilled off and the 30 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 . r r 2052 4 ■S'/ ' -y4- compound, melting point 135-137°C dec.
Example 42 [3S(i)]-3-[[I(Aminocarbonyl)amino]-2-thienylacetyl]-amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Method I: To a solution of 2 g of .(S) -3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 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. r 20524 £2 -8-er- Method II: To a suspension of 2 g of (+)-a-I(amino-carbonyl) amino]-2-thiopheneacetic acid, lOmM of (S) 3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyl-5 ammonium salt and lOmM of N-hydroxybenzotriazole in 50 ml of acetonitrile at 0°C is added (with stirring) lOmM of dicyclohexylcarbodiimide dissolved in 15 ml of acetonitrile. Stirring is continued for one hour at -15°C and for about 10 16 hours at room temperature. After filtering off the dicyclohexyl urea, the solvent is evaporated and the oily residue is dissolved in 50 ml of acetone. Adding the equivalent amount of potassium perfluorobutane sulfonate in 15 acetone yields crystalline product. Purification by column chromatography on HP-20, using water as an eluent, yields the title compound, melting point 220-223°C.
Example 4 3 [3S(_)]-3-[[[[(Methylamino)carbonyl]amino]-2-thienylacetyl]amino]-2-oxo-1-azetidinesulfonic acid, potassium salt (±) —a— [ [ (Methylamino) carbonyl]amino]-2-25 thiopheneacetic acid (0.54 g) and 1.00 g of (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (see Example 6A) are dissolved in 20 ml of acetonitrile and 0.5 cr of dicyclohexylcarbodiimide is added at a temperature of 0°C. After stirring for 8 hours the dicyclo-hexylurea is filtered off and after distilling off f 205240 r~GCi5 5f ' -sa- the solvent the oily residue is dissolved in 50 ml acetone and the equivalent amount of potassium perfluorobutane sulfonate is added. Crystalline product is filtered off and purification is performed on HP-20 using water as an eluent, melting point 205°C, dec. _ Example 4 4 [3S (1) ] -3- [It (Amin'ooxoacetyl) amino] -2-thienyl-acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt --: Following the procedure of Example 42, method II, but substituting (±)-a-[ (aminooxoacetyl)-amino]-2-thiopheneacetic acid for (1)-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-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 40, but substituting (R)-a- [ [ (4-ethyl-2, 3-.dioxo-l-pipera-zinylJcarbonyl]amino]benzeneacetic acid for (D)-a-[[(4-ethyl-2,3-dioxo-l-piperazinyl)carbonyl]-amino]-2-thiopheneacetic acid, yields the title compound, melting point 155-157°C, dec. 2 0 5 2 A 0 . GC155f "8 "if Example 4 6 3- (Acetylamino) -3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt Method I: A) 3- [ (N-Ace tyl-N-chloro) amino]-2-oxo-l-azetidinesulfonic acid, mixed sodium and potassi'um salt To a solution of 3- (acetylamino)-2-oxo-l-azetidinesulfonic acid, potassium salt C172 mg, see Example 23) in 'methanol (17 ml) containing 4% sodium borate decahydrate cooled to -15° to -10°C is added tert-butyl hypochlorite (110 jil) . 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 minimum 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 124°C, slow dec. ( -85-- ( €0155/ ' 5? ' B) 3-(Acetylamino)-3-methoxy-2-oxo-l-azetidine-sulfonic acid, potassium salt 3- [ (N-Acetyl-N-chloro)amino] —2-oxo-l-azetidinesulfonic acid mixed salt (37 mg) is dissolved in 1.5 ml of dry dimethylformamide and added to a solution of lithium methoxide (50 mg) in methanol (1 ml) at -78°C. After stirring for 15 minutes at -78°C, a 0.5 M solution of monobasic potassium phosphate (10 ml) is added, and the solution is then acidified to pH .4 with IN hydrochloric acid. To the solution is added tetrabutyiammonium 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 tetrabutyiammonium 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 iri vacuo gives an oil which crystallizes from methanol-ether. Trituration twice with ether gives the product as a colorless powder (11 mg); melting point 182-183°C, dec.
( A f ^ " -8^-Method II: A) 3-Amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 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, tetrabutyiammonium salt(60mg; see Example 4 9) in.methanol (2 ml) is added and the mixture is vigorously stirred for 15 minutes under a hydrogen atmosphere. Catalyst is removed by filtration through Celite on a millipore filter (0.5 my), 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-l-azetidine-sulfonic acid, potassium salt To a solution of 3-amino-3-methoxy-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (35 mg) in methylene chloride (10 ml) at 0°C is added propylene oxide (2 ml) and acetyl chloride (74 yl) . 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 205? /T>> eeres-f- of AG 50W-X2, K form, 0.6 meq/ral) . Removal of water from the eluate in vacuo yields the desired product (10 mg) .
Example 4 7 N- (3-Methoxy-2-oxo- 1-sulf o-3-azetidinyl) -2-phenyl-acetamide, tetrabutyiammonium salt A) N-Chloro-N- (2-ox6-l-sulfo-3-azetidinyl) -2-phenylacetamide, potassium salt To a solution of (S) -N- (2-oxo-l-sulfo-3- azetidinyl)-2-phenyiacetamide, potassium salt (50 mg; see Example 1) in methanol containing 4% sodium borate decahydrate (5 ml) cooled in a -5°C bath is added tert-butyl hypochlorite 15 (20 .yl) . After stirring for 32 minutes, the mixture is poured into 0.5 M pH 5.5 potassium phosphate buffer at 0°C. The resultant solution (pH 5.9) is adjusted to pH 4.5, concentrated in vacuo to remove methanol , and chromatographed 20 on HP-20AG, 100-200 mesh (100 ml). After washing the column with 0.5 M pH 5.5 buffer (100 ml) and water the desired product is eluted with 9:1 water:acetone. Concentration in vacuo gives 50 mg of the title compound.
B) N—(3-Methoxy-2-oxo-l-sulfo-3-azetidinyl)-2-phenylacetamide, tetrabutyiammonium salt To a stirring solution of lithium methoxide (160 mg) in 5 ml of methanol cooled to -78°C is 30 added a solution of N-chloro-N-(2-oxo-l-sulfo- 3-azetidinyl)-2-phenylacetamide, potassium salt ( ' f (14 9 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), 5 and washed three times with methylene chloride. Tetrabutyiammonium 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)., 10 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 14 9 mg of the product as an oil.
Example 48 N-(3-Methoxy-2-oxo-l-sulfo-3-azetidinyl)-2- phenylacetamide, potassium salt N-(3-Methoxy-2-oxo-l-sulfo-3~azetidinyl)- 2-phenylacetamide, tetrabutyiammonium 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 residual 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): u 1762, 1665 cm"1; NMR (CD-OD) 6 3.41 (S, 3H, OCH_), msx J 3.59 (S, 2H, CH2), 3.84 (ABq, J=6.3 Hz, 2H, H4), 30 7.30 (m, 5H, aromatic).
Analysis: Cal. for C1 2H13N206.1/2H20 Calc . : C , 3 9 . 8 8 ; H,3.62; EI, 7. 7 5 Found : C, 39.62; H,3.65; N,7.60 f ( Example 4 9 3-Methoxy-2-oxo-3-11 (phenylmethoxy) carbonyl ] amino] -1-azetidinesulfonic acid, tetrabutylammonium salt Method I: A) 2-0x0-3- [N-chloro-N- [ (phenylmethoxy)carbonyl] -amino]-1-azetidinesulfonic acid, tetrabutylammonium salt (S) -2-0x0-3- [ [ (phenylmethoxy)carbonyl] -amino]-1-azetidinesulfonic acid, tetrabutyiammonium 10 salt (0.9cp see Example 4) dissolved in 8 0 ml of methylene chloride is added to a mixture (cooled to 0-5°C) 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 15 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 20 of the extracts, drying (sodium sulfate), and removal of solvent iri vacuo yields the title compound as an oil (0.94 g).
B) 3-Methoxy-2-oxo-3-[[(phenylmethoxy)carbonyl] -25 amino]-1-azetidinesulfonic acid, tetrabutyi ammonium salt To a stirring solution of lithium methoxide (667 mg) in anhydrous methanol (10 ml) at -78°C is added a solution of 2-oxo-3-[N-chloro-N-[(phenyl-30 methoxy)carbonyl] amino]-1-azetidinesulfonic acid, tetrabutyiammonium salt (0.94 g) in dry dimethyl- 6C155I 7 O 2052 " 0 formamide (10 ml). After stirring the mixture at -78°C 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) : v (neat) 1767, 1720 cm"1; NMR (CDCl^) 6 3.40 max (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-l-azetidinesulfonic acid, potassium salt (400 mg) in water is added a 0.1 M tetrabutyiammonium bisulfate solution (10.9 ml, adjusted 20 to pH 4.3 with potassium hydroxide) . The mixture is extracted three times with methylene chloride, the extracts are combined, dried (Na2SC>4) and solvent is removed in vacuo to give a foam (625 mg) , having spectral characteristics approximating those 25 of the product of Method I. <=\! -o4- ""€€1 S 5-j 5 2^0 Example 5 0 3-Methoxy-2-oxo-3- [ [ (phenylmethoxy)carbonyl]amino] -1-azetidinesulfonic acid, potassium salt Method I: A) 2-0x0-3-[N-chloro-N- [ (phenylmethoxy)carbonyl]-amino]-1-azetidinesulfonic acid, potassium salt To a solution of (S)-2-oxo-3-[I(phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, 10 potassium salt (1.00 g; see Example 3) in methanol (90 ml) containing 4% sodium borate decahydrate at -10^C is added tert-butyl hypochlorite (420 yl) . After stirring for 2 hours at -10°C,.0.5 M monobasic potassium phosphate solution (100 ml) is 15 added and the pH is adjusted to 6 with 1 N hydrochloric acid. After concentration of the solvent in vacuo to 3 0 ml, the aqueous solution is chromatographed on HP-20AG, 100-200 mesh (200 ml) . After passage of a solution of monobasic potassium 20 phosphate • (5 0 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 25 point 173-175°C. c ■ GC155f— -& 2 05 7 B) 3-Methoxy-2-oxo-3-[ [ (phenylmethoxy)carbonyl]- ■amino]-1-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 J amino] - 1-azetidinesulfonic acid, potassium salt (857 mg) in dry dimethylformamide (13 ml). After 15 o minutes at -78 C the mixture is poured into 0.5 M monobasic potassium phosphate solution 10 (200 ml) and the pH is adjusted to 5.5 with IN hydrochloric acid- The aqueous mixture is washed with methylene chloride (three 100 ml portions), and tetrabutyiammonium bisulfate (1.169 g) is added. The product is extracted with methylene chloride 15 (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 tetrabutyiammonium 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, 0 K form, 0.6 meq/ml). Concentration of the eluate in vacuo yields an oil (30 mg), which is crystallized by scratching with acetone: v (KBr) 1760, 1725 cm"1; NMR (D_0) 6 3.48 max 2 (S, 3H, OCH3), 3.92 (S, 2H, H4); 5.20 (S, 2H, CH2) , 7.42 (S, 5H, aromatic), m.p. 196-198°. zlffl 4-^ Method II: A) l-Chloro-3- [N-chloro-N- [ (phenylmethoxy)carbonyl] -2-azetidinone A solution of 3-I[(phenylmethoxy)carbonyl]-5 amino]-2-azetidinone (4 4 0 mg. ; see Example 2C) in 4 0 ml of 4% raethanolic borax is cooled to 0°C and 0.5 ml of t^-butyl hypochlorite is added. After 30 minutes at 0°C, the solution is poured into 200 ml of cold water and extracted with two 10 10 0 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 1-chloro- 3- [N-chloro-N- [ (phenylmethoxy) carbonyl] amino] -2-azetidinone in 5 ml of tetrahydrofuran is cooled to -78°C 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 -78°C, allowed to warm 25 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 30 an oil that is applied to four 20 x 20 cm xl mm ( ( GGl55~f -9V- 20 5? silica gel plates. Development with, benzene-ethyl acetate (1:1) and isolation of the major UV-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 112-114°C.
C) 3-Methoxy- 2-oxo-3"- [ I (phenylmethoxy) carbonyl] -amino]-1-azetidinesulfonic acid, potassium salt A solution of 25 mg of 3-methoxy-3-[[ (phenylmethoxy) carbonyl ] amino]-2-azetidinone in 0.175 ml each of dichloromethane and dimethylformamide is stirred for 24 hours with 5 5.4 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 4 0 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 19 6-19 8°C, dec. r ' 52-4- Example 51 3- [ [1, 3-Dioxo-2-phenyl - 3- (phenylmethoxy) propyl ] amino ] -3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt A) 3- [ [1, 3-Dioxo-2-phenyl-3- (phenylmethoxy) propyl] -amino] -3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutyl ammonium salt Crude 3-amino-3-methoxy-2-oxo-l-azetidine-sulfonic acid, tetrabutyiammonium salt (431 mg, 10 see Example 74), containing borax, is dissolved in 3 0 ml of dry acetonitrile. Dry pyridine ( 317 yl) is added and the solution stirred well at -10°C under dry nitrogen, a-(Benzyloxycarbonyl)-phenylacetyl chloride (568 mg) in 3 ml of dry 15 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 20 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 25 3 ml of methylene chloride and chromatographed on a silica column using methylene chloride-methanol to give 4 70 mg of the title compound. ( ( -9&- 20524 B) 3-[[1, 3-Dioxo—2-phenyl -3- (phenylmethoxy) -propyl] amino] -3-methoxy-2-oxo-l-azetidinsulfonic acid, potassium salt 3-[ [1, 3-Dioxo-2-phenyl-3-(phenylmethoxy)-5 propyl] amino] -3-methoxy-2-oxo-l-azetidinesulf onic acid, tetrab-utylammonium salt (470 mg) is dissolved in 15 ml of 30% acetone: water and put through a Dowex 50WX2 (K+forra) column using the same solvent as eluent. The total eluate is evaporated in 10 vacuo to yield 34 5 mg of an amorphous solid which is lyophilized to an amorphous powder, melting point 100—120°C.
Anal, for C20HigOgN2SK Calc'd: C,49.37; H,3.94 N, 5 . 76; S,6.59 15 Found: C,49-08; H,4.00; N,5.58; S,6.29 Example 52 (i) -3- [ [ [ (Cyanomethyl) thio] acetyl ] amino] -3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt._3 20 To a solution of 3-amino-3-methoxy-2-oxo- 1-azetidinesulfonic acid, tetrabutyiammonium • salt (414 mg, see Example 74) in dry acetonitrile (50 ml) at -20°C is added diethylaniline (210 yl) and cyanomethylthioacetyl chloride (169 mg) . 25 After 10 minutes the solvent is removed under reduced pressure and a 0. 1M solution of tetrabutyiammonium 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 30 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 ( ( rtrsi -9?- 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 110-125°C.
Anal. Calc'd for C0H"nN^S_0CK: C,27.66; H,2.88; O 1U 3 2 0 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- [ (ami no carbonyl) amino ]-2-thiopheneacetic acid, yields the compound listed in Column II.
Column I 53. (+)-a-I(aminooxoacetyl)amino]-2-furanacetic acid 54 . (±) - a [ [ [ (cyanomethyl) amino]oxo- acetyl]amino]-2-thiopheneacetic acid 55. (R)-a-[(aminooxoacetyl)amino]-benzeneacetic acid 56. (R)-a-[(aminocarbonyl)amino]benzeneacetic acid J Column II [3S(i)—3—11[(aminooxoacetyl)-amino]-2-furanylacetyl]amino]— 2-oxo-l-azetidinesulfonic acid, potassium salt, melting point 212-215 C, dec. [3S (±)]-3-[[[[I(cyanomethyl)amino]-oxoacetyl]amino]-2-thienylacetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium'1 salt;' melting point 195-197 C, dec.' [3S(R*)]-3-[[[ (aminooxoacetyl) amino ]•-phenylacetyl]amino]-2-oxo-l-azetidine-sulfonic acid, potassium salt, melting point 207-209 C. [3S (R*)J-3-[[[(aminocarbonyl)amino]-phenylacetyl]amino]-2-oxo-l-azetidine-sulfonic gcid, potassium salt, melting point 225 C, dec. 2U52 4 Example 57 [3S ( — ) 3 —3— [ [2-(Methylthio) -1-oxopropyl] amino] -2-oxo-1-azetidinesulfonic acid, potassium salt Following the procedure of Example 31, Method II, but substituting (±)-2-(methylthio)- propanoic acid for (i)-a-azido-benzeneacetic acid, yields the title compound, mel-ting point 173°C,dec.
Example 58 [3S (R*)Q -3— CI [ [ 12 , 3—Dioxo-4 - I (phenylmethylene) amino] -1-piperazinyl] carbonyl ] amino]phenylacetyl ] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt (R) -a-1 [ 12 ,3-Dioxo-4- [ (phenylmethylene) amino] -1-piperazinyl]carbonyl]amino]benzeneacetic acid 15 (0.7 g) and (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 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 0°C. Stirring is continued 20 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 25 chromatography on HP-20 using water as eluent yields the title compound, melting point 19 3-194°C.
.. GC15 5£ 2 loo # i l ✓ A. ^ -srf- Example 59 [3S(Z)]-3-[[( 2-Ami no-4 - thiazolyl) [ (2-me thoxy-2-oxoethoxy) imino] acetyl] amino] -2-oxo-l-azetidine-sulfonic acid, potassium salt 5 (Z) -2-Amino-a- [ (2-methoxy-2-oxoethoxy) imino] - 4-thiazoleacetic acid (1.3 g) and (S)-3-amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (2.03 g; see Example 6A) is dissolved in 50 ml .of acetonitrile and 1.03 g of dicyclohexyl-10 carbodiimide dissolved in 5 ml of acetonitrile is added dropwise at-0°C. After 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 15 the equivalent amount of potassium perfluorobutane sulfonate. The title compound is isolated and purified by column chromatography on HP-20 using water as eluent, yielding the title compound melting point 195-198°C.
Example 60 [3S(R*) ] -3-[[[[[3-[I(4-Chlorophenyl)methylene] -amino] -2-oxo-l-imidazolidinyl] carbonyl] amino] -phenylacetyl]amino]-1-azetidinesulfonic acid , 25 potassium salt (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) in 100 ml of absolute diglyme (diethylene-glycoldimethyl ether), 1.5 g of (R)-a-[[[3-I[(4-3 0 chlorophenyl) methyl ene] amino] - 2-oxo-l-imidazolidinyl ] carbonyl]amino]benzeneacetic acid, an equivalent amount of dicyclohexylcarbodiimide, and Q.5 g of iol 2052 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 perfluorobutane sulfonate in acetone. The title compound precipitates and is filtered off, yielding 1.4 g of product melting point 217-222°C- Example 61 [3S(R*)]-3-[[[[[2-0x0-3- [ (phenvlmethylene) amino] -1-imidazolidinyl ] carbonyl] amino] phenylacetyl ] -amino]-1-azetidinesulfonic acid, potassium salt (S) -3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (2.25 g; see Example 6A) in 100 ml of dry dimethylformamide (DMF) , an equivalent of dicyclohexylcarbodiimide, 2.5 g of (R)-a-[[[2-oxo-3-[(phenylmethylene)amino]-1-imidazolidiny1 ] carbonyl]amino]benzeneacetic acid and 0.85 g of hydroxybenzotriazole are stirred together at ambiBnt temperature for 12 hours.
After this time, DMF is removed in vacuo and the residue is dissolved in 50 ml of acetone. 205240 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 224-226°C, dec.
Example 62 [3S(R*)]-3- [I [I [3- (Methylsulfonyl) - 2-oxo-l-imidazolidinyl] carbonyl] amino]phenylacetyl]-amino}-2-oxo-l-azetidinesulfonic acid, potassium salt 15 (s)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (2.25 g; see Example 6A) in 60 ml of dimethylformamide is stirred for 12 hours with 1.9 g of (R)-a-[ I [3-(methylsulfonyl) -2-oxo-l-imidazolidinyl ] carbonyl] amino]benzeneacetic 20 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 25 perfluorobutane sulfonate in 2 0 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 24 0-24 5°C, dec. —cci&frfr- -103-- 2052 a r\ Example 6 3 [3S(R*)]-3-[(Hydroxyphenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) in 100 ml of dry dimethylformamide is stirred for about 16 hours with 1.5 g of dicyclohexylcarbodiimide, 0.5 g of. hydroxybenzotriazole and 0.6 g of R-a-hydroxybenzeneacetic acid. The solvent is removed in vacuo and the residue is dissolved in 2 0 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 145-150°C, dec.
Example 64 [3S(S*)]-3-[(Hydroxyphenylacetyl)amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 63, 25 but substituting (S)-a-hydroxybenzeneacetic acid for (R)-a-hydroxybenzeneacetic acid, yields the title compound, melting point 195-197°C.
~GG±&5$ 2 0 5?'^ Example 65 [3S (±) ] -2-0XO-3- [ (phenyl ga-lfoacetyl) amino] -1-azetidinesulfonic acid, potassium salt (1:2) (S)-3-Amino-2-oxo-1-azetidinesulfonic acid, tetrabutyiammonium salt (2.25 g; see Example 6A) in 100 ml of dry diglyme., 2.4 g of triethylamine and 0.3 g of dimethylaminopyridine are cooled to 0°C. /(R)-a-(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 perfluorobutane sulfonate. After the addition of ether, the title compound precipitates and is filtered off, yielding 1.4 g of crude product; melting point 24 0-245°C dec . 2q water/acetone. 24 0-245°C dec., after recrystallization from Example 6 6 [3S(Z)-3-I[(2-Amino-4-thiazolyl) [ [ (diethoxy-phosphinyl )methoxy]imino]acetyl]amino]-2-oxo-2^ 1-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (2.25 g; see Example 6A) in 100 ml of dry dimethylformamide is treated with 1.87 g of (Z)-2-amino-a-[[(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 _G£1155-£— -lox- 20 52 removed in vacuo. The remaining oil is treated with an equivalent amount of potassium perfluorobutane 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 155-160°C/ dec.
Example 67 [3S (Z) ]—3— I [ (2-Amino-4-thiazolyl) [ [2- (1,1-dimethyl -ethoxy) -2-oxo-l-phenylethoxy] imino] acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt (S)-3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (2.25 g; see Example 6A) in 6 0 ml of dimethylformamide is stirred at room temperature with 2.4 g of (Z)-2-amino-a-[[2-(1,1-dimethylethoxy ) -2-oxo-l-phenylethoxy] imino] -20 4-thiazoleacetic acid, 1 g of hydroxybenzotriazole and 1.5 g of dicyclohexylcarbodiimide for 12 hours. The solvent is removed i_n vacuo and the residue is dissolved in 50 ml of acetone. The precipitated urea is filtered off and the mother liquor is 25 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 30 water/acetone (7:3) as eluent, yielding 1 g of product, melting point >250°C,dec. ( ( 2 0 52 Example 6 8 [3S(Z)]-3-[[( 2-Amino-4 -thiazolyl) [ ('lH-te trazol-5-ylmethoxy) imino] acetyl ] amino] -2-oxo-l-azetidinesulfonic acid/ potassium salt (S) -3-Amino—2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1-9 g; see Example 6A) in 60 ml of dimethylformamide is treated with 1.4 g of (Z) -2-amino-a- [ (lH-tetrazol-5-ylmethoxy) imino]-4-thiazoleacetic acid, 0.7 g of hydroxybenzotriazole and 1.4 g of dicycloh-exylcarbodiimide with stirring for 24 hours. After the solvent is removed in vacuo, the residue is dissolved in acetone and the precipitated 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 250°C, dec.
Example 69 [3S (Z) ] - 3- [ J (2-Amino-4-thiazolyl) [ (phenylmethoxy) -imino] acetyl] amino] -2-oxo-l-azetidinesulfonic acid , potassium salt (S) -3-Amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (1.5 g; see Example 6A) , 1.23 g of ( Z)-2-amino-a- [ (phenylmethoxy) imino]-4 -thiazoleacetic acid, 0.57 g of hydroxybenzotriazole 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 f .-G&tssr -iw- 2 0 5 2 A o 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 5 off and is purified by HP-20 column chromatography using water/acetone (9:1) as eluent, yielding 1 g of material, melting point 200°C, dec.
Example 7 0 [3S (Z) ] -3- [ I (2-Amino-^4-thiazolyl) [ (carboxymethoxy) -imino]acetyl]amino]-2-oxo-l-a2etidinesulfonic acid, potassium salt (1:1) I3S(Z) ] -3-[I(2-Amino-4-thiazolyl) 112-(diphenyl-methoxy) -2-oxoethox!yiimino] acetyl]amino] -2-oxo-l-15 azetidinesulfonic acid, potassium salt (1:1) (1.3 g; see Example 30) is mixed with 5 ml of anisole. At -15°C'25 ml of trifluoroacetic acid is added and the mixture is stirred for 10 minutes. Ether (100 ml) is added slowly at -10°C and subsequently 20 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 product is purified by chromatography on an HP-2 0 column, yielding 3.0 g of 25 the title compound, melting point 230-235°C, dec.
GC155f -lea- 2 0 5 2 4 f> Example 71 [3S(Z)]-3-1[(2-Amino-4-thiazolyl) [ [2-oxo-2-(phenylmethoxy) ethoxy] imino] acetyl] amino] -2-oxo-l-azetidine-sulfonic acid, potassium salt 5 Following the procedure of Example 28, but substituting (Z)-2-amino-a-[[2-oxo-2-(phenylmethoxy ) ethoxy] imino] -4-thiazoleacetic acid for (Z) -2-amino-a- [12- (diphenylmethoxy) -1,1-dimethyl-2-oxoethoxy]imino]-4-thiazoleacetic acid, yields 10 the title compound, melting point ca. 170°C, dec.
Example 7 2 [35(Z)]-3-[[[(2-Amino-2-oxoethoxy)imino](2-amino-4-thiazolyI) acetyl ] aminoh-2-oxo-l-azetidinesulf onic 15 acid, potassium salt Following the procedure described in Example 28, but substituting (Z)-2-amino-a-[(2-amino-2-oxoethoxy)imino]-4-thiazoleacetic acid for (Z)-2-amino-a-[[2-(diphenylmethoxy)-1,1-dimethyl-20 2-oxoethoxy] imino]-4-thiazoleacetic acid, yields the title compound melting point 205-210°C, dec.
Example 73 [3S(Z)]-3-[[(2-Amino-4-thiazolyl) (hydroxy imino)-25 acetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium salt A solution of 0.6 grams of 90% hydroxybenzotriazole in 100 ml of dimethylformamide is stirred for one hour with 10 grams of 4A 30 molecular sieves , filtered,and the filtrate added to a solution of 0.004 mole of (S)-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium ( salt (see Example 6A) in dimethylformamide. (Z) -2-Amino-a- (hydroxyimino) -4-thiazoleacetic acid (0.89 g) is added, followed by the addition of 0.91 g of dicyclohexylcarbodiimide.. The 5 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 10 on HP-20 resin yields '0.44 g of product, melting' point >240°C.
Example 74 3-Methoxy-2-oxo-3- [ (2-thienylacetyl) amino] -1 -azetidinesulfonic acid, potassium salt / A) 3-Ami no-3-me thoxy-2-oxo-1 -azetidine sulfonic acid, tetrabu'tylammonium salt (±) -3-Methoxy-3- [ [ (phenylmethoxy) carbonyl ] -20 amino]-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt ' (14 3 mg, see Example 49) is dissolved in 15 ml of dry methanol. Na_B,0 -10 H_0 u 2 4 7 2 (12 mg, 0.1 equiv.) is added, followed by 10% palladium on carbon (72 mg) . The mixture is 25 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. ( . ( -iiw- 2 0 52 B) 3-Methoxy-2-oxo-3- [ (2-thienylacetyl)aminoj-1-azetidinesulfonic acid,' tetrabutyiammonium salt 3-Methoxy-3-amino-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (102 mg) is dissolved 5 in 10 ml of dry acetonitrile. Dry pyridine (56.5 yl) is added and the solution is stirred well at -10°C under dry nitrogen. Thienylacetyl chloride (44 yl) in 1 ml' of dry acetonitrile is added dropwise. Jh 15 minutes the reaction 10 is complete as shown by thin layer chromatography. Potassium phosphate buffer (0.5 M, pH 5.5, 4.2 ml) and tetrabutyiammonium sulfate (8.5 mg, 0.1 equiv.) are added and most of the acetonitrile is removed in vacuo. The residue is diluted with water and 15 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 20 chloride-methanol, yielding 66 mg of the title compound.
C) 3-Methoxy-2-oxo-3-[(2-thienylacetyl) amino]'-1-azetidinesulfonic acid, potassium salt 3-Methoxy-2-oxo-3- [ (2-thienylacetyl)amino- 1-azetidinesulfonic acid tetrabutyiammonium 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 3 0 same solvent. The total'eluate is evaporated in vacuo to yield 95 mg of product which is GGi£5£- .$r_ *> ncn ^ 2052 lyophilized to give an amorphous powder, melting point 120-135°C.
Anal. Calc'd for C1(JH 1N2OgS2K: C,33.51; H,3.09; N, 7 . 8 2 ; S,17 . 89 Found: C,33.4 6 ; H,3.08; N,7.92; S,17.64 Example 75 [35 (Z) ] — 3— I I (2-Amino-4-thiazolyl)[ (carboxymethoxy) -imino]acetyl]amino]-2—oxo-1-azetidinesulfonic acid, 10 potassium salt [3S(Z)]-3-[I (2-Amino-4-thiazolyl) '[ [2-oxo-2-(phenylmethoxy) ethoxy] imino] acetyl] amino] -2-oxo-l-azetidinesulf onic acid, potassium salt (0.1 g; see Example 71) is dissolved in a mixture of 15 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. 20 m.p. 235°(dec.).
Example 7 6 3-[[(S)-[(Aminocarbonyl)amino]-2-thienylacetyl]-amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt, isomer A 25 To a solution of 3-amino-3-methoxy-2-oxo-l- azetidinesulfonic acid, tetrabutyiammonium salt (277 mg; see Example 46, method II, part A) in 15 ml of dry acetonitrile at -20°C is added pyridine (71 yl) 0.888 mmole) and (D)-2-amino-4-30 (2-thienyl)-5-(4 H)-oxazoline hydrochloride (166 mg). The mixture is stirred for 10 minutes and a second t^-Q 5 2 4 0 —G€rl aliquot of pyridine (71 yl) 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 5 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 10 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 3 5 mg of isomer A and 5 9 rug of isomer B is isolated.
Anal, for isomer A, m.p. 158-165°C.
Calc'd: cnHi3N4°7S2 • K" 1/2H20 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. 160-170°C(dec).
Calc'd for C-^H^N^O.^ • K • 1/2H20 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] -1-25 azetidinesulfonic acid, dipotassium salt To a stirred solution of crude 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (366 mg; see Example 74A) and 38 mg of borax in 35 ml of dry acetonitrile at -10°C 30 under nitrogen is added 0.53 ml of dry pyridine followed by a two minute addition of a solution of a-sulfophenylacetyl chloride monoetherate r ( (348 mg) in 8 ml of acetonitrile. After 20 minutes the. solvent is removed in_ vacuo and the residue is treated with 35 ml of 0.5 M pH 5.5 potassium phosphate buffer. Tetrabutyiammonium 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 of crude from a second run and the combined material is purified on a column 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 tetrabutyiammonium 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 20 5-210°C, dec.
Anal.. Calc'd for C^H^N^S^ - H20: C,29.51; H,2.89 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- (phenylmethoxy)-propyl] amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt (39 mg; see Example 51) is dissolved in methanol (5 ml). Anhydrous potassium ( 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 evaporated in vacuo to yield a glassy residue (34 mg) which is lyophilized to an amorphous powder, melting point I78-190°C,dec.
Anal, for C13H120gN2S K2* 0.5 H20: 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)-1-(methylthio)-2-oxoethoxy]imino]acetyl]-amino]-2-oxo-l-azetidinesulfonic acid, potassium salt . Following the procedure of Example 73, but substituting (Z)-2-amino-a-[[2-(1,1-dimethyl-ethoxy)-1-(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. -11J"- Bxample 80 (-) -3-Butoxy-3- [ [ (phenylmethoxy) carbonyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt A solution of 185 mg of 2-oxo-3-[N-chloro-N-[ (phenylmethoxy) carbonyl Jaraino] -1-azetidinesulfonic acid, tetrabutyiammonium salt (see Example 4 9A) in 1 ml of dimethylformamide is cooled to -78°C and 0.73 N lithium n-butoxide (3.8 ml) in n-butanol is added at -78°C. After 15 minutes, 0.5 M monobasic potassium phosphate buffer- is .added and the product extracted into dichloromethane (three 4 0 ml portions), dried over sodium sulfate, filtered and concentrated iri vacuo to give 179 mg of the corresponding tetrabutyiammonium salt of the title compound.
To a solution of 109 mg of the tetrabutyiammonium 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 6 6 mg of the title compound, melting point 186 .5-187.5°C, dec .
Example 81 [3± (E) ]-3-Methoxy-3-[[(methoxyimino) [ 2- [ [ (phenyl-methoxy)carbonyl]amino]-4-thiazolyl]acetyl]-amino-2-oxo-l-azetidinesulfonic acid, potassium salt A suspension of 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (Example 46, Method II, part A) (0.175 mmole) and sodium borate (0.0175 mmole) in 2 ml of dichloromethane at 0°C is treated with 28 jjl of pyridine and 0.175 mmole of (E)-a-(methoxyimino).-2- [[(phenylmethoxy)carbonyl]amino]-4-thiazolylacetyl chloride. After 1 hour, the 5 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 10 of the corresponding tetrabutyiammonium salt of the title compound.
The tetrabutyl ammonium salt (4 3 mg) is dissolved in 0.5 ml of acetone and 20 mg of potassium perfluorobutane sulfonate in 0.5 ml 15 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 144-146°C/ dec.
Example 8 2 13i(Z)]-3-Methoxy-3- [ [ (methoxyimino) [2- [ [ (phenyl-methoxy) carbonyl] amino] -4- thiazolyl] acetyl ] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 81, but 25 substituting (Z)-a-(methoxyimino)-2-[I(phenyl methoxy ) carbonyl]amino]-4-thiazolylacetyl chloride for (E)-a-(methoxyimino)-2- [ [(phenylmethoxy)-carbonyl] amino]-4-thiazolylacetyl chloride, yields the title compound, melting point 168-172°C, 30 dec. 230SZ4 -11?- Example 83 3-[[(R)-a-[[(4-Ethyl-2,3-dioxo-l-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-l-azetidinesulfonic acid, tetrabutyiammonium salt (Example 46, Method II, part A) in 30 ml of dry acetonitrile at -20°C under nitrogen is added 242 yl of dry pyridine followed by a solution of 352 mg of (R)-ct-[I(4-ethyl-2,3-dioxo-l-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 (Na^SO^) and evaporated leaving 54 6 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 17 3 mg) of the corresponding tetrabutyiammonium 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 -u- ' 2W52 applied to a column of HP 20-AG resin 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 171-172°C, dec.
Example 84 N- (3-Butoxy-2-oxo-l-sulfo-3-azetidinyl) -2-phenyl-acetamide, tetrabutyiammonium salt Following the procedure of Example 80 , but substituting N-chloro-N- (2-oxo-l-sulfo-3-azetidinyl) 2-phenylacetamide, tetrabutyiammonium salt (see Example 4 7A for preparation of the corresponding potassium salt) for 2-oxo-3-[N-chloro-N-[(phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, tetrabutyl ammo nium salt, yields the title compound as an oil: nmr (CDCl^) 3.62 (s, 2H,CgH^CH2) , 4.03 (ABq,2H,v=7cps, C-4 CH2), 6.98 (s,lH,NH) and 7.3 0 ppm (S,5H,CgH^).
Example 8 5 (R) -3-Methoxy-2-oxo-3- [ (phenylacetyl) amino] -1-azetidinesulfonic acid, potassium salt A 1M solution of dimethylformamide-sulfur trioxide complex is prepared by the slow addition of trimethylsilyl chlorosulfonate to dimethylformamide at 0°C followed by evacuation at 0.1 mm for 30 minutes at 0-25°C. 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 0°C. Cold 1M *G -11T- 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 5 is extracted twice with dichloromethane (discard) and 73 mg of tetrabutyiammonium bisulfate is * added. Extraction with dichloromethane (three 10 ml portions) gives a viscous oil after drying and evaporation in vacuo. Chromatography on 10 Mallincrodt CC-4 silica gel (_50:1) "using'2% methanol in dichloromethane" .as eluant gives 34 mg of (R)-3-methoxy-2-oxo-3-I(phenylacetyl) aminoj-1-azetidinesulfonic acid, tetrabutyiammonium salt. Ion exchange on Dowex 50W-X2 (K+, 10 15 equivalents) give the title potassium salt after lyophilization of the aqueous eluate: melting point 130°C, dec. , [a]^=+52° (C=0.5, water).
Example 86 (S)-3-[[[[(l-Ethyl-4-hydroxy-3-methyl-lH-pyrazolo-[3,4-b]pyridin-5-yl)-carbonyl]amino]phenylacetyl]-amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of Example 73, but 25 substituting a-[[(l-ethyl-4-hydroxy-3-methyl-lH- pyrazolo[3,4-b]pyridin-5-yl)carbonyl]amino]benzeneacetic acid for (Z)-2-amino-a-(hydroxyimino)-4-thiazoleacetic acid, yields the title compound, melting point 233-2 36°C, dec.
)XO ->T8- _GC15Sjr* 2057 4 Example 8 7 (R) -3-Ace tyl amino-3 -methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt A) 3-Ac e tyl ami no-1- [l-carboxy-2-methyl (propyl) ] -5 (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. 0]oct-2-ene-2-carboxylic acid (650. mg) and sodium bicarbonate (191 mg) in water is added a slurry 10 (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 temperature at 15 150-170°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 3.1) is adjusted to pH 2 with 1 N hydrochloric acid. After extracting 20 the aqueous solution five times with ethyl acetate, the combined extracts are dried (Na2SO^) and solvent is removed in_ vacuo to give an oil (48 7 mg) . Chromatography on silica gel yields the product (eluted in chloroform) as an oil 25 (381 mg) .
B) 3-Acetvlamino-1- [-1-(acetyloxy)-2-methyl(propyl)] ■ (3R)-3-methoxy-2-oxoazetidine The above azetidinone (464 mg) is dissolved 30 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 ( 12.1 (797 mg) is added- While 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 5 55-65°C for 15 minutes. The mixture is allowed to cool to room temperature, filtered through Gelite and the filter pad is washed well with acetonitrile. Solvent is removed in vacuo from the combined filtrate and washings. The 10 residue is taken up in water, and extracted four times with ethyl acetate. The combined extracts are dried (Na2SO^) and solvent is removed in vacuo to yield the desired product as an oil (382 mg) .
C) (R) -N- (3-Methoxy-2-oxo-l-azetidinyl)ac£tamide The above oil is dissolved in a methanol (10 ml) :water (1 ml) mixture, cooled in .an ice-methanol bath at -10° to -15°C and potassium 20 carbonate (194 mg) is added followed by sodium borohydride (53 mg) . After stirring at -15° to -8°C 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. 25 Exhaustive extraction with ethyl acetate, drying (Na2SO^) , and removal of solvent in vacuo yields an oil (2 24 mg) . Chromatography of the oil on silica gel, eluting with 5% methanol:95% methylene chloride giving an oil (169 mg) . The title 30 compound crystallizes from ether-pentane to give 131 mg of material, melting point 106-112°C (sintering 103.5°C) .
( ( Z$ e-> 4- D) (R)-3-Acetylamino-3-methoxy-2-oxo-1-azetidinesulfonic acid, potassium salt Under an argon atmosphere, 50 mg of (R)-3-acety1amino-3-methoxy-2-oxo-1-azetidine is placed in a flask and cooled to 0°C. AIM solution of dimethylformamide-sulfur 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 K-HPO. soultion and extracted 2 4 twice with 10 ml of methylene chloride. Tetrabutyiammonium 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 Na^SO^ and concentrated to afford 39 mg of product. The tetrabutyiammonium 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 EX4 6Ib and by isolation from natural sources (Ex. 165). ( -123.-Ex ample 8 8 it) -3- [ (Azidophenylacetyl) amino] -3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt A) (•_) -3- I (Azidophenylacetyl) amino] -3-methoxy-2- oxo-i-azetidinesuifonic acid, tetrabutyiammonium salt 3-Amino-3-methoxy-2-oxo-l-azetidinesulfonic acid tetrabutyiammonium salt (2Q2 mgs; see Example 74A) is dissolved in 20 ml of dry acetonitrile. 10 To the well stirred solution at -20°C under dry nitrogen are added dry pyridine CI67 pi] and a-azidophenylacetyl chloride (96 yl) . After 20. minutes, 0 . 5M pH 5.5 monobasic potassium phosphate buffer CI2 ml) is added and the acetonitrile 15 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 20 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 2 31 mg of the title compound.
B) (±)-3- [ (Azidophenylacetyl) amino]-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt (i) - 3 - [ (Azidophenylacetyl) amino] -3-methoxy-.2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (231 mg) in 30% acetone: water (15 ml) is 30 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 GC155-f -12^ 2 0 5 2 4- (.168 mg) as a 1:1 mixture of diastereomers.
Passage of this through, a 60 ml column of HP20-AG using water and water:10% acetone gives 71 mg of a 1:1 mixture of racemic diastereomers 5 and 7 0 mg of a 1:3 mixture of racemic diastereomers. The 1:1 mixture is lyophilized and dried in vacuo at 40°C to give the dried product as a hemihydrate, melting point dec. 130°C.
Analysis for C^2Hj2N5°6*K" H20: Calc'^: cr 35.9(1; 10 H, 3.26; N, 17.45; S, 7.98 Found: C, 35.94; H, 3.Q7; N, 17.24; S, 8.Q2 Example 8 9 3- [ (Azidophenylacetyl) amino] -3-methoxy-2-oxo-l-3_5 azetidinesulfonic, potassium salt, isomer A The 1:3 mixture of racemic diastereomers obtained in Example 8 8B 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 40°C, melting point 130°C, dec t as a mono-deuterate.
Example 90 [3± (R*) ] -3- [III (4-Ethyl-2 , 3-dioxo-l-piperazinyl) -25 carbonyl ] amino ] phenylacetyl ] amino] -3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt To a stirred solution of 3-amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (0.69 mmol; see Example 74A) in 30 ml of dry acetonitrile at -20°C under nitrogen is added 242 yl of dry pyridine followed by a solution of -12 S~ -QCTS3f~ 205 352 mg of (R)-a-I[(4-ethyl-2,3-dioxo-l-piperazinyl)-carbonyl] aminoj phenylacetyl chloride in 4 ml of acetonitrile- After 1 hour, 84 )jl of pyridine is added followed by 117 mg more of the acid 5 chloride in 1 ml of acetonitrile. The reaction is stirred for 2Q 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 10 three times with methylene chloride and the combined methylene chloride extract is dried (Na^SO^) and evaporated to a residue (54 6 mgl . Passage of this material through, a column of SilicAR CC-4, using methylene chloride and then 15 2%, 4%, and finally 6% methanol in methylene chloride provides two fractions (285 mg and 173 mg) of purified product in the tetrabutyiammonium 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 25 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 171-172°C, dec. 30 Analysis for CigH22N50gsK*H2°: 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 _G£155£ 2 0 5 2 4- 0 Lyophilization of the 21 mg fraction yields the title compound, melting point 171-172°C, dec.
Analysis for CigH22N50gSK-H20: Calc'd: C, 41.23 H, 4.37; N, 12.65 5 Found: C, 41.43; H, 4.11; N, 12.28 Treatment of the 285 mg fraction of tetra- 4- butylammonium salt with. Dowex 50-X2(.K ) provides 14 5 mg of potassium salt, which is combined with. the remaining 15 mg of the aforementioned 119 mg 10 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_. 1:1 J of diastereomers and an additional 4 2 mg of product as a mixture 15 (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 6 3 mg. 2 0 Example 91 " [3S (Z) ] -3-1 [ (Methoxyimino) [2- [I (phenylmethoxy) -carbonyl ]amino] - 4-thiazolyl] acetyl]amino] - 2-oxo-l-azetidinesulfonic acid, potassium salt To a solution of (S)-3-amino-2-oxo-1-25 azetidinesulfonic acid, tetrabutyiammonium salt (0.170 mmol; see Example 6A) and sodium borate (0.170 mmol) in 2 ml of methylene chloride at 0°C is added pyridine (62 yl) and (Z)-a-(methoxyimino) -2-[[(phenylmethoxy)carbonyl]amino]-4 -30 thiazoleacetyl chloride (0.51 mmol). The reaction mixture, after 40 minutes, is diluted with methylene chloride and water, followed by 0.1 M ( 52 4-0 tetrabutyiammonium sulfate buffered to pR 4 C5.1 ml). Th.e organic layer is separated and washed with water, adjusted to pH 2, water adjusted to pH 7, water saturated with sodium 5 chloride, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue is purified on SilicAR CC-4 silica gel CIO g) and the product eluted with 10% methanol/methylene chloride.
The tetrabutyiammonium salt, after dissolution in acetone/water, is passed through, an ion-exchange resin C8 ml, AG 50W—X2, K+ form 1Q0-200 mesh.). Removal of the water in vacuo from fractions 1-2 give 4Q mg of the title compound, melting point 172-174°C, dec.
Analysis for C-^^H^gN^OgS^K*H^O: Calc'd: C, 37.84; H, 3.33; N, 12.99; S, 11.87 Found: C, 37.95; H, 3.30; N, 12.73; S, 11.53 -2 0 Example 9 2 (-)-3-Butoxy-2-oxo-3-[(phenylacetyl) amino] -1-azetidinesulfonic acid, potassium salt A) 3- [Chloro (phenylacetyl) amino]-2-oxo-l-azetidine-25 sulfonic acid tetrabutyiammonium salt A solution of (S)-2-oxo-3-[ I (phenylmethoxy)-carbonyl]amino]-1-azetidinesulfonic acid, tetrabutyiammonium 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) -12$- 2052 at 0°C. After 1 hour 0.5 M monobasic potassium phosphate (.25 ml) is added and the mixture is extracted three times with methylene chloride (50 ml portions). The organic extracts are dried 5 over sodium sulfate, filtered and concentrated in vacuo to give 344 mg of the title compound.
B) (±) -3-Butoxy—2-bxo-3-p (phenyl acetyl) amino] -1-azetidinesulfonic acid, tetrabutylainmbhixfln salt 10 A solution of 3-Qchloro (phenylmethoxy). - carbonyl]amino]-1-azetidinesulfonic acid, tetrabutyl ammonium salt (344 mg) in dimethylformamide (5 ml) is added to 0.73 N n-lithium butoxide in n-butanol (6 ml) in dimethylformamide 15 (1 ml) at -78°C under an inert atmosphere. After 10 minutes the mixture is diluted with 0.5 M monobasic potassium phosphate solution (.17 5 ml) . After extracting three times with methylene chloride, the organic extract is dried over sodium 20 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- [ (phenylacetyl) ainino] -1-azetidinesulfonic acid, potassium salt (±)-3-Butoxy-2-oxo-3- [ (phenylacetyl)amino]-1-azetidinesulfonic acid, tetrabutyiammonium salt (4 3 mg) is dissolved in a water-acetone mixture 30 (.9:1) and placed on a cation exchange column (Dowex AGMP 50W-X2, 100-200 mesh, 5 g, K+ form). 7 -eets-sf 127 2052 The product is eluted with water and the eulate concentrated in vacuo to give 2Q mg of the title compound, melting point 122-125°C.
Analysis calc'd for ci5H]_gN2°6SK' !/2 H20: C' 44.66; 5 H, 4.96; N, 6.95.; S, 7.94 Found: C, 44.77; H, 4.76; N, 6.76; S, 7.75 Example 93 (i)-3-Ethoxy-2-oxo-3-T(phenylacetyl)aminoj-1-10 azetidinesulfonic acid, potassium salt 3-lChloro (phenylacetyl)amino]-2—oxo-1-azetidinesulfonic acid, tetrabutylammonium salt (2Q0 mg see example 92A) in dimethylformamide (4 ml) is added to 0.5 N lithium ethoxide in ethanol (12.20 ml) 15 at -78°C 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 20 sulfate, filtered and the solvent removed in vacuo. The residue is purified on SilicAR CC-4 silica gel (20 g) and the tetrabutyiammonium salt of the product (40 mg) is eluted with 2% methanol in methylene chloride. 25 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 30 in vacuo to give 25 mg of the title compound, melting point 94-9 6°C.
Analysis calc'd for C^H^IS^OgSK: C, 42.62; H, 4.10; N, 7.65; S, 8.74 Found: C, 40.36; H, 3.66; N, 6.77; S, 8.44 ( GGrb&Si— 205 2 4 0 Exadropl e 9 4 I3± (Z) ] -3-H C2-Ainino-4-tfcuiazolyl)(methoxy imino} -acetyl] aminoj -3-methbxy-2-bxd-l-azetidinesulfonic acid, potassium salt 5 3-Amino-3-methoxy-2-oxo-1-azetidinesulf onic acid, tetrabutyiammonium salt (see Example 46, method II, part Al is dissolved in acetonitrile (20 ml) and pyridine CI. mil and added to a vigorously stirring suspension of CZ)-a-Cmethoxy-10 imino)-2-amino-4-thiazol£acetyl chloride in acetonitrile (20 ml) cooled to Q-5°C. After stirring cold for 1 hour the mixture is diluted with 0.5 M monobasic potassium phosphate solution (100 ml) CpH of the mixture is 4.8) and solvent 15 is removed in vacuo. The residue is taken up in a minimal 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 20 elution with water. Further purification on HP-20 resin (20 0 ml) using water as the eluant gives 59 mg of the product as a powder after trituration with, acetonitrile-ether and then twice with ether. The product is an amorphous 25 powder that melts slowly and decomposes above 150°C.
Analysis calc'd for C^qH^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; 30 K' 10..11 "GClbb'f 205240 Example 95 [3R (R*) and 3S (_S*) ] -3-111 (Aminocarbonyl 1 amino] -phenylacetyl] amino]-3-methoxy-2-oxo-l-azetidinesulf onic acid, potassium salt A) Ct) -3-1 CAminophenyiacetyl) amino] -3 -methbxy-2-oxo-1-azetidinesulf onic' acid, inner salt (it) — 3— I (Azidophenylacetyl) amino] -3-me thoxy-2-oxo-l-azetidinesulfonic acid, potassium salt 10 (209 mg; see Example 88)1is dissolved in 60 ml of dry methanol. Anhydrous trifluoroacetic acid (0.6 ml) and 10% palladium on carbon (105 mgl are added and the mixture is hydrogenated for 1 hour. The catalyst is removed by filtration 15 and the filtrate is evaporated in vacuo to yield 271 mg of exude product.
B) J3R(R*) and 3S (S*) ] -3-I'[ T (Aminocarbonyl) amino] -phenylacetyl] amino]-3-methoxy-2-oxo-l-azetidine- 2 0 sulfonic acid, potassium salt (±)-3-[(Aminophenylacetyl)amino]-3-methoxy-2-oxo-l-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 approximately 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 160°C, dec.
Analysis for 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 ( ( -—GCI5.5f 2052 4 Exampl e "56 I3R (S*) and 3S (R* ) ]-3-TPI CAminocarbonyl) amino] -phenylacetyl] amino] - 3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt 5 Along with [3R(R*) and 3S(S*)]-3-[II (amino carbonyl) amino] phenylacetyl] amino-3-methoxy-2-oxo-l-azetidinesulfonic acid, potassium salt produced in Example 95,. there are produced 21 mg of J3R(S*1 and 3S CR* 11-3—111 (aminocarbonyl) -10 amino] phenylacetyl] amino] -3-methoxy-2-oxo-l-azetidinesulfonic acid/ potassium salt as a lyophilate, melting point 16Q°C dec.
Analysis for C^^^^O-^SK sesquihydrate: Calc'd: C, 35.65; H, 4.14; N, 12.81; S, 7.33 15 Found: C, 35.98; H, 3.87; N, 12.50; S, 7.32 Example 9 7 13^ (S* ) ] -3-Methoxy-3- 1111 [2-0X0-3-1 (phenylmethylene) -amino]-1-imidazolidinyl]carbonyl ]amino]-2-thienylacetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium salt To a stirred solution of 3-amino-3-me thoxy-2-oxo-azetidinesulfonic acid, tetrabutyiammonium 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 -20°C 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.
-GCT55J: " The reaction is stirred and allowed to Q°C over the course of 1 hour. zor 2 The reaction was diluted with a large volume of methylene chloride and then treated with 5 22 ml of 0.5 M pH 5.5 monobasic potassium phosphate buffer. The aqueous layer is washed with methylene chloride and the combined methylene chloride extract is washed with water, dried CNa2SO^), and evaporated to a residue (5Q8 mg} . This residue is chroma tographed 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 tetrabutyiammonium salt of the title compound.
To this salt (251 mg) in acetone is added a solution of 10 7 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/linm for 2 hours to give 95 mg of desired potassium salt as a mixture (ca. 1:2) of diastereomers having a melting point 200°C, dec.
Calc'd for C21H2JN608S2K: C, 42.85; H, 3.60; N, 14.28; S, 1Q.87 Found: C, 43.02; H, 3.74; N, 13.94; S, 10.71 i1 h — -13£- 52 4 0 Example 9 8 ( —ci.s) -4-Methyl-2-oxb-3-T[ (phenylmethoxy) carbonyl] -amino]-1-azetidihesiilfonic acid, potassium salt Al N-Benzyloxy-t.-boo*—allothreonine amide A solution of 6.9 g of d,1-t-boc-allothreonine and the free amine from 5.3 g of o-benzylhydroxyl-amine-HCl (M3.033 mole, ethyl acetate-sodium bicarbonate liberation) in 80 ml of tetrahydro-10 furan is treated with. 4.82 g of N-hydroxybenzotriazole and 6.5 g of dicyclohexylcarbodiimide in 20 ml of tetrahydrofuran. After stirring for about 16 hours at room temperature the slurry is filtered, concentrated in vacuo and chromatographed 15 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) (±—cis)-N-Benzyloxy-3-t-butoxycarbonylamino-4-methylazetidinone A solution of 6.8 g of N-benzyloxy-t-boc-allothreonine amide in 200 ml of tetrahydrofuran is stirred for about 16 hours with 5.24 g of 25 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 30 2.65 g of azetidinone. Rechromatography of the mother liquors and mixed fractions give an * "boc" is used to describe butoxycarbonyl GCT53T -13f- GCT53T— _ 205240 additional 0.6 g. Crystallization of a portion o twice from ether (-2Q C] gives the analytical sample of the title compound melting point 140—14 2°C.
CI ( ±—cis)-3-t-Butoxycarbonylamino-l-hydroxy-4 -methylazetidinone A solution of 3.2 g of cis-N-benzyloxy 3-t-butoxycarbonylamino-4 -methylazetidinone in 200 ml of 95% ethanol is stirred in an atmosphere 10 of hydrogen with. 0.7 g. of 10% palladium on charcoal. After 4Q minutes the slurry is filtered (uptake 24 9 ml] and the filtrate is evaporated and triturated with ether to give, in two crops, 2.05 g of solid, melting point 134-136°C.
D) ( + —cis-3-t-Butoxycarbonyl amino-4-methyl azetidinone A solution of 2.0 5 g of cis_-3-t-butyloxy-carbonylamino-l -hydroxy-4 -methyla zet idinone in 60 ml of methanol is treated with a total of 20 90 ml of 4.5 M ammonium acetate (40, 20 and 30. ml portions) and 4 5 ml of 1.5 M titanium trichloride (20, 10 and 15 ml portions) the second and third additions are made after 15 and 120 minutes, respectively. After 13 5 minutes the solution is 25 diluted with an equal volume of 8% sodium chloride and extracted with three 30 0 ml portions of ethyl acetate. The combined organic layer is washed with a mixture of 100 ml each of 5% sodium bicarbonate and saturated salt, dried, and 30 evaporated. Trituration with ether gives, 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 176-178.5°C.
E) (.+ -cis)-3-Benzyloxycarbonylamino-4-meth.ylazetidinane A solution of 1.55- g of cis-3-t-butoxy-carbonylamino-4-methylazetidinone in 4 ml each of methylene chloride and anisole is cooled to 0°C 5 and 5Q ml of cold trifluoroacetic acid is added.
After 90 minutes the solvents are evaporated in vacuo (benzene added and evaporated three times 1. The residue is dissolved in 25 ml of acetone, the initial pH (2.51 is raised to 7 with 5% sodium bicarbonate, and 2 ml of benzylchloroformate is added. The solution-is kept at 0°C and pH 7 for 4 hours and the acetone is removed in vacuo to give a slurry that is filtered. The filtrate is saturated with salt and extracted with methylene 15 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 20 of the title compound in fractions (100 ml each). 4-11. Crystallization of a small sample from ether gives the analytical sample, melting point 165-166°C.
E) ( ±-cis) -4-Methyl-2-oxo-3- [ p(phenylmethoxy) - carbonyl]amino]-1-azetidinesulfonic acid, potassium salt To a suspension of c.is-3-benzyloxycarbonyl-amino-4-methylazetidinone (0.75 g) in 7 ml each of dimethylformamide (dried with 4A sieves activated 30 at 320°C for 15 hours under argon flow) and methylene chloride (dried through basic A^O^) -ecrssr -13?- 205240 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 5 mixture is then stirrdd at room temperature under nitrogen for about 16 hours. The dimethylformamide is removed "in vacuo to give 4.6 g of residue which is dissolved in 300 ml of Q..5 M monobasic potassium phosphate solution (4Q°C for 10 10-15 minutes). The solution is cooled, passed through a column of HP-20 resin (.3 cm x 60 cm) with 400 ml of 0,5 M monobasic potassium phosphate 1 L of distilled water and (14:1) water:acetone to give 280 mg of product in fractions' 13 to 26 (1QQ ml 15 each]. Crystallization from MeOH: petroleum ether gives 757.5 mg of an analytical sample, melting point 214-215.5°C, dec.
Analysis calc'd for C^^^^SOgK: C, 40 .90 ; H, 3.72 N, 7.95; S, 9.10; K, 11.10 2 0 Found: C, 40.43; H, 3.60; N, 7.89; S, 8.69; K, 10 .82 Example 99 (3S-trans) -4-Methyl— 2-oxo-3- 11 (phenylmethoxy) cariionylj -amino]-1-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 133-135°C.
Analysis calc'd for C-^^j^^OgSK: 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 ■ T - :GC155r~ 2052 Example 1QQ (3 S-trans) - 4-Methyl - 2-oxo-3 -1 (phenylacetyl) aminoj -1-azetidinesulfonic acid, potassium salt A) (3S) -3-Amino-4-methyl-2-oxo-1 -azetidinesulf onic acid, tetrabutyiammonium salt C4S-trans) -4-Me thyl -2-oxo-3- [ I (phenylmethoxy 1 -carbonyl]amino]-1-azetidinesulfonic acid, potassium salt (352.4 mg; see Example 99] is dissolved in 2_o 20 ml of distilled water and treated with. 373.5 mg (1 mmole) of tetrabutyl ammonium hydrogen sulfate. After 10 minutes stirring at room temperature, the solution is extracted three times with. 10 ml portions of methylene chloride after saturation 15 with sodium chloride. The methylene chloride is dried over sodium sulfate and evaporated in vacuo to give 536 mg of the tetrabutyiammonium salt which is hydrogenated with 270 mg of 10% palladium on charcoal in 25 ml of dimethylformamide. The 2 0 mixture is filtered through Celite and washed twice with 2.5 ml portions of dimethylformamide to yield the title compound in solution.
B) (3S-trans)-4-Methyl—2-OXO-3- I (phenylacetyl)-25 amino]-1-azetidinesulfonic acid, potassium salt The crude (3S)-3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt from part A (the combined filtrate and washings) are treated at 0°C with 20 6 mg of dicyclohexylcarbodiimide, 153•mg of N-hydroxybenzotriazole, and 138 mg of phenylacetic acid. The reaction 52 4 0 mixture is stirred at Q°C for 1 hour and then at room temperature for 2 hours. The resulting precipitate is filtered, the filtrate is evaporated in vacuo, the residue is dissolved in 10 ml of 5 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} is a mixture of the potassium and tetrabutyiammonium s~alts of the title compound. 10 The solid is dissolved" in 5Q ml of 0.5 monobasic potassium phosphate arid'applied to an HP-20 column. Elution with water and- thjen water-acetone gives several fractions that are combined and evaporated to give the purified tetrabutyiammonium salt. An 15 aqueous solution of this material is passed through Dowex 50W-X2 (K+form) to give the title potassium salt (121.4 mg) . Trituration with, acetone-hexane yields 104.6 mg of the title compound, melting point 211-213°C. 2Q Analysis calc'd from ^' 41-72; H, 4.09; N, 8.11; S, 9.28; K, 11.32 Found: C, 41.70; H, 4.01; N, 8.07; S, 9.01; K, 11.02 Example 101' (cis) -4-Methyl-2-oxo-3—T (phenylacetyl) aminoj -1-azetidinesulfonic acid, potassium salt A solution of 320 mg of (±—cis)-4-methyl-2-oxo-3- [ I (phenylmethoxy) carbonyl]amino] -1- azetidine-30 sulfonic acid, potassium salt (see example 98) is prepared in 20 ml of water containing 483 mg of tetrabutyiammonium hydrogen sulfate and adjusted to pH 5.5.
GCl55f 2052 4.0 Extraction with, six 25 ml. portions of methylene chloride gives 517.3 mg of oil. A solution of this material in 15 ml of dimethylformamide is stirred with 400 mg of 1Q% 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, 169 mg of N-hydroxybenzotriazole and 24 7 mg of dicyclohexylcarbodiimide for 7.5 hours. The solvent is removed 10 in vacuo and the residue is dissolved in 2Q ml of acetone and filtered. The filtrate is treated with 25 ml of Q .Q44 M potassium iodide in acetone.
Dilution with an equal volume of ether gives a solid (330 mgl which is applied to a 50 ml HP-20 15 column in 20 ml of 0.05 M monobasic potassium phosphate. Elution with 20 0 ml of water followed by 1:9. acetone-water gives Rydon positive material in fractions (50 mil 6-10. Evaporation of fractions 7-9 gives 81 mg of solid. Recrystallization from 2 0 acetonitrile-water gives 4 6 mg of the title compound, which decomposes at >205°C. A second crop (6 mg) is obtained from the filtrate. A further 5 mg is obtained from fractions 6 and 10 by evaporation and recrystallization.
Analysis calc'd for C^2Hi3N2°5SK: ^2.84; 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 ; _ 2 0 52 4 0 Example 1Q2 £3S-J3g (z) ,4B].] -3-H (2-Amino-4-thiazolyl) (methoxyimino)-acetyl] amino] - 4 -me thyl-2-oxo -1 - a z e tidi nesul f on i c acid r pot ass jinn salt A) (3S—trans ) - 4-Methyl-2-oxo-3- I I (phenylmethoxy 1 -carbonyl] amino] -1-azet-i'dinesulfonic a'cidy tetrabutyiammonium salt (3S-trans) -4-Methyl-2-oxo-3-11 (phenylmethoxy ) -10 carbonyl]amino]-1-azetidinesulfonic. acid, potassium salt (352.4 mg; see Example 99) is dissolved in 20 ml of water and tetrabutyiammonium hydrogen sulfate (373.5 mg) is added. The aqueous solution is extracted three times with methylene chloride 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-[3ct(Z) ,46]]-3-1 I( 2-Amino-4-thiazolyl) (methoxv-2 0 imino)acetyl] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt A solution of 534.6 mg of (3S-trans)-4-methyl-2-oxp-3-I{(phenylmethoxy)carbonyl]amino]-1-azetidinesulfonic acid, tetrabutyiammonium 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 2 6.3 ml. The mixture is filtered and washed twice with 2.5 ml of dimethylformamide. The filtrate and washings 3Q (total ca. 25 ml) are stirred under nitrogen with 161 mg of (Z)-a-(methoxyimino)-2-amino-4-thiazole-acetic acid, 136 mg of N-hydroxybenzotriazole tl^l -IMf- and 164.8 mg of dicyclohexylcarbodiimide. The mixture is stirred under nitrogen for about 16 hours. The dimethylformamide is removed in vacuo and th.e gummy residue is dissolved in acetone and filtered to remove urea. To the filtrate is added a solution containing 272 mg (0.8 mmole) of perfluorobutanesulfonic acid, potassium salt in Q.8 ml of acetone. The slurry is diluted with.' an equal volume of ether and filtered to give 325.5 mg of crude product which is purified through chromatography on 75 ml of HP-20AG. Elution with 4 00 ml of water and 400 ml of (9:11 water: acetone mixture (50 ml fractions) gives 335 mg in fractions 3 to 10.
After, trituration with acetone-hexane, 9 7.3 mg of an analytical sample is obtained from fractions 3-5. Similar trituration of fractions 6-1Q gives an additional 9 0.4 mg of product as a solid.
Analysis calc'd for 2N5°gS2K: *-' 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, 13.9 5; K, 10.45 NMR(D 2 0) 1.57 (3H, d,j=7), 3.97 (3H,S), 4.30(1H, dof j=7.3), 4.7 0(1H, d. j=7), 6.95ppm(1H,S).
Example TO3 13S—[3a (Z) ,48] 1— 3— J[ (2-Amino-4-thiazolyl) [(1-carboxy-1-methylethoxy) imino] acetyl] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt A) N-Benzyloxy-t-boc-threonine amide A solution of 8.7 6 g of t-boc-threonine and the free amine from 6.4 g of O-benzylhydroxylamine HC1 (ethyl acetate-sodium bicarbonate liberation) in 100 ml of tetrahydrofuran is treated with 6.12 g GCJL55£ 205 2 4 0 of N-hydroxybenzotriazole and 8.24 g of dicyclohexylcarbodiimide in 2d ml of tetrahydrofuran.
The mixture is stirred under nitrogen for 2 6 hours, filtered, and evaporated in vacuo.
The residue is chroma tographed on a 30.0 g silica gel column (elution with chloroform and chloroform-ethyl acetate (3:11) yielding 7.2 g of compound. Crystallization from ether-hexane gives. 4.18 g of the title compound . - - B) ( 3S—trans) -N—Benzyloxy—3—t—butoxycarbonylamino-4-methylazetidinone A solution of 12.67 g of N-benzyloxy-t-boc-threonine amide, 11.5 g of triphenylphosphine, and 6.23 ml of diethylazodicarboxylate in 38 0 ml of tetrahydrofuran is stirred under nitrogen for about 16 hours. The solution is evaporated and chromatographed on a 9 00 gram silica gel column. Elution with chloroform-ethyl acetate 20 (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-Butoxy carbonyl amino-l~-hydroxy- 4-methylazetidinone A solution of 9.18 g of (3S-trans)-N-benzyloxy- 3-t-butoxy carbonyl amino-4-me thyl azetidinone in 300 ml of 9 5% ethanol is stirred in an atmosphere of hydrogen with 1.8 5 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.
/' I -14 £- D) C3S-trans) -3-t-Butoxycarbonylamino—4-methyl-azetidinone A solution of 4.9 8 of (3S-trans)-3-t-butoxy- carbony 1 amino-1-hydroxy-4-me thyl azetidinone in 20Q ml 5 of methanol is treated with 132 ml of 4.5 M ammonium acetate and then 6 6 ml of 1.5 M titanium trichloride and stirred for 4.5 hours. The 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 -Ben zyloxycarbonyl amino- 4 -me thyl -a zetidinone A solution of 3.3 g of C3S-trans) -3-t-butoxy- carbbnylamino-4-methylazetidinone in 10 ml each of dichloromethane and anisole is cooled to 0°C and 112 ml of trifluoroacetic acid is added. The solution is stirred for 9 0 minutes and evaporated 2 0 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. 25 The mixture is'stirred for 30 minutes at pH 7, diluted with 100.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 30 chloroforrn-ethyl acetate (4:1) gives 2.19 g of compound. Crystallization from ether-hexane yields 1.125 g of the title compound. -145- mm v F) (;3S-trans) —4-Meth.yl—2—oxo—3-.[1 (phenylmethoxy) -carbonyl] amino] -l-azetidiriesuifonic acid', tetrabutyiammonium salt A solution of 600 mg of (3S-trans)-3-5 benzyloxycarbonylamino-4-methylazetidinone in 2 ml of dimethylformamide is cooled to 0°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 80 ml 10 of cold 0.5 M monobasic potassium phosphate (adjusted to pH 5.51. The solution is extracted with three 50 ml portions of methylene chloride (discarded) and 8 68 mg of tetrabutyiammonium bisulfate is added. The resulting solution is 15 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- 13g (2 ) ,4 gj]—3— J I (2-Amino-4-thiazolyl) - [ (1-diphenylmethoxycarbonyl-l-methylethoxy) imino]-acetyl] amino] -4-methyl-2-bxo-i—azetidinesulfonic acid, potassium salt A solution of 1.54 g of ( 3S-trans) -4-methyl- 2-OXO-3- 11 (phenylmethoxy) carbonyl] aminoj -1-azetidine sulfonic acid, tetrabutyiammonium salt in 4 5 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 1.24 g of (Z)-2-amino-a-{ (.1-diphenylmethoxycarbonyl-1-methylethoxy)imino]-4-thiazoleacetic acid, 0.4 g of N-hydroxyhenzotriazole, and 580 mg of dicyclohexylcarbodiimide. The slurry is evaporated 5 in vacuo and the residue is triturated with 20 ml of acetone and filtered. The filtrate (plus 2 ml of washings) is treated with 8 68 mg of potassium perfluorobu-tanesulfonate in 3 ml of acetone. Dilution with. '75 ml of ether gives a 10 solid that is isolated by decantation of the mother liquor, trituration with ether, and filtration to give 0.91 g of tiie "title compound. The mother liquor is diluted with a further 100 ml of ether to give a second crop, 0.4 5 g, of the title compound.
H) [3S- [3a (Z) , 4 g]J-3- I f C2-Amino-4-thiazolyl) I (.1-carboxy-l-methyl ethoxy) imino] -acetyl ] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt 2Q A slurry of 140 mg of 13S- [3a (Z),43]]-3- I [ (2-amino-4-thiazolyl) [ (1-diphenylmeth'oxycarbonyl-1-methylethoxy) imino] acetyl] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt (first crop) in 0.5 ml of anisole is stirred at -12°C 25 under nitrogen and 2.5 ml of cold (-10°C) 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 -12°C, and allowed to warm to room temperature. The solid is isolated 2Q by centrifugation and washed twice with ether. A solution of this solid in 5 ml of cold water is -14 y- 2 0 5 ?:. 7 r immediately adjusted to pH 5.5 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 5 evaporation (acetonitrile added and evaporated three times) and trituration with ethar, m.p.^250°(dec.). Analysis calc'd for C13H15N508S2K2: C' 30-51'* 2.95; N, 13.69; S, 12.53; K, 15.28 Found: C, 29.63; H, 3.20; N, 12.96; S, 11.94; K, 12.78 NMR(D20) 1.46 (S, 6H) , ' 1.58 (1H, d/ j=1) 4.28(1H," d of q, j=7, 2.5), 4.67 (1H, d, j=2), 6.95ppm(S, 1H).
The remaining 1.22 g of I3S-l3a (.Zl,4pJJ-3-{I (2-amino-4-thiazolyl) I (1-diphenylmethoxycarbonyl-1-methylethoxy) imino]acetyl]amino—4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt (crops 1 and 2) are treated as above (4.2 ml anisole, 16 ml of trifluoroacetic acid, 13 minutes at 2q -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 10 4-133 25 Following the procedure of Example 11, but substituting the acid listed in column I for (Z) - 2-amino-a- [ I [hydroxy (phenylmethoxy) phosphinyl] -methoxy]imino]-4-thiazoleacetic acid, yields the compound listed in column II.
• • Column I j 104. 2-amino-a-[[2-(1,1-dimethylethoxy)-1-methyl-2-oxoethoxy] imino]-4-thiazoleacetic acid 105. (R)-a~[[(4-ethyl-2,3-dioxo-l-piperazinyl)-carbonyl]amino]-4-hydroxybenzeneacetic acid 106. (±)-a-[[ (4-ethyl-2,3-dioxo-l-piperazinyl)-carbonyl]amino]-2-furanacetic acid 107. (R)-a-[[(4-ethyl-2,3-dioxo-l-piperazinyl)-carbonyl]amino]-1,4-cyclohexadieneacetic acid 108 . (R)-a-[ [ [2,3-dioxo-4-[(phenylmethylene)amino]-1-piperazinyl]carbonylj amino]-4-hydroxybenzene acetic acid Column II 13S (Z)J — 3—UI'(2-amino-4-thiazolyl) [2- (1,1-dimethylethoxy)-l-roethyl-2-oxoethoxy]-imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt, hydrate (1:1); melting point 2B0°C, dec, [3S (R*)]-3-[[II(4-Ethyl-2,3-dioxo-4-piper-azinyl)carbonylj aminoj (4-hydroxyphenyl)-acetyl]aminoj -2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 197 C, dec. r I3S (±)J-3-III[(4-ethyl-2 ,3 -dioxo-4-piper-azinyl)carbonylj aminoj-2-furanylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 169-171°C. [3S (R*)]-3-[{1,4-cyclohexadien-l-yl[ [ (4-ethyl-2,3-dioxo-l-piperazinyl)carbonylj-amino]acetyl]amino]-2-o.xo-l-azetidine-sulfonic acid, potassium salt; melting point 185 C, dec. [3S(R*)]-3-[[[[[2,3-dioxo-4- I (phenylmethylene)-amino]-1-piperazinyl]carbonyl]amino](4-hydroxyphenyl)acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 194-197°C, dec.
Column 1 109. (Z)-2-amino-a-[(l-methylethoxy)imino]-4-thiazoleacetic acid 110. (Z)-2-amino-a-(phenoxyimino)-4-thiazoleacetic acid 111. (R)-a-[[[3-[[(4-hydroxyphenyl)methylene] ■ amino]-2-oxo-l-imidazolidinyl]carbonyl]-amino]benzeneacetic acid 112. (R)-a-[[[2-OXO-3-[(4-pyridinylmethylene)amino]-1 imidazolidinyl]carbonyl]amino]benzeneacetic acid 113. (Z)-2-amino-a-[[l-methyl-2-oxo-2-(phenylmethoxy) ethoxy]imino]-4-thiazoleacetic acid Column II [ 3S(Z)]-3-C[(2-amino-4-thiazolyl) [(l-methylethoxy) imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 195°C, dec. [3S(Z)]-3-[[(2-amino-4-thiazolyl)(phenoxyimino) acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 165 C, dec■ [3S(R*)]-3-[[[[[3— 11(4-hydroxyphenyl)methylene] amino] -2-oxo-i-imi'dazolidinyl] carbonyl] amino] -phenylacetyl]amino]-2-oxo-l-aEetidinesulfonic acid, potassium salt; melting point 236 C, dec. [3S(R*)]-2-OXO-3-[[t[[2-0x0-3- [ (4-pyridinylmethylene) amino] -1-imidazolidinyl]carbonyl]-amino]-phenylacetyl]amino]-1-azetidinesulfonic acid, potassium salt; melting point 230 C, dec. [3S(Z)]-3-[[(2-amino-4-thiazolyl) [ [ l-methyl-2-oxo-2-(phenylmethoxy)ethoxy]imino]acetyl]amino] 2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 110-115 C, dec.
• • Column I 114. (Z)-2-amino-a-[(cyclopentyloxy)imino]-4-thiazoleacetic acid 115. (R)-a-[[l-oxo-2-[[(phenylmethoxy)carbonyl]-amino]ethyl]amino]benzeneacetic acid 116. 2-furanacetic acid 117. (R)-a-[[(2-oxo-3-phenyl-1-imidazolidinyl)-carbonyl]amino]benzeneacetic acid 118. (R)-a-[[[2-OXO-3-(phenylmethyl)-1- imidazolidinyl]carbonyl]amino]benzeneacetic acid Column II [3S(Z)]-3-[[(2-amino-4-thiazolyl)[(cyclopentyloxy) imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt;, melting point 200 C, dec. [3S (R*)] -2-OXO-3- [ [phenyl [ [ [ [ (phenylmethoxy) carbonyl]amino]acetyl]amino]-Acetyl]amino]—1-azetidinesulfonic acid, potassium salt; melting point 260 C, dec.
(S) -3- [ (2-f.uranylacetyl) amino] -2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 110 C, dec. (3S)-2-OXO-3-[[[[(2-oxo-3-phenyl-l-imidazolidinyl)carbonyl]amino]phenyl-acetyl] amino]-1-azetidinesulfonic acid, potassium salt; melting point 217-222 C, dec. [3S(R*)]-2-OXO-3-[[[[[2-OXO-3-(phenylmethyl)-1-imidazolidinyl]carbonyl]amino]phenylacetyl] amino]-1-azetidinesulfonic acid, potassium salt; melting point 195-200 C Column I 119. a-[[[3-[(2-furanylmethylene)amino]-2-oxo-l-imidazolidinyl] carbonyl]amino]-4-hydroxy-benzeneacetic acid 120. (R)-a-[[[3-[[3-(2-furanyl)-2-propenylidene]-amino]-2-oxo-l-imidazolidinyl]carbonyl]-amino]benzeneacetic acid 121. (Z)-2-amino-a-[[[(ethylamino)carbonyl]oxy]-imino]-4-thiazoleacetic acid 122. (R)-a-[[[2 <3-dioxo-4-(phenylmethyl)-1- piperazinyl]carbonyl]amino]benzeneacetic acid 123.
(R)-a-[[[4-(1-methylethyl)-2,3-dioxo-l-piperazinyl] carbonyl]amino]benzeneacetic acid la Column II (3S)-3- [ [ [[[3-[(2-furanylmethylene)amino]-2-oxo-l-imidazolidinyl]carbonyl]amino](4r-hydroxyphenyl) acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 244 C, dec. [3S(R*)]-3-[[[[[3-[[3-(2-furanyl)-2-propenylidene]amino]-2-oxo-l-imidazolidinyl]-carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting, point' 195 C, dec. , [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[[(ethyl- ^ amino)carbonyl]oxy]imino]acetyl]amino]-2- 1 oxo-l-azetidinesulfonic acid, potassium salt; melting point 230 C, dec. [3S (R*) ] -3-{[ [ [ [2 ,3-dioxo-4- (phenylmethyl) -1-piperazinyl]carbonyl]amino]phenylacetyl]-amino]-2-oxo-l-azetidinesulfonic acid^ potassium salt; melting point 158-159 C, dec. [3S(R*)]-3-[[[[[4-(1-methylethyl)-2,3-dioxo-l-piperazinyl]carbonyl]amino]phenyl-acetyl]amino]-2-oxo-l-azetidinesulfonic O acid, potassium salt; melting point „ H 185-187°C Ul Column 1 124. (Z)-a-(methoxyimino)-2-furanacetic acid 125. (R)-a-[[[3-[[(dimethylamino)methylene]-amino]-2-oxo-l-imidazolidinyl]carbonyl] -amino]benzeneacetic acid 126. (R)-a-[[(3-ethyl-2-oxo-l-imidazolidinyl)-carbonyl]amino]benzeneacetic acid 127. (R)-a-[[[[[(4-methoxyphenyl)methoxy]carbonyl] amino]acetyl]amino]benzeneacetic acid Column II [3S(Z)]-3-[[(2-furanyl)(methoxyimino)-acetyl] amino —2-oxo-l-azetidinesulfonic acid, potassium salt) melting point 160 C, dec. [3S(R*)]-3-[[[[[3-[[(dimethylamino)-methylene]amino]-2-oxo-l-imidazolidinyl]-carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 195 C, dec. [3S(R*)]-3-[[[[(3-ethyl-2-oxo-l-imidazolidinyl]carbonyl]amino]phenyl-acetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium saltj melting point 185-190°C, dec. [3 S(R*)]-3-[[[[[[[(4-methoxyphenyl)-methoxy]carbonyl]amino]acetyl]amino]-phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 268 C, dec. o • "I Column I 128. (R)-a-[[[2-OXO-3-[[(phenylmethoxy)carbonyl]-amino]-1-imidazolidinyl]carbonyl]amino]-benzeneacetic acid 129. (Z)-2-amino-a-[(2-amino-l,l-dimethyl-2-oxoethoxy)imino]-4-thiazoleacetic acid 130. . (R)-a-[[[4-(1-methylethyl)-2,3-dioxo-l- piperazinyl] carbonyl]amino]-4-hydroxybenzene-acetic acid 131. (R)-a-[[[3 —(1-methylethyl)-2-oxo-l- imidazolidinyl] carbonyl]amino]benzeneacetic acid 132. (Z)-2-amino-a-[[2-(diphenylmethoxy)-1-methyl-2-oxoethoxy]imino]-4-thiazoleacetic acid Column II [3S(R*)]-2-OXO-3-t[[[[2-oxo-3-[[(phenylmethoxy) carbonyl]amino]-1-imidazolidinyl]-carbonyl]amino]phenylacetyl]amino]-1-azetidinesulfonic acid, potassium salt) melting point 175 C, dec. [3S(Z)]-3-[[[(2-amino-l,l-dimethyl-2-oxo-ethoxy)-imino](2-amino-4-thiazolyl)acetyl]-amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 210 C, dec. [3S(R*)]-3-[[[f[4-(1-methylethyl)-2,3-dioxo-l-piperazinyl]carbonyl]amino](4-hydroxyphenyl)acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 201-203 C, dec. [3S(R*)]-3-[[[t[3-(1-methylethyl)-2-oxo-l-imidazolidinyl] carbonyl]amino]phenyl-acetyl] amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 195-200°C, dec. [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[2-(diphenylmethoxy)-l-methyl-2-oxoethoxy]-imino]acetyl]amino]-2-oxo-l-azetidine-sulfonic acid, potassium salt; melting point 145-150 C Column I Column II 33. 5-methyl-3-phenyl-4-isoxazolecarboxylic acid (S)-3-[f(5-methyl-3-phenyl-4-isoxazolyl)-carbonyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt? melting point 230-232°C, dec. • m ro o f i N) O -155>- SGiSSS 205240 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-(diphenylmethoxy) -2-oxoethoxy] imino] acetyl] amino] -2-oxo-5 1-azetidinesulfonic acid, potassium salt, yields the compound listed in column II.
O <» Colunm I 134. [3S(Z)]-3-[[(2-amino-4-thiazolyl) [[2-(1,1-dimethylethoxy)-1-(methylthio)-2-oxoethoxy]-imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (see Example 79) 135. [3S(R*)]-2-OXO-3-[[[[[2-oxo-3-[[(phenylmethoxy) carbonyl]amino]-1-imidazolidinyl]carbonyl]-amino]phenylacetyl]amino]-1-azetidinesulfonic acid, potassium salt (see Example 128) Column II [3S(Z)]-3-[[(2-amino-4-thiazolyl)[[carboxy-(methylthio)methoxy]imino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt (Ii2)> melting point 165 C, dec. 3(S)— 3—[C[t(3-amino-2-oxo-l-imidazolidinyl)-carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 250 C, dec.
'J i-1 Ln IO O §■ Ui 3 r~h o' (' GC±55f~ 2 0 5?^ 0 Example 136 [3 a (,Z ) ,4a]-3-[[(2-Amino-4-thiazolyl) (methoxyimino) acetyl]amino]-4-methyl-2-oxo-i-azetidine-sulfonic acid, potassium salt A solution of 51.8 mg of (cis)-4-methyl-5 2-OXO-3-[[(phenylmethoxy)carbonyl] amino] -1- azetidinestilfonic 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 hydrogen 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 15 are combined and stirred for about 16 hours with 31 mg of (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid, 27 mg of N-hydroxybenzotriazole, and 31.5 mg of dicyclohexylcarbodiimide. The solution is evaporated in 20 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-2 5 tography on HP-20 AG (4 0 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 C10H12N5°6S2K: 30 : C,29.91; H,3.01; N,17.44 Found: C,29.30; H,3.31; H,16.66 NMR(D2q) 1.4 0(3H, d, j=7), 3.97 (3H,S), 4.46 (1H, apparent pentet, j=7), 5.37(1H, d, 7), 6.97 ppm(lH,S). -156*- ■ -GClS5-£ 205 7 Example 137 (3S-cis) -3—Amino-4-methyl-2-oxo~l~a-zetidinesulf onic acid A) t-Boc-1—allothreonine A suspension of 6.72 g of 1-allothreonine in 70 ml of 50% aqueous dioxane is treated with 9-45 ml of triethylamine and 18.1 g of t-butyl-10 pyrocarbonate. The resulting mixture is stirred at room temperature for 4 hours, and then diluted with 70 ml of water and 14Q ml of ethyl acetate. After thorough shaking, the layers are separated and the organic layer 15 is washed with 30 ml of 2:1 water:brine. Combined aqueous layers are then back-extracted with 70 ml of ethyl acetate- The aqueous layer is cooled in an ice bath and 10% potassium bisulfite solution is added to pH 2.3. The acidified 2Q solution is extracted with ethyl acetate (four 150 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-Meth.oxy—t-boc—1— allothreonine amide t-Boc-l-allothreonine (9.13 g) is dissolved in 85 ml of water and 41 ml of IN potassium hydroxide solution. Methoxyamine hydrochloride (5.22 g) and 8.67 g of l-ethyl-3 ,3-(dimethyl-aminopropyl)carbodiimide•HCl are added. The I *QC155J^- . -is,- 2 0 5 Z mixture is 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 5 layer is dried over anhydrous sodium sulfate and stripped of solvent to give 7.38 g of the title compound as a solid.
C) 0—Metha.nesulf onyl—N-methoxy-t-boc—1-alio threonine 10 amide N-Methoxy-t-boc-1 -allothreonine amide (7.32 g) is dissolved in 4Q m-1 of pyridine and cooled to -20°C under nitrogen. Methanesulfonyl chloride (3 ml) is added dropwise by syringe over a 15 5-minute period. The resulting mixture is slowly warmed to 0°C 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 HC1 solution, then 100 ml of 5% NaHCO^ solution. 20 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 solid.
D) (3 S.-cis) -3-t-B.utoxycarbonylamino-l.-me thoxy -25 4-methylazetidinone' O-Methanesulfonyl-N-methoxy-t-boc-1-alio-'. 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 6 5°C 3Q under nitrogen and stirred at this temperature for one hour. The reaction mixture is then ( (" r~n -m- 20514-3 filtered through. Celite and the filter cake is washed with ethyl acetate- The filtrate is concentrated and the residue is taken up in 2 5Q ml of ethyl acetate. The ethyl acetate 5 solution is washed with 10 0 ml of IN hydrochloric acid solution and 100 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 10 product.
E] (3S-cis) - 3-t—Butbxycarbonylamino—4-methyl-azetidinone Sodium (1.35 g) is dissolved in about 300 ml 25 of liquid ammonia at -50°C and 5-87-g of ( 3S~cis)- 3-t-Butoxycarboriyl amino-l-methoxy-4-methyl azetidinone in 3 5 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 quenched by adding 3.35 g of solid ammonium chloride in one portion. Ammonia is blown 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.
- GCl55f )6I 205": 4- F) (3 S-cis) -3-t-Butoxycarbonylamino-4-methyl-2-oxo-l-azetidinesulfonic acid, tetrabutyl ammonium salt I3S,4R] -3-t-Butoxycarbonylamino-4-methyl-5 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 stirring, an additional 1.8 g 10 of pyridine-sulfur trioxide complex is added and stirring is continued for 8Q hours. The reaction mixture is poured into 70Q ml of 0.5 M monobasic potassium phosphate solution and washed with methylene chloride (three 3Q0 ml ]_5 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 20 sulfate and stripped of solvent to give 10.7 6 g of the title compound as a gum.
G) (3S-cis) -3-Amino-4-methyl-2-oxo-l-azetidinesulfonic acid (3S~cis)-3-t-Butoxycarbonylamino-4-methyl- 2-oxo-l-azetidinesulfonic acid, tetrabutyl ammonium salt (10.7 6 g) is dissolved in 50 ml of 9 5-9 7% 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 ( ( -isa- mixture is stored in the freezer for about 16 hours and the frozen mixture is warmed to room temperature and stirred for an additional hour. The solid formed is filtered and washed with methylene 5 chloride to yield 9 82 mg of the title compound.
The filtrate is diluted with 1 liter of methylene chloride and kept at -20°C for 4 hours. The precipitate that forms is recrystallized from water-methanol-acetone to give an additional 10 167 mg of title compound.
NMR(D2o) 1-63 (3H, d, j= 6.5 cps), lR(nujol)1775 cm"1.
Example 138 13S- I3ct (Z) , 4qJ ] -3- rr2-Amino-4-thiazblyl) -methoxyimino) acetyl] amino-4-methyi-2-oxo-l-azetidinesulfonic acid, potassium salt 15 A solution of 201 mg of (Z)-2-amino-a- (me thoxy imino) -4-thiazoleacetic .acid and 153 mg of N-hydroxy-benzotriazole monohydrate in 3 ml of dimethylformamide is treated with 20 6 mg of dicyclohexylcarbodiimide. The mixture is 20 stirred at room temperature for 20 minutes under nitrogen and a solution of 180 mg of (3S~cis) -3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid and 0.14 ml of triethylamine in 2 ml of dimethylformamide is added (an additional 1 ml of 25 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-3Q sulfonate. Dilution with 10 ml of ether and filtration gives a solid product which is chroma-tographed on 200 ml of HP-20 resin eluting with 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 C,nH.-Nc0,SnK: C,29.91; H, 3.01; ± lz b b 2.
N,17.44 Found: C,30.03; H,3.21; N,17.06 NMR(D2Q) 1.40 (3H, d, j =6.5) , 3.98(3H,S), 4.48 1H, d of t }= 6.4, 5.5), 5.36 (1Hd, j=5.5) 6.97 (1H,S).
Example 139 (3S-trans)-3-Amino-4-methy1-2-oxo-1-azetidine-sulfonic acid A) Threonine, methyl ester, hydrochloride Under an atmosphere of nitrogen, a flask containing 15 500 ml of methanol is cooled to -5°C (ice/brine) and 13 0 ml (excess) of thionyl chloride is added at such a rate as to maintain the reaction temperature between 0° and 10°C. After recooling to -5°C, 59.5 g of 1-threonine is added and the mixture is allowed to reach 20 room temperature and stirred for 16 hours. The mixture is concentrated and evacuated at 10 ^ torr for 2 hours to yield a viscous oil. This material is used directly in the following step.
GCi&££ -16£- ^ ~ /■: r\ * .
B) Threonine amide ' The crude product from part A is dissolved in 2.5 1 of methanol and cooled to -5°C (ice/brine). The solution is saturated with ammonia gas, the 5 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 bicarbonate and 50 ml of water is added and the mixture is stripped to a viscous 10 oil.
C) ' Benzyloxycarbonyl thr eonine amide The crude product from part B (already containing the requisite amount of sodium 15 bicarbonate is diluted to a volume of 1 liter with water. To this rapidly stirring solution, 94 g (8 8 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 20 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 25 residue is then dissolved in 250 ml of hot ethyl acetate and 3 00 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 30 compound. -ley- 2 0 jser5 5f fT Q : D) Benzyloxycarbohyithreonine amide, O-lmesylate Under an atmosphere of argon, 100 g of benzyloxycarbonylthreonine amide is dissolved in 4 00 ml of anhydrous pyridine and cooled in 5 an ice/salt bath. To this stirring solution, 36.8 ml (54.5 g) of methanesulfonyl chloride is added over a 15 minute period. After 2 hours of stirring an additional 0.3 equivalents of methanesulfonyl chloride is added. The reaction 10 is then stirred for 1 hour 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 60°C for about 16 hours in a vacuum oven gives j_5 109 g of the title compound.
E) N-Sulfonyl benzyloxycarbonylthreonine amide, 0-mesvlate, tetrabutyiammonium salt A solution of 2-picoline (17.8 ml) in 2q 90 ml of methylene chloride is cooled to -5°C (ice-brine) and chlorosulfonic acid 5.97 ml is added at such a rate as to maintain the internal reaction temperature below 5°C. The resulting solution is added via canula, to a 25 suspension of 7.56 g of benzyloxycarbonylthreonine amide, G-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 ( ( -Geir55f 2 01 W organic layer is then washed once with 10Q ml of buffer solution and the combined aqueous phases are treated with 10.2 g of tetra-n-butyl-arnraonium hydrogensulfate and extracted with 5 methylene chloride Cone 300 ml portion and two 150 ml portions). 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—Amino—4-methyl-2-oxo—1-azetidine sulf onic 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 15 of N-sulfonyl benzyloxycarbonylthreonine amide, O-mesylate, tetrabutyiammonium 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, 20 diluted with 50 ml of water and 100 ml of methylene chloride and the phases split. The resulting organic phase is. dried over sodium sulfate and concentrated to yield crude C3S-trans)-3-benzyloxycarbonylamino-4-methyl-2-oxo-1-azetidine 25 sulfonic acid, tetrabutyiammonium 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 30 Celite with 50 ml of ethanol used as a rinse.
GC155f -i6y- 2 0 5X^0 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 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. > 218 "dec . , [a] ' = —41.1(C=1, 1^0) .
NMR(D2Q) 1.53(3H, d j-7), 4.80(2H,M).
Examples 140-143 Following the procedure of Example 13 8, but substituting (3S-trans)-3-amino-4-methyl-2-oxo-15 1-azetidinesulfonic acid for (3S-cis)-3-amino-4- methyl-2-oxo-l-azetidinesulfonic acid and the acid listed in column I for (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II. 140. (R)-a-[[[3-[(2-furanylmethylene)amino]-2-oxo-l-imidazolidinyl]-carbonylj amino]-benzeneacetic acid 141. (R)-a-[[(4-ethyl-2,3-dioxo-l-piperazinyl)-carbonyl]amino]benzeneacetic acid 142. (2)-2-amino-a-(hydroxyimino)-4-thiazoleacetic acid 14 3. [t) -ct- t (aminooxoacetyl) amino] -2-thiopheneacetic acid [ 3S— [ 3d (R* ) ,40]]-3-[[[[[3-[ {.2-f uranyl-methylene)aminoj-2-oxo-l-imidazolidinylJ -carbonyl]amino]phenylacetyl]aminoj-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 213 C, dec. [3S-[3a(R*),40]]-3-[[[[(4-ethyl-2,3-dioxo-l-piperazinyl )carbonylj aminoj phenylacetyl]-amino]-4~methyl-2-oxo-l-azetidinesulfonic acid, potassium salt; melting point 177 C,dec. [3S—[3a(z),40]]-3-f[(2-amino-4-thiazolyl)-(hydroxyimino)acetyl]aminoj-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt; melting point 2 30 C. [3S- [3a (±) ,40] ]t3«-I[[ (aminooxoacetyl) amino]-2-thienylacetyl] amino] r-4-itiethyl-2-oxo-l-azetidine .sulfonic acid, potassium salt; melting point 135 °C, dec. f ( 5 Anal, calc'd. for C_ AH_ , N^.0, nS_K -1 H_0: C, 38 .33; 2U Z± fa J.U 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 4 -w" Example 14 4 [3S- [3a(Z) ,4b]]~3-[[(2-Amino-4-thiazolyl) [[1,1-dimethyl-2-[(4-nitrophenyl)methoxy]-2-oxoethoxy]-imino]acetyl]amino]-4-methyl-2-oxo-l-azetidine-5 sulfonic acid, potassium salt To a slurry of (3S-trans)-3-amino-4-methyl-2-oxo-l-azetidinesulfonic acid (0.36 g; see example 139) in dry dimethylformamide (30 ml) under nitrogen at 26°C is added triethyl-10 amine (309 yl). After about 5 minutes a clear solution is obtained" and Qz)-2-amino-a-III, 1-dimethyl-2-1 (4-nitrophenyl )methoxy] -2—oxoethoxy] -imino]-4-thiazoleacetic'acid (0.816:g) is added followed by N-hydroxybenzotriazole (0.334 g) ^5 and dicyclohexylcarbodiimide (0.453 g). The mixture is stirred for twelve hours at 26°C, 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 g) 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). guts: 7 c -.I--5-* 2 0 ET t: * Example 145 [3a(Z),4g]-3-[[(2-Amino-4-thiazolyl) [ (1-carboxy-1-methylethoxy) imino] acetyl ] amino] -4-methyl-2- oxo-l-azetidinesulfonic acid, potassium salt (1:2) A) [3a(2) ,4a]-3-[12-Amino-4-thiazolyl) [ (1- diphenylmethoxycarbonyl-l-methylethoxy) imino] - acetyl ] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt • A solution of {cis)-4-methyl-2-oxo-3-[[(phenyl methoxy ) carbonyl] amino ] -1-azetidinesulfonic acid, tetrabutyiammonium salt (201 mg.; prepared from the corresponding potassium salt of example 98 as described in example 136) in 5 ml of dimethyl- formamide is stirred with 9 0 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 14 6 mg of (Z) -2-ammo-o- [ 1 -diphenyline thoxy carbonyl -1-methyl ethoxy) iminoj - 4-thiazoleacetic acid, 73 mg of dicyclohexylcarbodiimide 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 2Q of the title compound.
■GG±&5£ ^ 2 0 52 4 B) [3a(Z)/4g]-3-[I( 2-Amino-4-thiazolyl) [1-carboxy — 1-methylethoxy]imino]acetyl]amino]-2-methyl-4-oxo-1-azetidinesulfonic acid, potassium salt (1:2) A slurry of 186 mg of [3a (Z) ,4a]-3- [ [2-amino-4-thiazolyl)[(1-diphenylmethoxycarbonyl-l-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt in 0.6 ml of distilled anisole is cooled to -12°C and 3.0 ml of distilled trifluoroacetic acid (at -10^C) 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 -10°C 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 product,, as a hygroscopic solid.
Anal. Calc'd for C, .N^O S.-2K: C, 30.51; H, 2.95; 13 15 o o 2.
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 -mi- onr .
V/ "... '-v* ^ Example 14 6 [3S-[3a(Z),4b]]— 3—[[(2-Amino-4-thiazolyl)- 1(1-carboxy-1— methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid 5 [3S-[3a(Z),4f}]]-3-[[(2-Amino-4-thiazolyl)- [(1-carboxy-l-methylethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt (87.3 mg; see example 103) is dissolved in 1.38 ml of water, cooled to 0°C, 10 treated with 0.34 ml- of IN hydrochloric acid and the resulting crystals separated by centrifugation. The wet solid is dissolved in methanol, filtered, concentrated to about 0.5 ml and mixed with 1 ml of water, giving 55.9 mg of the title compound.
Example 14 7 [3S- [3a (Z) ,40] ]-3—[ [ (2-Amino-4-thiazolyl) [ (1-carboxy-l-methylethoxy) imino]-4-methyl-2-oxo-l-azetidinesulfonic acid, sodium salt 20 A 99-7 sample of J3S-[ 3a (Z )., 4 g] J - 3- [ [ (2-amino-4-thiazolyl) [(1-carboxy-l-methyl-ethoxy)imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid is mixed with 0.20 7 ml of IN sodium hydroxide and 25 the resulting mixture is gently warmed to dissolve the remaining 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 4 7.9 mg, a third from 0.24 ml of methanol and 0.24 ml of absolute ethanol gives 44.8 mg, and "GG±5Erf 171 2 0 52 4 0 a fourth from 0. 225 ml of methanol and 0.225 ml of absolute ethanol gives 38.8 mg. The solid is dried at 20°C and 0.01 mm of Hg for 18 hours and then equilibrated with atmospheric moisture for 2 4 hours, giving 4 0.9 mg of the title compound.
Example 14 8 [3S-[3a(Z) ,46] ]-3-[ [ (2-Amino-4-thiazolyl) I (1-carboxy-1-methylethoxy) imino] -4-methyl-2-oxo-l-azetidinesulfonic acid, disodium. salt A 3.00 g sample of [3S- [3a(Z),4B]]-3-[[(2-Amino-4-thiazolyl)- [ (1-carboxy-l-methyl-ethoxy) imino] acetyl ] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, (see example 146), is suspended in 30 ml of water and titrated with IN 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 2.3 8 g of solid. Partial equilibration (24 hours) with atmosphereic moisture gives 2.54 g of the title compound.
Examples 149-151 Following the procedure of Example 138, but substituting the compound listed in column I for (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II.
Column I 149. (R)-a-[[[2,3-dioxo-4-[[(phenylmethoxy)-carbonylj amino]-l-piperazinyl]carbonyl]-amino]benzeneacetic acid 150. (R)-a-[t[[(2 —furanylmethylene)amino]-2-oxo-imidazolidinyl]carbonyl]amino]benzeneacetic acid 151. (R)-a- [ [(4-ethy1-2,3-dioxo-l-piperazinyl)-carbonyl]amino]benzeneacetic acid Column II [3S-T3a (R*) , 4a]H4- [ [ [2-[ (4-methyl-2-oxo-l-sulfo-3-azetidinyl) amino]-2-oxo-l-phenylethyl]amino]carbonyl]-2,3-dioxo-l-piperazinyl] carbamic acid, phenylmethyl ester, potassium salt, melting point 191 C,dec [3S-[3a(R*),4a] ]-3-[[[[[3-[(2-furanylmethylene amino]-2-oxo-l-imidazolidinyl]carbonyl]-amino]phenylacetyl]amino]-4~methyl-2-oxo-1-azetidinesulfonic acid, potassium salt [3S—[3a(R*),4 a]]—3—£[[[(4-ethyl-2,3-dioxo-l-piperazinyl )carbonyl]amino]phenylacetyl]-amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt -175^ Gei-sSf"-" 2 C H 2 4 Example 152 [3S- I3a (Z) ,4a] ]-3- 11 (2-Amino-4-thiazolyl) C (1-carboxy-l-methylethoxy) imino] acetyl] amino] -4-methyl-2-oxo-l-azetidinesulfonic acid,■potassium salt (1:2) A) [3S-[3a (Z),4a]]-3-[I (2-Amino-4-thiazolyl)-I (1-diphenylmethoxycarbonyl-l-methylethoxy) imino]-acetyl ] amino] -4-methyl-2-axo-l-azetidinesulf onic acid, potassium salt .
A solution of 440 mg of (Z)-2-amino-a- [ (1-carboxy-l-me thyl ethoxy) imino]-4-thiazoleacetic acid and 153 mg of N-hydroxybenzotriazole mono-hydrate in 3 ml of dimethylformamide is treated with 206 mg of dicyclohexylcarbodiimide. The 15 mixture is stirred at room temperature for 30 minutes under nitrogen and a solution of 180 mg of (3S-cis ) -3-amino-4-me thyl-2-oxo-1-azetidinesulf onic acid, (see example 137) and 0.14 ml of triethylamine in 2 ml of dimethylformamide 20 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 and triturated with 12 ml of acetone. The slurry is filtered and the solid washed 25 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 30 washed with ether to give 656 mg of the title compound. -i7o n c ^ B) [3S- [3a (Z) ,4a] ] — 3— 11 (2-Amino-4-thiazolyl) -[ (1-carboxy-l-methylethoxy) imino]acetyl]amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt (1:2) A slurry of 656 mg of [3S- [3a(Z),4a] ]-3- [ [ (2-Amino-4-thiazolyl) [ (1-diphenylmethoxycarbonyl-1-me thyl ethoxy) imino] acetyl] amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt in" 2.3 ml of distilled anisole is cooled to -12°C 10 and 11.5 ml of trif luoroacetic acid (precooled to -10°C) is added. The solution is stirred for 15 minutes and 46 ml of ether followed by 23 ml of hexane are added. After 5 minutes at -10°C and stirring for 15 minutes at room temperature, 15 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 immediately 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 combined and lyophilized to give 239 mg of the title compound as a solid. Anal. Calc'd. for c13H1508N5S2K2•1/2 H20: 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, J=75), 1.46(6H,S), 4.48(lH,dof J = 75 * 5.5), 5.34(1H, d, j =5.5) 6.96ppm(1H,S) .
-GCT53T -17?- 7Qc?V Example 153 (±)-3-Amino-4,4-dijnethyl-2-oxo-l-azetidinesulfonic acid A) (±)-4,4-Dimethyl-2-oxo-l-azetidine-tertbutyl-diphenylsilane A solution of 40.5 ml of t-butylchlorodiphenyl silane in 112 ml of dimethylformamide is cooled to 0°C. To this is added 22 ml of triethylamine. 10 A solution of 12.87 g of 4,4-dimethy1-2-azetidinone 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 5°C under argon. This mixture is poured 15 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 10 0 ml portions of 0.5M monobasic potassium phosphate buffer, one 150 ml portion of sodium bicarbonate 20 solution, two 150 ml portions of water and one 150 ml portion of saturated sodium chloride solution. The solution is dried over sodium sulfate and concentrated in vacuo to yield 33.0 3 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-butyllithiura and 11ml of dry tetrahydrofuran is prepared at -50°C under argon in a 100 ml three-necked flask. A solution of 0.083 g of triphenylmethane in 1 ml of tetrahydrofuran is GC155f -17 8^ ^ ' 2 05 2 4 0 added. The resulting solution is cooled to -60°C, and 1.0 ml of diisopropylamine is added dropwise by syringe. This is stirred for 15 minutes and then cooled to -78°C. A solution of 2.3 g of 5 (-)-4,4-dimethyl-2-oxo-l-azetidine-tertbutyl- diphenylsilane in 8 ml of tetrahydrofuran is added slowly by syringe. The resulting solution is stirred for 20 minutes at -78°C, during which time heavy precipitation occurs- and uniform stirring 20 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 -78°C for 20 minutes, and 2 ml of trimethylsilyl chloride is added dropwise. 25 The reaction mixture is warmed to ambient temperature and stirred for 1 hour. Then the mixture is cooled to 0°C and poured into 150 ml of 0°C 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.
I ( . -17'- 20:: "I < C) (it) -3-Azido-4 ,4 -dimethyl-2-oxo-1-azetidine In a 50 ml three-necked flask, 1.52 g of (i)-3-azido-4,4-dimethyl-2-oxo-l-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 minutes 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 0°C, 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 iri 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-l-azetidine-sulfonic acid, tetrabutyiammonium salt To 0.100 g of (±)-3-azido-4,4-dimethy1-2-oxo-l-azetidine at 0°C 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 4 5 minutes. The solution is then poured into 20 ml of 0.5 M pH 5.5 monobasic potassium phosphate buffer.
G€ir5"5"f IVO 2 0 5240 This is washed with three 20 ml portions of methylene chloride (discarded) and Q.237 g of tetrabutyiammonium hydrogen sulfate is added to the aqueous solution. This was extracted with 5 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 concentrated in vacuo to yield 0.31 g 10 of oil, which appeared by nmr to be 50% dimethylformamide and 50% of "the title compound.
E) {.-) -3-Amino-4 , 4 -dimethyl-2-oxo-l-azetidine-sulfonic acid A solution of 0.155 g of (±)-3-azido-4,4 - dimethyl-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 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 20 rinsed with methylene chloride which is combined with the methanol solution. This clear solution is treated with 0.12 3 ml 9 7% formic acid. Upon addition of the acid the solution immediately becomes cloudy. After standing for 1 hour at 5°C, 25 the solid is filtered off to yield 0.0664 g of the title compound, m.p. 200-202°C(dec.) .
NMR(D 2 o} 1■6 4 (3H,S) , 1.68(3H,S), 4.42(1H,S), IR(KBr) 1765cm"1.
)S7 55 Example 154 [3± (z) ] -3- [ [ (2-Amino-4-thiazolyl) (methoxyimino) -acetyl] amino] -4 ,4-dimethyl-2-oxo-l-azetidinesulf onic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate (50 mg) and (Z) -2-amino-a-(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 (±) -3-amino-4 , 4-dimethyl -2-oxo-l-azetidinesulfonic acid (57 mg; see example 153) is added as a solid followed by triethylamine dropwise (0.05 ml). The reaction is stirred at ambient temperature for 16 hours. The dimethylformamide is removed under high vacuum at 30°C, 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-20AG column. Elution with 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 • K: C, 31.80; H, 3.40; N, 16.86; S, 15.43 Found: C, 29.47; H, 3.48: N, 14.98: S. 13 :-Ge±55£ -133*- 2 0 S 2 4 . (1) -4, 4-Dimethyl-2-oxo-3- [ (phenylacetyl)amino]-1-azetidinesulfonic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate 5 (45 mg) and phenylacetic acid (40 mg) in 0.5 ml of dimethylformamide is treated with dicyclohexylcarbodiimide (61 mg) under argon at ambient temperature. The resulting mixture is stirred for 1 hour and {-) -3-amino-4 ,4-dimethyl-2-oxo-10 1-azetidinesulfonic acid, (52 mg; see example 153) is added as a solid, followed by triethylamine dropwise (0.04 ml). The reaction is stirred at ambient temperature for 24 hours. The dimethylformamide is removed under high 15 vacuum at 30°C 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 2 0 yielding the title compound as a powder.
Anal, calc'd. for C^H^N^SK: 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 (D 2 0) 1.33 (S,3H), 1.58(S,3H), 3.68(S,3Hj, 4.70 (S,1H), 7.56ppm (broad S, 5H).
Example 156 (3S-trans)-3-[[(2-Amino-4-thiazolyl)oxoacetyl]-amino]-4-methyl-2-oxo-l-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° to -20°C) is added (2-amino-4-thiazolyl)glyoxylic acid, triethylamine Anal. Calc'd for CoH„N.0cS„K (372.42): C, 29.02; 9 9 4 6 2 H, 2.44; N, 15.04; S, 17.22; K, 10.50 20 Found: C, 28.87; H, 2.62, N, 14.85; S, 15.09; K, 10.81 Example 157 [3S(R*)]-3-[[[ (Aminoacetyl)amino]phenylacetyl] amino]-25 2-oxo-l-azetidinesulfonic acid, potassium salt, trifluoroacetate (1:1) salt The deprotection of 13S(R*)J-3-JI IIIII(4-methoxyphenyl)methoxy]carbonyl]amino]acetyl] amino]-phenylacetyl] amino]-2-oxo-l-azetidinesulfonic 30 acid potassium salt (see example 127) using trifluoroacetic acid and anisole yields the title compound, melting point 165°C, dec.
T T GCl55f~~ 182jL- 2052 Example 158 (35-trans) -3-Methoxy-4-methyl-2-oxo-3- [ [ (phenylmethoxy) carbonyl] amino] -l-azetidinesulfonic acid, potassium salt A) (35'-trans) -4-Methyl-3-iriethoxy-2-oxo-4-['[ (phenylmethoxy) carbonyl] amino] azetidine A solution of ,2.5 g (0.0106 mole) of (3R-trans) -4-methyl-2-oxo-3 [ [ (phenylmethoxy) -carbonyl] amino] azetidine (prepared from d-10 threonine in 12.6% yield essentially as described for the racemic cis isomer in Example 98C) in 112 ml of 4% borax in methanol is cooled to 0°C and 3.5 ml of t-butyl hypochlorite is added. After 20 minutes the 15 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 7 50 ml portions) , saturated salt, dried and evaporated to give 2 0 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 -78°C and diluted with 4 0 ml of dry tetrahydrofuran. Over 30 seconds a solution of the above chloro-25 amide in 20 ml of tetrahydrofuran (-78°C) are added via syringe. After 20 minutes at -78°C, 2 ml each of acetic acid and trimethyl phosphite are added. After 4 0 minutes at room temperature the solution is poured into 50 0 ml of water 3 0 and extracted with ethyl acetate (two 3 00 ml portions). The organic layer is washed with GCl55? — -18 3"- 2052 # 0 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 -I [(phenylmethoxy)carbonyl]amino-r-azetidinesulfonic acid, potassium, salt ■ A solution of 800 mg (0.00303 mole) of (3S-trans)-4-meth.yl-3rinethoxy-2-oxo-4-1 [ (phenylmethoxy) carbonyl] amino] azetidine in 2 ml of dimethylformamide is cooled to 0°C and 4 ml of dimethylformamide-sulfur trioxide complex is added. After 1 hour at 0°C and 4 hours at room temperature the 15 solution is poured into 8 0 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 20 extracted with dichloromethane to give 1.4 2 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 25 584 mg of crude product. Chromatography on HP-20 AG (2 00 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 10 4 mg of an analytical 30 sample.
Analysis calc'd for C-^H^^O^SK*H^O: 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, j=7), 3.46(3H,S), 4.22(2H, d of d, j=6), 5.18(2H,S), 7.43ppm(5H,S). r-GGiWf -18*- 20 5 Example 159 (3S-trans) -3-Methoxy-4 -me thyl-2-oxo-3-1 (.phenylacetyl) -amino] -1-azetidinesulfonic acid, potassium salt (3S-trans ) - 3 - amino- 3 -Methoxy- 4-methy1-2-oxo~i_ azetidinesulfonic acid, tetrabutyiammonium salt is prepared by the catalytic hydrogenation of (3S-trans) -3-methoxy-4-methyl-2-oxo-3- f f (.phenylmethoxy) carbonylj amino] -1-azetidinesulfonic acid, potassium salt Csee example 158).after conversion to the tetrabutyiammonium salt. Following the procedure of example 88, but utilizing (3S-trans)-37amino-3^nethoxy-4-methyl-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt and phenylacetyl chloride yields the title compound.
Anal, calc'd for c13H15N206SK: Cf42.61; H,4.31; N,7.65 Found: C,39.67; H,4.09; N,7.30 NMR (D2Q) 1.29 (BH, d, j=7) 3.45(3H,S), 3.73(2H,S), 4.36 (2H, d of d, j=6), 7.38ppm (5H,S).
Example 160 [ 3S- [3o(Z),4g]]-3-[I (2-Amino-4-thiazolyl) [ [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-araino-a- [ [2- (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 155-160°C, dec. —eetssf -is?- ■ 205 24 Example 161 [3S-[3a (Z) ,4b]3~3-I [ I (Carboxymethoxy) imino] (2-amino-4-thiazolyl) acetyl] amino] -4 -methyl-2-oxo-1-azetidinesulfonic acid, dipotassium salt The deprotection of I3S-_[3a (Z1, 4 gj J-3-Ji (_2-amino-4-thiazolyl) 112-(diphenylmethoxy)-2-oxoethoxy]-imino] acetyl ] amino] -2-methyl-4 -oxo-l-azetidine-sulfonic acid, potassium salt (see example 160) using trif luoroacetic acid and anisole yields the title compound, which decomposes a.t:>250°C - Example 162 (S) -3-1 [ [ (2 , 6-Dichloro-4-pyridinyl) thio] acetyl] -amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Acylation of (S)-3-amino-2-oxo-l-azetidine-sulfonic acid, tetrabutyiammonium salt (see example 6A) with I(2,6-dichloro-4-pyridinyl)thio]acetic acid, 4-nitrophenyl ester, followed by treatment with potassium perfluorobutane sulfonate, yields the title compound, melting point 212-214°C.
Example 163 I3S;(R* ) ] -3-1 III C4-Amino-2 , 3-dioxo-l-piperazinyl) -carbonyl] amino] phenyl ace tyl] amino] - 4 -me thyl -2 -oxo-l-azetidinesulfonic acid, potassium salt [3S(R*)]-14-[112- I (4-methyl-2-oxo-l-sulfo-3-azetidinyl) amino]-2-oxo-l-phenylethyl]amino]-carbonyl1-2 ,3-dioxo-l-piperazinyl]carbamic acid, phenylmethyl ester, potassium salt (see example 149) is hydrogenated using gaseous hydrogen and 10% palladium on charcoal as catalyst, yielding the title compound, melting point 165°C, dec. 205::. 4 Example 164 [3S (Z) ] -3- [ J (2-Amino-4-thiazolyl) ][Cl-carboxy-l-methylethoxy) Imino] acetyl]amino]-2-oxo-1-azetidine-' sulfonic acid, sodium salt (1:2) [3S{Z)]-3-I t (2-Amino-4-thiazolyl) [12- (diphenylmethoxy) -1,l-dimethyl-2-oxoethoxy] imino] acetyl]amino] -2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (see example 28) is deprotected using trifluoroacetic acid and anisole to yield the title compound, melting point 18 5°C, 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-a - [ Ihydroxy (phenylmethoxy) phosphinyl ] -methoxy]imino]-4-thiazoleacetic acid, yields the compound listed in column II.
Column I 165. 2,6-dimethoxybenzoic acid 166 . (Z)-2-amino-a-[[4-(diphenylmethoxy) -4-oxobutoxy]imino]-4-thiazoleacetic acid 16 7. 2- [ [ [phenylmethoxy)carbonyl]-amino]methyl]benzoic acid 168. a-[ [ [5-hydroxy-2-(4-formyl-l-piperazinyl)pyrido12,3-d]-pyrimidin-6-yl]carbonyl]amino]-benzeneacetic acid Column II (S)-3-[(2,6-dimethoxybenzoyl)amino]-2-oxo-1-azetidinesulfon^c acid, potassium salt; melting point 180 C, dec. [3S(Z)]-2-[[ (.2-amino-4- thiazolyl) [ [4 -(diphenylmethoxy)-4-oxobutoxy]imino]-acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt? melting point 125-130 C, dec. , < (S)-2-OXO-3-[[2-[[[(phenylmethoxy)carbonyl] amino]methyl]benzoyl]amino]-1-azetidinesulfonic acid, potassium salt; melting point 234.5 C, dec. (3S)-3-[I[I[5-hydroxy-2-(4-formyl-l-piperazinyl)pyrido[2,3-d]pyrimidin-6-yl]carbonyl]amino]phenylacetyl]amino]-2-oxo-l-azetidinesulfonic acig, potassium salt; melting point 265-270 C, dec.
GCT5"?f :24^ Example 169 (-trans) -3-Amino-4--ethyl-2-oxo-l-azetidinesulf onic acid A) t - Bo c - N -me thoxy - 6 —t hr e oe thyl serin ami d e threo-D,L-6-Ethylserine (1.33 g) is dissolved in 10 ml of 2N potassium hydroxide and 5 ml of t-butanol- After the addition of 2.4 6 g of di-t-butylpyrocarbonate the two-phase mixture is stirred for 4 hours at ambient temperature. O-Methylhydroxylammonium chloride (1.25 g) is added and the pH is adjusted to 4 with IN hydrochloric acid. l-Ethyl-3-(3-dimethylamino-propyl )carbodiimide , hydrochloride CI.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 5 0 ml portions of ethyl acetate. The ethyl acetate extracts are combined and dried over MgSO^. Removal of the solvent in vacuo yields 1 g of the title compound- B) t-Boc-O-methanesulf onyl-N-me thoxy-B-threo-pr op ionamide t-Boc-N-methoxy-B-threoethylserinamide (10.5 g) is dissolved in 6 5 ml of pyridine. Methanesulfonyl chloride (4.65 ml) is added dropwise at 0°C.
After stirring for 3 hours at ambient temperature, the reaction mixture is poured into 200 g of ice and 300 ml of IN hydrochloric acid. The pH is adjusted to 4 with concentrated hydrochloric acid. After extraction with three 8 5 ml portions . m -Jrtfs- 205240 of ethyl acetate the combined extracts are dried over MgSO^ and concentrated in vacuo. The residue is treated with carbon tetrachloride and concentrated again. Stirring with ether followed 5 by filtration yields 6.9 g of the title compound.
C) (trans) - 3 - t_-Bu t o;ky c ar h onyl amino- 4 - e thyl -1 -me thoxy-2-a zetidinone Anhydrous potassrium carbonate (4.15 g) and 125 ml of dry acetone are brought to reflux and 3.4 g of t-Boc-O-methanesulfonyl-N-methoxy-0-threo-propionamide in 25 ml of acetone is added.
After 1 hour the reaction mixture is cooled and filtered, and the filtrate is concentrated 15 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 (trans) -3-t-Butoxycarbonylamino-4-ethyl-l- methoxy-2-azetidinone (3 g) is added to 170 ml of liquid ammonia at -78°C under nitrogen and 1.68 g of sodium is added in 5 portions with stirring over a 5 minute period. Stirring is 25 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 th.e title compound. -laa*- \°lS^O.
E) (trans ) -3-t-Butoxycarbonylamino-4--ethyl—2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt To a 2 ml of absolute pyridine in 20 ml 5 of dry dichloromethane is added trimethylsilyl-sulfonyl chloride ("3.7 ml) in 5 ml of dry dichloromethane. The addition is accomplished at -30°C, under nitrogen, over a 10 minute period. After stirring at ambient temperature 10 for 30 minutes, the flask is evacuated to yield a pyridine-sulfur trioxide complex. (.trans) -3-t-Butoxycarbonylamino-4-ethyl-2-azetidinone (2.67 g) and 20 ml of dry pyridine are added to the flask which, is then placed in an oil 15 bath preheated to 90°C. After 15 minutes a clear solution is obtained and poured into 200 ml of a 1M solution of dibasic potassium phosphate. After the addition of 2 7 g of dibasic potassium phosphate and 100 ml of water a' clear solution 2 0 is obtained. The solution is extracted with two 60 ml portions of ethyl acetate. Tetrabutyiammonium hydrogensulfate is added to the aqueous layer, and the aqueous solution is extracted with three 100 ml portions of dichloromethane 25 and the combined organic layers are dried over MgSO^. Concentration in vacuo yields 6.9 g of the title compound. -193.- 5 2 4 F) (trans ) -3-Amino-4-ethyl-2-oxo-l-azetidinesulfonic acid (trans)-3-t-Butoxycarbonylamino-4-ethyl-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium 5 salt (6.75 g] in 4Q ml of 98% formic acid is stirred for 3 hours at ambient temperature. Dichloromethane (60 ml) is added and the mixture is refrigerated for.-about 16 hours. The resulting precipitate is separated by filtration and then dried in vacuo to yield 0.85 g of the title compound, melting point 185°C, dec.
Example 170 (trans,Z)-3 —11(2-Amino-4-thiazolyl) [(1-carboxy-15 1-methylethoxy) imino]acetyl]amino]-4-ethyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt A) (trans,Z)-3-I[(2-Amino-4-thia zolyl) [(1-diphenylmethoxycarbonyl-l-rnethylethoxy) imino] -2 0 acetyl] amino]-4-ethyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt (trans)-3-Amino-4-ethyl-2-oxo-l-azetidinesulf onic acid (0.55 g) and 335 mg of triethylamine are dissolved in 50 ml of dry dimethylformamide. 2^ (z)-2-Amino-a-[(1-diphenylmethoxycarbonyl-l- methylethoxy ) imino] -4-thiazoleacetic acid (1.14 g) is added with stirring at 0°C followed by 4 50 mg of hydroxybenzotriazole and then 0.69 g of dicyclohexylcarbodiimide. After stirring for about 16 hours at 0°C the flask is evacuated. Dry acetone (25 ml) is added to the solid with I £C±55±- -19&- 2 0 52 4 0 stirring. The mixture is filtered and 0..94 g of potassium perfluorobutanesulfonate is added to the filtrate followed by 100 ml of ether.
After standing for 1 hour at 0°C the solid is filtered, washed with ether and dried in vacuo yielding 1.58 g of the title compound.
B) (trans_, Z) -3-11 (2-Amino- 4 -th ia zoiyl) [(1-carboxy-1-me thyl ethoxy) imino J acetyl! amino'] -4 -ethyl-2-oxo-l-azetidinesulfonic acid, dipotassium salt To a suspension"of 1.31 g of (trans),z)-3-[I (2-amino-4-thiazolyl)3 (1-dipheny lmethoxycarbonyl-1-methylethoxyl imino J 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 -15°C.
After stirring for 2 hours at -10°C a clear solution is obtained. At -30°C 80 ml of dry ether is added and the resulting precipitate tj- is filtered, and then treated with 5 ml of water.
The pH is adjusted to 5.5 with IN potassium hydroxide at 0°C, 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 160°C, dec. -195"- GC155f__ 20T74 0 Example 171 [3S (,Z) ] -3-j [ (2-Amino-4-thiazolyl) [ (4-hydroxy-4-oxobutoxy) imino] acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt 5 The deprotection of I3S(Z)]-3-I[(2-amino-4- thiazolyl) [14- Cdiphenylmethoxy) -4-oxobutoxy] imino] -acetyl] amino] -2-oxo-l-azetidinesulfonic acid, potassium salt Csee example 16 6) using trifluoroacetic acid and anisole yields the title compound, 10 melting point >200°C.
Example 172 CS) -3-1 [2- (Aminomethyl ) benzoyl] amino] -2-oxo-l-azetidinesulfonic acid', inner salt 15 The deprotection of (S)-2-oxo-3-[ [2-[I [ (phenyl methoxy) carbonyl] amino]methyl]benzoyl] amino] -1-azetidinesulfonic acid, potassium salt using hydrogen gas, palladium on charcoal, and hydrochloric acid, yields the title compound, melting point 20 162-165°C.
Example 17 3 (S) -3- [ I [2- (4-Formyl-l-piperazinyl) -5-hydroxy-pyrido [2 , 3-d] pyrimidin-6-yl] carbonyl ] amino] -2-25 oxo-l-azetidinesulfonic acid, potassium salt (S) -3-Amino-2-oxo-l-azetidinesulfonic acid , tetrabutyiammonium salt (see example 6A) is coupled with 2-(4-f ormyl-l-piperazinyl)-5-hydroxy-6- I ( 4-nitrophenoxy) carbonyl] pyrido [2 , 3-d] pyr imidine 3Q and treated with potassium perf luorobutanesulf onate in acetone to yield the title compound, melting point 290°C, dec.
I eei-s-5-f—- '-»* 2052 4 Example' 174 (3S-trans ) -a- [ I (.4 -Meth.yl-2-oxo-l-s.ulf o-3-azetidinyl} -amino] carbonyl] benzeneacetic acid', dipbtassium salt (3S-trans), -3-Amino-4 -methyl-2-oxo-l-azetidine-5 sulfonic acid (see example 139) is coupled with a-(carboxyl)benzeneacetyl chloride and treated with triethylamine and potassium perfluorobutanesulf onate to yield the title compound, melting point 147°C, dec.
Example 175 13S-trans] -3-Amino-4 —cyclohexyl-2-oxo-l-a zetidine-sulfonic acid A) a— (t—Butoxycarbonylamino) -0—cyclohexyl—8-hydroxy-threo-propionic acid B-Cyclohexyl-a-amino-6-hydroxy-threo-propionic acid (15 g) is suspended in 150 ml of acetonitrile and 70 ml of water. Triethylamine (17.8 g) is 20 added and the mixture is heated with stirring to 60°C. At this temperature a clear solution is obtained and 21.0 g of di-t-butylpyrocarbonate is added and stirring at 60°C is continued for 1.5 hours. The solvent is removed in vacuo 25 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 Na^SO^ and evaporated to dryness . The remaining 30 crystalline material is filtered with petrol ether, yielding 20.4 g of the title compound, melting point 113-115°C. -19>- 2-0 5- •B) a - Ct-Bu-toxycarhonylamino),-g-cyclohexy l-S-hydroxy-N-me thoxy-threo'-propionamide a- (t-Butoxycarbonylamino) -0-cyclohexyl-8-hydroxy-threo-propionic acid (20.2 g) and 7.6 g of O-methylhydroxylamine hydrochloride are suspended in 350 ml of water and 175 ml of t-butanol. The pE of the mixture is adjusted with potassium carbonate to 4 .l-Ethyl-3- (3-dimethyl aminopropyl)carbodiimide C16.4 g) 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 Na2SO^ and evaporated to dryness. The remaining crystals are filtered off with petrol ether yielding 18.6 g of the title compound, melting point 125-127°C.
C) a- (t-Butoxycarbonylamino) -g-cyclohexyl-8-(methanesulf onyl oxy) -N-methoxy-threo-propionamide a- (t-Butoxycarbonylamino) -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 0°C 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, . "GCl55f J) .0 ml of ethyl acetate is added and the pH is adjusted to 3 with dilute sulfuric acid- The organic layer is separated, dried with. Na2SC>4 and the solvent is removed in vacuo. The remaining solid is collected with petrol ether yielding 19.0 g of the title compound, melting point 150-152°C.
D) [ 3 S-trans 3 —3 — ("t-Butoxycarbonylaminol -4 -10 cyclohexyl-1-me thoxy— 2-a z etidi none a- (t-Butoxycarbonylamino) -B-cycloh.exyl-6-(methanesulfonyloxy)-N-methoxy-threo-propionamide (18.7 g) is dissolved in 500 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 in vacuo and the remaining oil is dissolved in 30 ml of ethyl acetate. Upon the addition of 2 0 petrol ether, the title compound precipitates and is filtered off (12.9 g), melting point 110—112°C.
E) [3S-trans3-3-(t-Butoxycarbonylamino)-4-25 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 QZISSI 52 10 amount of 0.0.25 g sodium is added and stirring is continued for 5 minutes. Ammonium chloride (0.89. g) is added and the ammonia is removed. The residue is extracted with warm ethyl acetate.
The organic extract is evaporated to dryness and the remaining crystals of the title compound are filtered with petrol ether, yielding 0.5 g, melting point 130.-132°C.
F) T3S-trans 1 -3- (t-Butoxycarbonylamino) -4-eyelohexyl-2 —oxo—1-azetidinesulfohic acid, pyridine salt I3S-trans] -3- ('t-Butoxycarbonylamino) -4-cyclohexyl-2-azetidinone (5.3) is dissolved in ^5 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 6 hours at room temperature. Removal of the solvent in vacuo 20 yield 11.3 g of the title compound as an oil.
G) [3S-trans] — 3— (t—Butoxycarbonylamino)—4-cyclohexyl-2-oxo—1-azetidinesulfonic acid, tetrabutyl ammonium salt [3S-trans] -3- (t-Butoxycarbonylamino).-4- cyclohexyl-2-oxo—1-azetidinesulfonic acid, pyridine salt (11.3 g) is dissolved in 250 ml of water. Tetrabutyiammonium hydrogensulfate (.9.0 g) is added with stirring and the pH is 3Q adjusted to 6.5 with IN potassium hydroxide. The aaueous solution is extracted twice with -GG±€r5-f- Zloo -18$ - 2 0 5 .0 ml portions of methylene chloride. The organic portions are dried with Na2SO^, filtered and the solvent is distilled off, yielding 8 g of the title compound, melting point 135-138°C.
H) [3S—transj — 3-Amino-4-cyclohexyl—2—oxo-1-azetidinesulfonic acid [ 3S - trans] - 3 (t-Butoxycarbonylamino 1-4-cyclohexyl-2-oxo-l-azetidinesulfonic acid, tetrabutyiammonium salt (3.8 g) is stirred in 20 ml of formic acid for 3 hours, followed by the addition of 20 ml of methylene chloride. The precipitated title compound (1.0 g) is filtered off, melting point 217-219°C.
Example 176 [3S-[3a(Z) , 48]3-3-[[(2-Amino-4-thiazolyl) -(methoxyimino) acetyl] amino] -4-cyclohexyl-2-oxo-l-azetidinesulfonic acid, potassium salt I3S- trans J -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-a-(methoxyimino)-4-thiazoleacetic acid (0.2 g) , 0.16 g of hydroxybenzotriazole and 0.42 g of dicyclohexylcarbodiimide are added. Stirring is continued for 4 8 hours at ambient temperature. The precipitated urea is filtered off and the solvent is removed in vacuo. The residue is 7.o! \ dissolved in 10 ml of acetone and 0.41 g of potassium perfluorobutanesulfonate is added.
After the addition of 50 ml of ether, the title compound precipitates and is filtered.
Column chromatography using HP-20 and water/ acetone (9:1] as eluent, yields 0.36 g of product, melting point 200-205°C (after lyophilization).
Example 177 [3S- [3a (Z) , 4 g] ] -3- [T (2-Amino-4-thiazolyl )T (1- carboxy-l-methylethoxy) imino] acetyl] amino] -4-cyclohexyl-2—oxo-l-a zetidinesulf onic acid, dipotassium salt A) [3S-[3a (Z) ,4g]]-3-[ [ (2-Amino-4-thiazolyl) [(1-diphenylmethoxycarbonyl-l-methylethoxy) imino] -acetyl] amino] -4-cyclohexyl-2-oxo-l-azetidinesulfonic acid, potassium salt [3S-trans] -3-Amino-4-cyclohexyl-2-oxo-1-20 azetidinesulfonic acid (0.2 g; see example 175) is dissolved in 30 ml of dimethylformamide and 0.09 g of triethylamine with stirring. Hydroxybenzotriazole (0.12 g), 0.30 g of (Z)-2-amino-a-[ (1-diphenylmethoxycarbonyl-l-methylethoxy) imino] -25 4-thiazoleacetic acid and 0.33 g of dicyclohexylcarbodiimide 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 30 oil is dissolved in 5 ml acetone, treated with 0.3 g of potassium perfluorobutanesulfonate and ( I i < GCl55f -2031- _ 205Z4-. poured into 10.0 ml of ether with stirring.
I3S-I3a (Z),4 B] ] -3-1 [ (2-Amino-4-thiazolyl)-I (1-diphenylmethoxycarbonyl-l-methylethoxy) -imino] acetyl] aminoH -4-cyclohexyl-2-oxo-l-azetidine-5 sulfonic acid, potassium salt CO . 61 gl precipitates and is filtered off.
B) [3S- [ 3a (Z) , 4 g] ] -3-11 C2-amino-4-thiazolyi) [ (1-carboxy-1-meth.ylethoxylimino] acetyl] amino] -4-10 cyclohexyl-2-oxo—1-a zetidinesulf onic' acid, dipotassium- salt [3S-I3aCZ) ,4B]]-3-[I C2-Amino-4-thiazolyl) -[ CI-diphenylmethoxycarbonyl-l-methylethoxy) imino] -acetyl]amino] -4-cyclohexyl-2-oxo-l-azetidinesulfonic 15 acid, potassium salt (0.61 g) is suspended in 6 ml of anisole, cooled to -15°C and 5 ml of trifluoroacetic acid is dropped in with stirring. The temperature is maintained for one hour and then lowered to -30°C. About 100 ml of dry 20 ether is added at such a rate, that the temperature does not exceed -10°C. The precipitated compound is filtered off and chromatographed using HP-20 resin and water/acetone C9:l) as eluent, yielding 0.3 g of the title compound, melting point 25 11-120°C, dec. (after lyophilization) .
GC155f -2G3.- 2057 4-J Example' 17 8 [3S-f3a,4 g] -4-Cyclohexyl-3-J'['I113-f (2-furanyl-methylene) amino] -2-oxo-i-imidazolidinyl] carbonyl] -ami no ] pheny 1 acetyl ] amino] - 2 -oxo-1 -azetid inesulfon ic 5 acid, potassium salt [3S-trans] -3-Amino-4-cyclohexyl-2-oxo-l-azetidinesulfonic acid C.0.1 g; see example 175) is dissolved in a -mixture of 30 ml of dry dimethylformamide and 0.0 5 g of triethylamine 10 with stirring. I[11C2-Furanylmethylene) amino]- 2-oxo-l-imidazolidinyl] carbonyl] amino] phenylacetic acid (Q.14 g) , 0.0 6 g hydroxybenzotriazole and 0.17 g of dicyclohexylcarbodiimide are added and the solution is stirred for 5 days at room 15 temperature. The solvent is removed in vacuo, the residue is dissolved in 10 ml of acetone and the precipitated urea is filtered off. The mother liquor is agitated with 0 .15 g of potassium perfluorobutanesulfonate and diluted 20 with 50 ml of ether. The precipitate is filtered, and chromatographed with HP-20 resin using water/acetone (9:1) as eluent yielding 0.14 g of product, melt: (after lyophiliaztion). 0.14 g of product, melting point 195-20Q°C, dec. -20ifr 205240 Example 179 [ 3S-13a.(R* ) -j.4.6] ] -4 -Cyclohexyl-3-113- C4-ethyl-2 ,3-dioxo-l-piperazlnyl) —1. 3-dioxo-2-phenylpropyl] -amino]-2-oxo-l-azetidinesulfonic' acid, potassium salt 13S-trans] -3-Amino-4-cyclohexyl-2-oxo-l-azetidinesulfonic acid (0.1 g; see example 175) is dissolved in 30 ml of dimethylformamide and 0.5 g of triethylamine with stirring. (JR)-a-[ [ (4-ethyl-2 , 3-dioxo-l-piperazinyllcarbonyl] -amino]benzeneacetic acid, 0.06 g of hydroxybenzotriazole and Q.-.17 g of dicyclohexylcarbodiimide are added, and the mixture is stirred for about 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 perfluorobutanesulf onate 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 175-180°C (after lyophilization).
Example 180 (trans, Z)-3-[I(2-Amino-4-thiazolyl) (methoxyimino)-acetyl] amino2-4-ethyl-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of example 170, part A, but substituting ( Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid for (Z)-2-amino-a-[(1-diphenylmethoxycarbonyl-l-methylethoxy) imino]-4-thiazoleacetic acid, yields the title compound, melting point 190°C, dec. -2 05- 2 0 C 2 *.
Example 181 (±)-(trans)-3-Amino-2-oxo-4-phenyl-1-azetidinesulfonic acid A) (±) - (trans) -2 -oxo-4-phenyl-! -azetidine—ter t-butyldiphenyisilane A solution of tert-butylchiorodiphenylsilane (20.56 gl in dimethylformamide (45 mil iff cooled to 0°C under argon and treated with triethylamine (10.4 mil and then ..(±l-2-oxo-4-phenyl-l~azetidine. After several hours at 0°C, the resulting mixture is treated with, additional triethylamine (.1 mil and tert-butylchlorodiphenylsilane (.2.11 g) , and allowed to stir for 65 hours at 5°C. 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 bicarbonate solution (50 ml), water (two 50 ml portions) saturated sodium chloride solution and dried (Na^SO^). 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. ee±5 -20<- ZQZ % ■■ B) (±) — (trans)—3—az.ido—.2—oxo-4-phenyl—1—a-zet-idine-tert-butyldiphenylsilane A 50 ml flask equipped with, stirring bar, gas inlet, and septum is flame dried under argon 5 and charged with n-butyl lithium (0.65 ml of a 1.6 M solution in hexane] which is cooled to -40.°C and dissolved in tetrahydrofuran (2 ml) . Diisopropylamine 10.16 ml) is added dropwise, the resulting mixture is stirred for 30 minutes 10 and cooled to -78°C; . A solution of (il-(trans)- 2 -oxo-4-pheny 1-1-aze tidine-ter t-butyl dipheny 1 silane (4 00 mg) in tetrahydrofuran (1.5 ml) is added dropwise over about 5 minutes. After stirring an additional 20 minutes the solution is treated 15 with p-toluenesulfonyl azide (204 mg) in tetrahydrofuran (0.5 ml). The resulting mixture is stirred 10 minutes at -78°C and treated dropwise with chlorotrimethylsilane (0.4 ml). After an additional 10 minutes of stirring, the cooling-20 bath is removed and the reaction mixture is stirred at ambient temperature for 2.5 hours.
Then, while cooling at 0°C, ethyl acetate (20 ml) is added followed by pH 4.5 phosphate buffer (8 ml). The organic layer is washed with additional 25 buffer (two 8 ml portions), 5% sodium bicarbonate solution (three 10 ml portions), 50% sodium chloride solution (10 ml), saturated sodium chloride solution (10 ml) and dried (Na^SO^). Filtration and concentration in_ vacuo yields 30 500 mg of oil which is flash chromatographed with 5% ethyl acetate-hexane, yielding the title compound (25 3 mg).
.JGbT5%£~~ w t'«-> C1 C-). - (trans) -3-azido—2-oxo-4-phenyl—1-azetidine A solution of 17 g of crude (±)-(trans) -3-azido-2-oxo-4-phenyl-1-azetidine-tert butyl-diphenylsilane is dissolved in methanol (240 ml) and treated dropwise at 0°C with concentrated HCl (35 ml) . The cooling bath is removed, the reaction stirred at ambient temperature for 1 hour, and recooled to 0°C whereupon saturated sodium bicarbonate solution is added to neutrality. The resulting mixture is extracted with ethyl acetate (one 3 00 ml portion and four 100 ml portions) and the organic extracts are washed with 1:1 5% sodium bicarbonate 50% sodium chloride solution, saturated sodium chloride solution, and dried (Na2SO^). 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 4 60 mg of the title compound.
D) (i)-(trans)-3-azido-4-phenyl—1-azetidine-sulfonic acid, tetrabutyiammonium salt A solution of (±)-(trans)-3-azido-2-oxo-4-phenyl-l-azetidine (30 0 mg) in dimethyl- 9 c . O formamide (3 ml) is cooled to 0 C under argon and treated dropwise with a complex of dimethylformamide 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 -GerSlif - 2 0 5 2 4 and poured into 80 ml of 0.5 M monobasic potassium phosphate (pH 5,5} . The solution is extracted with dichloromethane (.discarded) and 541 mg of tetrabutyiammonium bisulfate is added. The resulting mixture is extracted with dichloromethane and the organic extracts are washed with 10% sodium chloride solution and dried CNa^SO^I. Filtration and concentration ih vacuo affords 80.0 mg of oil; approximately 40% the desired product, the remainder dimethylformamide. This mixture is used without purification in the next step.
E) (+) - (trans) -3-amino-4-phenyl-l-azetidine-sulfonic acid A solution of (±)-(trans)-3-amino-4-phenyl-1-azetidinesulfonic acid, tetrabutyiammonium 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 evacuated and fresh hydrogen is introduced.
After an additional 4 5 minutes the reaction is complete, and the system is flashed with nitrogen. After several days at room temperature in dichloromethane-methanol (4 :1, 20 0 ml) catalyst aggregation is complete and filtration is accomplished. The filtrate is concentrated in vacuo to 18 ml and 0.2 ml of 9'7% formic acid is added. After cooling at 5°C for several hours the resulting solid is filtered and washed with dichloromethane to afford, after drying, 150 mg of the title compound as a solid. Analysis calc'd for CgH^gN^O^S: 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 G£155f — p-a* m i r ~ Example 182 ( + )-(trans)-2-Oxo-4-phenyl-3—I(phenylacetyl)aminoj-1-azetidinesulfonic acid/ potassium salt A) (±) -trans-2-oxo-4-phenyl — 3—I(phenylacetyl) amino] -1-azetidinesulfonic acid tetrabutyiammonium salt A solution of ,N-hydroxybenzotriazole mono-hydrate (52 mg) and phenylacetic acid C46 mg) in dimethylformamide (0.3 ml) is treated with solid dicyclohexylcarbodiimide ("70 mg) under argon at 0°C. The cooling bath is removed and the resulting mixture stirred at ambient temperature for 1 hour. After dilution'with additional dimethylformamide (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 dimethylformamide. The filtrate was added to 20 ml 0.5 monobasic potassium phosphate (pH 4.5), the mixture washed with three 8 ml portions of ethyl acetate (discard), and 10 5 mg of tetrabutyiammonium 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 chioride solution (10. ml) and dried (Na2SO^). Filtration and concentration in vacuo yields (after heating at 32°C under high vacuum) 165 mg of oil; approximately 40% is the desired compound, and 60% dimethylformamide. -20-r- B) Ct),-trans-2-oxo-4-phenyl-3- j (phenylacety 1) -amino] -1-azetidinesuifonic acid',' potassium -gait A solution of (+) -trans-2-oxo—4-phenyl-3-I (phenylacetyl) amino] -1-azetidinesulfonic acid tetrabutyiammonium salt in 1.5 ml acetone is treated with 41 mg (0. .121 mmolel of potassium perf luorobutanesulfonate.' Dilution with 12 ml of ether affords a glass, which upon trituration with ether affords 4 3 mg of solid which contains about 20% of an impurity containing a tetrabutyiammonium moiety. The solid is dissolved in 5Q% aqueous acetone and passed through a Dowex 50W-X2 K ion-exchange resin (1 ml).
Removal of solvent yields a solid which is washed with acetone, hexane and dried (60°C, high vacuum) . The yield of the title compound is ]5 mg.
Analysis calc'd for C,_H^^N_0CS-K: C, 51.23; H, 3.80; - 17 Id 2 b 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 18 3 (±) - ( trans , Z ) -3- 112—Amino-4-thiazolyi) (methoxyimino) -acetyl] amino]-2-oxo-4-phenyl-l-azetidinesulfonic acid, potassium salt A solution of N-hydroxybenzotriazole hydrate (52 mg) and (Z )-2-amino-a-(methoxyimino)-4-thiazoleacetic acid (69 mg) in dimethylformamide (0.3 ml) is treated with solid dicyclohexylcarbodiimide (70 mg) under argon, at ambient I a.// 2 G r 3 4 0 temperature. The resulting mixture is stirred for 1 hour, (±)~(trans)-3-amino—2-oxo—4-pheny1-1-azetidinesulfonic acid (75 mg; see example 181) is added as a solid, followed by triethylamine 5 dropwise CO.0 5 ml). The reaction is stirred at ambient temperature for 2 3 hours. The dimethylformamide is removed under high vacuum at 30°C, and the.residue triturated with 2 ml of acetone and filtered. The filter cake is washed with 10 additional acetone (.two 3 ml portions) and potassium perfluorobutanesulfonate (8 6 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 15 of the title compound as a solid.
Analysis for C, cHn .Nc0^.Sn • K - 15 14 5 6 2 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, 15.0 7; S, 13.87; 20 ' K, 7.5 Example 184 (cis)-2-Oxo-4-phenyl-3- T(phenylacetyl)amino]-1- azetidinesulfonic acid, potassium salt A) N-Benzvlidene-2,4-dimethoxybenzylamine 12.0 g of 2 , 4-dimethoxybenzylarnine hydrochloride is added to 100 ml of IN sodium hydroxide solution and the mixture is extracted with 125 ml of ethyl 30. acetate. The organic layer is dried over anhydrous sodium sulfate and stripped of solvent to give -Ge±55-r -21$- 2 0 .2 g of 2,4-dimethoxybenzylamine as an oil.
This amine is dissolved in 150 ml of Benzene; 6.47 g of benzaldehyde and C.G" g of g-toluene-sulfonic acid monohydrate are added. The mixture 5 is heated under reflux removing water with a Dean-Stark separator and in two hours the calculated amount of water (1-1 ml) separates out. The mixture is cooled to room temperature. Upon further cooling the benzene solution 10 deposits some precipitate. Benzene is removed under reduced pressure and 60 ml of petroleum ether is added to the residue. An oily layer separates out with more precipitate. Benzene (10 ml) is added to make the layers homogeneous 15 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-l- (2,4-dimethoxybenzyl)-20 2-oxo-3-azidoazetidine a-Azidoacetic acid (1.62 g) is dissolved in 25 ml of methylene chloride under ..nitrogen. To this solution are added 3.24 g of triethylamine and 1.0 2 g (4.0 mmole) of the imine 25 N-benzylidene-2,4-dimethoxybenzylamine dissolved in 10 ml of methylene chloride. The mixture is cooled in an ice-bath and 3.36 g of trifluoroacetic anhydride is added slowly; the solution becomes dark colored. After stirring for 1 hour 30 in an ice-bath, the mixture is warmed to room temperature and stirred an additional 15 minutes.
ZOi' The solution is then washed with water (60 ml) , 5% NaHCO^ solution (two 50 ml portions1, and IN HCl solution (60 ml). The organic layer is dried over anhydrous sodium sulfate and stripped of 5 solvent to give 1.72 g of crude product as dark gum. The gum is treated with charcoal several times and the resulting Brown mixture is chromato' graphed on 40 g silica gel eluting with 1:1 petroleum ether:ethyl acetate. The combined 10 fractions yield crystal upon quick-freezing in a dry ice-acetone bath. Using this as a seed the product is recrystallized from petroleum ether-ethyl acetate to give 817 mg of the title compound as needles which melt upon warming to 15 room temperature.
C) (-)-(cis)-4-Phenyl-2-oxo-3-azidoazetidine (-)-(cis)-4-Phenyl-l- (2,4-dimethoxybenzyl)-2-oxo-3-azidoazetidine (737 mg) is dissolved in 20 25 ml of acetonitrile and heated to 80°-83°C under nitrogen. To the resulting solution are added over a 1 hour period 9 43 mg of potassium persulfate and 570 mg of potassium monohydrogen phosphate, both dissolved in 25 ml of water. 25 After the addition, the mixture is further heated at 80°-83°C for 7 hours. The mixutre is cooled and the pK is adjusted to 6-7 by adding solid potassium monohydrogen phosphate. Most of the acetonitrile is removed under reduced pressure 30 and the resulting mixture is extracted with r-212f- PGi 201 ■ 60 ml of chloroform. The chloroform layer is washed with water (.6 0 ml). , dried over anhydrous sodium sulfate and stripped of solvent to give a crude product as an oil- The crude product is 5 chroma to gr pah ed on 4 0 g of silica gel eluting with 1:1 petroleum ether-ethyl acetate. The combined fractions yield crystals and the product is recrystallized from petroleum ether-ethyl acetate to give 267 mg of the title compound.
D) (.-) - (cis) -4—Phenyl-2—oxo—3—azido—-1—azetidine-sulfonic acid," tetrabutyl' ammonium salt Ct) - (cis ) -4-Ph.enyl-2-oxo-3-azidoazetidine (162 mg) is cooled to 0°C under nitrogen and 15 3.5 ml of ca-Q . 5M dimethylformamide-sulfur trioxide -complex solution in dimethylformamide is added dropwise via syringe. The resulting clear solution is stirred at 0°C for 15 minutes. The mixture is then poured into 50 ml of 0. 5M 20 monobasic potassium phosphate solution and washed with methylene chloride (three 50 ml)portions). tetra-n-Butylammonium bisulfate (29 2 mg) is added to the aqueous solution and the mixture is extracted with methylene chloride (six 5Q ml 25 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. -2 IS"- E) (±) - (cis) —2-0x0-4-phenyl-3—1 (phenylacetyl) amino] -1-azetidinesulfonic acid,' potassium salt (+) - (cis) -4-Phenyl-2-oxo-3-azido-l-azetidine-sulfonic acid, tetrabutyl ammonium salt (293 mg) is dissolved in 4 ml of ethanol and hydrogenated with 8Q mg of platinum oxide catalyst at one atmosphere. After 1 hour stirring the catalyst is filtered through-a Millipore filter with Celite; some catalyst- 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 dimethylformamide. N-Hydroxybenzotriazole monohydrate (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 perfluorobutane 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 qive 26 mg of the title compound.
Analysis calc'd for C^-^H^Oc-^SK• 2H2O: C, 46.99; H, 4.41; N, 6.45 Found: C, 47.24; H, 4.19; N, 6.34 .' Example 185 (cis, Z) -3-1 H2-Amino—4-thiazolyl). (methoxyimino) -acetyl] amino] -2-oxo-4-phenyl-l-azetidines,ulf onic acid, potassium salt 5 (cis) -2-0x0-4—pheny 1-3-1 (phenylacetyl) amino] - 1-azetidinesulfonic acid, potassium salt (56Q mg; see example 184, part D) is dissolved in 5 ml of ethanol and hydrogenated with 11Q mg of platinum oxide catalyst at one atmosphere. After one 10 hour stirring the catalyst is filtered through a Millipore filter with Celite; catalyst particles pass through the filter to give a hlack filtrate. Ethanol is removed under reduced pressure and the residue is dissolved in 4 ml of dimethyl-15 formamide. N-Hydroxybenzotriazole monohydrate (168 mg), 221 mg of the (Z)-2-amino-a-(methoxyimino) 4-thiazoleacetic acid and 227 mg of dicyclohexylcarbodiimide are added and the mixture is stirred for about 16 hours under nitrogen. The slurry 20 is evaporated iii 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 perfluorobutane sulfonate. Upon adding 15 ml of ether a 25 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 C..-H, ,OfNcS.K-l 1/2 Hn0: - 15 14 6 5 2 2 C, 36.73; H, 3.49; N, 14.28; S, 13.07; K, 7.97 Found: C, 36.65; H, 3.00; N, 13.99; S, 13.48; K, 8.30 2 C Example' 186 (cis) -3-Amino-2 -oxo-4 -(2-phenylethenyll -1-azetidinesulfonic acid A) N- (3-phenyl -2-propertylidene? -4-methoxyaniiine g-Anisidine (12.32 g) is dissolved in 160 ml of methylene chloride and 20 g of anhydrous - magnesium sulfate is'- added. The mixture is cooled in an ice bath, and 13.22 g of trans -10 cinnamaldehyde is added. The mixture is stirred under nitrogen for 2 hours and then filtered. The filtrate is evaporated to give a solid. The crude product is recrystallized from methylene chloride-petroleum ether to give 20 .96 y of the 15 title compound as a solid.
B) (1)-(cis)-3-Azido-l- (4-Methoxyphenyl)-2-oxo-4-(2-phenvlethenvl)azetidine 2-Azidoacetic acid (24.26 g) is dissolved 2<-1 in 100 ml of methylene chloride and cooled in an ice bath. To this solution is added 4 8.57 g of triethylamine and 14.24 g of N- (3-pheny1-2-propenylidene)-4-methoxyaniline dissolved in 250 ml of methylene chloride. To the resulting 25 solution is added 50.41 g of trifluoroacetic anhydride dropwise over a one hour period. After stirring for one hour in an ice bath, the 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 \ 3-/ g. ^2 with, water (750 ml), 5% sodium bicarbonate solution (two 75Q ml portions), and IN HC1 solution (750. ml). The organic layer is dried over anhydrous sodium sulfate and stripped of 5 solvent to give a solid. The crude product is recrystallized from ethyl acetate to give 11.39 g of the title compound as a solid.
C) (±) - (cis) -3—Azido—2-0x0-4—C2-phe-nylethenyl) 10 azetidine To a solution of 10.22 g of eerie ammonium nitrate in 13 ml of water at 0°C is added 1.99 g of (i) - (cis) -3-azido-l- (.4-methoxyphenyl) - 2-oxo-4-(2-phenylethenyl)azetidine is dissolved in 65 mi 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), 20 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. 25 Fractions (.50 ml each) 11-16 are combined and evaporated to give 802 mg of the title compound as an oil. -2H- 20 524 D) Ci) ~ (cis) -3-Azido—2-o:xo-4 - (2—phenylethenyl) -1-azetidinesulfonic acid, tetra-n-5utylammohiiim salt (i) - (.cis ) -3-Azido-2-oxo-4- (2-phenylethenyl) -azetidine (334 mgl is dissolved in 3 ml of dimethylformamide and 8 68 mg of pyridine-sulfur trioxide is added. The mixture is stirred at room temperature for 40 hours under nitrogen and then poured into 200 ml of 0.5 M monobasic potassium phosphate solution and washed with 30 ml of methylene chloride. tetra-n-Butyl ammonium bisulfate (5 30 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 15 (two 100 ml portions), dried over anhydrous- magnesium sulfate and stripped of solvent to give 8 24 mg of the title compound as a gum.
E) (i)-(cis)-3-Azido-2-oxo-4—(2—phenylethenyl)-1-2 0 azetidinesulfonic acid (±)-(cis)-3-Azido-2-oxo-4-(2-phenylethenyl)-1-azetidinesulfonic acid, tetra-n-butylammonium salt (300 mg) is dissolved in 4 ml of tetrahydrofuran and stirred rapidly. To the mixture is added 25 600 mg of zinc dust followed by 0.8 ml of IN monobasic potassium phosphate solution. The mixture is heated to 4 5°C and stirred at this temperature for 3 hours . The mixture is then filtered and the filtrate is taken in 40 ml of 30 methylene chloride and 10 ml of water. The GG±55T A small amount of acetonitrile is added to dissolve the precipitate 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 (15 ml each) 2-13 are combined and evaporated to give 101 mg of the title compound as a foam.
Example 187 ( + )-(cis,Z)-3-[[(2-Amino-4-thiazolyl) (methoxyimino)-2 0 acetyl]amino]-2-oxo-4-(2-phenylethenyl)-1-azetidinesulfonic acid, potassium salt A solution of 68 mg of (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid and 51 mg of N-hydroxybenzotriazole monohydrate in 2 ml 25 of dimethylformamide is treated with 69 mg of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 30 minutes under nitrogen, (cis)-3-Amino-2-oxo-4-(2-phenylethenyl)-1-azetidinesulfonic acid (90 mg; see example 186) 30 and 34 mg of triethylamine are added and the mixture is stirred for 20 hours under nitrogen.
The slurry is evaporated in vacuo and triturated with. 10 ml of acetone. The slurry is filtered and the filtrate is treated with 113 mg of potassium perfluorobutanesulfonate. Dilution with 30 ml of ether and filtration gives 169 mg of a solid product, which, is dissolved in a small amount of ca. 10% acetonitrile/water and applied on 34 ml of HP-20 resin, eluting with 150 ml of water, then 10% acetone/water. Fractions (15 ml each) 16-19 "are combined and stripped of solvent to give 110-mg of the title compound as a solid.
Analysis calc'd for H2^: ^0.23; 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 - (methoxycarbonyi) - 2-oxo-1-azetidinesulfonic acid A) I(4-Methoxyphenyl)imino]acetic acid, methyl ester A dry 3-necked, 1 liter flask equipped with a nitrogen inlet and stirring bar is charged with 56 . 88 g of MgSO^ 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. After stirring an additional 20 minutes at 0°C, f i ■* y v' /'•" the reaction mixture is suction filtered, dried over sodium sulfate, filtered and concentrated in vacuo to one-quarter volume. Hexane (300 ml) is added, and the solution is concentrated to an 5 oil which semi-solidifies on standing under high vacuum at 5°C.
B) (cis) -3- (1, 3-Dihydro—1-, 3—dioxo—2H—isoindol-2-yl) -4-methoxycarbonyl-2-oxo-l— (4-trie thoxy phenyl) 10 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-Methoxy-phenyl)imino]acetic acid, methyl ester (21.09 g) 15 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 20 stirred for 1.5 hours at 0°C, 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 25 solution (50 0 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. -22a- ,GG-t5~5f 2 0 r 2 4 0 C) (cis)-4- (Methoxycarbonyl) -1- (4-methoxyphenyl) -2-OXO-3- f J (phenylmethoxy) carbonyl] aminoj azetidine A dry 50Q ml flask equipped with nitrogen inlet, stirring bar, and septum was charged with 18.65 g of (.cis 1-3- (1, 3-dioxo-2H-isoindol-2-yl) -4-methoxycarbonyl-2-oxo-l- (4-methoxyphenyl) azetidine and 325 ml of dichloromethane. The resulting suspension is cooled to -3Q°C, and methyl hydrazine (3.52 mil is added dropwise. The reaction is warmed to Q°C and stirred for 1 hour. An additional 0.4 ml of methyl hydrazine is added and the mixture is stirred for 10 minutes. This sequence is repeated with a total of 2.9 equivalents of methyl hydrazine (7.7 ml) has been added. The solvent was removed iii vacuo; 200 ml of fresh dichloromethane is added, and the mixture is again concentrated. This sequence was repeated two 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 0°C (nitrogen atmosphere) and treated with diisopropylethyl amine (17 ml) followed by benzyl chloroformate (7 ml) dropwise. The reaction is stirred at 0°C for 30 minutes, then at ambient temperature for 1.5 hours. The mixture is wash.ed 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), ( I _ -22£- dried (sodium sulfate)., and filtered. . Concentration in vacuo, yields a foam which on trituration with ether yields 9. .9 g of the title compound as a solid.
D) (.cis ) -4- (MethoxycarBonyl) -2-oxo-3— I I Cphenyl-methoxy)carbonyi]amino]-T-azetidine A solution of eerie ammonium nitrate (8.59 g) in 60 ml of 1:1 acetonitrile-water is treated with a slurry of 2 g (cis1-4-10 (methoxycarbonyll-1- (4-methoxyphenyll-2-oxo-3- I [ (phenylmethoxy)carbonyl]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 15 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% sodiuir. bicarbonate (three 70 ml portions) . The basic washings are back-washed with ethyl 20 acetate (50 ml) and the combined organic extracts are washed with aqueous sodium sulfite, 5% aqueous sodium carbonate (100 ml), 5% sodium chloride solution (two 100 ml portions), saturated sodium chloride (two 50 ml portions), and stirred 25 over Darco G-60 charcoal for 30 minutes. Sodium sulfate is added, and the mixture is filtered and concentrated in vacuo to yield an oil which on trituration with ether yields 685 mg of the title compound as a solid. 6et5^r~ 202240 E) (cis ) -4- (Methoxycarbonyl) -2-oxo-3-IJ (phenylmethoxy) carbonyl] amino"] -l-azefldihesulf onic acid, tetrabutyiammonium salt A mixture of (cis)-4-(methoxycarbonyl)-2-5 oxo-3- [ I (phenylmethoxy) carbonyl] amino] -1-azetidine (100 mg) and 17 2 mg of a pyridine-sulfur trioxide complex in 1 ml of pyridine is stirred under argon for 3 hours at 80°C. The reaction mixture is poured into 70 ml of 0.5 M monobasic potassium 10 phosphate (pH .5.5} and extracted with four 30 ml portions of dichloromethane (discard). Tetrabutyiammonium hydrogen sulfate (122 mg) is added to the aqueous layer which is then extracted with dichloromethane (four 30 ml portions). The organic 15 extracts are washed with 8% sodium chloride solution, dried over sodium sulfate and filtered. Concentration in vacuo yields 18 6 mg of the title compound as a viscous oil.
F) (cis ) -3-Amino-4- (methoxycarbonyl) -2-oxo-l-azetidinesulfonic acid A solution of 18 6 mg of (cis)-4-(methoxycarbonyl) -2-OXO-3- 11 (phenylmethoxy) carbonyl] amino] -1-azetidinesulfonic acid, tetrabutyiammonium salt in 2 ml of methanol is hydrogenated over 10% palladium on charcoal (9 5 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 staae is necessary to induce crystallization).
After crystallization commences, the mixture is allowed to stand for about 16 hours at 10°C. ( 2tff24C The resulting solid is washed with dichloromethane, hexane, and dried invvacuo, yielding 50 mg of the title compound.
Example 189 (cis ) -3- I I2-Amino-4-thia zolyl) III- (diphenyl-methoxycaf&onyl) —l-roe thyl ethoxy] imino] acetyl] -amino ] -4 - (methoxycarBonyl) -2-dxo-l-azetidinesulf onic acid, potassium salt 10 A solution of N-hydroxybenzotriazole hydrate (34 mg) and 1Q1 mg of 2-amino-a— III— (diphenylmethoxy carbonyl )-1-methylethoxy]imino]-4-thiazoleacetic acid in 0.5 ml of dimethylformamide is treated with solid dicyclohexylcarbodiimide 15 (4 5 mg) and the mixture is stirred under argon for 45 minutes (ambient temperature). (cis)-3-Amino-4-(methoxycarbonyl)-2-oxo-l-azetidinesulfonic acid (45 mg; see example 188) is then added as a solid followed by triethylamine (0.03 ml) 20 dropwise. The reaction is stirred at ambient temperature for about 16 hours. The dimethylformamide is removed under high vacuum at 30°C and the residue is triturated with acetone. The supernatant is treated with potassium 25 perfluorobutanesulfonate (67 mg). Dilution with ether produces a solid which is washed with ether and dried in vacuo to yield 9 3 mg of the title compound. -22?- _GC15^§— 2 0 52 Example 190. (cis ) -3-11 (2-Amino-4 —thiazolyl} I(1-carbaxy-l-methylethoxy) imino] acetyl] amino] -4 - (methoxycarbonyl) -2-oxo-i-azetidinesuTfonic acid, 5 dipotassium salt A slurry of (cis)-3-[I2-Amino-4-thiazolyl)-III- (diphenylmethoxycarbonyll-1-methylethoxy). -imino] acetyl] amino]'-4-(methcrxycarbony 11-2-oxo-l-azetidinesulfonic acid, potassium salt in Q.4 ml 10 0f anisole is stirred at —12°C under argon, and 0.9 ml of cold (-10°C) trif luoroacetic 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 -10°C, then 15 15 minutes at ambient temperature. The solid is isolated by centrifugation and washed with ether. The pH of a suspension of this material in 0.5 ml of cold water is adjusted to 6 with IN potassium hydroxide and then applied to a 20 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 C^^H^^K2N^OgS2: C, 31.15; H, 2.81; N, 12.98 25 Found: C, 29.08; H, 3.03; N, 12.19 - kcir>i r 2 OS 2 Example 191 (S ) - (trans ) -3-Amino-4-ethynyl-2-oxo-l-azetidine-sulfonic acid A) 2-(Trimethvlsilyl)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 10 methylmagnesium bromide in ether. The mixture is stirred for 140 -minutes yielding the title compound.
B) (S) -(trans)-4-[2-(trimethylsilyl)ethynyl]-2-15 oxo-3-[(triphenylmethyl)amino]azetidine To a flame dried 250 ml 3-necked flask is added 6.00 g of (S)-(cis)-4-(methylsulfonyl)-2-oxo-3-[(triphenylmethy1)amino]azetidine. The flask is flushed with nitrogen, and then 20 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 25 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). 30 When the addition is complete the cold bath is removed. After 45 minutes, a solution of 3.5 g ( '22^ - 2 2 6 d.- rri 5 5 f 4- 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/dichlorometh'ane 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 cis and trans isomers C) (S) - (trans) -4-ethynyl-2-oxo-3-.[ (triphenyl-methyl)amino]azetidine (S)-(trans)-4-[2-(trimethylsilyl)ethynyl]-2-0x0-3-[ (tr iphenvlmethyl) amino] azetidine (2.-97 g) is dissolved in 30 ml of dichloromethane and 330 mg of tetrabutyiammonium fluoride (containing 20-25% water) is added. After 20 minutes, the solvent is removed in_ 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; o 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-l-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 80-85°C 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 in 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 >180°C). 23/ ^23^t?- Example 19 2 [ 3S- [ 3a ( Z ) , 4 6 ] ] -3- [ [ ( 2-A.mi.no-4- thia zoly 1) (methoxyimino) ace tyl ] amino]-4-ethynyl-2-oxo-l-azetidine-sulfonic acid, potassium salt ( Z) -2-Amino-a - (methoxy imino)'-4 - thiazoleacetic acid (100 mg), 85 mg of N-hydroxybenzo-triazole monohydrate, and 113 mg of dicyclohexylcarbodiimide 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-l-azetidinesulfonic acid (9 5 mg; see example 191) is added as a solid with 1.0 ml of dimethylformamide and 56 yl 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 >230°C.
^GC15 5-£- 2C -' (' ( ^33.
Example 193 (S)-3~[[[(2, 5-Pichlorophenyl)thio]acetyl]amino]-2-oxo-l-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-Dichlorophenylthio&cetic 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, tetrabutyiammonium bisulfate (612 mg) is added, the pH was raised to 3 with IN potassium hydroxide solution, and after extracting a total of three times with dichloromethane, the combined extracts are dried (Na2S0^), and solvent is removed iri 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 C^^H^N^Cl2K: C, 31.21; H, 2.14; N, 6.62; CI. 16.75 Found: C, 27.90; H, 2.11; N, 5.84; CI, 18.0 4 ee±^5~f ~ :12- ^33 set51 '-12 6e-— 2 Q r 2 4 0 Example 194 (3S-trans)-3-[[[(2,5-Dichlorophenyl)thio]acetyl]-amino] -4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt 5 (3S-trans)-3-Amino-4-methyl-2-oxo-l- azetidinesulfonic acid (250 mg; see example 139) is dissolved in dimethylformamide (2 ml) with triethylamine (193 yl). 2,5-Dichlorophenyl-thioacetic acid (285 mg), N-hydroxybenzotriazole 10 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. 15 ' The filtrate is washed with ethyl acetate, layered with dichloromethane and tetrabutyiammonium bisulfate (4.2 mmol) is added. After a total of three extractions with dichloromethane, the combined extracts are dried (Na2SO^) and 20 solvent is removed rn 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 25 ether, and dried to give a powder (30 6 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 water:methanol mixture. 30 Trituration of the residue with acetone gives a powder (233 mg); melting point 212-213°(dec.).
Anal . Calc'd for C^^ lN2<^>5('^2^2K: C, 32.95; H, 2.54; N, 6.41; CI, 16.21; S, 14.66 Found: C, 32.91; H, 2.60; N, 6.42; CI, 16.50; 35 S, 13.77 205240 Examples 195-196 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-methy1-2-oxo-1-azetidinesulfonic acid and the acid listed in column I for (Z)-2-amino-a-(methoxyimino)-4-thiazoleacetic acid, yields the compound listed in column II. 195. 196.
(R)-((aminooxoacetyl)amino](4-hydroxypheny1)acetic acid (R)-[(aminooxoacetyl)amino] ■ phenylacetic acid [3S-[3a(R*),4S]]-3-[[[ (aminooxoacetyl)amino] -' (4-hydroxyphenyl)acetyl]-amino]-4-methyl-2-oxo-l-azetidinesulfonic acid, potassium salt [3S- [3a (R* ) , 48] ] - 3- [ [ [-(aminooxoacetyl) amino]-phenylacetyl]amino]-4-methyl-2-oxo-1-azetidinesulfonic acid, potassium salt; melting point 187°C, dec.
Example 19 7 [3S(R*)J-3—[{[(Aminooxoacetyl)amino] (4-hydroxy-phenyl )acetyl]amino]-2-oxo-l-a2etidinesulfonic acid, potassium salt Following the procedure described in Example 28, but substituting (R)-[(aminooxoacetyl) -amino](4-hydroxyphenyl)acetic acid for (Z)-2-amino-a-I[2-(diphenylmethoxy)-1,1-dime thy1-2-oxoethoxy]-imino]-4-thiazoleacetic acid, yields the title compound melting point 128°C, dec.
Example 198 [3S (R* ) 3 — 3 —.[ [ (2-Amino-4-thiazoly 1) [ [ [3 - [ (2-furanyl-methylene)amino]-2-oxo-l-imidazolidinyl]carbonyl]-amino]acetyl]amino]-2-oxo-l-azetidinesulfonic acid, potassium salt Following the procedure of example 6, but substituting (R)-2-amino-a-[[[3-[(2-furanyl-methylene)amino]-2-oxo-l-imidazolidinyl]carbonyl]-amino]-4-thiazoleacetic acid for aminothiazoleacetic acid yields the title compound, melting point >2 50°C.
I ~aei55f- '•^3f ^ 2 0 Z 2 Example 195 Biological Production of EM5117 9 Liter Fermentation Chromobacterium violaceum SC 11,378 A.T.C.C. 5 No. 31532 is maintained on the following sterilized agar medium (A) : Grams Yeast Extract / 1 Beef Extract 1 NZ Amine A 2 Glucose 10 Agar 15 Distilled to 1 liter The pH is adjusted to 7.3 before sterilization 15 at 121°C for 30 minutes.
A loopful of surface growth of the microorganism is used, to inoculate each of three 500 ml Erlenmeyer flasks, each containing 100 ml of the following sterilized medium (B) : 20 Grams Oatmeal 20 Tomato Paste 2 0 Tap H20 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 rprn; 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 -GUI bit [ ** the following sterilized medium (C): Grams Oatmeal 20 Tomato Paste 20 5 Glucose 30 Tap 1^0 to 1 liter The pH is adjusted to 7-0 before sterilization of 121°C for 15 minutes.
After inoculation, the flasks are incubated at 10 25°C on a rotary shaker (30 0 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. 31532 is used to inoculate 20 each of five -500 ml Erlenmeyer flasks each containing 100 ml of sterilized medium (B). The flasks are then incubated at 25°C on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After the appropriate 25 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 30 at 25°C on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After the -GCT55f—— ^3# ^ 2 C ;r : 2 4 C 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 5 the fermentation is continued under the following conditions: temperature - 25°C; pressure - 10 psig; aeration - 10 cfm; agitation - 155 rpm. Dcon is added as needed asv antifoam agent. After approximately 18-24 hours the fermentation is 10 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 0.005 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 in vacuo to 50 0 ml.
The concentrated material is extracted with 4 00 ml of n-butanol. The upper phase is —eei55T" 2 0 F? '"■) A f\ discarded and the lower phase is concentrated to dryness in vacuo. The residue is dissolved (to the extent possible) in 150 ml of methanol. The insoluble material is discarded and the methanol 5 solution is concentrated to dryness in vacuo, yielding 38.6 g of crude antibiotic.
The crude product is dissolved in 10 ml of methanol-water (1:1) and chromatographed on a 500 ml column of cross-linked dextran gel (Sephadex G—101 in 10 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 15 is concentrated i_n 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 20 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 5 76 mg of material.
'Zb.o _ 20524 The 576 mg .of residue i's dissolved in '4 ml of water and the pH adjusted to 5 with, about 1 ml of 0.1 N sodium hydroxide. The solution is chromatographed on a column of alkylated cross-5 linked dextran gel (Sephadex LH-20) in water, eluting at 1 ml /minute and collecting 10 ml fractions. Active fractions (38-44) are combined and concentrated, giving 4 59 mg of - residue.
Three hundred and forty eight (348) mg of 10 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.
Ac-tive fractions (36-43) are combined and concentrated giving 186.4 mg of material. Chromatography 20 (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 18 6.4 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 dissolved 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 -GCTSSF 2 012 4 yields 189.0 mg of crystalline solid. Recrystallization of this material is accomplished by dissolving it in 0.38 ml of water and adding 3.4 2 ml of methanol. The resulting mixture is cooled on ice and filtered, yielding 145 mg of crystals. Two further recrystallizations in this manner from water-methanol, 1:9, yields 95.9 mg of EM5117, potassium salt, m.p. 194°(dec.).
Optical rotation in water at 21°C (c=l) A (mn) . la] 589 579 54 6 436 365 +94 .3 +98 .6 +113.1 +203 +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 " 30 50 2.5 7 Nutrisoy Flour Glucose Yeastamine CaC03 Distilled H^O to 1 liter MEDIUM E Yeast Extract Malt Extract Glucose Glycerol Distilled H^0 to 1 liter Adjust pH to 7.3 before sterilization Grams 4 10 30 2 -eexSsf' MEDIUM F Glycerol L-asparagine KH_PO„ 2 4 Na_HP0. 2 4 Glucose MgS04.7H20 Yeast Extract Tap H20 to 1 liter MEDIUM G (NH4)2S°4 L-asparagine Glucose Glycerol KH2po4 K2HP04 MgSO -7H20 Yeast Extract Distilled H20 to 1 liter Adjust pH to 7.0 MEDIUM H k2hpo4 kh2po4 Na Citrate MgS04 (NH4)2S°4 Glucose Yeast Extract Distilled ho to 1 liter Grams 10 5 1 2 50 0.2 2.5 Grams 2 5 50 10 3 7 0.2 2.5 Grams 7 3 0.5 0.1 1 . 30 2.5 2 V,: GGi&o-Jr-"" 2 C 52 MEDIUM I Nutrisoy Flour (nh4)2so4 Glucose Yeast Extract CaC03 Distilled H^O to.1 liter MEDIUM J Gerber1s Baby .Oatmeal Contadina Tomato Paste Glucose Tap H^O to 1 liter Adj ust pH 7.0 MEDIUM K Gerber1s Baby Oatmeal Contadina Tomato Paste Glucos e Tap H20 to 1 liter Adjust pH 7.0 MEDIUM L Yeast Extract Malt Extract Glucose MEDIUM M Amberex (10 03) Glucose Tap H20 to 1 liter Grams 10 5 50 2.5 5 Grams 20 .5 20 Grams 5 20 20 Grams 4 10 34 Grams 5 30 MEDIUM N _jse±-55~f' -2S£$/L 2 C Amberex Cerelose Tap E^O to 1 liter Grams 5 33 • GQi-5-Erf-^ —=-23-6"— Example 19 6 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 1 Beef Extract. 1 NZ amine A ■/" 2 Glucose 10 Agar 15 Distilled H^O to 1 liter The pH is adjusted to 7.3 before sterilization at 121°C 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) : 20 Grams Yeast Extract 4 Malt Extract 10 Dextrose 4 Distilled 1^0 1 liter The pH is adjusted to 7.3 before sterilization at 121°C for 15 minutes.
After inoculation, the flasks are incubated at 25°C 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 0 5 2 4 0 sterilized medium (C): Grams Yeast Extract 5 Glucose 10' Distilled H^O 1 liter The medium is sterilized at 121°C for 15 minutes.
After inoculation, the flasks are incubated at 25°C on a rotary shaker (300 rpm; 2 inch stroke) for 18 hours. At this time the contents of the 10 flasks are pooled and the broth is centrifuged yielding approximately 9 liters of supernatant broth.
Isolation and Purification (small scale) 15 Activity from the broth supernatant (10 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 AG1-X2(Cl~)), washed 20 with water and eluted with 5% sodium chloride in aqueous 0.01 M NaH^PO^. The eluate is -concentrated iri vacuo .
The residue is adsorbed on 250 g of charcoal which is washed with water. EM5210 is eluted 25 with methanol-water (1:1). The active fractions are combined and concentrated in vacuo yielding crude EM5 210.
The crude EM5210 is chromatographed on a 280 ml column of strong base anion exchange 30 resin (Bio.Rad AG 1-X2 (CI-)) using a linear gradient prepared from 1 liter of water and 205? 50 1 liter of 2M pyridinium acetate (pH 4.5). The active fractions are combined and concentrated in vacuo.
The partially purified EM5210 is further 5 ■ 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 10 is prepared by passing EM5210 through a column of cation-exchange resin (Dowex 50-X2) in the potassium form.
Isolation and Purification (large scale) 15 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 1-X8 (Cl~) ) - The resin is washed with water and eluted with 5% sodium chloride 20 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 20-4 0 25 mesh Darco) in water. The column is washed with water and the EM5210 is eluted with methanol-water, 1:1. Active fractions are combined and concentrated. The residue (16 g) is dissolved in water and chroma tographed on a column (600 ml) 30 of strong base ion exchange resin (Bi'o.Rad AG 1-X2(Cl~), 200-400 mesh), eluting with a linear j ^GeT55f 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 5 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 10 and freeze-dried, yielding 120 mg of the sodium salt of EM5210.
To transform the sodium salt to the lithium salt an ion-exchange resin (Dowex 50 W-X2, lithium form) column is used. The sodium salt (100 mg) 15 is dissolved in 0.5 ml of water, applied to the column and eluted with water. The active fractions are combined and directly freeze-dried, yielding 95 mg of the lithium salt of BM5210 or as an amorphous solid.
The free acid (inner salt) of EM5210 is 20 prepared by passing a base salt of EMS210 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 7 0 (IT1") and eluted with water 25 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) 30 gives two major ninhydrin positive spots by paper chromatography (Whatman No. 1, butanol-acetic S€-155f 5 2 4 0 acid-water (5:1:4), and one weak ninhydrin positive spot that quenches UV-excited fluorescence. The two major ninhydrin positive spots are D-glutamic acid and D-alanine.
Physical characteristics of EM5210 1) UV spectrum of the sodium salt in water: end absorption 2) IR - Major peaks of the lithium salt in KBr: 10 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 (s) 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): X ( nra) [ a ] 589 +73° 578 +79° 546 +91° 436 +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 Tomato Paste 20 2-Sc> ; &CT55f" r*T" gf»-v / ..,, -'4 MEDIUM D (continued) Grams Tap to 1 liter Adjust pH to 7.0 before sterilization of 121°C for 15 minutes.
MEDIUM E Grams Yeastamine ... 5 Cerelose 11 Tap H20 to 1 liter Sterilization at 121°C.for 15 minutes.
MEDIUM F Grams Glucose 5 Tartaric Acid 2 Yeast Extract 0.5 (NH4)2P04 1 (NH4)2S04 2 K_HPO . 0.5 2 ■ 4 NaH_PO. 0.5 2 4 MgS04-7H20 0.2 CaC03 1 Distilled 1 liter Adjust pH to 6.0 before sterilization of 121°C for 15 minutes.
MEDIUM G Grams Nutrisoy flour 30 Glucose 50 Yeastaraine 2.5 CaCC>3 7 Distilled H20 to 1 liter OS-/ _£G±55'fl ^ 2 0 52 4 0 MEDIUM G (continued) Sterilization at 121 C for 30 minutes, MEDIUM H Grams NZ Amine A 10 Cerelose 33 Yeastamine 2.5 Tap 1^0 to 1 liter Sterilization-at 121°C for 15 minutes.
MEDIUM I Grams Nutrisoy flour 15 Soluble starch 15 Glucose 50 CoCl2.6H20 0.005 CaCo^ 10 Distilled H^O to 1 liter Sterilization at 121°C for 30 minutes 7 sers5T I * Biological Activity The following methodology is used to determine the minimum inhibitory concentration (hereinafter referred to-as MIC) of the 8-lactams of this invention.
The test organisms are grown in approximately -20 ml of Antib-iotic 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 37°C for 18 to 20 hours. These cultures are assumed to 9 contain 10 colony forming units (hereinafter CPU) per milliliter. The cultures are diluted 4 1:100 to give a final inoculum level of 10 15 CFU; dilutions are made with K-10 broth*.
The compounds are dissolved in the appropriate diluent at a concentration of 1000 yg/ml. Twofold dilutions are made in K-10 broth resulting in a range from 1000 yg/ml to 0.5 y g/ml . 1.5 ml 20 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 yg/ml to 0.0 5 yg/ml. Organism growth control plates containing agar 25 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 4 inoculum level of 10 CFU on the agar surface.
( ( -GC15-5JL 0&- The plates are incubated at 37°C for 18 hours and the MIC's are determined. The MIC is the lowest concentration of compound inhibiting growth 5 of the organism.
*K—10 broth is a yeast beef broth containing: Beef extract 1.5 g Yeast extract 3.0 g Peptone v 6.0 g Dextrose 1.0 g Distilled water q.s. 1 liter **K—10 agar Beef extract 1.5 g Yeast extract 3.0 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 6-lactams of this invention are tested against various organisms. The number following each organism refers to the number of 25 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 yg/ml. The symbol "N.T." means not tested. n Organism Staphylococcus aureus , Staphylococcus aureus , Staphylococcus aureus , Staphylococcus aureus , Streptococcus faecalis , Streptococcus agalactiae , Micrococcus luteus, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Klebsiella aerogenes , Klebsiella pneumoniae , Proteus tnirabilis, Proteus rcttgeri, Proteus vulgaris, Salmonella typhosa, Shigella sonnei , Enterobactev cloacae, Enterobactev aerogenes, Citrobacter freundii, Serratia mavcescens , Pseudomonas aeruginosa, Pseudomonas aeruginosa , Acimtobaotev calcoaccticus 1276 2399 2400 10165 9011 9287 24 95 8 2 94 10857 10896 10909 10440 9527 3855 8479 9416 119 5 844 9 8236 10078 9518 978 3 954 5 8329 , 8333 • • M.I.C. (yg/ml) Product of. Example 5 6 1.6 0.8 1 . 6 6 . 3 0.8 1. 6 12.5 6.3 12 . 5 25 6.3 50 100 50 100 12.5 100 50 100 7 8 9 3.1 6.3 3.1 3 .1 3.1 3.1 6.3 3.1 6.3 6.3 6 . 3 6.3 12 . 5 12.5 12.5 - - 100 100 1.6 6.3 3.1 3.1 6.3 12.5 3.1 6.3 100' , 100 100 — 100 100 100 - 100 100 50 - - - 100 100 50 50 100 100 100 100 100 50 - - 100 100 - .
- - N) O tn 0 _n -h v C.'J Orga nism Staphylococcus aureus, 127 6 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 2400 Staphylococcus aureus, 10165 Streptococcus faocalia, 9 011 Streptococcus agalactiae, 9 2 87 Micrococcus luteus, 24 95 Escherichia coli, 8294 Escherichia coli, 10857 Escherichia coli, 10896 Escherichia coli, 10909 Klebsiella aerogenes, 104 4 0 Klebsiella pneumoniae, 9527 Proteus mirabilis, 385 5 Proteus rettgeri, 84 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 8449 Enterobactev cloacae, 8 236 Enterobactev aerogenes, 100 7 8 Citvobacter fveundii, 9 518 Serratia marcescens, 978 3 Pseudomonas aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 3 29 Acinetobacter calcoaccticus, 8333 12 12 . 5 6.3 6.3 100 1 . 6 1.6 0.4 0 ; 8 1.6 0 . 4 0.4 0.1 0 . 2 <0 .05 0.4 0.1 0.4 0 .4 0.8 3.1 12.5 0 . 8 12.5 25 O ' > M.I.C. (pq/ml) Product of Example 13 14 . 16 17 18 12.5 100 3.1 12.5 50 6.3 50 6.3 12.5 50 12 .5 6.3 50 12.5 12.5 - - 50 12.5 12.5 50 - 100 ' - - - - 12.5 1.6 12.5 6.3 6.3 50 0.8 50 100 0.4 - - - - 50 0.8 - - - - 50 3.1 -j 100 100 - 0.2 ' 100 100 - 0.4 - - - - 12.5 0.2 - . - - - 12.5 0.1 - 1.6 <0.05 - - 0.1 - 12.5 <0.05 0.8 • - - - - - 0.4 - - - - 100 0.8 - - - - - 3.1 - - - - - 3.1 100 6.3 100 50 0.4 50 50 50 - 12.5 - - - 100 100 50 6.3 is) C: f 1 I £-■ o t • Organism Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Streptococcus faecalis Streptococcus agalactiae, Micrococcus Escherichia Escherichia Escherichia 19 luteits coli, coli, coli , coli.
Escherichia Klebsiella aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Salmonella typhosa, Shigella sonnei, Enterobacter cloacae, Enterobactev aerogenes, Citrobacter freundii, Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas aeruainosa, AcinetobaciBr calcoaceticus 1276 2399 2400 10165 9011 9287 2495 8294 10857 10896 10909 10440 9527 3855 8479 9416 1195 8449 8236 10078 . 9518 9783 9545 8329 , 8333 411 o M.I.C. (tig/ml) Product of Example 21 22 . 23 24 26 3.1 12.5 50 50 6.3 1.6 1.6 3.1 12.5 100 50 6.3 1.6 1.6 3.1 12.5 100 50 3.1 1.6 3.1 6 . 3 100 100 100 6.3 - - • 50 50 3.1 1.6 12.5 50 3.1 0.1 0.8 6 . 3 6.3 50 • T- 12 t 5 0.8 3.1 0.8 - i- -• 0.4 6.3 0.8 100 - - <0.05 3.1 12.5 1.6 100 - 100 0.2 6 . 3 0.8 100 N.T. 100 <0.05 1.6 50 0.8 - - - 0.4 6.3 0.8 - - - <0 .05 0.4 100 0.8 - - - 0.2 0.4 12.5 <0.05 - ■ - 100 0.4 0.4 50 1.6 f - - <0.05 0.2 50 0.8 100 - - <0.05 0.4 12.5 0 .8 100 - - 0.2 6.3 100 1.6 100 - -! 0.4 6.3 - 0.8 - - - 0.8 50 6 . 3 100 - - 0.4 50 50 100 100 100 0.8 50 3.1 6.3 - 50 0.4 0.4 - 100 3.1 12.5 50 — — — 50 IO o f H Organism Staphylococcus a~ trews, 127 6 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 24 00 Staphylococcus aureus- 10165 Strep tococcus facc.alis, 9011 Streptococcus agalactiae, 9 28 7 Micrococcus lutcus, 24 95 Escherichia coli, 8 294 Escherichia coli, 10857 Escherichia coli, 1089 6 Escherichia coli, 10909 Klebsiella aerogenes, 10440 Klebsiella pneumoniae, 9 52 7 Proteus mirabilis, 38 55 Proteus rettgeri, 84 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 84 49 Entcrobactcr cloacae, 8 236 Enterobacter aerogenes, 10 078 Cl trobactfrcundii, 9 518 Serratia marccscnns, 9783 Pseudomonas aerugi)iosa, 9 54 5 Pseudomonas aeruginosa, 8 329 Acinetobacter calcoaceticus, 8 333 27 O ( ► H.I.C. (tig/ml) 28 29 31 32 33 12.5' - 12.5 1.6 1.6 1.6 3.1 - 12.5 3.1 0.8 1.6 3.1 - 6.3 1.6 1.6 3.1 '100 - 100 6.3 12.5 3.1 12.5 - - - 0 .4 12 . 5 0.4 1.6 0.4 0.2 0.4 3.1 12.5 6.3 1.6 0.8 1. 6 12.5 0.4 12.5 - ; - - - 1 . 6 0.2 6.3' 0 '' 3.1 3.1 12.5 3.1 0.8 12.5 : 50 100 - 50 - 1. 6 0.1 3.1 50 50 50 100 0.4 12.5 - - - - 1 . 6 <0.05 6.3 100 100 ■ - - 1. 6 <0 . 05 6.3 100 100 100 - 0 . 2 <0 . 05 0.8 100 - - 1 . 6 0.1 12.5 100 6.3 3.1 12 . 5 0.8 <0.05 3.1 50 - ■ - 100 3.1 0 . 2 6.3 100 - - - 6 . 3 0 . 8 6.3 - - - - 50 0 . 8 12.5 - - - - ' ■1 . 6 - - - - - 12.5 0 . 8 50 - - . - 3.1 0 .8 12.5 12.5 12.5 50 3.1 50 - - - - — 100 — — — — — N) O Organism Staphylococcus aureus, 1276 Staphylococcus aureus, 239 9 Staphylococcus aureus, 24 00 Staphylococcus aureus, 10165 Streptococcus faecalis, 9011 Streptococcus agalactiae} 9 28 7 Micrococcus luteus, 24 95 Escherichia coli, 8 29 4 Escherichia colij 108 57 Escherichia coli-, 10896 Escherichia coli, 10.90 9 Klebsiella aerogenes, 1044 0 Klebsiella pneumoniae, 95 27 Proteus mirabi'lia, 3 85 5 Proteus rettgeri, 84 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 84 49 En terobac ter a loacae, 8 23 6 Enterobacter aerogenes, 10078 Citrobacter frcwidii, 9518 Serratia marccsccns, 9 78 3 Pseudomonas aeruginosa, 9545 Pseudomonas aeruginosa, 8 3 29 Acinetobacter calcoaceticus, 8 3 33 • H M.I.C. (ug/ml) Product of Example 37 39 40 41 42 43 44 6.3 0 . 8 3.1 1.6 6.3 3.1 6 . 3 0.8 3.1 1.6 6.3 12.5 3.1 1 . 6 0 . 8 3.1 1.6 12 . 5 12 . 5 6 . 3 100 3.1 6.3 3.1 100 100 - 50 0 - - - 100 0.4 0.4 0.8 1.6 1.6 3.1 1 . 6 1.6 0.8 1.6 3.1 6.3 12.5 3.1 - - 1.6 - 50 3.1 50 12.5 0.2 50 ' 100 3.1 - 100 1.6 .50 6.3 - - 0.2 100 50 1.6 - - 0.8 - • - 50 6 . 3 - - 0.4 - - 3.1 - - <0.05 50 100 3.1 - 100 <0.5 6.3 •50 1.6 100 <0.5 50 - 50 3.1 - - <0.5 100 0 1.6 - - 0.4 100 100 3.1 - - '0.8 100 50 12 .5 - - 1.6 - 50 6.3 - - 1.6 - • . - 1.6 50 100 50 100 12.5 0.2 100 50 12.5 3.1 - - 6.3 25 - 100 100 >0 & IS) o H Ln Ul H- Organism Staphylococcus aureus, 127 6 Staphylococcus aureus, 2 399 Staphylococcus aureus, 24 00 Staphylococcus aureus, 10165 Streptococcus, faecalis, 9011 Streptococcus agalactiae, 9 287 Micrococcus luteus, 249 5 Escherichia coli, 8294 Escherichia coli, 10857 Escherichia coli, 108Q6 Escherichia coli, 109 09 Klebsiella aerogenes, 1044 0 Klebsiella pneumoniae, 9527 Proteus mirabilis, 3 85 5 Proteus rattgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 8 44 9 Enterobacter cloacae8236 Entcrobacter aerogenes, 100 78 Ci trobac ter freu?iclii, 9518 Serratia marcescens, 9783 Pseudomonas aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 3 29 Acinetobacter calcoaceticus, 8333 45 • • M.I.C. (yig/ml) Product of Example 46 48 50 . 51 52 53 54 100 12.5 12.5 12 .5 50 12.5 3.1 100 12 .5 50 12 . 5 3 .1 100 12 .5 50 100 12. 5 6.3 100 50 100 100 100 - - - ■- - . - 100 50 6.3 12 .5 6.3 3.1 1. 6 50 3.1 6.3 1.6 ,12.5 6.3 1.6 - 100 12.5 6.3 - 50 100 , 0 •• -0 12 . 5 6.3 100 - — .25 12.5 6.3 N.T. 12.5 100 - 12.5 3 .1 - 100 6.3 - 50 12 . 5 .6.3 - - 12.5 3.1 - 50 100 - 100 12 . 5 3 .1 - - - 100 . - 6 . 3 - 100 6.3 1.6 - 100 12.5 3.1 100 - - - - - 100 •. 50 12 . 5 100 100 - - 50 . 50 100 100 - - 50 12.5 - 50 100 50 12.5 6 . 3 - - - - - - 100 100 — - 100 * — — \V\ to Organism Staphylococcus aureus, 127 6 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 240 0 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae, 9 28 7 Micrococcus luteus, 24 9 5 Escherichia coli, 8 29 4 Escherichia coli, 108 57 Escherichia coli, 10896 Escherichia coli, 10909 Klebsiella aerogoies, 104 4 0 Klebsiella pneumoniae, 9 527 Proteus mirabilis, 3855 Proteus rettgeri, 84 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 84 4 9 Entt'.robactar cl oanac, 8 236 Enterobacter aerogenes, 10 078 Citrobacter frewidii., 9 518 Serratia marcescens, 9 78 3 Pseudomonas aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 3 29 Acinetobacter calcoaccticus, 83 3 3 • O M.I.C, (yg/ml) Product of Example 56 57 58 59 60 61 62 12.5 0.8 12.5 1.6 3.1 3.1 12.5 0.8 6.3 1.6 1 . 6 1.6 12,5 50 1.6 6.3 3.1 3.1 3.1 - 100 12.5 50 50 50 - 100 50 - 100 100 50 12.5 6.3 0.8 1.6 0.2 0.2 0.4 6 . 3 1.6 3.1 0.4 0.8 1.6 - - 0.8 1.6 0.8 1. 6 6.3 - - <0 .05 1.6 <0.05 <0.05 0.4 100 - 0.2 6.3 0.2 0.2 3.1 100 - 0.1 0.8 0.1 0.1 1.6 - - 0 .8 3.1 0.8 1 . 6 12.5 - - 0.1 0.8 0.2 0.2 1.6 100 - 0.1 1.6 1.6 0.8 0.8 50 100 0.1 0.1 3.1 1.6 0.8 - - <0.05 3.1 0.2 <0.05 0.1 100 - 0.1 0.8 0.8 0.4 1.6 100 - 0.4 1.6 0.4 0.4 3.1 - - 0.8 3.1 1.6 0.8 6.3 - - 1.6 3.1 3.1 3.1 12.5 - - 1.6 0.4 0.4 6.3 50 - 1.6 1.6 1.6 50 50 0.8 6.3 0.4 0.2 3.1 - - 12.5 - 6.3 6.3 100 - 100 100 6.3 — o Ni O ft ■ % Organism Staphylococcus aureus, 1276 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 24 00 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae, 9 28 7 Micrococcus luteus, 249 5 Escherichia coli, 8294 Escherichia coli, 10857 Escherichia coli, 10896 Escherichia coli, 10909 yClebsiella aerogenes, 104 4 0 Klebsiella pneumoniae, 9 5 27 Froteus mirabilis, 3 85 5 Proteus rettgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 8 44 9 Envobactpr cloae.ae, 8 2 36 Entcj.robact?r aerogenes, 100 78 Citrobacter frewidii, 9518 Serratia marcescens, 9783 Pseudomonas aeruginosa, 95 4 5 Pseudomonas aeruginosa, 8 3 29 Acinetobacter calcoaceticus, 8333 • O M.l.c. (yg/ml) Product of Example 64 65 . 66 67 68 69 70 3.1 100 50 ' 3.1 — 6.3 12.5 50 3.1 100 12.5 - 12.5 50 3.1 100 12 .5 - - - - • 50 - - - - ■- - 6.3 50 3.1 1.6 12.5 0.1 12.5 - 0.8 0.4 0.1 100 - - 12.5 12.5 1.6 12.5 1.6 100 - 1.6 1.6 1.6 0.8 1.6 100 - 12.5 6.3 ■ 6.3 12.5 3.1 100 - 3.1 1.6 0.8 1.6 0.4 - 12 . 5 ' 6.3 1.6 1.6 - - 3.1 1.6 0.4 6.3 0.8 - - 0.8 0.4 0.4 1.6 1. 6 100 100 0 . 2 0.2 <0.05 0.2 <0 .05 - - 0.1 0.2 0.8 0.4 0 . 8 100 - 3.1 0.2 0.2 3.1 0.4 100 - 12.5 6.3 1.6 6.3 0 . 8 - - 12.5 6.3 0.8 12.5 0.8 - - 12.5 1.6 1. 6 - - 12.5 6.3 12.5 1.6 100 100 12 .5 6.3 50 6.3 12.5 6.3 6.3 3.1 1.6 1.6 - - - 100 50 100 12.5 o M . I .C. (ug/ml) Product of Example Organism 71 72 73 74 76 77 78 79 Staphylococcus aureus, 1276 3 .1 12.5 6.3 12.5 6.3 - - Staphylococcus aureus, 2399 3.1 12 . 5 6.3 6.3 - - Staphylococcus aureus, 2400 3 .1 12.5 12.5 6.3 — - 12.5 Staphylococcus aureus, 10165 50 50 12.5 50 12.5 — - - Streptococcus faecalis, 9011 50 100 - - - - - 100 Streptococcus agalactiae, 9287 1.6 3.1 3.1 12.5 3.1 - - 3.1 Micrococcus luteus, 249 5 3.1 6.3 6.3 3.1 0.8 - . 50 1.6 Escherichia coli, 8 294 0.8 3.1 50 1.6 Escherichia coli, 10857 3 .1 1. 6 2 5 3.1" 100 ' 12.5 0.4 Escherichia coli, 10896 3.1 12.5 1.6 6.3 1.6 Escherichia coli, 10909 12 . 5 0.8 12.5 12.5 0.8 6.3 0.4 Klebsiella aerogenes, 1044 0 50 1 . 6 50 3.1 100 3.1 Klebsiella pneumoniae, 9527 12.5 0.8 1.6 50 0.8 Proteus mirabilis, 3855 12.5 1.6 12 . 5 50 3.1 0.2 Proteus rettgeiK, 8479 1.6 0 .1 1.6 50 3.1 50 <0 .05 Proteus vulgaris, 9416 3.1 1.6 . 25 100 3.1 50 50 0.2 Salmonella typhosa, 1195 12.5 0.8 1.6 50 0 .2 Shigella sonnei, En t>-roh .7c ter c loacac., 8449 0.8 12.5 0.8 12.5 1.6 8236 50 0.8 100 12.5 12.5 1.6 Enf'.-'rebac ter aevogo^ies, 10078 100 1 . 6 100 6.3 100 •25 6.3 Citrobacter freundii, 9518 50 12 .5 50 12.5 3.1 Serratia marcescens, 9783 100 50 6.3 50 3.1 Pseudomonas aeruginosa, 9545 12.5 6.3 50 12.5 6.3 6.3 3.1 Pseudononas aeruginosa, 8329 - 50 100 - 100 50 Acinetobacter calcoaceticus , 8333 - 100 50 - 12.5 —■ — — m to is) O c.
H-u-Lr Hi » ".1 o • O Organism Staphylococcus aureus, 1276 Staphylococcus aureus, 2 39 9 Staphylococcus aureus} 240 0 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae, 9 287 Micrococcus luteus, 24 9 5 Escherichia coli, 8 294 Escherichia coli, 10 857 Eaoherichia coli, 10 89 6 Escherichia coli, 10909 Klebsiella aerogenes, 104 40 Klebsiella pneumoniae, 9527 Proteus mirabilis, 385 5 Proteus rettgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei84 49 Enterobacter cloacae, 82 36 Enterobactev aerogenes, 100 78 Citrobacter freundii, 9 518 Serratia marcescens, 9 78 3 Pseudomonas aevuginosa, 9 54 5 Pseudomonas aeruginosa, 8 329 Acinetobacter calcoaceticus, 8 333 M.I.C. (yig/ml) Product of Example 88 89 90 91 92 9 3 9.4 ,12.5 12 . 5 3.1 - 50 100 12 .5 3.1 - 50 100 12.5 50 6.3 - 50 100 50 50 100 - 50 — ■ — - — 100 - — 12.5 12.5 12.5 0.8 - 3.1 ■ 0.8 0.4 12. 5 1.6 100 3.1 - - 12.5 - — , .L V 100 12 . 5 12 . 5 0 . 8 50 - _ 50 50 6 . 3 - - _ 100 50 1.6 - - — - - 12.5 - - — 100 100 100 1.6 - 100 100 100 6.25 - — 50 50 3.1 - 100 3.1 100 50 0 .1 100 _ — 50 100 50 1.6 - — 50 - - 3.1 - - 50 • - - 6.3 - - - . - - 12 . 5 - - - - 12 . 5 - — - 6.3 - _ — 100 3.1 100 - - , _ 50 — - - — - - - — o M.i.c. (ug/ml) Product of Example Organism Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Streptoooccus faecalis, Streptococcus agalactiae, Micrococcus luteus, Escherichia coli, Eaoherichia coli, Escherichia coli, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Proteus rettgeri, Proteua vulgaris, Salmonella typhosa, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter frewidii, Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, 95 ■ 96 97 98 99 100 101 1276 50 - 6 . 3 100 100 50 2399 50 - 6.3 50 100 50 2400 50 - 12.5 50 100 50 50 10165 100 100 100 9011 - - - — - - - 9 287 6.3 12 . 5 1.6 100 100 12.5 ' 2495 .6.3 6 . 3 1 . 6 - 100 50 8294 - 6.3 - - , ,100 , 100 10857 100 0.2 - — 50 50 10896 12.5 50 1. 6 - - 50 100 10909 6.3 50 0 .8 - - 50 10440 50 - 6 . 3 - 9527 50 - 1 . 6 - - 100 100 3855 50 100 3.1 - 8479 100 6 . 3 - - 50 100 9416 50 - <0 . 05 _ — 100 - 1195 50 — 1.6 - — 50 100 8449 12.5 50 3.1 - — 50 100 8236 • 50 50 6 . 3 - — - - 10078 . 50 100 12 . 5 - — - • V — 9518 50 50 3.1 - — 100 — 9783 50 12 . 5 - - 50 » 9545 ; 50 50 6 . 3 - 8329 ' - - - - 8333 : 50. 50 — - - — , is) O & JP i- M.I.C. (liq/ml) Organism Staphylococcus aureus, 1276 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 2400 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae, 9 287 Micrococcus luteus, 24 9 5 Escherichia coli, 82 94 Escherichia coli, 10 857 Escherichia coli, 10 896 Escherichia coli, 10909 Klebsiella aerogenes, 104 40 Klebsiella pneumoniae, 9 5 27 Proteus mirabilis, 3855 Proteus rettgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 1195 Shigella sonnei, 8449 Enterobacter cloacae, 82 36 Enterobacter aerogenes, 10078 Citrobacter freundii, 9 518 Serratia marcescens, 9 78 3 Pseudomonas^ aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 32 9 Acinetobacter calcoaceticus, 8 33 3 Product of Example 102 103 104 105 106 107 108 100 12 . 5 3.1 12.5 3 11 1.6 100 - 6.3 3.1 6.3 1.6 3.1 50 - 6.3 3.1 12,5 1,6 3.1 ■ - - 50 50 50 50 - - 50 100. - 50 3.1 50 1 .6 1.6 0,8 0,8 0.8 3.1 12.5 1 . 6 3.1 12,5 ■ 3,1 0.8 0 .1 0.4 6 . 3 0.4 0,8 . 3,1 , 0.8 <0.05 <0.05 0 . 2 0.1 o.,i 0.1 <0 .05 0.1 0 . 2 6 . 3 0.8 1,6 1.6 0 . 2 <0 .05 <0 .05 1 ,6 0.1 0.2 Q . 4 0.1 0 . 2 0 . 4 0 .4 0,8 3.1 0 .8 0 .1 <0.05 3.1 0.1 0,2 0.4 0 .05 0.1 <0.05 1 . 6 0 .1 0,1 0 .4 0.2 <0 .05 <0.05 0 . 2 <0.05 <0 .05 0.1 <0.05 <0.05 <0.05 0 .8 <0.05 <0,05 0.1 <0.05 <0 .05 <0.05 0 .8 0.1 0,1 0.4 0.1 0.1 0 . 2 3.1 0.4 0.4 0.8 0.4 0.2 0.4 6.3 0.8 0.8 1.6 1.6 0.4 0.4 0.8 1,6 6.3 1.6 0.1 0.4 12 . 5 1.6 6,3 3.1 0.8 0.8 0.2 12 . 5 3,1 12,5 6,3 1.6 0.8 0.8 3 .1 0.2 0,8 0.2 0.8 100 3 .1 50 3.1 12,5 12.5 12.5 12 . 5 100 — 50 50 M.I.C. (pg/ml) Organism Staphylococcus aureus, 127 6 Staphylococcus aureus, 2 39 9 Staphylococcus aureus j 2 400 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae} 9 287 Micrococcus luteus, 2 49 5 Escherichia coli, 8 294 Eaoherichia coli, 10 857 Escherichia coli, 10 89 6 Escherichia coli, 10909 Klebsiella aerogenes, 104 40 Klebsiella pneumoniae, 9 5 27 Proteus mirabilis, 3855 Proteus rettgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 8 449 Enterobacter cloacae, 8 2 36 Enterobacter aerogenes, 100 7 8 Citrobacter freundii, 9 518 Serratia marcescens, 9 78 3 Pseudomonas aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 329 Acinetobacter calcoaceticus, 8 333 Product of Example 109 110 Ill 112 113 114 115 12 . 5 1 . 6 1.6 1.6 3.1 12 i 5 1.6 12.5 1. 6 1 . 6 1 . 6 3.1 12.5 0.8 6.3 1 . 6 3 . 1 1.6 3.1 6.3 0 .8 100 12.5 50 6.3 3.1 50 50 100 50 sa 100 1.6 0 . 4 0.2 0.1 0.4 a,8 Q . 8 0 .8 0.4 0.8 0.8 Q . 8 0.2 3.1 0.8 12 .,5 6 . 3 0.8 ' 1.6 - .0.2 0 . 2 <0.05 <0.05 0.4 : 0.2: 6.3 0.8 6 . 3 1 .6 0.2 12. 5 Q .8 - 0 . 2 3.1 0 . 8 0.1 6.3 0.4 - 1.6 6 . 3 1.6 3.1 - 0 . 2 6.3 1 . 6 0 . 2 6.3 1.6 100 0.2 1.6 1.6 0.4 6.3 0.4 ■p* <0 .05 0.4 3.1 0.8 Q. 4 <0.05 1QQ 0.2 0 . 4 0 .4 <0.05 1.6 <0.05 6.3 0.1 6.3 3.1 0.4 3.1 0.4 - 0.8 12.5 3.1 0.4 12.5 1.6 - ' 1.6 6.3 1.6 3.1 - • 3.1 12.5 3.1 6.3 - . 1.6 12 . 5 3.1 0.8 6.3 12.5 6 . 3 3.1 6.3 0.4 0.8 0.8 0.8 3,1 0,8 12.5 12 . 5 12.5 100. - 100 — 2 5 " — # M.I.C. (pg/ml) Product of Example Organism Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Streptococcus faecalis. Streptococcus agalactiae, Micrococcus luteus, Escherichia coli, Escherichia coli, Escherichia coli, Escherichia coli, Klebsiella aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Proteus rettgeri, Proteus vulgaris, Salmonella typhosa, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundiij Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, 116 117 118 119 120 121 122 1276 6.3 3.1 1.6 6.3 3.1 0.8 2399 6.3 3.1 1. 6 6.3 3.1 1.6 2400 6.3 6.3 3.1 3.1 3.1 12.5 3.1 10165 12.5 12 . 5 0. 100 100 6.3 9011 - 50 100 100 100 - - 9287 3,1 0 . 2 0 .4 <0 .05 <0.05 3.1 0.8 2495 12.5 1.6 3.1 0.8 0.8 6.3 0.4 8294 100 3 .1 .12.5 0.2 3 .,1 , 3.1. 6.3 10857 50. <0.05 0.1 <0 .05 <0.05 1.6 <0.05 10896 50 0.8 1 . 6 0.2 0.8 3.1 1.6 10909 50 0.2 0 . 8 <0 .05 0.2 1.6 0.4 10440 100 3.1 12,5 0.4 6.3 6.3 6.3 95 27 100 1.6 6 . 3 0.1 0.8 3.1 0.2 3855 100 0 . 8 3 . 1 0.4 3.1 3.1 1.6 8479 1.6 6 . 3 Q . 2 3.1 0.2 0.8 9416 100 <0.05 0 . 2 <0 . 05 0.1 3,1 <0.05 1195 100 1 .6 6 . 3 0.2 1.6 1.6 0.8 8449 100 1.6 6.3 0.2 1.6 3.1 1.6 8236 100 3.1 6 . 3 0.8 3.1 6.3 3.1 10078 • - 6 . 3 2 5 0.8 6.3 12.5 12.5 9518 1 , 6 6.3 0.4 1.6 50 3.1 9783 12.5 1,6 12.5 0 .8 6.3 6.3 9545 12.5 6 . 3 1 . 6 0.4 0.2 6.3 3.1 8 329 . - 100 50 6.3 3.1 1QQ 8 333 . 50 100 50 6.3 1QQ .25 • O Organism Staphylococcus aureus, 1276 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 2400 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae, 9 28 7 Micrococcus luteus, 24 9 5 Escherichia coli, 8 2 94 Escherichia coli, 10 857 Escherichia coli, 10 896 Escherichia coli, 10909 Klebsiella aerogenes, 104 40 Klebsiella pneumoniae, 9 5 27 Proteus mirabilis, 3 85 5 Proteus rettgeri, 84 79 Proteus vulgaris, 9416 Salmonella typhosa, 119 5 Shigella sonnei, 8449 Enterobacter cloacae, 82 36 Enterobacter aerogenes, 100 7 8 Citrobacter freundii, 9 518 Serratia marcescens, 9 78 3 Pseudomonas aeruginosa, 9 54 5 Pseudomonas aeruginosa, 8 329 Acinetobacter calcoaceticus, 8 33 3 M.I.C. (pg/ml) Product of Example 123 124 125 126 127 128 129 3.1 12 . 5 6.3 3.1 3.1 0.8 50 3.1 12 . 5 6.3 3.1 3.1 3.1 6.3 12.5 6.3 3.1 3.1 6.3 12.5 50 5Q 50 - - 100 100 Q 5Q 50 1.6 0.8 0 . 4 0 . 8 1.6 Q ,8 1.6 1.6 3.1 3.1 3.1 6.3 3.1 Q.8 1.6 6.3 12.5 - . 25 , 0.4 <0.05 6.3 0.1 0.4 Q, 2 0.2 0.4 12.5 3.1 6.3 6.3 0.8 0.1 12.5 0.8 1.6 - 1.6 0.1 1.6 12,5 5Q 0.8 0.2 0.8 6.3 3.1 0.2 0.2 6 . 3 0 . 4 3.1 — 12.5 <0 .05 <0 .05 1. 6 1 . 6 3.1 12,5 0.05 <0 .05 3.1 <0 . 05 0.2 a,8 0.2 0.2 .6 . 3 3.1 6.3 — 12.5 <0 .05 0.4 12.5 3.1 6.3 — 12,5 0.4 0.8 100 12.5 .12,5 — 12,5 0.4 • 3.1 100 50 50 - 5Q Q. 8 .0.8 100 12 . 5 - 12.5 1.6 : 1.6 12.5 - 3.1 0 . 8 12.5 1.6 3.1 0.4 6.3 12.5 - 100 12.5 . 50 - 50 . 0 M , I. C . ()jg/ml) Product of Example Organism 130 131 132 133 134 135 136 Staphylococcus aureus, 1276 6 . 3 12.5 6.3 - 3.1 100 Staphylococcus aureus, 2399 6 . 3 12 . 5 3 .1 — 3.1 100 Staphylococcus aureus, 2400 6 . 3 6.3 12.5 — 3.1 50 Staphylococcus aureus. 10165 100. 50 50 12.5 — 50 - Streptococcus faecalis, 9011 100 50 — — 5Q T* Streptococcus agalactiae, 9 287 12.5 0.4 0.4 12.5 50. 0-, 8 3,1 Micrococcus luteus, 2495 3 .1 3.1 1 . 6 IQO 6,3 6,3 Escherichia coli, 8294 1.6 3.1 - 3,1' •" 6 ,3 Q. 2 Escherichia coli, 10857 0.2 0 . 2 CO o 50 0.8 ' a,8 <0 ,Q5 Escherichia coli, 10896 1.6 6 . 3 3.1 100 3.1 12.5 Q. 2 Escherichia coli, 10909 0.4 1 . 6 0.8 - Q.8 0..8 0.1 Klebsiella aerogenes, 10440 1.6 50 6.3 - 3,1 3.1 0,4 Klebsiella pneumoniae, 9527 0.2 3.1 1.6 - a.8 a,8 Q.l Proteus mirabilis, 3855 0.8 6.3 1 . 6 - 0,4 Q.4 <0,05 Proteus rettgeri, 8479 0.2 6.3 <0 .05 100 <0 .05 a,4 <0,05 Proteus vulgaris, 9416 0.2 0.4 1 . 6 50 0.4 Q.4 <0.05 Salmonella typhosa, 1195 0.4 12.5 0.4 - 0.4 Q, 8 <0 .05 Shigella sonnei, 8449 0.8 12 . 5 1. 6 - 1.6 3,1 Q .2 Enterobacter cloacae, 8236 3.1 1 . 6 - 0 .8 3,1 0,2 Enterobacter aerogenes, 10078 3.1 50 6.3 - 3,1 6.3 0.4 Citrobacter freundii. 9518 6.3 12.5 - 3.1 12.5 0.2 Serratia marcescens, 9783 : 6.3 12.5 - 6.3 Q.4 Pseudomonas aeruginosa, 954 5 0 . 8 3.1 100 6.3 Q.8 0,8 Pseudomonas aeruginosa, 8329 12.5 - 50. 12.5 Acinetobacter calcoaceticus, 8333 50 100 — 5Q c\ o a Organism Staphylococcus aureus , 1276 Staphylococcus aureus, 2 39 9 Staphylococcus aureus, 2400 Staphylococcus aureus, 1016 5 Streptococcus faecalis, 9011 Streptococcus agalactiae , 9 28 7 Micrococcus luteus, 24 9 5 Escherichia coli, 82 94 Escherichia coli, 10 857 Escherichia coli, 10 89 6 Escherichia coli, 10909 Klebsiella aerogenes, 104 40 Klebsiella pneumoniae, 9527 Proteus mirabilis, 38 55 Proteus rettgeri, 8 4 79 Proteus vulgaris, 9416 Salmonella typhosa, 1195 S}\igella sonnei, 8449 Enterobacter cloacae, 8 2 36 Enterobacter aerogenes, 10078 Citrobacter freundii, 9 518 Serratia marcescens, 9 78 3 Pseudomonas aeruginosa, 9 545 Pseudomonas aeruginosa, 8 329 Acinetobacter calcoaceticus, 8 333 • (II M.I.C. (ijq/ml) Product of Example 138 140 141 142 143 144 14^ 100 50 6.3 _ - 50 50 N.T. '50 6.3 - 50. 50 12.5 100 12,5 - - - 1.6 0.4 6.3 3.1 Q.8 50 6.3 6 .3 6.3 3.1 12.5 1.6 Q.l 1. 6 1.6 1.6 1 .-6 • 50 ' 0.4 <0.05 <0.05 <0.05 1.6 0.4: Q.4 0.2 0.1 0 .4 1. 6 1.6 1.6 6.3 0.2 <0 .0.5 0 .1 0 .1 0.8 0.8 6.3 <0.05 0 .2 0 .8 1. 6 1.6 3.1 - 0.8

Claims (3)

WHAT WE CLAIM IS: f) A. J. P. & S. I(L Z -ft
1. A compound of the formula v R1-NH- -CH- 'R, n m NH-S03M o wherein Ri is hydrogen or acyl or the group Rj-NH- is protected amino; |V\ © |< hydrogeiA Gy tK CAHPIa j V is a leaving group;^and R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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. «
2. A process for preparing a compound having the structure V . R . ANH - CH C // o -NHSO^M® wherein A is a protecting group; V is a leaving group; R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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; and M® is hydrogen or a cation characterised by sulfonating a compound having the formula ANH - CH c o' V ■ C ' NH, ■ R, •R- 2 0 5 2 A Q -27,Y wherein A, V, R3 and R4 are as herein defined.
3. A compound having the structure v /R4 ANH - CH C ^3 // O C NHSO?M® 3 wherein A is a protecting group; V is a leaving group; R3 and R4 are the same or different and each is hydrogen, alkyl, cycloalkyl having from 3 to 7 carbon atoms, phenyl or substituted 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; and M® is hydrogen or a cation. v J. P. & S. 1 a- C(A(wa 3 ^ compound as claimed in any -one of claims-1 to 3■ substantially as hereinbefore described with reference to any example thereof. oated this day of 19^3 A. J. FARK & SON per ■ agents for the appw0ant6 & AUG 1983
NZ205240A 1980-02-07 1981-02-05 Sulphonamide derivatives,being starting materials for producing beta-lactams NZ205240A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11927680A 1980-02-07 1980-02-07
US18889380A 1980-09-29 1980-09-29

Publications (1)

Publication Number Publication Date
NZ205240A true NZ205240A (en) 1984-07-31

Family

ID=26817179

Family Applications (2)

Application Number Title Priority Date Filing Date
NZ196202A NZ196202A (en) 1980-02-07 1981-02-05 Beta-lactam antibiotics (of azetidine-sulphonic acid type)
NZ205240A NZ205240A (en) 1980-02-07 1981-02-05 Sulphonamide derivatives,being starting materials for producing beta-lactams

Family Applications Before (1)

Application Number Title Priority Date Filing Date
NZ196202A NZ196202A (en) 1980-02-07 1981-02-05 Beta-lactam antibiotics (of azetidine-sulphonic acid type)

Country Status (29)

Country Link
JP (3) JPS56125362A (en)
KR (1) KR860001289B1 (en)
AR (1) AR245932A1 (en)
AU (1) AU569407B2 (en)
CA (1) CA1338670C (en)
CH (2) CH653993A5 (en)
DD (1) DD156180A5 (en)
DE (1) DE3104145C2 (en)
DK (1) DK166280C (en)
ES (1) ES8205397A1 (en)
FI (1) FI80271C (en)
FR (1) FR2509299B1 (en)
GB (2) GB2071650B (en)
GR (1) GR74151B (en)
HK (1) HK57785A (en)
IE (2) IE51392B1 (en)
IL (1) IL62082A (en)
IT (1) IT1135360B (en)
KE (1) KE3539A (en)
LU (1) LU83117A1 (en)
MY (1) MY8600179A (en)
NL (1) NL192924C (en)
NO (2) NO161065C (en)
NZ (2) NZ196202A (en)
PH (3) PH24729A (en)
PT (1) PT72465B (en)
SE (3) SE457954B (en)
SG (1) SG39185G (en)
YU (1) YU44829B (en)

Families Citing this family (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0050965A1 (en) * 1980-10-23 1982-05-05 Takeda Chemical Industries, Ltd. Beta-lactamase inhibitory composition
WO1982001873A1 (en) * 1980-12-05 1982-06-10 Takeda Chemical Industries Ltd 1-sulfo-2-oxoazetidine derivatives and process for their preparation
US4673739A (en) * 1980-12-05 1987-06-16 Takeda Chemical Industries, Ltd. 4-carbamoyloxymethyl-1-sulfo-2-oxoazetidine derivatives and their production
JPS57131758A (en) * 1980-12-05 1982-08-14 Takeda Chem Ind Ltd 1-sulfo-2-oxoazetidine derivative, its preparation and use
WO1983000690A1 (en) * 1981-08-25 1983-03-03 Takeda Chemical Industries Ltd Azetidine derivatives and process for their preparation
US4675397A (en) * 1980-12-05 1987-06-23 Takeda Chemical Industries, Ltd. 1-sulfo-2-oxoazetidine derivatives and their production
MX7096E (en) * 1980-12-05 1987-06-19 Takeda Chemical Industries Ltd METHOD FOR PREPARATION OF 2-OXOAZETIDINE DERIVATIVES
JPS58210061A (en) * 1982-05-31 1983-12-07 Takeda Chem Ind Ltd 1-sulfo-2-oxoazetidine derivative, its preparation and use
US4782147A (en) * 1980-12-05 1988-11-01 Takeda Chemical Industries, Ltd. 1-sulfo-2-oxoazetidine derivatives and their production
US4572801A (en) * 1981-04-30 1986-02-25 Takeda Chemical Industries, Ltd. 4-Carbamoyloxymethyl-1-sulfo-2-oxoazetidine derivatives and their production
CA1181075A (en) * 1981-07-13 1985-01-15 David M. Floyd CRYSTALLINE ANHYDROUS FORM OF [3-S-[3.alpha.(Z),4 .beta.]]-3-[[(2-AMINO-4-THIAZOLYL) [(1-CARBOXY-1-METHYLETHOXY)IMINO]ACETYL] AMINO]-4-METHYL-2-OXO-1-AZETIDINESULFONIC ACID
FR2515182B1 (en) * 1981-10-23 1986-05-09 Roussel Uclaf NOVEL PRODUCTS DERIVED FROM 3-AMINO 2-OXO AZETIDINE 1-SULFAMIC ACID, THEIR PREPARATION PROCESS, THEIR APPLICATION AS MEDICAMENTS AND THE INTERMEDIATE PRODUCTS NECESSARY FOR THEIR PREPARATION
FR2538389B2 (en) * 1981-10-23 1986-05-16 Roussel Uclaf NEW PRODUCTS DERIVED FROM 3-AMINO 2-OXO AZETIDINE-1-SULFAMIC ACID, NEW PROCESS FOR THE PREPARATION OF OPTICALLY ACTIVE PRODUCTS, APPLICATION OF NEW PRODUCTS AS MEDICAMENTS AND PRODUCTS NECESSARY FOR THEIR PREPARATION
IL67451A (en) * 1981-12-25 1987-10-20 Takeda Chemical Industries Ltd 4-cyano-2-azetidinones and production thereof and process for the preparation of 4-carbamoyl-2-azetidinones using them
EP0088488B1 (en) * 1982-01-22 1986-10-01 Beecham Group Plc Antibacterial agents, their preparation and use
EP0086563A1 (en) * 1982-01-22 1983-08-24 Beecham Group Plc Antibacterial agents, their preparation and use
US4443374A (en) * 1982-02-01 1984-04-17 E. R. Squibb & Sons, Inc. Process for preparing (3S)-3-[[(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]-acetyl]amino]-2-oxo-1-azetidinesulfonic acid, and 4-substituted derivatives
WO1983002942A1 (en) * 1982-02-19 1983-09-01 Yoshioka, Kouichi Process for preparing 2-azetidinone-1-sulfonic acids, and starting materials therefor
AU564150B2 (en) * 1982-04-30 1987-08-06 Takeda Chemical Industries Ltd. 1-sulfo-2-azetidinone derivatives
EP0096296B1 (en) * 1982-06-03 1987-07-29 F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft 1-sulfo-2-oxoazetidine derivatives
DE3377061D1 (en) * 1982-06-03 1988-07-21 Hoffmann La Roche Process for the preparation of 1-sulfo-2-oxoazetidine derivatives
US4704457A (en) * 1982-06-21 1987-11-03 E. R. Squibb & Sons, Inc. Process for the preparation of (3S)-3-[[[2-(protected amino)-4-thiazolyl]-oxoacetyl]amino]-2-oxo-1-azetidinesulfonic acid and 4-substituted derivatives thereof
US4939253A (en) * 1982-08-04 1990-07-03 E. R. Squibb & Sons, Inc. 2-oxoazetidin-1-yloxy acetic acids and analogs
US4681937A (en) * 1982-09-27 1987-07-21 E. R. Squibb & Sons, Inc. 3-acylamino-2-oxo-1-azetidinyl esters of phosphonic acids, phosphoric acid and phosphoric acid esters
US4551276A (en) * 1982-10-06 1985-11-05 E. R. Squibb & Sons, Inc. 2-Oxoazetidin-1-yloxymethyl sulfonic acid and analogs
US4533495A (en) * 1982-12-30 1985-08-06 Takeda Chemical Industries, Ltd. Method of producing 2-azetidinone-1-sulfonic acid compounds
US4548747A (en) * 1983-05-02 1985-10-22 E. R. Squibb & Sons, Inc. 3-Acylamino-1-sulfonylaminocarbonylmethoxy-2-azetidinones
US4652651A (en) * 1983-05-31 1987-03-24 Hoffmann-La Roche Inc. Process for the manufacture of 1-sulpho-2-oxoazetidine carboxylic acid intermediates via catalytic ester cleavage
US4670554A (en) * 1983-05-31 1987-06-02 E. R. Squibb & Sons, Inc. ((3-Acylamino-2-oxo-1-azetidinyl)oxy)methyl)phosphinic acids
US4501697A (en) * 1983-06-17 1985-02-26 E. R. Squibb & Sons, Inc. 4-[[(Amidomethyl)oxy]methyl]-2-oxo-1-azetidinesulfonic acid salts
JPS601162A (en) * 1983-06-17 1985-01-07 Dai Ichi Seiyaku Co Ltd Azetidine-1-sulfonic acid derivative
US4587051A (en) * 1983-07-18 1986-05-06 E. R. Squibb & Sons, Inc. Desulfonation process for preparing 2-azetidinones
US4596777A (en) * 1983-08-10 1986-06-24 E. R. Squibb & Sons, Inc. Process for preparing (3S)-3-[[[2-(protected or unprotected amino)-4-thiazolyl]acetyl]amino]-2-oxo-1-azetidinesulfonic acid and 4-substituted derivatives thereof
US4551277A (en) * 1983-11-09 1985-11-05 E. R. Squibb & Sons, Inc. 4-(3-Acylamino-2-oxo-1-azetidinyl)-4-oxo-2-butenoic acid
JPS60112789A (en) * 1983-11-21 1985-06-19 Otsuka Chem Co Ltd 3-acylamino-4-alkyl-2-azetidinone-1-sulfonic acid derivative, tis preparation, and drug containing it
JPS60226855A (en) * 1984-04-26 1985-11-12 Nippon Kayaku Co Ltd N-benzyloxycarbonyl-l-threoninamide hemihydrate and preparation thereof
US4771135A (en) * 1984-05-22 1988-09-13 Eli Lilly And Company Process for carbon-carbon bond formation at the C-4 position of 3-acylaminoazetidinones and products and starting materials therefor
US4918185A (en) * 1984-05-22 1990-04-17 Eli Lilly And Company Process for carbon-carbon bond formation at the C-4 position of 3-acylaminoazetidinones and products and starting materials therefor
US4610824A (en) * 1984-10-09 1986-09-09 E. R. Squibb & Sons, Inc. Hydrazide derivatives of monocyclic beta-lactam antibiotics
US4680409A (en) * 1984-10-09 1987-07-14 E. R. Squibb & Sons, Inc. Hydrazide containing derivatives of 2-amino-4-thiazole-acetic acid
US4647660A (en) * 1985-05-02 1987-03-03 E. R. Squibb & Sons, Inc. 3-acylamino-2-oxo-1-azetidinesulfonic acids
US4632985A (en) * 1985-06-03 1986-12-30 E. R. Squibb & Sons, Inc. 3-acylamino-2-oxo-1-azetidinesulfonic acids
US4754041A (en) * 1985-06-17 1988-06-28 E. R. Squibb & Sons, Inc. 3-acylamino-2-oxo-1-azetidinesulfonic acids
US4638062A (en) * 1985-06-17 1987-01-20 E. R. Squibb & Sons, Inc. 3-acylamino-2-oxo-1-azetidinesulfonic acids
FR2588867B1 (en) * 1985-10-23 1988-12-09 Roussel Uclaf NOVEL PRODUCTS DERIVED FROM THE 3-AMINO 2-OXO AZETIDINE SERIES, THEIR PREPARATION PROCESS AND THEIR APPLICATION
MX2007000526A (en) * 2005-05-09 2007-03-28 Sicor Inc Process for making aztreonam.
JP5648250B2 (en) * 2007-11-21 2015-01-07 国立大学法人東京工業大学 Polymer organic compounds and organic compounds having sulfonated nitrogen-containing heterocycles, methods for producing them, ion exchangers, electrolyte membranes, pharmaceuticals, catalysts, membrane electrode assemblies, fuel cells using them
EP2804589A1 (en) 2012-01-16 2014-11-26 Mahmut Bilgic Pharmaceutical formulations comprising aztreonam
PL3122745T3 (en) 2014-03-24 2019-08-30 Novartis Ag Monobactam organic compounds for the treatment of bacterial infections
DK3353175T3 (en) 2015-09-23 2020-09-14 Novartis Ag Salts and solid forms of a monobactam antibiotic
WO2019026004A2 (en) 2017-08-02 2019-02-07 Novartis Ag Chemical process for manufacturing monobactam antibiotic and intermediates thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6046955B2 (en) * 1977-12-30 1985-10-18 武田薬品工業株式会社 Antibiotic G-6302
JPS55164672A (en) * 1979-06-08 1980-12-22 Takeda Chem Ind Ltd Azetidine derivative and its preparation
EP0021678B1 (en) * 1979-06-08 1984-11-07 Takeda Chemical Industries, Ltd. 1-sulpho-2-oxoazetidine derivatives, their production and pharmaceutical compositions thereof
WO1982001873A1 (en) * 1980-12-05 1982-06-10 Takeda Chemical Industries Ltd 1-sulfo-2-oxoazetidine derivatives and process for their preparation
JPS57131758A (en) * 1980-12-05 1982-08-14 Takeda Chem Ind Ltd 1-sulfo-2-oxoazetidine derivative, its preparation and use

Also Published As

Publication number Publication date
AU569407B2 (en) 1988-01-28
IE810230L (en) 1981-08-07
AU6698581A (en) 1981-08-13
HK57785A (en) 1985-08-09
IL62082A (en) 1986-08-31
GB8333191D0 (en) 1984-01-18
DK166280B (en) 1993-03-29
NO170015B (en) 1992-05-25
DE3104145A1 (en) 1981-12-17
DE3104145C2 (en) 1999-05-12
FI80271C (en) 1990-05-10
IT1135360B (en) 1986-08-20
DK52381A (en) 1981-08-08
PH24729A (en) 1990-10-01
NO161065C (en) 1989-06-28
CA1338670C (en) 1996-10-22
DD156180A5 (en) 1982-08-04
IT8119587A0 (en) 1981-02-06
JPH0623188B2 (en) 1994-03-30
YU44829B (en) 1991-04-30
SE457954B (en) 1989-02-13
LU83117A1 (en) 1982-09-10
FR2509299B1 (en) 1985-08-30
KE3539A (en) 1985-07-12
NL192924B (en) 1998-01-05
NO810410L (en) 1981-08-10
GR74151B (en) 1984-06-06
MY8600179A (en) 1986-12-31
SE8100861L (en) 1981-08-08
GB2071650A (en) 1981-09-23
JPH0586023A (en) 1993-04-06
KR830005131A (en) 1983-08-03
SE8602193D0 (en) 1986-05-14
IE51392B1 (en) 1986-12-24
SE8602193L (en) 1986-05-14
FR2509299A1 (en) 1983-01-14
IE862297L (en) 1981-08-07
GB2071650B (en) 1984-12-05
PH23316A (en) 1989-07-14
JPH0670006B2 (en) 1994-09-07
PH23317A (en) 1989-07-14
JPS56125362A (en) 1981-10-01
SE8602194L (en) 1986-05-14
NL8100571A (en) 1981-09-01
NO860225L (en) 1981-08-10
SE500216C2 (en) 1994-05-09
FI810352L (en) 1981-08-08
PT72465B (en) 1982-02-04
CH651020A5 (en) 1985-08-30
IL62082A0 (en) 1981-03-31
SG39185G (en) 1985-12-13
ES499171A0 (en) 1982-06-01
NL192924C (en) 1998-05-07
KR860001289B1 (en) 1986-09-10
GB2139618A (en) 1984-11-14
GB2139618B (en) 1985-05-01
IE51393B1 (en) 1986-12-24
AR245932A1 (en) 1994-03-30
AU4574885A (en) 1985-11-07
NO170015C (en) 1992-09-02
ES8205397A1 (en) 1982-06-01
NZ196202A (en) 1984-07-31
DK166280C (en) 1993-08-30
CH653993A5 (en) 1986-01-31
AU548896B2 (en) 1986-01-09
SE8602194D0 (en) 1986-05-14
YU31981A (en) 1984-02-29
FI80271B (en) 1990-01-31
PT72465A (en) 1981-03-01
JPH02160764A (en) 1990-06-20
NO161065B (en) 1989-03-20

Similar Documents

Publication Publication Date Title
NZ205240A (en) Sulphonamide derivatives,being starting materials for producing beta-lactams
US4775670A (en) 2-oxo-1-azetidinesulfonic acid salts
US4794108A (en) 1-carboxymethoxy acetidinones and their production
EP0048953B1 (en) Beta-lactam antibiotics
EP0014476B1 (en) Optically active acylated cephalosporin analogs, processes for their preparation and pharmaceutical compositions comprising them
US4529698A (en) Process for preparing a 2-oxo-1-azetidinesulfonic acid salt
CA1340253C (en) B-lactam antibiotics
EP0308416A1 (en) N-1 substituted sulfonylaminocarbonyl, c-4 substituted monobactams
JPH03123771A (en) Novel production of optically active 3-substituted azetidinone compound
EP0191989B1 (en) Antibiotic isoxazole derivatives, their production and use
EP0187500B1 (en) Monobactams
KR930001405B1 (en) Process for preparing 2-(4-substituted amino-3-oxo-isoxazolydin-5-oxo)-tetra hydrofuran carboxyl acid derivatives
JPH0240669B2 (en)
US4883869A (en) Novel beta-lactams containing amino acid substituents
EP0187355A1 (en) Process for preparing beta-lactam antibiotics
JPH08291124A (en) New physiologically active substance hydrostatin a and its production and use
JPH0330595B2 (en)