CA1108135A - Antibacterial agents - Google Patents

Abstract

Abstract of the Disclosure The specification discloses a steroselective total synthesis of certain novel substituted .DELTA.2,3-1, 4-morpholine-2carboxylic acids possessing ? fused .beta.-lactam ring in the 1,6-position and carrying a substituent cis to carbon 5 in the 7-position of the fused ring system represented by the general formula I

wherein X is amino, azido or acylamido and Z represents optionally substituted C1-C6 alkyl, aryl, aralkyl or heterocyclic. When X is acylamino, these acids (and their pharmaceutically acceptable salts and physiologically hydrolyzed esters) are potent antibacterial agents. The compounds having the above general formula represent a new family of .beta.-lactam antibiotics. They can be considered nuclear analogs of cephalosporins in which the sulphur atom of the dihydrothiazine ring is replaced by an oxygen atom and shifted from position 5 to position 4 of the .beta.-lactam ring system as numbered in the formula above.

Description

The present in~ention relates to a steroselective total synthesis of certain novel substituted a2 '3-1, 4-morpholine-2-carboxylic acids possessing a fused ~-lactam ring in the 1,6-position and carrying a substituent cis to car~on 5 in the 7-position of the fused ring system represented by the general formula ~C~::25-Z

wherein X is amino, azido or acylamido and Z represents optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic. When X is acylamino, these acids (and their pharmaceutically acceptable salts and physiologically hydrolyzed esters) are potent antibacterial agents.
Also included in this invention are various novel intermediates useful in preparing the active ~-lactam derivatives desribed above and various processes for the production of the intermediates and active com-pounds.
The compounds having the above general form-ula represent a new family of ~-lactam antibiotics. They can be considered nuclear analogs of cephalosporins in which the sulphur atom of the dihydrothiazine ring is replaced by an oxygen atom and shifted from position 5 to position 4 of the ~-lactam ring system as numbered in the formula above.

~1~3~3~ ' Sheehan has used the term 0-cepham for the structure 7 ~ 0 o 0-Cepham [J.C. Sheehan and M. Dadic, J. Heterocyclic Chem., 5, 770 (1968)] and we propose the use of the term 0-2-isocepham for the basic system having the formula 7 6 ~
~ 0 2 ; ~ .
The numerical prefix indicates the position of the hetero-atom.
There is thus provided by the present invention the novel 0-2-isocephem carboxylic acid compounds having 10 the formula H H
~ 7 R-NH l l 0 ~ N ~ CH2-S-Z

II
wherein R is an acyl group and Z represents optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic, and easily cleavable esters and pharmaceutically acceptable salts of said acids and esters.

- 2 -

3~

The acyl group R can be chosen from a wide variety of organic acyl radicals which yield products of : improved properties and is preferably an acyl radical which is contained in a naturally occurring or bio-synthetically, semi-synthetically or totally-syntheti- -cally produced pharmacologically active N-acyl derivative of 6-arninopenicillanic acid or 7-aminocephalosporanic acid. Examples of suitable acyl groups which can be used have been defined in our related Belgian Patent 837,265, but it should be noted that this is not intended to be an exhaustive list of all the possible acyl groups which may be used.
A preferred class of acyl groups are those of the forrnula O
Ar'-CH-C

. .
: wherein Ar' is a radical of the formula Im Rm Rn~

R

(~ o i~8~3~

in which ~m, ~n ~nd R are alike or different and each is hydrogen, hydroxy, (lower)alkyl, cyano, (l~wer)alkoxy, chloro, bromo, iodo, fluoro, trifluoromethyl, nitro, amino, (lower)alkylamino, di(lower)alkylamino, (lower)-alkanoyl, tlower)alkanoyloxy such as p-acetoxy or phenyl and Y is amino or a group cbtained by reacting the amino group with acetaldehyde, formaldehyde or acetone, fluoro, chloro, bromo, iodo, hydroxy, (lower)alkanoyloxy, carboxy, guanidino, 3-guanyl-1-ureido, 3-(2-furoyl)ureido, 3-benzoyl-ureido, sulfo, sulfoamino, ureido, thioureido, (lower)-alkoxy, cyano, cyanamino or indanyloxycarbonyl. Parti-cularly preferred Ar radicals are phenyl, p-hydroxyphenyl,

4-hydroxy-3,5-dichlorophenyl, 3-chloro-4-hydroxyphenyl, o-, m- or p- aminomethylphenyl, 2-thienyl, 3-thienyl, l-cyclohexenyl and 1,4-cyclohexadienyl. Particularly preferred Y groups are amino, hydroxy and carboxy.
Set forth below are formulae of the most preferred acyl groups of this class:

~ IN-CO- ; ~o ~ CN-CO-, (~-? ` O

Cl ~ Cl H~/ \~--CN-CO- HO
C;S~CH^CO- ;

N-CO- H-CO-NH2 ~IH2 ~CH-CO- ;

E3--CH-CO- ICH_CO_ OH

~3~ H-CO- ; ~:H-CO-OH OH
.

~ IH-co_ ~CH-co-.

5 --CH-CO- ~CIH-CO-S I ; COOH
COOH

~-CO- ~ 3-CO-COOH
,~

Of most interest are the acyl groups of the above class where the acid ArCH(X)COOH is of the D-series.
Other particularly preferred acyl groups for the compounds of formula I are N ' C-CH2CO- ; ~ CO--: OCH3 N = N
~ N-CH CO- ; ~
N = C N ~ S~H2CO-;

- U

: ~ C~3 where U and V are alike or different and each is hydrogen, chloro or fluoro;

- 6 `:
:

llQ8135 ~(:H2C{l- CH2CO- ' -~H2CO- ,~--CH2C-~C-CH2CO~ CH-CO-I
O _ C -NH-C= O

OCH2CO- . ~SCH2CO- ' 9~2NH2 2CO- , Cl-C7 alkyl-CO-~CO- ' ~CH2CO-OC2~15 ~3_ . ~C~12CO~
CH2CO , N~,N

Cl-C7 slkoxy-CO-, O~CO- ~ I

~CH2C-IIH-CH2CO-, I
NH ~OC~2NHCO- ' OH
O--C-- C--H ~h C \~ CH2CO~
~N~/--CH2CO-; ' ;

~ , ~CO-' CO- .~ SCH2CO- , ~_<CH2NH2 CH2NH2 ~C~12CO- 9nd~--CH2CO-, --8-- . .

l~J8~135 Substituent Z in formulae I and II above may be Cl-C6 alkyl, aryl, aralkyl or heterocyclic, any of said groups being optionally substituted by one or more substituerts. The alkyl group may be a monova-lent saturated aliphatic hydrocarbon radical having from 1 to 6 carbon atoms and a straight or branched chain, e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl or hexyl. The aryl group may be a mono-, bi- or polycyclic aromatic hydrocarkon radical, e.g., phenyl, l-naphthyl, 2-naphthyl or 2-phenanthryl. The term aralkyl as used herein includes monovalent aryl-substituted aliphatic hydrocarbon radicals o~ the formula aryl-(ALK)m~ in which aryl is as defined above, m is an integer of 1 to 4 and ALK represents a straight or branched chain alkylene radical, e.g., -(ALK)m- may be methylene, ethylene, propylene, butylene, l-methylpropylene, 2-ethylethylene and the like. Eeterocyclic Z substituents may be heteromonocyclic or heterobicyclic residues of aromatic character as well as appropriate partially or wholly saturated residues.
A preferred group of compounds of formulae I and II are those in which Z is Cl-C6 alkyl, aryl selected from phenyl or naphthyl, aralkyl of the formula aryl-(ALK)m- in which aryl is phenyl or naphthyl, m is an integer of 1 to 4 and ALK represents a straight or branehed ehain alkylene radieal, or heteroeyelie seleeted from a S- or 6- membered heteroeyclic ring eontaining 1 to 4 atoms seleeted from N, O or S, said alkyl radi~al being optionally substituted by one or more substituents seleeted from hydroxy, halo, amino, nitro, di(Cl-C4 alkyl) amino, earboxy, sulfo or eyano and said aryl, aralkyl or heteroeyelic radicals being optionally substituted by one or more substituents selected from halo, Cl-C4 alkyl, Cl-C4 alkoxy, eyano, earboxyl, amino, nitro, C3-C4 eycloalkyl, C2-C4 alkenyl, trifluoromethyl, hydroxy, hydroxymethyl~ Cl-C4 alkylthio, Cl-C4 ~ alkylamino, di(Cl-C4 alkyl) amino, mercapto, phenyl, :: benzyl, alkoxyalkyl of up to 4 earbons or -(CH2)nCOOH
. in which n is an integer of 1 to 4. Examples of suit-able heterocyelic radicals include thienyl, furyl, ~ pyrazolyl, imidazolyl, isoimidazolyl, triazolyl, i tetrazolyl, thiazolyl, thiadiazolyl, oxazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl and triazinyl.
A most preferred group of compounds of formulae I and II are those in which Z is an optionally substituted 5- or 6- membered heterocyclic ring eontaining 1 to 4 atoms seleeted from N, O and S, the substituents being preferably those mentioned above. Ecpecially preferred heterocyelie rings are optionally substituted ' triazole, thiadiazole, oxadiazole or tetrazole radicals. Within this group the most preferred Z substituents are 1,2,3-triazolyl-5-yl, 2-methyl-1,3,4-thiadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl, l-N-methyltetrazolyl, l-carboxymethyltetra-zol-5-yl and 1-carboxyethyltetrazol-5-yl.
The term "(lower)alkyl" as used herein means both straight and branched chain aliphatic hydro-carbon radicals having from one to ten carbon atoms such as methyl, ethyl, proyl, isopropyl, butyl, isobutyl, t-butyl, amyl, hexyl, 2-ethylhexyl, heptyl, decyl, etc.
Similarly, where the term "(lower)" is used as part of the description of another group, e.g. "(lower)-alkoxy", it refers to the alkyl portion of such group which is therefore described above in connection with "(lower)alkyl".
The pharmaceutically acceptable salts re-ferred to above include the nontoxic carboxylic acid salts, e.g. nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and salts with nontoxic amines, e.g. trialkylamines, procaine, dibenzylamine, N-benzyl-~-phenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine, N-alkylpiperidine and other amines which have been used to form salts of penicillins and cephalosporins. When a basic group is present, as when it occurs in the 7-acyl group, the present invention also includes the pharmaceutically acceptable`acid addition salts, e.g. salts with mineral ~8~S

acids such as ~ydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric and salts with organic acids such as maleic, acetic, citric, oxalic, succinic, benzoic, tartaric, fumaric, mandelic, as-orbic ar,d malic. ~he term "pharmaceutically acceptable salts" is also meant to include nontoxic acid addition salts of the easily cleavable esters referred to above. The compounds which contain a basic group in radical R may also be present in the form of an internal salt, i.e. in the form of the zwitterion.
The easily cleavable esters referred to above include ester groups which are removable by methods, e.g. chemical or enzymatic hydrolysis, txeat-ment with chemical reducing agents under mild condi-tions, irradiation with ultraviolet light or catalytic hydrogenation, which do not result in any appreciable destruction of the remaining portion of the molecule.
Examples of suitable esters include those disclosed in U.S. Patents 3,284,451 and 3,249,622 and U.K.
Patents 1,229,453 and 1,073,530. Esters which have been used previously in penicillin and cephalosporin chemistry include for example benzhydryl, p-nitrobenzyl, benzyl, trichloroethyl, silyl such as trimethylsilyl, phenacyl, p-methoxybenzyl, acetonyl, phthalidyl, indanyl and (lower)alkyl such as methyl, ethyl and t-butyl.
Particularly preferred easily cleavable esters are those which are hydrolyzed under physiological condi-tions such as pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl.

1~8~

As the 0-2-isocephem compounds of the present invention may possess one or more asymmetric carbon atoms, the invention includes all of the pos-sible enantiomeric and diastereomeric forms of th~ com-pounds of the general formula II shown above. Resulting mixtures of isomers can be sepaxated into the individual isomers according to methods which are known per se, e.g. fractional crystallization, adsorp-tion chromatography or other suitable separation processes. Resulting racemates can be separated into the antipodes in the usual manner after introduction of suitable salt-forming groupings, e.g~ by forming a mixture of diastereoisomeric salts with optionally active salt-forming agents, separating the mixture into diastereoisomeric salts, and converting the separated salts into the free compounds, or by frac-tional crystallization from optically active solvents.
It will be appreciated that certain of the compounds of this invention exist in various states of solvation and the anhydrous as well as solvated forms are within the scope of the invention.
The free acid compounds of general formula II and general formula I wherein R is acylamido and physiologically hydrolyzed esters thereof together with the pharmaceutically acceptable salts of such free acids and esters are useful as antibacterial agents. The remaining acids, esters and salts of formula I are valuable inter-mediates which can be converted into the pharma-cologically active compounds of formula II as by the prscesses described below.
Preferred compounds of formula II are those in which R is an organic acyl group. Use of the acyl groups mentioned in Belgain patent 837,265 as well as those mentioned above results in active end-products having advantageous pharmacological properties.
A preferred embodiment of the present invention consists of the compounds of formula H H
R-NH ~ o '.; I I I

. C02H
II

in which R is an organic acyl group as defined above and Z represents a 5- or 6-membered heterocyclic ring containing N, O or S, said heterocyclic ring being optionally substituted by one or more , ,i' , ~ 8135 substituents selected from halo, Cl-C4 alkyl, Cl-C4 alkoxy, cyano, carboxyl, amino, nitro, C3-C4 cycloalkyl, C2-C4 alkenyl, trifluoromethyl, hydroxy, hyd.roxymethyl, Cl-C4 alkylthio, Cl-C4 alkyl-amino, di (Cl-C4 alkyl) amino, mercapto, phenyl, benzyl, alkoxyalkyl of up to 4 carbons or ~CH2)nCOOH
in which n is an integer of 1 to 4, and easily cleavable esters and pharmaceutically acceptable salts thereof.
A most preferred subclass within the above compounds of Formula II consists of the D-isomers of the acids in which R is ~-amino-~-(p-hydroxyphenyl)acetyl, ~-amino-~-(3-chloro-4-hydroxyphenyl)acetyl, ~-amino-~-(3,5-dichloro-4-hydroxyphenyl)acetyl, -amino-~-(2-thienyl)acetyl, ~-amino-~-(3-thienyl)acetyl, ~-amino-a-(1-cyclohexenyl)acetyl, ~-amino-~-(1,4-cyclohexadienyl)-acetyl, ~-hydroxyacetyl, ~-hydroxy-~-(2-thienyl)acetyl, a-hydroxy-~-(3-thienyl)acetyl, ~-hydroxy-~-(l-cyclohexenyl)-acetyl or a ~-hydroxy-~-(1,4-cyclohexadienyl)acetyl, or pharmaceutically acceptable salts thereof.

11~8~35 Another preferred embodiment of the present invention consists of the compounds of formula H H
R-NH ~ o ~ ' I I I . ~.
~ ~\ ';
O ¦ CH2-S-Z
Co2H

wherein R is an organic acyl group as defined above and Z is a triazole, thiadiazole, oxadiazole or tetrazole radical, said radical being optionally substituted by one or more substituents selected from halo, Cl-C4 alkyl, Cl-C4 alkoxy, cyano, carboxyl, amino, nitro, C3-C4 cycloalkyl, C2~C4 alkenyl, trifluoromethyl, hydroxy, hydroxymethyl, Cl-C4 alkylthio, Cl-C4 alkylamino, di (Cl-C4 alkyl)amino, phenyl, benzyl, mercapto, alkoxyalkyl of up to 4 carbons or -(CH2)nCOOH in which n is an integer of 1 to 4, and easily cleavable esters and pharmaceutically acceptable salts thereof.
A most preferred embodiment of the present invention consists of the compounds of formula II in which Z is 1,2,3-triazol-5-yl, 2-methyl-1,3,4-thiadiazol-5-yl, 2-methyl-1,3,4,oxadiazol-5-yl, l-N-methyltetrazol-5-yl, 1-carboxymethyltetrazol-5-yl or l-carboxyethyl-tetrazol-5-yl.

., .

~ . . .

~ 8~3S

The most preferred ~ompounds of this group of the Formula II are those in which R is Ar'-CH-CO-Y

wherein Ar' is phenyl, p-hydroxyphenyl, 4-hydroxy-3,5-dichlorophenyl, 3-chloro-4-hydroxyphenyl, o-, m- or p-aminomethylphenyl, 2-thienyl, 3-thienyl, l-cyclohexenyl or 1,4-cyclohexadienyl and Y is amino, hydroxy or carboxy.
Another preferred subclass within this group consists of the D-isomers of the acids in which R is a-amino-a-(p-hydroxyphenyl)acetyl, a-amino-a-(3-chloroo 4-hydroxyphenyl)acetyl, a-amino-a-(3,5-dichloro-4-hydroxyphenyl)acetyl ~-amino-a-(2-thienyl)acetyl, a-amino-a-(3-thienyl)acetyl, a-amino-a-(l-cyclohexenyl)-acetyl, a-amino-a-(1,4-cyclohexadienyl)acetyl, a-hydroxy-acetyl, a-amino-a-(2-thienyl)acetyl, a-hydroxy-a-(3 thienyl)acetyl, a-hydroxy-a-(l-cyclohexenyl)acetyl or ~-hydroxy-~-(1,4-cyclohexadienyl)acetyl, or pharma-ceutically accepta~le salts thereof.
The present invention further provides var-ious novel intermediates useful in the synthesis of the 7-acylamido 0-2-isocephem compounds of formula II
described above. .
Preferred embodiments of the present inven-tion are the novel intermediates having the formula H H
O
:~ ~ N ~

C2R"
IV

wherein Z is optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic and R" is hydrogen or an . easily cleavable ester carboxyl-protecting group, and salts thereof. The preferred Z substituents are as defined above in connection with the compounds of formulae I and II.
Other preferred embodiments of the present invention are the intermediates having the formula H2N~ _ --f ~ ~ N ~
CO R~ CH2S Z

III

~ .

wherein Z is optionally substituted Cl~C6 alkyl, aryl, aralkyl or heterocyclic and R" is hydrogen or an easily cleavable ester carboxyl-protecting group, and salts thereof. The preferred Z substituents are those mentione~
above as beinq preferred in connection with the compounds of formulae I and II.
The intermediates of formulaeIII and IV may be in the form of the free carboxylic acid or a salt thereof or in the form where the carboxyl group is pro-tected in a conventional manner such as preferably by esterification. ~he protecting group is selected so that it may be removed by methods which do not result in any appreciable destruction of the remaining portion of the molecule. Preferred carboxyl protecting groups are the easily cleavable esters as defined above including in particular benzhydryl, p-nitrobenzyl, trichloroethyl, silyl including especially trimethylsilyl, phenacyl, p-methoxybenzyl, acetonyl, (lower)alkyl such as methyl, t-butyl or ethyl, benzyl, triphenylmethyl, methoxymethyl, acetoxymethyl, phthalidyl, indanyl and pivaloyloxymethyl.
The novel 7-acylamido compounds of formula II
may be prepared by N-acylating a 7-amino intermediate of the formula H H

H N
2 ~ \

~ N ~
O ¦ CH -S-Z
C2R" 2 III

~3 ~

wherein Z is optionally substituted ~l-C6 alkyl, aryl, aralkyl or heterocyclic and R" is hydrogen or an easily -~
cleavable ester carboxyl-protecting group, or a salt thereof, with an acylating acid of the formula R-COOH

wherein R is an organic acyl group, or with its functional equivalent as an acylating agent for a primary amine and, if desired, (a) when R" is a carboxyl-protecting group, converting the 7-acylated ester to the free acid compound or a physiologically hydrolyzed ester or a pharmaceutically acceptable salt of said acid or ester, or (b) when R" is hydrogen, converting the

7-acylated carboxylic acid to a physiologically hydrolyzed ester or a pharmaceutically acceptable salt of said acid or ester and, if desired, resolving a resulting isomer mixture into its component isomers.
The 7-amino starting materials of general : formula III are of use primarily as intermediates in preparing the pharmacologically active N-acyl deriva-tives of formula II. The free acids, physiologically hydrolyzed esters and pharmaceutically acceptable salts of said acids and esters of formula III, however, do possess some antibacterial activity per se against various pathogenic microorganisms.

~, .

.
' ~ ~ .

~ .

li~B135 The 7-acylamido 0~2-isocephem compounds cf formula II are prepared by N-acylation according to known methods of the 7-amino group of intermediate III with an acylating acid of the formula R-COOH

wherein R is an acyl group, or with its functional equi-valent as an acylating agent for a primary amino group.
The acylating agents for preparing the products of formula II are known, readily preparable by known methods or described herein as well as in our Belgian Patent 837,265.
Intermediate III may be acylated either in the form of the free carboxylic acid (or salt thereof) or as an easily cleavable ester (or acid addition salt thereof).
Preferred esters include benzhydryl, benzyl, p-nitrobenzyl, trichloroethyl, silyl (especially trimethylsilyl), phenacyl, p-methoxybenzyl, acetonyl, (lower)alkyl including partic-ularly methyl, ethyl and t-butyl, triphenylmethyl, methoxy-methyl, acetoxymethyl, pivaloyloxymethyl, phthalidyl and indanyl. The procedures for preparing esters of carboxylic acids are disclosed in the literature and are well-known to those skilled in the art of penicillin and cephal-~ osporin chemistry. Methods for preparing certain of the ; more preferred easily cleavable esters, i.e. the pival-oyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl and ~.

. ' 13~

phenacyl esters, are disclosed in U.S. Patent 3,284,451 and in U.K. Patent 1,229,453. Preparation of phthalidyl esters of penicillins and cephalosporins is described in South African ~atent Applications 72/3799 and 72/3800 The free acid form of intermediate III may also be converted to a silyl ester, e.g. trimethylsilyl ester, as by the methods described in the literature, e.g.
U.S. Patent 3,249,622. The silyl ester carboxyl-protecting group may be easily removed following the acylation reaction by hydrolysis or alcoholysis.
Prior to the acylation reaction, any re-active substituents on the acylating acid or derivative thereof, e.g. hydroxy, carboxyl or mercapto, may be pro-tected by use of suitable protecting or blocking groups which are well-known to those skilled in the art of ~-lactam chemistry, e.g. as by acylation or silylation.
When the acylating agent contains an amino functional group in the acyl moiety, the amino group is protected by a conventional amino-blocking group which may be readily removed at the conclusion of the reaction. Examples of suitable amino-protecting or blocking groups include t-butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1-naphth-carbonyl, trichloroethoxycarbonyl, 2-ethoxycarbonyl-l-methylvinyl and 2-methoxycarbonyl-1-methylvinyl. A
particularly valuable amino-blocking group is a proton, ~J~35 as in the acylating agent of the formula ~3 fH -C-c 1 NH2 HCl Preferred amino-protecting groups are t-butoxycarbonyl, carbobenzyloxy, the proton and a ~-diketon or B-ketoester as in l~.K. Patent 1,123,333 or U.S. Patents 3,325,479 and 3,316,247, e.g. methyl acetoacetate, or a ~-ketoamide as in Japan 71/24714. When the t-butoxy-carbonyl, carbobenzyloxy, ~-ketoester, ~-diketon or ; ~-ketoamide protecting groups are employed, it is preferred to convert the acylating acid containing the blocked amino group to a mixed anhydride, e.g.
with ethyl or isobutyl chloroformate, before reaction ~ :
with compound III or a salt thereof. After the acylation coupling reaction, the amino-protecting group and any other functional protecting groups used may be removed by methods known ~ se to form the desired product of formula II. With respect to amino-protecting groups, the t-butoxycarbonyl group may be removed by use of formic acid, the carbobenzyloxy group by catalytic hydrogenation, the 2-hydroxy-1-naphthcarbonyl group by acid hydrolysis, the trichloroethoxycarbonyl group by .~

:' :
.~

.~ 8~5 treatment with zinc dust in glacial acetic acid, ~he proton by neutralization, etc.
Acylation of a free amino group of a cephalo-sporin or penicillin nucleus is a well-known reaction, and any of the functional equivalents of the carboxylic acid RCOOH ~ommonly used in penicillin or cephalosporin chemistry as acylating agents for primary amino groups may be employed in acylating intermediate III. Examples of suitable acylating derivatives of the free acid include the corresponding acid anhydrides, mixed anhydrides (e.g. alkoxyformic anhydr~des), acid halides, acid azides, active esters and active thioesters. The free acid may be coupled with compound III after first reacting said free acid with N,N'-dimethylchloroformininium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360(1965)] or by the use of enzymes or of an N,N'-carbonyldiimidazole or an N,N'-carbonylditriazole [cf. South African Specification 63/26843 or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide:
cf. Sheehan and Hess, J.A.C.S., 77, 1967 (1955)~, or of alkylylamine reagent [cf. R. Buijle and H. G. Viehe, Angew. Chem. International Edition, 3, 582, (1964)] or ~813~

of an isoxasolium salt reagent ~cf. R. B. Woodward, R. A. Olofson and H. MAyer, J. Amer. ~hem. Soc., 83, ; 1010 11961)], or of a ketenimine reagent Icf. C. L.
Stevens and M. E. Munk, J. Amer. Chem. Soc., 80, 4065 (1958)l or of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S. 3,651,050) or of diphenylphosphoryl azide lDPPA; J. Amer. Chem. Soc., 94, 6203-6205 (1972)] or of diethylphosphoryl cyanide [DEPC;
Tetrahedron Letters No. 18, pp. 159S-1598)] or of diphenyl phosphite ITetrahedron Letters No. 49, pp. 5047-5050 (1972)]. Other examples of suitable amide coupling reagents which have been described in the literature include (CH3)2SCH2CCHBr/DMSO (J. Chem. Soc. (C) 1904 (1969), HCCOCH3 (Rec. Trav. Chim. 74 769 ~1955), (CH3)2C(OCH3)2 (Chim. Ther. 2, 195 (1967), SiC14 (J. Org. Chem. 34, 2766 (1969), TiC14 (Can. J. Chem.
48, 983 (1970), (PNC12)3 (J. Org. Chem. 33, 2979 (1968) SO3-DMF (J. ~. Chem. 24, 368 (1959), ion exchange resins (Helv. 44, 1546 (1961) and J.C.S. C, 874 (1969) and ~ Ie (J, Chem. Soa. 4650 (1964).

N

:, An equivalent of the acid chloride is the corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a guasi-aromatic five mem~ered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temper-ature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole~
Dicarboxylic acids yield dimidazolide. The by-product, imidazole, precipitates and may be separated and the imidazolide isolated but this is not essential. A
preferred acylating agent for preparing 7-acylamido ~; compounds containing an ~-amino substituent, e.g.
~-aminobenzyl, ~-amino-~-thienylmethyl, etc. is the - 20 N-carboxy anhydride (Leuch's anhydride). In this structure the group which activates the carboxyl group also serves to protect the amino group. Another ` preferred acylating agent for introducing a side .

il~813~

chain containing an ~-amino functional group is the acid chloride hydrochloride, of the formula Ar-CH-COCl NH2-HCl which also serves a dual function of carboxyl activation and amino protection. Mention was made above of the use of enzymes to couple the free acid with compound III. Included in the scope of such processes are the use of an ester, e.g. the methyl ester, of that free acid with enzymes provided by various micro-organisms, e.g. those described by T. Takahashi et al., J.A.C S , 94(11), 4035-4037 (1972) and by T. Nara et al., J. Antibiotics (Japan) 24(5), 321-323 (1971) and in U.S. 3,682,777. A particularly preferred coupling agent for coupling the acylating acid with compound III
(or a salt or ester thereof) is N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as described in J A.C.S., 90, 823-824 and 1652-1653 (1968) and U.S.
Patent 3,455,929.
The particular process conditions, e.g. tem-perature, solvent, reaction time, etc. selected for the coupling reaction are determined by the nature of the reactants and acylation method used and are known to those skilled in the art.

;, ' '''''' ~-- ~' The acylating agents which may be used to form the N-acyl compounds of formula II are known in the literature along with methods fc,r their synthesis or are disclosed in the examples which follow. In those cases where the acylating agent contains one or more asymmetric carbon atoms and thus exists in optically active forms, the compounds obtained using such an acylating agent are ordinarily obtained in racemic form. When the separate optical isomers are desired, the acylating agent can be resolved in a conventional manner such as by reacting the free acid with cinchonine strychnine, brucine or the like, fractionally crystal-lizing to separate the diastereoisomeric salts and separately acidifying the solid phase and the liquid phase to liberate the optical isomers.
The 7-acylamido compounds of the present in-vention may be isolated in any of the ways customarily employed for the isolation of corresponding cephalosporin compounds. Formation of a desired pharmaceutically acceptable carboxylic acid or acid addition salt is carried out by known methods, e.g. reaction of the acid of compound II (or ester in the case of acid addition salts) with an appropriate base or acid.

13~

A compound of ormula lI in the form of the free acid or a salt thereof may be converted to a pharma-ceutically acceptable ~al~ thereof or to a physiologic-ally hydrolyzed ester or pharmaceutically acceptable salt thereof. Similarly, the product of formula II
in the form of an easily cleavable ester or salt thereof may be converted to the free acid product or a pharma-ceutically acceptable salt thereof by removal of the esterifying group to form the free acid, e.g. by acidic or alkaline hydrolysis, by enzymatic hydrolysis (as with human or animal serum), by hydrogenolysis or by treatment with chemical reagents known to remove partic-ular blocking groups, e.g. sodium thiophenoxide as in U.S. 3,284,451, and subsequent treatment of the free acid with an acid or base to form a pharmaceutically acceptable salt.
The easily cleavable esters of the compounds of formula II are useful as intermediates in the produc-tion of the free acid product. The pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl esters are also useful as active antibacterial agents since on oral administration they are rapidly hydrolyzed to the active metabolite. These esters are of partic-ular interest because they provide on oral administration different rates and amounts of absorption and give dif-fering concentrations of the active antibacterial agent in blood and tissues.

The 7-amino intermediates of general foxmula III may be prepared by selectively reducing a 7-azido intermediate of the formula H H

O CH~S-Z
C2R' IV' wherein Z is optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic and R" is an easily cleavable ester carboxyl-protecting group. The carboxyl-protected compound may, if desixed, be cleaved to produce the free-acid intexmediate III which can be converted to a salt by methods known per se.
Preferred reducing agents for use in pre-paring the intermediates of formula III include chemical reducing agents such as zinc and ammonium chloride, aluminum amalgam and hydrogen sulfide in the presence of a base, e.g. triethylamine or ammonia. Catalytic hydrogenation may also be employed with such catalysts as noble metals, preferably platinum or palladium including derivatives thereof such as oxides, hydroxides, and halides, or Raney nickel, said catalysts being option-ally supported on a conventional carrier such as carbon or diatomaceous earth. Catalytic hydxogenation is performed ~ .

~ ~)~35 with a non-reducible inert solvent, e.g. methanol, ethanol or ethyl acetate, and preferably at atmospheric or slightly ele~ated pressure at room temperature.
Compound III in the carboxyl-protected form or a salt thereof may be used directly as a starting material in the N-acylation process discussed above.
Alternatively, the protected intermediate may be de-blocked to form the free carboxylic acid which may then be option-ally converted to a salt or to another carboxyl-protected form, e.g. a physiologically hydrolyzed ester or salt thereof. By proper selection of reduction conditions and protecting groups, azido intermediate IV' may be converted either simultaneously or in stepwise fashion to the 7-amino free acid III. Thus, if mild hydro-genation conditions are used, e.g. catalytic hydro-genation with 10% Pd-on-charcoal or a mild chemical reducing agent such as H2S in the presence of a base such as triethylamine or ammonia, the azido group may be reduced without concomitant removal of esters resistant to such conditions, e.g. benzyl or p-nitrobenzyl. If stronger reducing conditions are used such as 30%
Pd-on-diatomaceous 2arth, both the azido group and most reducible esters will be simultaneously reduced.

.~
' :

~ 8~3~

A preferred embodiment of the present invention is the process comprising the consecutive steps of 1) selectively reducing a 7-azido intermediate of the formula IV' to produce a carboxyl-protected 7-amino intermediate of formula III and/ if desired, removing the carboxyl-protecting group to produce the corresponding free acid intermediate of formula III or optionally a salt thereof; and 2) N-acylating intermediate III or a salt thereof with an acylating acid of the formula R-COOH
where R is an acyl group/ or with its functional equivalent as an acylating agent for a primary amine and, if desired, (a) when R" is a carboxyl-protecting group, converting the 7-acylated ester to the free acid compound or a physiologically hydrolyzed ester or a pharmaceutically acceptable salt of said acid or ester, or (b) when R" is hydrogen, converting the 7-acylated carboxylic acid to a physiologically hydrolyzed ester or a pharmaceuti-cally acceptable salt of said acid or ester and, if desired, resolving a resulting isomer mixture into its component isomers.

~3~

The 7~-azido intermediates IY' may be pre-pared by two alternative methods. In one procedure a dihalide intennediate of the formula H H

C ~X) (CH2X' ) C2R ~
V

wherein Y' represents a displaceable leaving group, preferably a group such as halo or sulfonyloxy, ; e.g., alkyl- or substituted alkylsulfonyloxy or aryl- or substituted arylsulfonyloxy, and most preferably a group selected from halo, -OSO2-(lower)-alkyl including especially -OSO2CH3, -OSO2CF3 and -OSO2C6H4CH3(para), X and X' which may be the same or different each represent a halogen atom, preferably bromine or iodine and most preferably iodine and R' is an easily cleavable ester carboxyl-protecting group is reacted in an inert organic solvent in the presence of an acid acceptor with a nucleophile of the formula Z-SH

wherein Z is optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic, or a salt thereof, :' ~ .

to form a thiolated intermediate of the formula Y' ., .
H H

~N~C (X) (CH2-S-Z) VI

Intermediate VI is then converted to the desired 7B-azido compound by cyclization with base in an inert : organic solvent.
The dihalide starting material V may be used in either of its isomeric forms ~ , .
H H ¦
N3 _ _ ) ~ X

CH2X' 2R ' Va or Y' H H
: N ~ CH2X' .' I I
.~ ~ N
O
C2R' , Vb ' s or as a mixture of isomers. Formula V above is in-tended to represent either of the individual isomers or the mixture. Any dihalide including a mixed dihalide, e.g. X ~ Cl, X' - Br, may be used but the most preferred compound is the diiodide. Compound V
is reacted in an inert organic sol~ent, e.g., methylene chloride, with the desired thiol or a salt thereof. Since an acid HX is given off during the reaction, an acid acceptor, preferably an organic base such as pyridine or a trialkylamine, is used.
Compound V, the thiol and the acid acceptor are preferably employed in approximately equimolar amounts. The temperature for the displacement step is not critical, but best results are obtained at room temperature or below, most preferably at a temperature of about 0C.
Cyclization of intermediate VI is carried out in an inert organic solvent, preferably a polar organic colvent such as dimethylsulfoxide or dimethyl-formamide, with a suitable base. The base used in the cyclization step may be selected from a wide variety of bases including especially those of the following categories:
(a) anion~ derived from carboxylic acids having a PKa Of between about 3.5 and 5.5;

.. - ' - :

3L~.'D8135 ~b) tertiary organic amines such as a trialkyl-amine (e.g., triethylamine~, pyridine, n-methylpiperidine, n-methylmorpholine, etc.;
(c) alkali metal hydrides, e.g., sodium or potassium hydride; and (d) organolithium compounds including especially lithium alkyls, e.g., met~yl, lithium or butyl lithium.
Most preferred cyclization bases are acetate and formate anions, e.g., from alkali metal, ammonium or substituted ammonium formates or acetates. The most preferred base is acetate anion. The base is pre-ferably used in a molar excess relative to compound VI. While the preferred temperatures for this step are room temperature or below, the temperature is not critical. The leaving group Y' in formula Vl should be one which is efficiently displaced under the conditions of the base cyclization reaction and is preferably halo (chloro, bromo, fluoro, Dr iodo) or sulfonyloxy. The most preferred leaving group is the mesylate group.
Preparation of intermediate V is described in the text below under the heading "Preparation of Starting Materials". Briefly summarized, the reaction scheme is as shown in Flow Sheet I below:

' ;- ~L~ 3~ ~

HON ~bo~yl ~oee tion ~s y eta ~zat~on C-C~CH3 ~ >
C02R ' HONO'~\ nitroso ~ reduction >
CO2R' CH3 H2N O ~ Schiff base :~ ~ C ~ ~ form~tion as _ >
~ C02R' CH3 clnnamaldehyde .; ' ., .

lactam formation as with : ~o ~ azidoacetyl halide >

C02R ' ~ . , H H
`~ N ~ -ozonolysis C~ ~c ' ' ' CO2~' 3 :' 1 ' ~ 31 3 N3 _ 6elective aldehyde ~/ 0~ , reduction >
.. ~ N

C02R ~

H H CH OH conver~ion of N3 - ~ 2 hydroxyl group to a ~ore activ~ted _ I u ~ leaving group as by ~ , N ~ O halogenation or ~ ~H3 esterification with CO2R' a ~ulfonic scid derivative 3 ~ de-ketalization >

~N ,~ o OSO CH hydroxyl activation N H H 1 2 3 ns with triflic 3 ~ snhydride >
N

C02R ' N H ~ 2 3 ~llene form3tion 3 - - J ~ith bsse >

C2R ' ~8 halo~enation , ~ V
~,~C--CH2 ~02R l Flow Sheet 1 81:~S

An alternative method ~or preparing the 7~-a~ido intermediates of formula IV' involves reacting an intermediate of the formula H H
N
r~N I
' 0~ \~CH2Y"

VII

wherein Y" represents a displaceable leaving group and R~ is an easily cleavable ester carboxyl-pro-tecting group in an inert organic solvent in the ; presence of an acid acceptor with a thio of the formula HS-Z

wherein Z represents an optionally substituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic group, or a salt thereof.
Leaving group Y" in formula VII may be any nucleophilic group which is displaceable by the thiol. Examples of suitable leaving groups include halo (chloro, bromo, fluoro or iodo) and sulfonyloxy, i.e., alkyl- or substituted alkylsulfonyloxy or aryl-or substituted arylsulfonyloxy. Preferred leaving groups are halo, -OSO2-(lower~alkyl including especially -OSO2CH3, -OSO2CF3 and -OSO2C6H4CH3(para).
A most preferred leaving group is the mesylate group.

~1~8135 The nucleophilic displacement reaction is conducted in an inert organic solvent, e.g., methylene chloride r in the presence of an acid acceptor, pre-ferably an organic base such as pyridine or a trialkyl-amine. ~he heterocyclic thiol, acid acceptor and intermediate VII are preferably used in approximately equimolar quantities. The temperature is not critical, but best results are obtained at room temperature or below, most preferably at a temperature of about 0C.
The preferred starting materials of formula VII used in the above process include Y" = halo or sulfonyl-oxy .
A preferred method of preparation of these starting materials involves the steps of (1) cyclizing in an inert organic solvent, e.g., dimethylformamide, a dihalide inter-mediate of formula V with a base R2COO
in which R2 in hydrogen or (lower) alkyl to produce an intermediate of the formula H H
N3 _ _ " ~~
~ l ' ~ N ~ H2OCOR2 C 2R' :', in which R2 is hydrogen or (lower) alkyl and;
.
,,.,~

~2) subjecting said intermediate to acid hydrolysis to produce an intermediate of the formula H H
N3 _ ~ l ; o ~ CH2 ; and C2R ' either (a) esterifying the 3-hydroxymethyl intermediate with a sulfonic acid derivative, e.g., methanesulfonyl chloride, p-toluenesulfonyl chloride or triflic anhydride, in the pre-sence of an organic base and in an inert organic solvent, e.g., methylene chloride, to produce the desired 3-sulfonyloxy deriva-tive of formula VII; or (b) halogenating the 3-hydroxymethyl intermediate with a phosphorus halide, e.g., phosphorus trichloride, or phosphorus tribromide, to produce the desired 3-halomethyl derivative of formula VII.

~L3~ii The present invention also provides an alter-native method for preparing the novel 7-acylamido compounds of formula II; which process comprises reacting in an inert organic solvent in the pre-sence of an acid acceptor a 7-acylated compound of the formula H R
R-NH ~
~
~N~` CH2Y"
C02R"
VIII

wherein R is an acyl group, Y" is a displaceable leaving group and R" is hydrogen or an easily cleavable ester carboxyl-protecting group, or a salt thereof, with a thiol of the formula HS-Z ~:

wherein Z is optionally ~ubstituted Cl-C6 alkyl, aryl, aralkyl or heterocyclic, or a salt thereof and, if desired, (g) when R" is a carboxyl-protecting group, converting the 7-acylated ester to the free acid compound or a physiologically hydroylzed ester . ;, ~` or a pharmaceutically acceptable salt of said acid or ester, or (b) when R" is hydrogen, converting the - 7-acylated carboxylic acid to a physiologically hydrolyzed ester or a pharmaceutically acceptable salt of said acid or ester; and, if desired, resolving a resulting isomer mixture into its component isomers.

The nucleophilic displacement of the Y" group in compound VIII may be carried out using the same general reaction conditions, i.e., solvents, temperature range, acid acceptors, as described above in connection with thiolation of compound V.
The 7-acylated starting materials for the above process may be prepared by the procedures disclosed in the art.
While Y" may be any nucleophilic leaving group which is displaceable by the desired thiol, the preferred starting materials are compounds wherein Y" is halo (chloro, bromo, iodo or fluoro) or sulfonyloxy, i.e., alkyl- or substituted alkyl-sulfonyloxy or aryl- or substituted arylsulfonyloxy.
Examples of preferred leaving groups include halo, -S2 -(lower)alkyl, -OSO2CF3 and -OSO2C6H4CH3(para).
- A most preferred leaving group is the mesylate group.
Starting materials of formula VIII may be prepared from 73-azido compounds of formula VII by the consecutive steps of (1) Selectively reducing intermediate VII
as by catalytic hydrogenation with a noble metal catalyst, e.g., 10~ Pd-on-charcoal, or by the use of chemical reducing agents such as zinc and ammonium chloride, aluminum amalgam or hydrogen ~ ' - 4~ -~813~

sulfide in the presence of a base ~uch as ammonia or triethyl2mine to pro-duce a 7-amino intermediate of the formula H H
: 2 f / N ~ CH2-Y" ; and CO R' (2) N-acylating said 7-amino intermediate or a salt thereof with an acylating acid of the formula R-COOH
!, ~ wherein R is an acyl group, or with its functional : equivalent as an acylating agent for a primary amine, , and, if desired, converting the carboxyl-protected 7-acylamino compound to the corresponding free acid or to a salt thereof as by the methods disclosed above in connection with the de-blocking of compound II.

`,'' , 13~

The pharmaceutically active compounds of the present invention are potent antibacterial agents useful in the treatment of infectious diseases in poultry and animals, including man, caused by many Gram-positive and Gram-negative bacteria. The active compounds are also of value as nutritional supplements in animal feeds and as agents for the treatment of mastitis in cattle.
The novel medicaments provided by the pre-sent invention may be formulated as pharmaceutical compositions comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier or diluent. The compounds may be administered both orally and parenterally. The pharmaceutical preparations may be in solid form such as capsules, tablets or dragees, or in liquid form such as solutions, suspensions or emulsions.
In the treatment of bacterial infections in man, the active compounds of this invention may be administered parenterally or orally in an amount of from about 5 to 200 mg~Kg./day and preferably about 5 to 20 mg./Kg./day in divided dos-age, e.g. three or four times a day. They are administered in dosage units containing, for example, 125, 250 or 500 mg. of active ingredient with suitable physiologi-cally acceptable carriers or excipients.

.

~13~ii Illustrative examples of the preparation of starting materialS and compounds of the present invention follow. These examples are given in illustration of, but not in limitation of, the present invention. All temperatures are in degrees Centrigrade. DMF represents dimethylformamide, THE
stands for tetrahydrofuran and EEDQ is the amide - bond forminq reagent having the structure OEt ;~ CO2Et '.:
The ~-lactam compounds prepared in the -., 10 examples which follow all have the hydrogen atoms at ,. carbons 6 and 7 cls with respect to each other and, unless indicated, the products are racemic mixtures ; in the sense that they are composed of equal parts of the two isomers having the following structures:

H H H H
>N~ ,~N<

", C--O-- C--O--::
.

1~813S

pREPARATION OF STARTING MATERIALS
. _ . . .

Preparation 1 A. 2 3 OS 2 3 H H H H
3 ~ Br ~ -C~32Dr N ~ CD2Dr ~ Dr Benzyl Oximino-Acetoacetate CH3C - Cl - CO2CH20 O NOH
1.1 The procedure was essentially the same as that used to make the corresponding ethyl ester by H. Adkins and J. Reeve, JACS 60, 1328 (1938).
In a three necked one liter flask fitted with a thermometer, a dropping funnel and a magnetic stirrer ; were placed 173 g. (0.9 mole) of benzyl acetoacetate lThe benzyl acetoacetate was prepared as described by Baker et al., J. Org. Chem. 17, 91 (1952)] and 130 ml.

of glacial acetic acid. The contents were cooled in an ice bath and a solution of 69 g. (1 mole) of sodium 11~J1813~

nitrite in 130 ml. of water ~as added over a period of half an hour; the temperature was kept at 0 to 10C. After the reaction mixture was stirred for one hour at room temperature, 400 ml. of water was added and the stirring was continued for an additional two hours. The reaction mixture was extracted three times with 200 ml. portions of diethyl ether. The diethyl ether extracts were combined, washed once with water, three times with saturated sodium bicar-bonate solution and once with brine. After drying over anhydrous sodium sulfate, the diethyl ether solu-tion was evaporated leaving [1.1] as a clear oil which solidified upon trituration with petroleum ether (30-60) to give 186.5 g. (93.2%) of white solid.
Its NMR spectrum was consistent with the assigned structure. Generally the product was used as such in subsequent reaction but it can be recrystallized from toluene, m.p. 81-82C.

_nzyl Oximino-Acetoacetate Ethylene Xetal CH3CI~ CO2CH20 ketalization~ CH3 -~C~ Cl CO2CH20 o NOH ~ NOH
1.1 2.1 :' In a two liter flask fitted with a Dean Stark water separator and a condenser were placed 186.5 g.

.

8~3~

(0.85 mole) of benzyl oximino-acetoacetate ~ , 62 g.
(1 mole) of ethylene glycol, 800 ml. of benzene (reagent grade) and 2 g. (10.5 m~ole) of p-toluenesulfonic acid monohydrate. The reaction mixture was boiled at reflux ~ntil 15 ml. of water was removed (3 houIs). ~he benzene solution was washed once with saturated sodium bicar-bonate solution and once with brine. After drying over anhydrous sodium sulfate, the benzene solution was evaporated, leaving 212 g. (94% yield) of benzyl oximino-acetoacetate ethylene ketal 12.1] as a light yellow oil!
Its NMR spectrum was consistent with the assigned structure. Generally, the product was used as such in subsequent reactions but one of the isomers can be crystallized2 from toluene-petxoleum ether (b.p. 30-60C.); m.p. 52C.
Anal- Calc'd- for C13H15N5 C, 58-86; H~ 5.70; N~ 5-28-Found: C, 58.97 ~, 5.68: N, 5.12.
1. A mixture of the syn and anti isomers.
2. Only 35% of the oil could be crystallized.

1~08~3~

Benzyl Amino-Acetoacetate Ethylene Ketal Selective ~ -reduction ~
CH3/C\- 11 - CO2CH20 CH3/ ~~ FH ~ C2CH20 NOH ~__J 2 2.1 3.1 Freshly prepared aluminum amalgaml (from 27 g.
of aluminum foil) in a three-necked one liter flask was , covered with 500 ml. of diethyl ether. The flask was fitted with a mechanical stirrer, a condenser, and a dropping funnel. A solution of benzyl oximino-aceto-acetate ethylene ketal [2.1] (132.5 g.; 0.5 mole) in 300 ml. of wet diethylether2 was added dropwise at such a rate as to maintain boiling at reflux. After stirring for four hours, the reaction mixture was filtered through a Buchner funnel. The filtrate was evapor-ated leaving 110 g. of yellowish oil. The oil was dissolved in 800 ml. of dry diethylether and dry hydrogen chloride gas was bub~led into the solution ~` 20 until no further precipitation occurred. The white ; precipitate was filtered off and washed once with diethylether and then dried in vacuo. This provided j 108 g. of benzyl aminoacetoacetate ethylene ketal hydrochloride [3.1]; m.p. 157-158~C.

`:
.

3~

Anal. Calc'd. for C13H17N04-HCl: C, 54.26; ~, 6.31;
N, 4.87.
Found: C, 53.9~; H, 6.19;
N, 4.60.

To obtain the free base, the hydrochloride salt was suspended in 500 ml. of diethylether and con-centrated ammonium hydroxide was added with shaking until the solid went into solution. The diethylether layer was separated and washed twice with brine. After drying over anhydrous sodium sulfate, the solvent was ; evaporated leaving 90 g. (71% yield) of colorless oil.
1. The aluminum amalgam was prepared essentially as described in A.I. Vogel ("Practical Organic Chemistry", 3rd, Edn., Longemans Green & Co., London, (1957), p. 198) except for the following modification:
(a) 5% NaOH was used.
(b) The second washing with ethanol was omitted.

.

.,,, ., ..

~Q ~3 (c) Dry diethyle~her was used for washing snd m2st of the water must be d~ained.
2. ~he d~ethylether was saturated with water by sh~king with water in 8 separato~y funnel.
3. The product can be stored as the hydsochloride salt.

Schiff Base Formation And B-Lactam Form2tion ' 0 ; ~ Schiff base ~O
~ ~ formation ~ N~ ~
CH ~ - CH - C0 CH20 ~ CH3 :` NH2 C02CH20 3.1 ~ ~ 4.1 H H

0 ~ 3 ~2CH20 5.1 Sn ~ one litc flask fitted with a Dean Stark wate:
: separator and a condenser were placed 70.3 g. (0.28 mole) , : benzyl aminoacetoaceta~e ethylene ketal t3.1], 37 g. (0.2a ~ mole) cinnamaldehyde, a~d 750 ml. of methylene chlorid~

a3 .

~Q8~35 (reagent ~radel. The mixture ~as boiled at reflux for 30 minutes and then 400 ml. of methylene chloride were distilled off. The concentrated solution was then dried over anhydrous sodium sulfate and then eva-porated to dryness in vacuol. The residual oil was checked by NMR to ensure that Schiff base formation was complete before continuing on to the next step.
The freshly prepared Schiff [4.1~ was diluted with 600 ml. of methylene chloride2 and cooled to 02C.
(ice-salt bath). Triethylamine (31.1 g.; 0.308 mole) was added and then a solution of 36.2 g. (0.308 mole) of azidoacetyl chloride3 in 362 ml. of methylene chloride2 was added dropwise at 0C. over a period of one hour. The reaction mixture was stirred for an additional hour at room temperature4 and then evapor-ated on a rotary evaporator at reduced pressure while being heated on a 35C. water bath5. The residue was diluted with 500 ml. of diethylether and filtered.
The filtrate was washed twice with brine and dried over anhydrous sodium sulfate. Evaporation of this solution yielded 117.5 g. (94% yield) of styryl ~-lactam[5.1]. Its NMR and IR spectra are consistent with the assigned structure and indicate the presence of a mixture of isomers, diasteriomeric at the carbon to the carbonyl of the benzyl ester.
1. This evaporation must be done to ensure complete Schif~ ~ase ~orm~tion.
2. All the me~hylene chlorlde used $n the cyclo-addition reaction was re~gent gr~de which was first dried over molecular sieve (Type 4A) and then over anhydrous calcium chloride. ~t ~ -storet thereafter over molecular sieve (Type 4A) . .
3. J. H. Boyer and J. Horner, J. Amer~ Chem. Soc., (1955), 77, 951.
4. ~he reection mixture can be ~ept overnite at 0 if necessary.
5. This operation is necessary to ensure complete ~-lactam formation.

H H

Cd3 2.(CH3)25 ~ OCH3 C02C~2 ; 5.1 6.1 Styryl ~-lactam 15.1] (117.5 g.; 0.262 mole~ was dissolved ln one liter of methylene chloride (reagent srade), cooled to -50 to -60C. in a dry ice-acetone bath, and ozonized until a faint blue-green color appeared. ~he solution was then flushed with nitrogen until the color faded. Methylsulfide tl ml.) was 3 ~ i ~dded to the -50C. solution, which was then allowed to 610wly reach 25 as the cooling bath gradually -melted. ~t was kept overnite at room temperature under nitrogen and then it was washed twice with 1% ~od~um bicarbonate solution, twice with brine, driet over anhydrous sodium sulfate, and evaporated to dryness. ~he resulting oil triturated four t~mes with 100 ml. portions of petroleum ether (b.p.
30-60C.) o remove benzaldehyde. The oil was then triturated carefully with diethylether whereupon lt solidified. The solid was filtered off and ; dried to provide 75 g. (71.5%) of aldehyde 16.1] as a m~xture of isomers diasteriomeric at the carbon a to the carbonyl of the benzyl ester. Recrystalliza-tlon of [6.1] from ether ~ave white crystals, m.p.
101-102C. (co~rected).

~nal. Calc'd. for C17H18N406 : C, 54.54; H, 4-84;
N, 14.96.
Found : C, 54.75; H, 4.87;
` N, 14.89.

N ~ O ~l ~ }~
3 `ff' /--~ Na~4 3 ~J,~
d~><~3 T~F o~ ~3 CO2CH20 o C02CH2~
7.1 8.1 The ~ldehyde 16.1~ (116.3 g.; 0.31 mole) was d~ssolved ~n 600 ml. of T~F (reagent gr~de) and the solution w8s then cooled to -10C. (ice-methanol bath). Sodium borohydride (5.88 g.; 0.155 mole) was added and the reaction mixture was stirred 1 hour. 10% ~queous hydro-chloric acid was added until the mixture was slight~y ~cidic, then 600 ~1. brine was added. The THF layer was separated and the aqueous phase was extracted twice w~th 250 ml. port~ons of diethyle~her. The combined organic phases were washed twice wi~h 400 ~1. portions of brine, dried over ~nhydrous sodlum sulfate, and evaporsted ln va~uo to yield 117.3 g. of crude alcohol [7.1l ~s an orange oil, This oil w~s used ~s 6uch in the next rea~tion.
A solution of methanesulfonyl chloride ~37.8 g.;
0.34 mole) in 100 ml. of methylene chloridel w~s added dropwise at 0C. (ice-water bath) to a stirring solu-tion of alcohol ~7.1] (105.6 g,; 0.28 mole, triethyl-a~ine (56.6 g.; 0.34 mole) and one liter of methylene chloridel. A~terwards, the reaction was stirred for 30 hours at 25C, It was then washed twice wlth brine (500 ~1. portions), dried oves anhydrous sodlum sulfate, and eva~orated in vacuo The result~ng oil was dissolved in methylene chloride, treated with norite, znd then filtered over approximately 200 g. of activity I silica gel. The silica gel was then washed with approximately 2 liters of methylene chloride. The filtrate was evap-orated to dryness and the resulting oil (116 g.) was covered with diethylether. It crystallized on stsnding to give 87.2 g. (80% from ~6.1]~ of mesylate [8.1] as off-white solid, m p, 97-99C. (corrected~
1. The methylene chloride used was reagent grade which had been further purified by pass~ng over 8 column of calcium chloride and then storing ~; ~ver molecular sieve (Type 4A) ~a ~ 3~ ' - H H ~S02CH3 N3 ~ r~~ de-Ketal- ~ OH
I ¦ O O ization N

~o2c~32es ~/ C02t:H20 8.1 ~ ~/ 9.1 , H H 2 3 3 ~ OSO2CF3 o ~ ~ H3 10.1 :, A mixture o~ mesylate [8.1] (3.19 g.; 6.43 mmole) and 30 ml. of 95% trifluoroacetic acid was stirred at 25 for 2 hours. The mixture was diluted with 300 ml.
of brine and extracted three times with methylene chloride (100 ml. portions). The combined extracts were washed three times with water (50 ml. portions, unt~l neutral), dr~et (anhydrous so~iu~ sulfate) and evaporated to dryness in vacuo lea~ing 3.17 g. of a brown Dil. NMR spectra of this oil indicate the presence of >90% enol (9.1].

Crude enol l9.1~ (48.0 g.; 0.~17 mole) and tr~fl~c ~nhydrlde (33.0 g.; 0.117 mole) were dissol~ed ~n 50n ~1. of methylene chlor~de ~nd the solution was the~
cooled to O-C. ~ce-water bath). A solution of trl-ethylam1ne (11.8 g.; 0.117 ~ole) in 80 ml. of methylene chloride2 was added d~opw~se o~er a per~od of 40 ~inutes.
When the additlon was complete, the ice-water bath was removed and the mixture was stirred ~t 25 for 45 :: :
m~nutes. The ~ixture was then poured into 300 ~lo of ~ce WE~er and washed with colt water untll the pH of the washings was approximately 6. The extract was dr~ed (anhydrous sodlu~ sulfate) and ev~porated ~n vecuo ~ -to give 54.0 g. of crude tr~late 1lO.l] as a dark sed :~
oil. This oil was dissolved in 400 ml. of benzene (US~) ~nd passed th~ough 2 1 1/2" pad of activity II~ s~lica gel. The pad was washed with 1 1. of benzene. Evapor-ation of the benzene gave 38.3 g. of a yellow oil. This oil was carefully triturated with 50 ml. of absolute ethanol and then cooled at 0C. for 2 hours. ~he sesult~n~ whi_e solid was filte_ed off ~nd driet in vac~ to give 19.5 g. o~ tr~flate ~1~.1] as one i~omer, m~p. 57-59C. (corrected).
Anal. Calc'd. for C17l~l7F3N4O4S2: C, 37.67; H, 3.14;
~ , 10.33; S, 11.82.
Found: C" 37.40; H, 3.12;
N~ 10.43; S, 11.73.
~o ~ 3~' 1~ Triflic anhydride was prepared e~ follows:
170 g. (lOC ml.) CF3S03~ ( FLUOROCH~lIC ACID" - t- ademark -3M Company) and 135 g. P2Qs were m~xed carefully, shaken well, snd stored 18 hours protected fr~m istu~e. The pro~uct was distilled from the resulting sol~d m~8 us~ng a fla~e; the fraction boiling 80-90~C. was collected. Re-dist~llation of this fraction y~elded 119.45 g. (74%) of tr~fl~c ~nhydr~de boiling 82-84C.
2. The methylene chloride used was reagent grade lo which hsd been further purified by pass~ng over a column of calcium chloride end then stored over molecular s~eve (Type 4A).

H H. 2 3 H ~ S2~3 ~: ~3 - - I orgsnic ~ N3 - J
~ OS02CF3 base ~ ~ ~ ~CH2 0 ~ ~ CH3 ~ ~ C

co2CH2~ ~ C02C~12P

H OS02CH3 H 0sa2CH3 +3 ~ ~ Br C02CH2~ COZcH20~
~2.1 13.1 3~

Triethylamine (1 g.; 0.01 mole) was added to a stirred solution of triflate llO.l] (5.42 g.; 0.01 mole) in 55 ml. of methylene chloride (A.R.) at room temPera-ture. After stirring for five minutes (at which point TLC shows complete formation of allene [11.1~, a solution of bromine (10 ml. of lM solution in CC14; 0.01 mole) was added dropwise. After addition of the bromine, the mixture was concentrated, absorbed onto Activity I
silica gel and dry column chromatographed on Activity I
silica gel by eluting with methylene chloride (VSP).
This yielded one fraction (unifoxmly one spot by TLC) weighing 2.5 g. (45%). Its IR, W , and NMR spectra were consistent with the expected dibromide structure [13.1].

Anal. Calc'd. for C16Hl2Br2N4O6 N, 10.15.
Found: C, 35.25; H, 2.97;
N, 10.02.

- 6~ -B . OSO~ CH 3 OSO CH

~ ¦ ~nd~ ~2I
O ~CH2I ~ I

A solution of triethylamine (101 mg., 1.00 mmole) in 1.4 ml. of methylene chloride was added with stirring to a solution of triflate from Preparation lA above [10.1] (542 mg., 1.00 mmole) in 5.4 ml. of methylene chloride at 0C. After allowing the solution to warm to 24D over 15 minutes, a solution of iodine (254 mg., 1.00 mmole) in 7.5 ml . of methylene chloride was added with stirring over 30 minutes, then washed with water, dried, decolorized, filtered and the solvent evaporated in vacuo to give the diiodide [23.1] (588 mg.; 91%
yield) in greater than 95~ purity. The IR and NMR
spectra were consistent for the proposed structures.

Anal. Calc'd. for C16H16N406I2 N, 8.67; I, 39.28; S, 4.96.
Found: C, 29.76; H, 2.47;
N, 8.61; I, 39.37; S, 5.18.

Other suitable intermediates of general formula V in the specification and claims may be prepared by:
(1) substituting another easilY cleavable ester group for the benzyl ester of starting material 1.1; or (2) esterifying compound 7.1 with another sulfonic acid derivative or halogenating compound 7.1; or (3) halogenatins allene 11.1 with a halogenating agent other than bromine or iodine, e.g. BrCl. ~ -Preparation 2 . ~
p-Nitrobenzyl 7~-azido-3-methylsulfonyloxymethyl-~3-0-2-isocephem-4-carboxylate H H
N3 ~ ~

N ~ CH20S02CH3 A solution of methanesulfonyl chloride (0.50 ml., 6.5 mmole) in 10 ml . of methylene chloride was added dropwise with stirring to a solution of p-nitrobenzyl 73-azido-3-hydroxymethyl- A3-o-2-isocephem-4-oarboxylate ;

~8~3~

(2.41 g., 6.43 mmole), triethylamine (0.97 ml., 7.0 mmole) and 75 ml. of methylene chloride at -10C. After 1/2 hour at -10 and 1 hour at 24, the ~olution was washed with 5~ hydrochloric acid, 2% s~dium bicarbonate, and water (85 ml. each), then the solvent was evaporated in vacuo to give the mesylate title product, 2.86 g.
(98~ yield), as a yellow foam. The NMR of the product was in agreement with the proposed ~tructure.
The p-nitrobenzyl 7~-azido-3-hydroxymethyl- ~3-0-2-isocephem-4-carboxylate starting material used above may be prepared as follows:
1. The diiodide intermediate of the formula .'~ 0502CH3 ~3 ~ Il N ~ CH2I

Co2cH24~3No2 was prepared from p-nitrobenzyl acetoacetate according to the procedures of Preparations lA and B (Starting Materials) described above.
2. The diiodide intermediate (6.6 g., 9.6 mmole) was cyclized with potassium formate (2.54 g., 30 mmole~

1~ 135 in a solution of 100 ml. DMF and 0.1 ml. water at 0.
After stirring for S hours with the cooling bath removed, the mixture was poured into 100 ml. of cold water and extracted with methylene chloride. After washing with water containing a little NaCl, drying and evaporatiOn in vacuo, p-nitrobenzyl 7~-azido-3-formyloxymethyl- ~ -0-2-isocephem-4-carboxylate was recovered (5.3 g.) as a brown oil.
3. To a solution of 5.3 g. of the 3-formyloxymethyl intermediate in 53 ml. of acetone was added 26 ml. of water and 3.2 ml. of 12M HCl. The mixture was stirred at 24 for 7 hours, then poured into 100 ml. water and extracted with methylene chloride. The combined extracts were washed with water containing a little sodium chloride, dried and evaporated in vacuo to give 3.6 g.
of a brown oil. The oil was absorbed from methylene chloride onto 18 g. of silica gel and placed on a 72 g.
silica gel column (grade 3, 5% ether). The column was eluted with 200 ml. of ether, then with ether/ethyl acetate 3:1. The major component ~Rf 0.20) gave, on evaporation of the solvent in vacuo, a yellow solid which was recrystallized from acetone-ether to give the 3-hydroxymethyl starting material of this example, 950 mg. (17.5~ yield from the diiodide). m.p. 147-148.
''', . .

11~ 135 Anal. Calc'd- for ClsH13N5O7: -N, 18.66.
Found: C, 48.11; H, 3.61;
N, 18.81.

By replacement of the methanesulfonyl chloridein the procedure above with other sulfonic acid derivatives and the p-nitrobenzyl ester with other easily cleavable esters, suitable intermediates may be prepared having the formula N H H
3 \ ~

~ ,~ ~ CH2Y"
: 2 in which Y" is sulfonyloxy and R' is an easily cleavable ester residue.
' Preparation 3 ~ Benzyl 7~-azido-3-bromomethyl- ~3-0-2-isocephem-;~ 4-carboxylate N3 \H H
n o~ ~CH2Br CH2c6H5 ~' .

13~

To a solution of benzyl 7~-azido-3-hydroxy-methyl- ~3-0-2-isocephem-4-carboxylate in benzene is added about an e~uimolar amount of pyridine and a slight molar excess of phosphorus tribromide.
There is produced the 3-bromomethyl title product.
The benzyl 7~-azido-3-hydroxymethyl- ~3-0-2-isocephem-4-carboxylate used above may be prepared by the method disclosed in Preparation 2 by substi-tuting thebenzyl ester of the diiodide intermediate for the p-nitrobenzyl ester used therein7 Other suitable 7/3-azido-3-halomethyl esters may be prepared by the above method by appropriate vari-ation of the ester acetoacetate starting material and the halogenating agent.

: Preparation 4 Benzyl 7~-phenoxyacetamido-3-methyl-sulfonyloxy-methyl- ~3-0-2-isocephem-4-carboxylate C6H50CH2CO~H~

o \ ~ \ CH20502CH3 .

3~

Benzyl 7~-azido-3-methylsulfonyl-oxymethyl- .
~3-0-2-isocephem-4-carboxylate ~Preparation 2) is dissolved in methylene chloride and cooled to 0C. Two equivalents of triethylamine are added and, while stirring and cooling, H2S gas is passed through the solution until it is saturated. The solution is allowed to come to room temperature and concentrated to give benzyl 7~-amino-3-methyl-sulfonyl-oxymethyl- ~3-0-2-isocephem-4-carboxylate.
The ester is reacted with equimolar amounts of phenoxyacetic acid and EEDQ in methylene chloride to give the title product.
Other N-acylated compounds of the formula H H
R-NH~
. I I
, o CH2oso2cH3 .
. may be prepared by substituting for the phenoxyacetic ~cid used above another desired acylating agent, preferably one which will produce a starting material in which R is one of the acyl groups mentioned as being preferred in connection with the novel end-products of formula II.

~ 3 Prep~r~tion 5 l-carboxymethyl-1~2,3,,4-tetrazole-5-thiol ~nd its ti-soc~um salt I

N N
Il 11 HS C\ /N

A) Recrystallization of l-methYl-5-mercaptotetrazole Procedure:
1. One hundred and ten grams of l-methyl-5-mercaptotetrazole is slurried in 350 ml. of bo~ling chloroform. A near solution is obtained.
2. The hot solut~on (50-60) is rapidly filtered -; by vacuum through a heated ~uchner fu~nel (11 cm. SS
No. 604 paper containing 1/4 to 113 inch of packed filter aid ("Supercel"). The filter pad is washed 7 with 50 ml. of 50-60 C. chloroform which is added to the filtrate.
3. The filtrate is cooled to approximately 0-6 C.
ant kept at 0-6 C. for 2 hours. The crystals whlch have formed are collected by filtration at 0-6 C. and washcd with 60 ml. of 0-6 C. chloroform which is ~dded to the filtrate. The crystals (cut A) are air drieA at 37-45 C. for 18 hours.

* Trademark.

4. The filtrate i8 concentrsted on the rotary v8cuum evflporator t60~ C. bath) to ~pproxi-mately one-half volume. This glurry is coolet to 0-6 C. end kept ~t 0-6 C, for 2 hours. The cry~tals are c~llected by filtration at 0-6 C., washed wirh 40 ml. of 0-6 C. chloroform which is edded to the filtrate. The crystals (cut B) a~e air dried at 37-45 C. for 18 hours. Crystal cuts A and B are compos~ted to give an approximate 65%
we~ght yield.
5. The filtrate of cut B, Step 4 may be reworked tw~ce as described in Step 4 to o~tain an additional 15% secovery.

B) Preparation of l-carboxymethYl-1,2,3,4-tetrazole-5-thiol and its di-sodium salt 1. ~lve hundred ml. o~ subst2ntlally dry and pure tetrahydroruran ln a 2-llter ~ neck flask with ~tlrrer ls cooled ln a salt-acetone-lce bath to approximately -10 C. Dry nitrogen gas i9 blown on the llquld surrace.
2. ~lve hundred ml. of 15.06~ (1.6 N) b~tyl lithlum ln hexane (Foote Mineral Co.) ls added over a ten minute perlod under dry nitrogen and ~tlrring to the tetrahydrofuran. me near solutlon is cooled to -5 to -10 C.

~ 3 5 3. Fort~ ælx and rOur tenths gram (46.4 g,) Or l ~ethyl-5-mercaptotetrazole (recrystallized a~ QboYe) ls dlssolved ln 200 ml. Or substantlally pure and dry tetra~ydro~uran. qhe solutlon 18 ~lltered lr cloudy and then cooled to 5 to 10 C.
4. The cooled solution o~ step ~ 18 added over 10 minu~e~ wlth stlrring and under dry nit~o-gen to the butyl llthium solution. The temperature should be maintalned at -5 C. to ~10 C. maxlmu~.
Preclpltates may form.
5. Ihe mixture 1~ stlrred under dry nltrogen and 0 C. to ~10 C. for one halr hour.
6, Anhydrous carbon dioxlde gas ls bubbled through at a rapld rate and wlth rapld stirrlng ror 15-30 minutes at approxlmately amblent temperature (0 to 10 C.) to no higher than ~20 C.
7, m e white prec~pitate whlch rorms ls 8ultab~y collected by rlltratlon in an area Or low humidlty. The preclpltate ls wa~hed with about 75 ml. of tetrahydroruran.
8. The preclpltate ls dlssolved in 250 ml. ~ -of water (pH 8.5-9.5). A second layer of tetra-hydrofuran may be present. Thls may be removed ~n the Yacuum rotary evaporator (50 C. bath~, i ,

9. The a~eous solutlon is adJusted to pH 1.6-2.0 with concentrated hydrochlorlc acid.

10. me acld aqueou~ solutlon ls extracted twice wlth 250 ml. portlons Or ethyl acetate.
Each 250 ml. ethyl acetate extract ls back extracted wlth 100 ml. portions Or water. m e water extracts are dlscarded. The ethyl acetate extracts (rree o~ any water layer) are rlltered and compo~lted.

11. m e comblned e~yl acetate extracts are concentrated to dryness on the vacuum rotary evaporator (60 C. bath),

12. m e cry~tals ln the flask are bolled with ~00 ml. Or chloroform for about 2 mlnutes. The hot slurry (50-60 C.) ~s vacuum ~iltered through a heated ~uchner ~mnel (11 cm-SS~604 paper), m e crystals are washed with about 75 ml. o~ 50 C.
chloroform. The crystal~ are alr dried at room temperature rOr about 3 hour~ and then made about 100-200 me~h,

13. The 100-200 mesh crystals are treated with bolllng chloro~orm exactly as described ln step 12 (the hot chloroform removes ~ost o~ the unreacted l-methyl-5-mercaptotetrazole). Yield:
approxlmately 45 to 50 gram~ Or crystalline 1-car~oxymethyl-1,2,3,4-tet~azole-5-thiol. These crystalc ~ J~ ~ 3~

uay contaln 0.02 to O.O5 moles Or l-methyl-5-aercaptotetrazole .
1~ . me crystals o~ fitep 1~ are slurrled ~lth 250 ml~ Or ethyl ether at room temperature ~or ~-5 m~nutes. ~he mlxture ls filtered. The lnsoluble~ (O.5-5%~ may be a contaml~atlng symmetrical mercaptotetrazole ~etone o~ t~e ~ollowlng tentatlve structure:

N - ~ O N ~ N
~C ~N CH2 - C - CH2 - N ~ ~N
SH SH

CAUTIO~: Thls compound EXPLODES at approximately 205-210 C.
15. Ihe ether flltrate o~ 8tep 14 1~
evaporated to dryness on the vacuum rotary evaporator (5O C. bath). Approximately ~2 to 48 grams Or crystalllne l-carboxymethyl-1,2,3.4-tetra-zole-5-thiol containing approximately 0.01-0.05 mole Or l-methyl-5-mercaptotetrazole 16 recovered.
16. The crystals are dicsolved ln 420 ml.
absolute ethanol (approxlmately lOO mg./ml.).
he solutlon ls warmed to 50-60 C.
17, TQ the hot ~olutlon Or step 16, 310 ml.
Or a 41% sodlum 2-ethylhexanoate (SEH) solutlon ln i~opropanol ls added wlth very rap~d stlrr~ng over a lO m~nute period. A crystalllne precipltate ror~s. The ~.lxture ls ~lurried at 50-60 C, for 20 minutes.

:

J~135 ~ 8 . me mixtuFe 1~ riltered hot (50-60 C.) through a heated Buchner ~unnel ~11 cm-SS-No. 60 paper). m e crystals are washed wlth 75 ml. o~
50 C. ethanol.
19. ffl e ethanol damp crystal~ of ætep 18 - are slurried in 200-~00 ml. o~ ethanol. The ~lurry 15 passed through a 200 mesh screen. lhe slurry ls heated to 50-60 C. ror 5 minutes wi~h rapld ~lrring (unreacted di-~odlum l-methyl-5-mercaptotetrazole is very so~uble ~n hot ethanol).
20. qhe crystals are collected at 50-60 C.
on a 11 cm-SS No. 604 paper ln a heated Buchner ~unnel. The crystals are washed wlth 75-100 ml.
ethanol and vacuum dried at 50-60 C. for 24-48 hours. yield: 40-48 grams Or dl-sodium l-carboxymethyl-1,2,3,4-tetrazole-5-thiol (free of 1-methyl-~-mercaptotetrazole as observed by h~).

.

1~ 13~ i l-CarboxYethy1-1,2.3.4-tetrazole-5-~hiol .

N - N

HS ~ /N

(CH2) 2-C02H

R) 2-CarboethoxyethYlisocYanate ~ -alanine ethyl ester hydrochloride (93.6 g.), triethylamine (123.5g) and methylene chloride ~400 ml) were mixed together and cooled to -10 C. Carbon disulfide (46.S g) dissolved in 150 ml. of chloroform was added to the above solution during a two-hour period while keeping the temperature at about -10 C.
~fter the addition was complete, the temperature was allowed to warm to 10 C. for a~out 10 minutes. The 501ut~0n was again cooled to 10 C. and 66.3 g of etllyl chlorof~rmate in 60 ml of chloroform was added dropwise over a 40-minute period with stirring. The temperature was ~llowed to rise to room temperature for 30 min~tes and again cooled to 0C. an additional ,., 61.6 9 of triethylamine was ~dded a~ 0 C. and then the 601ut~0n was stirred at room temperature for 3 hours.
~ he mixture was treated with water and the organlc phase collected, washed w~th 2 x 250 ml of 2N HCl, then 2 x 250 ml of NaHC03, then 2 x 250 ml of water, The osgan~c phase was dried over Na2S04 and the solvent 7~

1 ~ 81 3 ~ ' removed in vacuo to produce 93.7 ~ ~f ~n oil found to be the desired product. The IR and NMR spectra were consistent with the structure.
B) l-Carbox~ethyltetrazol-5-thio' &odium azlde (29.7 g) was dissol~gd ~n 400 ml ~f water and heated to 60 C in a nitrogen ~tmosphere.
2-Car~oethoxyethylisocyanate (46.9 g) dissolved in ~0 ml of Skellysolve B (essentially n-hexane) wa adde~ to t~e heated sodium azide ~olution. The ~olut~on was stirred for about 150 minutes at about 70-72 C., then cooled to 30 C. ~n an ice bath.
50~ sodium hydroxide solution was added until the pH
was 12. The mix~ure was heated for forty minutes at 70 C. and cooled to 15 C. in an ice ~ath~ The pH
was adjusted to 2 using conc~ Hcl and then extracted wlth ethyl acetate t4 x 150 ml). The ethyl acetate extracts were washed with water, then dried over 80dium sulfate. The sol~ent was evaporated 'n vacuo ~nd the product was collected as crystals from methylene chloride to yield 19.5 g of ti~le product.
Substitution in the procedure for the pre-paration of 1-carboxyethyltetrazol-5-thiol for the ~-alanine ethyl ester used therein of ~n equimolar quantity of an appropriately substituted ~mino acid ester of 3 ~o 4 carbon atoms produces the correspond~ng l-carboxy tC3-C4 alkyl)tetrazol-5-thiol, e.g., 1-carboxypropyltetr~zol-5-thiol and l-car~oxy-bu~yltetrszol5-thiol.

* Trademark.

i~l`Q~.;3~

E X A M P L E_S

Example 1 ; .
p-Nitrobenzyl 7~-azido-3~ methyltetrazol_5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate N N
I I O N N

o,),_ ~fLC~2S~

C02CH2 ~)_ N2 ~ ~:
~ ` ' "

To a solution of p-nitrobenzyl 7~-azido-3-methylsulfonyloxymethyl- ~3-0-2-isocephem-4-carboxYlate (4.53 9., 10 mmole) and triethylamine (1.4 ml., 10 mmole) in 90 ml. of methylene chloride was added 10 mmole of l-methyltetrazole thiol. The solution was stirred at 24C. for 16 hours, then washed with 5% HCl and water (100 ml. each), dried and the solvent evaporated in vacuo to give the product as a yellow oil. Upon recrystalli-zation from ethyl acetate, the title product was obtained in 7~% yield; m.p. 150-152C. The NMR was consistent with the proposed structure.

' . .

7 ~ _ Example 2 p-Nitrobenzyl 7B-azido-3-(2-methyl-1,3,4-thiadiazol-~-ylthlome~hyl)- ~3-0-2-isocepnem- ~

~,./\o ~ , o.~ CN2 5~5 1 co 2 cH 2~ N0 2 The procedure of Example 1 is repeated except that the l-methyltetrazole thiol used therein is replaced :
by 10 mmole of 2-methylthiadiazole thiol. The title product was isolated as a yellow oil in 95~ yield.
The NMR and IR spectra of the product were in agreement with the proposed structure.

Example 3 p-Nitrobenzyl-7~-azido-3-~2-methyl-1,3,4-oxadiazol-5-ylthiomethyI)- ~3-0-2-isocephem-4-carboxylate O ~ C 2 ~ O

C02CH2~ N2 , _ 79 _ 313~

A solution of p-nitrobenzyl 7~-azido-3-methylsUl-fonyloxymethyl- ~3-0-2-isocephem-4-carboxylate (1.36 g., 3.0 mmoles) and triethylamine (0.38 ml., 3.0 mmoles) in 50 ml. of dichloromethane was treated with 2-methyl-1,3,4-oxadiaæole-5-thiol (0.35 g., 3.0 mmoles). The reaction mixture was stirred at room temperature for 48 hours and then washed with 10% HCl, water and brine.
The solution was dried over sodium sulfate and evaporated in vacuo giving crude product. The crude product was purified by dry-column chromatography on silica gel - (60 g., Activity III) eluting with 15% ethyl acetate in ether. There was obtained 1.0 g. of pure title product in 70% yield. The NMR spectrum was in agreement with ;~ the proposed structure. ~ ;

Example 4 If the general procedures of Examples 1-3 are repeated using equimolar weights of 1,2,3-triazole-5-thiol, l-carboxymethyl-1,2,3,4-tetrazole-5-thiol and 1-carboxyethyl-1,2,3,4-tetrazole-5-thiol, respectively, in place of the thiols used therein, there are produced p-nitrobenzyl 7~-azido-3-(1,2,3-triazol-5-ylthiomethyl)-A3 o 2 isocephem 4 carboxylzte, p-nitrobenzyl 7~-azido-~` , 3-(1-carboxymethyl-1,2,3,4-tetrazol-5-ylthiomethyl)-3-0-2-isocephem-4-carboxylate and p-nitrobenzyl 7~-azido-3-(l-carboxyethyl-l,2,3,4-tetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate, respectively.

Example 5 p-Nitrobenzyl 7~-amino-3-(1-methyltetrazol-S-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate O N N

N ~ CH2-S J ~ l CO2CH2 ~ - N

, Hydrogen sulfide was slowly passed into a solution of the 7~-azido ester of Example 1 (340 mg., 0.72 mmole) and triethYlamine (77 mg., 0.76 mmole) in 10 ml. of methylene chloride for 50 min. One ml. of 10% HCl ~, was added and the mixture concentrated in vacuo. The ` residue was mixed with 25 ml. methylene chloride and ; washed with 25 ml. each of 10% NaHCO3 and dilute NaCl.
The methylene chloride solution was dried, treated with charcoal, filtered and evaporated ln vacuo. The re-sulting residue was mixed with methylene chloride, the sulfur filtered off and the solvent evaporated ln vacuo to give the title product, 276 mg. ( 86% yield), as a .81 yellow tar. The IR and NMR spectra were consistent with the proposed structure.
Repeating the above procedure but using the benzyl 7~-azido ester instead of the p-nitrobenzyl ester gave benzyl 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxylate.

Example 6 p-Nitrobenzyl 7B-amino-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)- ~3~0-2-isocephem-4-carboxylate H N H H

2 ``" r ~ oL C~~S ~ C~

c02CH2 ~r NO2 The procedure of Example 5 is repeated with the p-nitrobenzyl 7~-azido-3~ methyltetrazol-5-ylthio-methyl)- ~3-0-2-isocephem-4-carboxylate used therein replaced by an equimolar weight of p-nitrobenzyl 7,3-azido-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxylate. The title product is produced as a yellow solid in 85% yield. The IR
and NMR spectra of the product were consistent with the proposed structure.
' .

Example 7 p-Nitrobenzyl 7~-amino-3-(2-methyl-1,3,4-oxadiazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate ~8135 H2N H _ ~ N N

~ ~ C~2-S ~ ~ c~3 C02CH2~ N2 Hydrogen sulfide was bubbled through a solution of the 7B-azido ester of Example 3 (0.99 g., 2.1 mmole) and trithylamine (0.32 ml., 2.3 mmole) in 50 ml. of dichloromethane for 1 min. and the mixture was then stirred for an additional 45 min. Nitrogen was then bubbled through to remove excess H2S and the solvent was evaporated in vacuo to leave a residue of title product.
, .
Example 8 Pivaloyloxymethyl 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate The title compound is produced according to the method of Example 2 of U.R. Specification 1,229,453 by replacing the 7-aminocephalosporanic acid used therein by 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid.

:`
'.~' '~, 13~

The respective acetoxymethvl; methoxymethyl, acetonyl and phenacyl esters of 7~-amino-3-(1-nethyltetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid are prepared by substituting in the method above for the chloromethyl pivalate used therein an equimolar weight of chloromethyl acetate, chloromethyl methyl ether, chloroacetone and phenacyl bromide, respectively. .

Example 9 p Nitrobenzyl 7~-(2-thienylacetamido)-3~ methyl-tetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate ~, .

~ CH2CONS `O CNH3 N

C52-5 --( ¦

C2 2 ~ NO2 A solution of 2.0 mmole each of p-nitrobenzyl 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)- ~3-0-2-isocephem -4-carboxylate, 2-thienyl acetic acid and EEDQ in 20 ml. of methylene chloride was stirred at 24C. for 2 hours. The resulting solution was washed with 5% NaHCO3, 10% HCl (2x) and dilute NaCl (20 ml.
each), dried and the solvent evaporated in vacuo to give the product as a yellow oil. The product was crystallized from methylene chloride/ether to give the title product in 53% yield; m.p. 165-168 (dec.).
The IR and NMR of the product were consistent with the proposed structure.

Example 10 p-Nitrobenzyl 7~-(2-thienylacetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate s ~Lc~2s1\, C~3 C02CH2 ~ N02 The procedure of Example 9 was repeated with the 7~-amino ester used therein rePlaced by p-nitrobenzyl 78-amino-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate.
The product was absorbed onto 6 g. of silica gel and placed onto a 24 g. column of silica gel. The column was eluted with methylene chloride followed by ethyl acetate. The major fraction was obtained as a yellow foam in 55% yield. Upon recrystalliza-tion from methylene chloride/ether, the title product was obtained; m.p. 127-129C. ~he IR and NMR spectra were in agreement with the proposed structure.
. ~ .

Anal. Calc'd. for C24H21N5O6S3: C, 49.05; H, 3.60;
N, 11.92; S, 16.37.
Found: C, 47.42; H, 3.54;
N, 11.87; S, 16.20.

Example 11 p-Nitrobenzyl 7~-phenoxyacetamido-3-(1-methyltetrazol-S-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylate :~ .

. ~, ~C~2_s~ ! :

CO2CH2 ~ 2 ; A solution of p-nitrobenzyl 7~-amino-3-(1- -methyltetrazol-5-ylthiomethyl)- ~3-9-2-isocephem-4-carboxylate (270 mg.). EEDQ (150 mg.) and phenoxyactic acid (92 mg.) (0.60 mmole each) in 6 ml. of methylene chloride was maintained at 24C.
for 2 hours. This solution was diluted to 15 ml.
with methylene chloride, washed with 5% NaHCO3 (25 ml.), 10% HCl (2 X 25 ml.) and saturated MaCl (25 ml.), an~ then dried and absorbed onto 1.7 g. of silica gel (grade III). The silica gel was placed on a chromatographic column containing 3.4 g. of silica gel (grade III). The column was eluted with ether changing to ether-methylene chloride tl:l) after fraction 4 (all fractions were 5 ml.). TLC of fractions 8-17 showed them to contain one compound (Pf=0.11 on silica gel eluted with ether). These fractions were combined and evaporated in vacuo to gi~e the title product as a yellow tar, 236 mg. (67% yield). The .
.
-~
, ~ .

~ - 87 -.

11~8~;~
i IR and NMR spectra of the product were consistent with the proposed structure.

Example 12 Benzyl 7~-phenoxyacetamido-3-(1-methyltetrazol-5-ylthiomethyl~- ~ 3-0-2-isocephem-4-carboxylate The procedure of Example 11 was repeated except that the p-nitrobenzyl startin~ material was replaced by the correspondinq benzyl ester. The title product was obtained upon recrystallization from methylene chloride-ether (1:1); m.p. 86-88C. U.V.~ meaxH 284 ; (=10,400). The NMR and IR spectra of the product - were consistent with the proposed structure.

Example 13 p-Nitrobenzyl 7B-(2-thienylacetamido)-3-(2-methyl-1,3,4-oxadiazol-5-yl)- ~3-0-2-isocephem-4-carboxylate ; I I H H

CH2CONH~ CH2 sl~ o J~ CH3 C2 CH2 ~3 N02 : ., .~ - .

11~ 35 The p-nitrobenzyl ester residue of Example 7 was dissolved in 50 ml. of dichloromethane and treated with 2-thienylacetic acid (0,31 g., 2.2 mmoles) followed by EEDQ (0.54 g., 2t2 mmoles).
The mixture was stirred at room temperature for 16 hours and then washed with water, 1% NaHCO3, water, 10% HCl, water and brine. It was then ~ried over sodium sulfate and evaporated in vacuo. ~he residue was purified by dry-column chromatography on silica gel (50 g., activity III) eluting with 25~ ethyl acetate in ether to give 0.61 g. of pure title product in 50~ yield. The NMR of the product was in agreement with the proposed structure.

Example 14 7B- (2-Thienylacetamido)-3-(1-methyltetrazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid :
The p-nitrobenzyl ester from Example 5 (1.00 mmole) in 50 ml. of ethyl acetate and 25 ml. of n-butanol to which 10 ml. of 0.1 M HCl and S00 mg. of 20% palladium hydroxide-on-diatomaceous-earth had been added was hydrogenated on a Parr apparatus at 24C and 50 p.s.i.

..:

11~ 35 for 3 hours. The catalyst was filtered off and the ~olution extracted with 1% NaHCO3 (containing some MaCl) (3 X 25 ml.). The aqueous extract was cooled to 0C., acidified to pH 1 with 10~ HCl and saturated with NaCl. The acidified aqueous was extracted with methylene chloride (4 X 50 ml.). The methylene chloride was concentrated in vacuo to 100 ml. and after standing, the title product was collected as a colorless solid in 18.5% yield; m.p. 182-184 ,....~
(dec.). U.V. ~mhax 280 (~=8500). The IR spectrum was consistent with the proposed structure.
';
Anal. Calc d- for C16H16N6 5 2 N, 19.26.

Found: C, 44.10, H, 3.71;

N, 19.07.

A sample of the title product ~called BC-L70) ; after solution in water and dilution with Nutrient ~roth was found to exhibit the following Minimum Inhibitory Concentrations (M.I.C.) in meg./ml. versus the indicated microorganisms as determined by over-night incubation at 37C. by tube dilution.

M.I.C. in mc~./ml.

. Or~an~sm BC-L70 ~eph~lexin CePhalothin D~ pneumonlae A9585 .016 ..25 .13 ~5% 6erum*
Str. pyosenes A9604 .016 .25 . .06 ~57. serum* -~
S. eureus Smith~ A9537 .13 1 .13 S. sureus Smith~ A9537 1 1 .5 ~50% 8erum S. sure~s BX1633-2 A9606 .5 2 .25 et 10 dil'n S. aureys BX1633-2 A9606 2 4 .5 et 10 ' dil'n S. ~ureus meth.- A15097 1 8 resist.; at 10-3 dil'n Sal. enteritidist A9531 .03 4 .5 ~ :
E. coli Juhl~ A15119 1 8 16 E. col~$ A9675 16 8 63 . pneumoniae$ A9977 .25 4 4 K. pneumoniae$ A15130 8 16 16 Pr. mirabil~s$ A9900 .13 4 Pr. morgani~ A15153 32 125 125 P8. aeru~inosa~ A9843A 125 125 125 Ser. marcescens~ A20019 125 125 125 Ent. cloacae A9656 125 125 125 Ent. cloacae A9657 1 4 4 Ent. cloacae A9659 125 125 125 * 50% Nutr~ent Broth - 45% Antibiotic Assay Broth at 10 4 d~lue~on.

Example 15 7~-(2-Thienylacetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid The procedure of Example 14 was repeated up to the point of preparing the acidified aqueous extract with the p-nitrobenzyl ester starting material re-placed by p-nitrobenzyl 7B-(2-thienylacetamido)-3-(2-methyl-1,3,4-thiadiazol-5-ylthiomethyl)- ~3-o-2-isocephem-4-carboxylate. The acidified aqueous was extracted with tetrahydrofuran/ethyl acetate (9:1) (2 X 50 ml.).
The organic extract was washed with saturated NaC1 (50 ml.), dried, treated with charcoal and the solvent evaporated ln vacuo to give a brown oil.
The oil was crystallized from ethyl acetate/ether to give the title product in 39% yield; m.p. 100-105C. (dec.). U.V. ~mHaF/H2O 276 (= 12,500). The IR and NMR were consistent with the proposed structure.

Anal. Calc~d. for cl7Hl6N4o5s3-l/2 CH3CO2C2H5: ~
C, 45.96; H, 4.06; N, 11.29; S, 19.37.
; Found:
C, 45.69; H, 3.97; N, 11.30; S, 19.63.

. .

M.I.C. data for the product (called BC-L71) is shown in the following table.
M.I.C. in mc~./ml.
~ BC-JJ71 Cephalexin ~ephalothin D. pneumoniae A9585 .016 5 .06 ~5% ~erum* .008 .5 .06 Str. pyogenes A9604 .016 .25 .06 ~5% serum* .008 .13 .06 S. ~ureus Smith i A9537 .25 1 .13 .06 ~5 .06 S. aureus Smith ~ A9537 2 2 .5 ~50X serum 2 2 .5 S. aureus BX1633-2 A9606 .5 2 .25 at 10-3 dil'n .13 1 .13 S aureus BX1633-2 A9606 4 4 ,5 a~ 10-2 dil'n 2 2 .2S
S. aureus meth.- A15097 4 16 res~st.; at 10-3 2 16 dil'n Sal. enteritidis ~ A9531 06 2 13 E. coli Juhl $ A15119 8 8 8 E. coli ~ A9675 322 16 32 K. pneumoniae i A9977 ,5 4 K. pneumoniae ~ A15130 32 166 16 Pr. mirabilis ~ A9900 1 8 Pr. morganii ~ A15153 132 125 125 Ps. aeruginosa ~ A9843A 125 125 125 Ser. mascescens ~ A20019 125 125 12$
Ent. cloacae A9656 125 125 125 Ent. cloacae A9657 4 4 4 Ent. cloacae A9659 125 12255 1225 * 50% Nutrient Broth - 45% Antibiotic Ass~y Broth at 10 4 ~ilution.

Q~13~

Example 16 7~-~2-Thienylacetamido)-3-(2-methyl-1~3,4-oxadiazol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid The p-nitrobenzyl ester of Example 13 (0.60 g., 0.15 mmole) was dissolved in 125 ml. of ethyl acetate and 40 ml. of n-butanol. The solution was treated with 1.05 ml. of IN HCl (1.05 mmole) and hydrogenated over 0.60 g. of 20% palladium hydroxide on carbon for 3 1/2 hours at 50 p.s.i.g. The mixture was then filtered, washing well with ethyl acetate, and evaporated in vacuo. The residue was slurried with ether and extracted with 1% NaHCO3 (2 X 50 ml.) followed by 25 ml. of water. The aqueous extracts were acidified in the cold with 10% HCl and extracted with ethyl acetate (3 X 30 ml.). The solution was dried over sodium sulfate and evaporated ln vacuo. The solid residue was slurried with ether, filtered and dried giving 69 mg. (15% yield) of the title product; m.p. 170-173. U.V.~ max = 278 nm (F = 12,453).

20Anal. Calc'd. for C17H16N4O5S2: , N, 12.84.
~`` Found: C, 43.95; H, 3.57;
N, 11.35;
residue: 3.05.

M.I.C. data for the product (called BC-L76) is shown in the following table.

M,l.C. in mc~./ml.
~anism BC-L76 Ccphalexin Cephalothin D. pneumoniae A9585 .016 .~5 .016 ~5% ~erum*
Str. pyogenes A9604 .008 .13 .03 +5% ~erum*
S. aureus Smith ~ A9537 .06 .5 .06 S. aureus Smith ~ A9537 1 1 .25 ~50% serum S. aureus 8X1633-2 A9606 .5 2 .13 at 10-3 dil'n S. aureu3 BX1633-2 A9606 .5 4 .13 at 10-2 dil'n S. aureus meth.- A15097 2 32 2 resist.; at 10-3 dil~n Sal. enteritidis ~ A9531 .5 2 .13 E. coli Juhl $ A15119 8 8 16 E. coli ~ A9675 63 16 63 K. pneumoniae i A9977 1 4 2 K. pneumoniae ~ A15130 63 8 32 Pr. m~rabilis ~ A9900 .5 4 .5 Pr. mor~anil ~ A15153 125 ~125 >125 Ps. ~eru~inosa $ A9843A ~125 >125 ~125 Ser. marcescens ~ A20019 >125 ~125 ~125 Ent. cloacae h9656 ~125 >125 ~125 Ent. cloacae A9657 16 , 4 8 Ent. cloacae A9659 ~125 ~125 ~125 * 50% Nutrient Broth - 45% Antibiotic Assay Broth at 10 4 dilution.

13:~

7~-phenoxyacetamido-3~ me~hyltetrazol-5-ylthio-methyl~ 3-0-2-isocephem-4-carboxylic acid A solution of the p-nitrobenzyl ester prepared according to Example 11 (~3~ mg., 0.405 mmole) in 12 ml. of tetrshytrofuran (peroxide free) and 2.4 ml. of ethanol with 140 mg. of 10% palladium-on-charcoal was hydrogenated (Parr Shaker) at 24 and 50 p.s.i. for 4 hours. The cstalyst was filtered off and the solvent evaporatet in vacuo.
The residue was dissolved in 25 ml. of ethyl acetate and washed with 25 ml. each of 10% HCl and water. The acqueous layers were combined and extracted with 10 ml. of ethyl acetate (which was washed with 10 ml. of water). The com~ined ethyl acetate was extracted with 1% NaHC03 ' ~ 35 (2 x 10 ml.). The combined bicarbonate extracts were acidified with 10% HCl and extracted w~th ethyl acet~te. The ethyl acetate extract was wa~hed with water ~nd ~aturated NaCl nd then drled and evaporated ~n vacuo to give 67 mg.
(37% yield) of crude title product. The product was crystallized from chloroform to give title product with m.p. 136-138 (dec.). U.V. ~ MaeH

276 (~=10,600).

Anal. Calc d. for C18 18 6 6 N, 18.83; S, 7.18.
Found: C, 46.70; H, 3.94 N, 17.74; S, 6.98;
Residue: 1.16.

.

M.l.C, data for the product (called ~C-L62) is shown in the following table.

1~ 35i .
M.I.C._ in mc~ . Iml .
Or~anism BC-L62 Cephalexin Cephalothin D. pneumoniae A9585 .13 .13 .03 ~SX ~erum*
Str. pyogenes A9604 .13 .06 .03 ~5Z serum*
S. sureus Smith i A9537 .25 .25 .06 S. aureus Smith ~ A9537 1 1 .25 ~50% ~erum S. ~ureus BX1633-2 A9606 2 2 .13 at 10-3 dil'n S. aureus BX1633-2 A9606 16 4 .25 at 10-2 dil'n S. aureus meth.- A15097 4 16 reslst.; at 10-3 dil'n Sal. enteritidis ~ A9531 .5 4 .13 E. coli Juhl $ A15119 4 8 16 E. coli ~ A9675 32 16 32 K. pneumoniae i ~9977 2 4 K. pneumoniae ~ A15130 32 16 32 Pr. mirabilis ~ A9900 2 4 .5 Pr. mor~anii ~ A15153 63 125 >125 Ps. aeruginosa ~ A9843A ~125 ~125 ~125 Ser. ~arcescens ~ A20019 ~125 ~125 >125 Ent. cloacae A9656 ~125 ~125 ~125 Ent. closcae A9657 32 4 4 Ent. cloacae A9659 125 >125 ~125 * 50% Nutrient Broth - 4S% Antibiotlc Assay Broth ~t 10 4 dilution.

11~ 3~ ~ .

Example 18 am~no-a-phenvlacetamido]-3~ methyltetrazol- -5-YlthiomethY~ 3-O-?-lsocephem-4-carboxyllc ~cid (h~drochloride salt) H-CONH ~ 0 IH3 2 o~ ~CH2-S ~
C02H HCl N--N

A mixture contain~ng p-nitrobenzyl 7~-amino-3-(l-methyltetrazol-5-ylthiomethyl)- A3-0-2-isocephem-4-carboxylate (2.8 g., 6.25 mm~le), EEDQ (1.55 g., 6.25 mmole) and D(-) -azidophenyl acetic acid ~1.11 g., 6.25 mmole) in 200 ml. methylene chloride was kept at room temperature (protected from moi~ture by ~ calcium chloride drying tube) for 16 hours; It wa6 then washed successively with 10% HCl, water, 5% NaHC03 and brine, dried (Na2S04) ~nd evapor~ted in vacuo to dryness leaving a yellow amorphous solid. This was ~uspended in ether ~nd filtered to glve 3.67 g. of a solid whlch was identlfied by IR and ~MR a6 p-nitrobenzyl 7~ azido-~-phenylacetamido]-3-(1-methylt~trazol-5-ylthiomethyl)-3-0-2-isocephem-4~c~rboxylate; m.p. 132-134C.
'"
~ .
_99_ 11~8135 An~l. Calc'd- for C25H22N107S C~ 49-S0; H~ 3~65;
N, 23.09.
Found: C, 49.47; H, 3.64;
N, 22.86.

A mixture consistlng of the above ester (0.40 g., 0.65 mmole) snd 20% Pd(OH)2 on Celite t0.50 g.) in 50 ml. ethyl ~cetate, 50 ml. n-butanol And 6.5 ml. of .
O.lN HCl was 6haken on a Parr apparatus under 60 p.~
of hydrogen for 6 hours. The solid was filtered off on a Celite pad which was washed well with absolute ethsnol. The combined filtrate6 were evaporated in VACUO at 40C. to leave ~ yellow flmorphous ~olid. This was triturated with ether and collected by filtratio~
to give 0.21 g. solid; m.p. 110-120 (deco~p,), U.V.
= 274, ~ maX ~7550. The IR and NMR ~pectra of the product were in agreement with.the proposed structure.
M.I.C. data for the product (called BC-L78) ls shown in the following table, Purity of the sample was estin~ted to be bout 25%.

.~ .
.

-100- . ' 35 ~
: i M.l.C. ln mc~.~ml.
Or~anism BC-L78 ~ Cephalothir D. pneumoniae A9585 0.25 0.25 .03 ~SZ serum~
Str. pyogenes A9604 0.13 0.13 .03 ~5X serum*
S. ~ure~s Smith ~ A9537 2 0.5 .06 S. aureus Smith ~ A9537 8 1 0.5 ~50% serum S. sureus BX1633-2 A9606 8 2 0.25 ~t 10-3 dil'n S. ~ureus BX1633-2 A9606 63 8 0.5 at 10-2 dil~n S. sureus meth.- A15097 (37) 63 32 resist.; at 10-3 (28)>125 li5 63 - dil 'n Sal. en~erltidis ~ A9531 0.5 2 0.25 E. coli Juhl ~ A15119 2 8 16 : :
E. coli ~ A9675 16 16 63 K. pneumoniae ~ A9977 1 4 K. pneumoniae ~ A1513~ 8 16 16 ~r. mirabilis ~ A990n 1 4 Pr. ~organ~i ~ A15153 32 ~125 ~125 Ps. aeruginosa i A9843A >125 >125 ?125 Ser. ~arcescens ~ A20019 >125 >125 ~125 Ent. cloacae A9656 ~12~ 125 ~125 Ent. cloacae A9657 4 4 4 Ent. cloacae A9659 16 ~125 ~125 * 50% Nutrient Broth - 45% Antib~otic Assay Broth nt 10 4 dilution.

-w~

Exsmple 19 . Repe~ting the general N-acylatlon procedures of Ex~mple6 9, 1~ 11, 12, 13 , or 18 to react the following ~cylating agents with 7~-amino-3~ methyltetrazol-5-ylthiomeehyl)-~\3-0-2-i~ocephem-4-carboxylic acid (or ~n ester or salt thereof), the following products ~re obt~ined after removal of sny functional blocking groups.
.,,,~ ' .
AcYlatin~ ARent Product ~ixed anhydride of 7~-(2-Aminomethylphenyl-potassium 2-(1-carbo- scetamido)-3-(1-methyltetrazol-methoxypropen-2-ylamino- 5-ylthiomethyl)-~3-0-2-methyl)phenyl~ceta~e with lsocephem-4-carboxylic acid isobutyl chloroformate ~-benzoylureidophenyl- 7~ -Benzoylureidophenyl-~cetic acid ~cetamido)-3-(1-methyltetrazol-S-ylthiomethyl)- ~3-o-2-. .
. lsocephem-4-carboxylic acid , ~
2,6-dimethoxybenzoyl ~ ,6-Dimethoxybenzamido)-; chloride . 3-(1-methyltetrazol-5-ylth~o-. ~ethyl)- ~ -0-2-isocephcm-4-carboxylic acid ~ .
, .
~ -10~- -. - . . .

Acylatin~ A~ent Product D-~nhydro-o-carboxy- 7~-(D--Hydroxyphenylacetemido)~
~ndelic acid 3~ methyltetrazol-5-ylth~o-~ethyl)- A3_0_2_isocephem-4-carboxylic acid 3 benzyl-1,2,4-oxadiazole- 7~-~N-(Phenylacetimidoyl)-S-one-4-acetic ~cld sm$noacetamido~-3-(1-methylte-trazol-5-ylthiomethyl)-~ -0-2-~socephem-4-carboxylic ~c~ d .
; ~aler~c acid 7~-Valeramido-3-(1-methylte-trazol-5-ylthiomethyl)- ~3-o-2- ~:
- 160cephem-4-carboxylic ~cid phenylace~ic acid 7B-Phenylacetamido-3-(1-methyltetrezol-5-ylthiomethyl)-A3 0 2 isocephem-4-carboxylic acid 3-thienylacetyl chloride 7~-(3-Thienylacetamldo)-3-(l-methyltetrazol-5-ylthio-methyl)-~3-0-2-isocephem-4-' carboxylic acid ' a-carboxybenzyl-phenyl- 7~ carboxY-~-Phenyl-~cetic acid acetamido]-3-~1-methyltetrazol-~: 5-ylthiomethyl)- ~3-0-2-isoccphem-4-c~,rboxylic ~c~d .

8~ 3~

~c~laeinr~ ~ent Product ~-hydroxyphenylacetic 7~-(o-Hydroxyphenylacet~mido)-cld 3-(l-methyltetrszol-5-ylthio~ethyl~-~3-0-~-isocephem-4-carboxyl$c acid cysnoacetlc acid 7~[cyanoacetamidol-3-(l-methyltetrazol-5-ylthiomethyl)-/\3-0-2-isocephem-4-carboxyllc acid ~-cyanopropionic ~cid 7~-(a-cyanopropionamido)-3-~l-methyltetrazol-5-ylthio-methyl)-~3-0-2-isocephem-4-c~rboxyl$c ~cid ~-(2H)-tetrazoleacetic 7~-[2-(2H)-tetrazolylacetsmido~-ac$d 3-(1-methyltetr~zol-5-yl~hiomethyl)-: ~3-0-2-isocephem-4-carboxylic acld 3-(o-chlorophenyl)-5- 7~-[3-(o-chlorophenyl)-5-methyl-4-isoxazole- ~ethylisoxazol-4-ylcarboxamidol-carboxylic scid chloride 3-(l-methyltetr~zol-5-ylthio-methyl)- ~\3-0-2-$socephem-4-csrboxylic acid l-(lH)-tetrazolylacetyl 7~-ll-(lH)-tetrazolylacetamido]-; chlor~de. 3-(l-meehyltetr~zol-5-ylthi methyl)-~3-0-2-isocephem-4-carboxylic acid l~Q~3~ ~

EX~

~ ollowing the ~cyl~t~on ~ethots of the preceeding examples and in particulsr those d~sclosed in U.S. ~atent 3,546,219, the compounds listed below sre prepared by reacting 7~-a~lno-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-c~rboxylic acid (os an ester or salt ~heseof), wi~h the ~pproFriate scylsting agent.

RYCO-CO~ ~ fH3 N
o~N~

RY_ phenyl p-acetamidophenyl p-methoxyphe~yl p-methylphenyl 2-methoxy-5-methyl~henyl m-chlorophenyl o-nitrophenyl 2,4-d~chlosophenyl a-naphthyl 2-phenanthryl p-aminophenyl 2-thienyl p-~imethyl~minophenyl.

' A r ~ #13 x~mple 21 Follow~ng the acyls~on methods of ~he preceeding cx~mples ~nd in particular those di~closed in U.Kc Patents 1,296,081 and 1,294,541, ~he compounds l~sted ~elow are prepsred by re~cting 7~-a~ino-3-tl-~ethyltetrazol-S-ylthiomethyl)-~3-0-2-isocephem-4-carboxylic ~cid or sn e8ter or 8alt thereof with an 2cylating agent of the formNl~

Ra -CR-COOH

X--C
H-N-C-R
Il . X' or 2 funct~onal equivalent thereof.

X C ~CH25~

X ' Ra X X' ~
phenyl 0 ~mino ~H2 2-Shienyl 0 i~ino ~H2 ~; 3-thienyl 0 imino NH2 trophenyl 0 ~mino NH2 O ~mino NH2 ~-aminophenyl p-methylplleny~ 0 imino ~H2 ~chlo~ophenyl 0 imino N~2 B~, Ra X X' R~
p-methoxyphenyl O ~mino N~2 p-hydroxyphen~1 0 imino ~2 p-di~hylamlno- O ~mino NH2 phenyl 3,4-dimerhoxy- 0 ~mino N~2 phenyl ~-methoxyphenyl O i~o N~Z
p-ace~amidcphenyl O imino ~H~
m-hydroxyphen~-1 0 ;~;no ~2 3~5-dichl~ro-4- 0 Imino NH2 hyds~xyphenyl 3-chloro-4- 0 imino ~H2 : hydrox~phenyl phenyl O 0 2-fury~
2-thienyl 0 ~ 2-f 3-~hienyl 0 ~ 2-fury~
phenyl O O phenyl 2-tbienyl 0 0 phenyl phenyl 0 0 2-thieny~
p-chloroph~nyl O 0 2-uryl p-hyd~oxyphen~l O 0 2-~ury 3-chloro-4- 0 0 2-fury~
hydrox~phenyl 3~5-dichloro-~- 0 0 2-furyl hyd~oxyphenyl : ~fl7 il~8~3 Ra X Xi R~
m-aminophenyl O 0 2-furyl p-methylphenyl O 0 2-furyl p-dimethylamino- O O ~-furyl phenyl p-me~hoxyphenyl O 0 2-furyl ~-hydroxyphenyl O 0 2-furyl p-acetamidophenyl O 0 2-furyl m-nitrophenyl O 0 2-furyl phenyl O O CH3 2-thienyl 0 0 CH3 3-thienyl 0 0 CH3 phenyl O O -CH2-C6H5 phenyl O O
phenyl O O
phenyl O O

phenyl O ~l3C ~
N~o CH3 phenyl O O ~ ~
~O~CH3 phenyl O N~S~

ir~i8 ~ 35 Ra X X ' Ri phenyl O o phenyl O O N~O~Lc6Hs phenyl O O ,¢~

phenyl S 0 2-furyl 2-thienyl S 0 2-fu~yl 3-thienyl S 0 2-furyl p-hydroxyphenyl S O CH3 phenyl O imino phenyl phenyl O imino 2-thienyl phenyl O imino 2-fury 3-thienyl 0 imino phenyl phenyl O imino l I

' , ' 1 ~ 81 3 5 Ex~mPle ?2 When 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)-/\3-0-2-isocephe~-4-carboxylic acid or an ester or salt thereof is acylated according to the procedures above and ln particulsr those tisclosed in U.S. 3,692,779 with an acid chloride of the formula RlTN - CH2COC 1 ~,~ ~0~0 ; there are produced the compounds listed below.

.

CH
R -~ -CH2CONH~ N--N
O ~ 2 ~

' R~

benz)~l ;

813;~ii 1 ~,S ; ~ ; ~,~ ;

N~ N3 ; ~

C6H5 ~1~3 ; 3 ~bL ; ~;

dichlor om~ thyl ;
n-propyl;
cyclopentyl;
., , cyclohe~cyl;
p-chlosobenzyl;
~ phenyl : 2-thie~yl;
3-thienyl. ~ :~

... .

.

~8~13 EX8mD1 e 2 3 When the 7-acylamido-3~tl-m~thyltetrazol-5-ylthlo~ethyl)- ~3-0-2-isocephem-4-car~oxylic acid compounds of Example 28 ~se hydrogenated as by the process of U,S, Pate~t 3,692,~79, these are produced the compounds listed bel~wO

Ri- o ~~~C~2CO~Y.~ l H3 o~ ~ C~12 ~
co2~ N- N

where Ri is as de~ ned in Example 22.

Example 24 When 7~-amino-3-(1-~ethyltetrazol-5-ylthio~ethyl)-~3-0-~-isocephe~-4-car~oxylic acid o~ an ester or salt thereof is acylated according to the procedures of the above examples (an~ in p2rticulas the prccedures disclosed in U,S. Patent 3,646,024) with an acid chloride of the formula ; R -CH-CO-C1 R It ' .ere sre prsduced the compounds listed below.

~ 81~5 '~

Ra-CH-CONH CH3 R~ N NH ~ 1_N
o o~N~CH~~S_~
Ra Ri phenyl phenyl phenyl 2-thienyl phenyl 2-furyl 2-thienyl phenyl 3-thienyl 2-$uryl p-hydroxyphenyl phenyl . . .

ExamPle 25 ~ :

When 7B-amino-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-car~oxylic acld or an ester or salt theseof is acylated ~ccording to the procedures sbove ~nd in particular sccoTding to the methods of U.S.
Patent 3,778,436 with ~n acylat~ng agent of the formula R -CH-COOH
.,, S-C-R
i 11 O

or a functlonal equivalent thereof, there are produced the compounds l$sted below 1 1~ 8 ~ 3 5 !

l~-CH-CON}I~-- 2 R R~
.

3,4-d~ethoxyphenyl ~thyl 2-thienyl p-methylphenyl 2,4-dichloro?henyl ethyl S-methyl-3-phenyl- ethyl ~80~azol-4-yl 2-thienyl ethyl 2-furyl 2-furyl phenyl phenyl 1,4-cyclohexadien- ~ethyl l-yl -il4-1~0~L3 ~ -~!i , Preparation of 7~-t~-~2-AminomethYl ~ Ey~hexadien~
cetamido~-3-(1-methyltetrazol-5-ylthiometh~l~- L~3 0-2-isoce~hem-4-carboxylic acid ~CH2CONH ~f o ~ C02H N - N

,.~ ~ ' .

A. a-(2-Amlnomethvl-1~4-~yclohexadienyl~aceti~ acid A solution of 16.5 g. ~0.1 mole) of o-aminomethyl- '`!
phenylacetic acld in 1.5 1 of llqu~d ammonia (which had been treated with 50 ~. of Li to remove a trace of :~
moisture) was slowly diluted with 500 ml. of dry t-BuOH.
To the solution was addet in sm~ll portions 3.4 g. (0.5g,-: atom) of Li over a period of 4 hours and ~he mixture i .~"
i was stirred for 16 hours at room temperature removing :
.. the liquid ammonia in a hood and finally evapora~ed to dryness.below 40 C. The residue was dlssolved in 500 ml.
of water and ~he solution was chromatographed on a column , of IR-120 (H~, 700 ml.) res~n ~nd el~ted with lX
NH40H solution. N~nhydr~n positive frac~,lons of the eluate were combined and evaporated to dryne~.
The residue wa ~ashed with four 50 ml. portions of hot acetone ~nd recrystallized from 500 ml. of ethanol-water (1:1) to give 11.2 8. (677.) of a-(2-amin~methyl-1,4-cyclohexad~enyl)acetic acld as colorless needles.
M.p. 183C.

IR: ~ nu~ 1630, 1520, 1380, 1356 cm 1.

~MR ~D2O ~ R2C3 2.72 (4H, ~, H2C_ ), 3-01 (2H, s, CH2CO), 3.20 (2H, 5, CH2-N), 5.78 (2H, s, H_ C=).

Anal. Calcd. for CgH13N02 C, 64.65; H, 7.84;
N, 8.38.
Found: C, 64.77; ~, 8.06;
N, 8.44.

8. a-[2-(t-Butoxycarbonvlaminom thvl~-1,4-cyclohexadienyl]-scetic acid ' To a ~tirred solution of 8.0 g. (0.048 mole) of a-(2-aminomethyl-1,4-cyclohexadienyl)acetic acid and 3.8 g. (.096 ~ole) of NaOH in 150 ml. of water wa.c added a solution of 10.3 g. (0.072 mole) of t-butoxycarbonylazide ln 80 ml. of THF and the mixture was stirred.for 18 hours -1~6-~08~3~

~t r~om temper~ture. The THF was removed u~der reduced pressure and the residual solution was ~a~hed with eeher (2 x 100 ~1.), acidifiet with 6 N HCl ~nd extracted w~th ether (3 x 100 ml.). The combined extraces were washed with ~ater (2 x 100 ~1.) and ~ saturatet NaCl solution ~lO0 ml.), dr~ed with Na2S04 and evaporated to dryness. The oily residue was triturated with n-hexane to give 10.5 g. (82%) of colorless p~wder ~elting at 113C.

IR: ~/ ma~ 3370~ 1715, 1640, 1530, 1280, 1160 cm 1.

NMR: ~ CpC13 1.45 (9H, s, t-Bu-~), 2.73 (4H, s, H2C ~C C)~

3.16 (2H, s, CH2C0), 3.76 (2H, d, 6Hz, CH2N) 4.90 (lH, ~, NH), 5.66 (2H, s, H> C=), 10.6 (lH, br-s, C00~, Anal. Calcd. for Cl4H21N04: , ; .
Found: C, 63.13; H, 8.21; N, 5.26.

C. 7B-lG-t-ButoxYcarbonYlaminomethy~ _clohexadienvl)-acetamido~-3-(l-methyltetrazol-5-ylthiomethyl)-~3-0-2-isoce~hem-4-c2rboxylic a _d ' To a stlrred solution of equimolar amounts of ~-12-(t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl)acetic scid and 2,4-din~trophenol in ethyl acetate is added an equ~molar emount of N,N'-d~cyclohexylcarbodiimide. The ~eac~ion mixture ls stir~et a~ room tempersture for 3 hours. She ~eparated d~cyclohexylurea is filtered off. ~he filtrate is evaporated to dryness to give the ~ctivsted ester which is dissolved in eetrahydr ~uran. To this solut~on is added a solution of 7B-~mino-3-(1-~ethyltetrazol-5-ylthio~ethyl)- ~3-o-2-~ocephe~-4-carboxylic acid and triethylamine in ~pproxim~tely a 1:2 molar pro?ortion, respectively, relative to the a-[2-(t-butoxycarbonylam~nomethyl)-1,4-cyclohexadienyl]scetic acid. The mixture is stir~ed ~t room temperature for several kours and concentrated in V2C~O. The concent_ate is washed with ether, ac~difiet with dilute mineral acit and estracted with ethyl acetate.
The extracts are washed with water and sa~urated NaCl solution and dried to gi~e the title product.

D. 7B~ (2-Amino~eth~l-l,~-cvclohexadionvl~acetamito~-3-(l-m2ehvltetrazol-S-YlthicmethYl~- G3-0-2-isoce~he~-4-carbox~ acid A solution of 7B-~c-(2-t-bu~oxyca-Donyl2mino~.ethyl-1,4-cyclohaxadienyl)aceta~ido]-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isoce?hem-4-ca bo~ylic acit ~n trifluoroacetic acid is stirred at 0C. for one ho~r.

1~C1 8~3S `~

~o the solution ~s ~tded dry ethes un~il a prec~pitate forms. The precipitate is collected by filtration, ~u~pended ~n water Jn~ ad~usted ~o pH6 to give the title produc~.

ExamPle 27 7-~ ~a-(2-Am-inomeehv~ -cyclohe~xenvl~acetamidol-3 tetrazol-5-Ylthio~ethvl~ 3-0-2-isoce~he~-~-c~r~oxvlle acid ~ CH2-s~

A. ~?-(N-t-~tox~ar~o~Ylaminsmethvl~ c~clohe,~en--acetic acid A solution of ~-[2-(t-butoxycarbonylaminome~hyl)-1,4-cyclohexadienyl3-acetic acid (1.33 g., 5 mmoles) ln 3~. a~mon~ hydroxi~e (10 ml.) was hydrogenated at 40 psi with pallad_us2 o~ cha-eoal (lOZ, O.~ g.). A
th~oret~cal amount of hydrogen w~s taken up in 3 hours.

~he ca~alyst was removed and the filtrate uas acidified ~o pH 2 w~th dil. ~Cl a~t extracted with ethyl ~cetate 11~

3L~

(2 x SO ml.). The co~bined extracts were washed with ~ater (20 ml.), dr~ed with Na2S04 ~nd evfipor~ted ~nder redueed pressure to afford an oil (1.34 g.) which ~ol~di-f~ed on ~tanding for several days. Recrystall~zation from n-hexsne - ethyl ~cetate gave 1.2 g. t~tle product as colorless pris~s melting st 118-119C.

IR ~ nu~ol 3450, 173~, 1660, 1510 cm NMR:~ ppm 3 1.58 (9H, s, t-butyl-~), 1.50 - 1.90 (4H, m, -CH2-), 1.90 - 2.20 t4H, m, llylic methylene-H), 3.18 (2H, s, C~2-CO), 3.78 (2H, d, 6 Hz, CH2-N), 5.00 (lH, br-s, NH), 8.98 (lH, br-s, COOH).

: Anal. Calcd. for C14H23N04: C, 62-43; H, 8-61;
N, 5.20 Found: C, 62.12; H, 8.77;
N, 5.37.

B. 7~-[~-(2-t-~utoxycarbonYlaminomethv-l-cYclohexenvl) scetamido~-3-(1-methvltetrazol-5-ylthiomethvl)-~3-0-2-isoceDhem-4-carbDxylic acid : To a stlrred solution of equimolar amounts of 2-(N-t-butoxycarbonylaminomethyl)-l-cyclohexen-l-yl]
acetic acid snt 2,4-dinitrophenol ~n erhyl acetate is .

1~8i~

added an equimolar a~ount of NIN'-d~cyclohexylcarbo-diimide. The reactlon mixture is fitirret for 1 hour at room temperature and the precipitated d~cyelohexylurea 1~ filtered o~f. The filtr2te is cooled to 5-C. and poured into a cold ~olution of 7~-amino-3-(1-methyl-te~razol-5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic acid snd excess triethylamine in 50% ~queous T~F. The mixture is stisred overnight at room temperature and washed with ether. The aqueous layer is acidif~ed with tilute HCl to precipitate the title produet.

, C. 7~ Aminomethyl-l-cyclohexenyl~acetamido~-3-(l-methyltetrazol-5-vlthiomethYl~- A3-o-2-isocePhem-4-~arboxYlic acid ' ' .

A solution of 7~-[c-(2-t-butoxycarbonylaminomethyl-l-cyclohexenyl)acetamido]-3-(1-methyltetrazolo5-ylthio-~ethyl)- ~3-0-2-isocephem-4-c~r~oxylic acid in trifluoro-~cetic ac$d is stirret at 0C. for 1.5 hours. The mix-ture is diluted wi~h ethe. to separate the trifluoro-ecet~te salt which is dissolved in water and neutralized to give the title p~oduct.

Exam~le 28 ~' When the p-nitrobenzyl 7~-amino-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxyl~te of Ex~mple 15 ls replaced by sn equimolar weight of p-~21 1 ~ 8 i 3 5 nltrobenzyl 7~-smino-3-(1,2,3~triszol-5-ylthio-~ethyl)-~3-0-2-~socep~em-4-c~rboxylete, p-niero-benzyl 7~-amino-3-(1-carboxymethyl-1,2,3,4-tetrazol-S-ylth~omethyl)-~3-0-2-isocephem-4-carboxylate ~nd p-nitrobenzyl 7~-amino-3-(l^carboxyethyl-1,2,3~4-tetrazol-5-ylthi~methyl)-~\3-0-2-isocephem-4-carboxylate, respectively, there are produced after catalytic hydro-genation as in the procedure of Example 20 7~
thienylacetamido)-3-~1,2,3-triszol-5-ylthiomethyl)-~3-0-2-isocephem-4-car~oxylic acid, 7~-(2-thienylacet-~mido)-3-(1-carboxymethyl-1,2,3,4-tetrazol-5-ylthio-methyl)-~3-0-2-isocephem-4-carboxylic acid snd 7B-(2-thienylacetamido)-3-(1-carboxyethyl-1,2,3,4-tetrazol-5-ylthiomet~.yl)-~3-0-2-isocephem-4-carboxylic acid, respectively.

Example 29 When the 7B-amino-3-(1-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxylic acid (or ester or salt thereof) in the procedures of Examples ll , 12 , 18 and 19-27. is replaced by an equimolar amount of 7~-amino-3-(1,2,3-triazol-5-ylthiomethyl)- ~3-0-2-isocephem~4-carboxylic acid, 7~-amino-3-(2~methyl-1,3,4-thiadiazol---l22-~0813~ i 5-ylthiomethyl)- ~3-0-2-isocephem-4-carboxylic arid, 7~-amino-3-(2-methyl-1,3,4-oxadiazol-5-ylthiomethyl)- ~3-0-2 isocephem-4-carboxylic acid, 7~-amino-3-(1-carboxymethyl-tetrazol-5-ylthiomethyl)- ~3-0--2-isocephem-4-carboxylic acid or 7B-amino-3-(1-carboxyethyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxylic acid (or an ester or fialt thereof, any reactive functional groups other than the 7- ~
amino group being suitably protected if necessary), respec- ~ -tively, there are produced (after any necessary deblocking of functional protecting groups) the corresponding 7~-acyl-amino carboxylic acids of each of the above-named nuclei.

Example 30 .;
When the d-amino products of any of the aforementioned Examples pertaining thereto are reacted with acetone accord-ing to the procedure of U.S. Patent 3,303,193, there are ob-tained the corresponding 0-2-isocephem derivatives of the formula Ra-CIH c----n CH ~
HN N CH CH O

C C N
H3C / ~ H3 O I CH2-S-Z

COOH

where Ra is as defined in the above-mentioned examples and Z represents the appropriate alkyl, aryl, aralkyl or heterocyclic group of the selected nucleus, or pharmaceutically acceptable salts thereof.

, ~

8i3 .

Ex~mPle 3l 7~-Phenox~acetamido-3~ methYltetrazol-5-Ylthio- -meth~ 3-0-2-~socePhem-4-carboxYlic acid (~lternate process) A mixture o~ benzyl 7~-phenoxyacetamido-3-meehylsulfonyloxyme~hyl- ~3-0-2-lsocephem-4-c~rboxylate tO.22 g., 0.43 mmole; preparet accordi~g to preparation 4 ~bove), triethylam~ne (0.07 ml., 0.5 mmole) and l-methyltetrazole thiol ~O.OS g., -0.5 mmole) ~n methylene chloride ~50 ml.) was stisred at room temperat~re for 16 hours, washed with lOZ HCl ~20 ml.) and brine ~2 x 50 ~l.), dried and co~centr~ted to give a semi-solid (0.24 g.) which was purlf~ed by column chromatography to afford 0.15 g. of pure benzyl 7~-phenoxyacetamido-3-~l-methyltetrazol-5-ylthiomethyl)-~3-0-2-isocephem-4-carboxylate ~s a wh~te sem~-solid.
The product was shown by IR snd NMR to be identical with the product of Example 18 Anal. Calc'd. for C25H24N6065 N, l5.66.
~ound: C~ S5.89; H, 4.55;
N, 15.34.

The benzyl ester ls subjected to catalytlc hydrogenation followin~ the procedure of Example -~
23 to give the title product.

Claims (10)

Div. I

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

1. A process for preparing a 7-amino 0-2-isocephem intermediate of the formula III

wherein Z is optionally substituted C1-C6 alkyl, aryl, aralkyl or heterocyclic and R" is hydrogen or an easily cleavable ester carboxyl-protecting group, or a salt thereof; which process comprises selectively reducing a 7-azido intermediate of the formula IV' wherein Z is as defined above and R' is an easily cleavable ester carboxyl-protecting group and, if desired, removing protecting group R' to produce the corresponding free acid intermediate of formula III and, if desired, converting the free acid form of intermediate III to a salt thereof.

2. The process of Claim 1 wherein the 7-azido intermediate is reduced by catalytic hydrogenation with a catalyst selected from palladium, platinum or Raney nickel, said catalyst being optionally supported on a carrier, or by chemical reduction with a reducing agent selected from zinc and Div. I

ammonium chloride, aluminum amalgam or hydrogen sulfide in the presence of a base.

3. The process of Claim 1 wherein R" is hydrogen or an easily cleavable ester carboxyl-prctecting group and Z
represents 1,2,3-triazol-5-yl, 2-methyl-1,3,4-thiadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl, 1-N-methyltetrazol-5-yl, 1-carboxymethyltetrazol-5-yl or 1-carboxyethyltetrazol-5-yl.

4. The process of Claim 3 wherein R" is hydrogen.

5. The process of Claim 3 wherein Z is 1-N-methyl-tetrazol-5-yl.

6. Compounds having the formula wherein R" is hydrogen or an easily cleavable ester carboxyl-protecting group and Z represents optionally substituted C1-C6 alkyl, aryl, aralkyl or heterocyclic, or salts thereof, whenever prepared or produced by the process of Claim 1 or by an obvious chemical equivalent thereof.

7. Compounds having the formula Div. I

wherein R" is hydrogen or an easily cleavable ester carboxyl-protecting group and Z represents optionally substituted C1-C6 alkyl, aryl, aralkyl or heterocyclic, or salts thereof, whenever prepared or produced by the process of Claim 2 or by an obvious chemical equivalent thereof.

8. A compound having the formula wherein R" is hydrogen or an easily cleavable ester carboxyl-protecting group and Z represents 1,2,3-triazol-5-yl, 2-methyl-1,3,4-thiadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl, 1-N-methyltetrazol-5-yl, 1-carboxymethyltetrazol-5-yl or 1-carboxyethyltetrazol-5-yl, or a salt thereof, whenever prepared or produced by the process of Claim 3 or by an obvious chemical equivalent thereof.

9. A compound of Claim 8 wherein R" is hydrogen, whenever prepared or produced by the process of Claim 4 or by an obvious chemical equivalent thereof.

10. A compound of Claim 8 wherein Z is 1-N-methyltetrazol-5-yl, whenever prepared or produced by the process of Claim 5 or by an obvious chemical equivalent thereof.