CA1100129A - Cephalosporin compounds - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention relates to a new class of cephalosporin compounds having in the 3-position a 5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthiomethyl group and to a process for the preparation thereof character-ized by reacting a 3-acetoxymethylcephalosporin compound with a 3-mercapto-5-oxo-6-hydxoxy-4,5-dihydro-1,2,4-tria-zine. The 3-triazinylthiomethylcephalosporin compounds have excellent broad spectrum gram-positive and gram-negative antibiotic activity.

Description

The present invention relates to a new class of cephalosporin compounds having in the 3-position a 5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthiomethyl group and to a process ~or the preparation thereof. These compounds have excellent broad spectrum gram-positive and gram-negative antibiotic activity.
The present invention provides novel cephalo-sporin compounds of -the formula I

0 \ ) ~ 0 OOR

2 R~ ~

in which Rl is hydrogen or lower alkyl; R2 is hydrogen, an alkali metal cation, or a readily removable ester forming ` O
group; and R is hydrogen or the group R'-C- in which R' is hydrogen; Cl-C6 alkyl; Cl-C3 haloalkyl; Cl-C3 cyanoalkyl;
Cl-C3 azidoalkyl; Cl-C3 hydroxyalkyl; ~-nitrobenzyloxy;
4-amino-4-carboxybutyl; a 4~substituted-amino-4-carboxybutyl ester for the formula . 11 '.
A~ C CH~-(CH2) 2-C~12-- NH
A ' in which A is diphenylmethyl, p~nitrobenzyl, benzyl, 2,2,2-:
trichloroethyl, t-butyl, or p-methoxybenzyl, and A' is ~ .
",~
- , ' .

C2-C4 alkanoyl, C2-C~ haloalkanoyl, benzoyl, halobenzoyl, 2,4-dinitrophenyl, or phthaloyl;
or R' is a group of the formula a~ ~ ~o~(Z)m~CH
a' o_:~

in which a and a' independently are hydrogen, Cl-C4 lower alkyl, Cl-C~ lower alkoxy, halogen, hydroxy, or aminomethyl;
Z is O or S; and m is O or l;
or R' is a group of the formula P-C~
Q
in which P is 2-thienyl, 3-thienyl, l-tetrazyl, or a phenyl group of the formula a _ .
20 ~ ~O _ a' o 9 in which a and a' are as defined above; and Q is hydroxy, formyloxy, acetoxy, carboxy, sulfo, amino, or -NHY in which Y lS benzyloxycarbonyl, t-butyloxycarbonyl, O O R'''O
.. .. . ..
-C-NH-C-NH2, or -C-N---C-V in whih R''' is hydrogen or ll NH

Cl-C3 alkyl, and V is phenyl, halophenyl, furyl, mono- or di-(Cl-C3 alkyl)amino, mono- or diphenylamino, or R''' and V taken together form a heterocycle, R''' being -(CH2)n-in which n is 2 or 3, and V being -NR " "-, in which R'''' is hydrogen, methanesulfonyl, or Cl-C3 alkyl; or R' is a group of the formula R' '-CH?- in which R'' is 2-thienyl;

3-thienyl; 2-furyli 2-oxazyl; 2-thiazyl; l-tetrazyl; ben-zotriazolyl; 1,3,4-thiadiazolyl 2-thioi 1,2,5-thiadiazolyl-3-thio; 1,3,4-oxadiazolyl-2-thia; pyridyl-thia; 1-(4-cyano~-1,2,3-triazolyl; or 1-(3-cyano)-1,2,4-triazolyl.
The present invention also provides a process for preparing the cephalosporin compounds of formula I
which comprises reacting a 3-acetoxymethylcephalosporin compound of formula II

I--T I~
0~ N\ ~ CH2 O~C CHs II

wherein R and R2 are as defined above, with a triazinyl-thio derivative of the formula \N/ ~

R~
wherein Rl is as defined above;
:: optionally acylating the compounds so obtained wherein R is hydrogen or Q LS amino;
and if desired removing the amino and/or carboxy protecting groups.

i In the above definition of Rl, the term "lower alkyl" means an alkyl group having from l to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec~butyl, or isobutyl. Preferably, Rl is methyl or ethyl, and, more preferably, Rl is methyl.
¦ Examples of the resulting 3-substituent of the cephalosporin in which Rl is as defined above include 5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthiomethyl; 4-methyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthio-lO methyl; 4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthiomethyl; 4-n-propyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4~
triazin-3-ylthiomethyl; 4-isopropyl-5-oxo-6-hydroxy-4~5-di-hydro-1,2,4-triazin-3-ylthiomethyl; 4-n-butyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthiomethyl; 4-sec-butyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthio-methyl; and 4-isobutyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-; triazin-3-ylthiomethyl.
~ ~ R2 of formula I is hydrogen, an alkali metal cation, such as lithium, sodium, or potassiumj or a readily removable ester forming group. The term "a readily re-movable ester forming group" refers to the commonly em-ployed carboxyl1c acid protecting groups used to block the C4 carboxylic a~id group of the cephalosporin molecule.
5uch~groups are readily removable by conventional tech-n1ques, and include, ~or example, C~-C6 tert-alkyl, C5-C6 tert-alkenyl, C5-C6 tert-alkynyl, benzyl, diphenyl-methyl, ~-nitrobenzyl, ~-methoxybenzyl, 2,2,2-trichloro-ethyl, phenacyl, trimethyls1lyl, Cl-C5 alkanoyloxymethyl, phthalidyl, and othe~ like cleavable moieties.

X-4273A ~5_ , .

læ~

Examples of C4-C6 tert-alkyl groups include, for example, t-butyl, t-amyl, and t-hexyl. Examples of C5-C6 tert-alkenyl groups are t-pentenyl and t-hexenyl. Examples of C5~C6 tert-alkynyl groups are t-pentynyl and t-hexynyl.
When R2 is a readily removable ester forming group, it is preferred that it be t~butyl, diphenylmethyl, benzyl, p-nitrobenzyl, ~-methoxybenzyl, 2,2,2-trichloroethyl, tri~
methylsilyl, acetoxymethyl, pivaloyloxymethyl, or phthalidyl. Most preferably, when R2 is a readily re-movable ester forming group, it is ~-nitrobenzyl, acetoxy-methyl, or pivaloyloxymethyl.
Most preferahly, however, R2 is hydrogen or an alkali metal cation. These R2 substituents define those compounds of formula I which are most active antibiotical-ly . .':
Cleavage of the ester moiety to the free 4-carboxyl~function is desirable to produce a cephalosporin in which R2 is hydrogen or an alkali metal cation. Cleavage is accomplished by conventional treatment. This includes, for example, treatment of the ester with an acid such as trifluoroacetic acid, or hydrochloric acid, or with zinc and acid, such as formic acid, acetic acid, or hydro-`~ chloric acid. Cleavage likewise can be accomplished by hydrogenating the ester in the presence of palladiumJ
rhodium, or a compound thereof, in suspension or on a carrier such as barium sulfate, carbon, or alumina.
In the foregoing definition of the group R', theterm "Cl-C6 alkyl" refers to straight and branched chain alkyl hydrocarbon groups such as methyl, ethyl, _-propyl, isopropyl, n-butyl, sec butyl, _-amyl, isoamyl, n-hexyl, and 2,3-dimethylbutyl. The term "Cl-C3 haloalkyl" refers to such groups as chloromethyl, bromornethyl, 2-iodoethyl, 2-chloropropy], and 3-bromopropyl. The term "Cl-C3 cyano-alkyl" refers to such groups as cyanomethyl, 2 cyanoethyl, 3~cyanopropyl, and 2-cyanopropyl. The term "Cl~C3 azido-alkyl" refers to such groups as azidomethyl, 2~azidoethyl, 3-azidopropyl, and 2-azidopropyl. The term "Cl-C3 hydroxy-alkyl" refers to such groups as hydroxymethyl, 2-hydroxy-ethyl, 3-hydroxypropyl, and 2-hydxoxypropyl.
In defining the 4-substituted-amino-4-carboxy-butyl ester group, the term A' includes the groups "C2-C4 alkanoyl", "C2-C4 haloal~anoyl", and "haloben~oyl". The term 'lC2-C4 alkanoyl" refers to acetyl, propionyl, or butyryl. The term "C2-C4 haloalkanoyl" refers to chloro-acetyl, bromoacetyl, 2-chloropropionyl, or 3-bromobutyryl.
The term "halobenzoyl" refers to chloro and bromo substi-tuted benzoyl groups such as 4-chlorobenzoyl, 4-bromo-benzoyl, and 2,4-dichlorobenzoyl.
As used herein, the texm "halogen" and the term "halo" each refers to fluoro, chloro, bromo, or iodo. The term "Cl-C4 lower alkyl" refers to the straight and branched chain lower alkyl hydrocarbon groups such as methyl, ethyl, _-propyl, isopropyl, _-butyl, and t-butyl. The term "Cl-C4 lower alkoxy" includes methoxy, ethoxy, isopropoxy, -~
or n-butoxy.
The followiny are illustxative of the group O
ll R'-C-NH- in Formula I above: formamido, acetamido, pro-pionamido, butyramido, a-methylpropionamido, valeramido, a methylbutyramido, trimethylacetamido, caproamido, heptyl-amido, chloroacetamido, bromoacetamido, fluoroacetamido, iodoacetamido, ~-chloropropionamido, ~-bromopropionamido, B-chlorobutyramido, ~-~luorobutyramido, cyanoacetamido, a-cyanopropionamido, ~-cyanopropionamido, ~-cyanobutyramido, azidoacetamido, ~-azidopropionamido, ~-azidobutyramido, 3-azidobutyramido, hydroxyacetamido, a-hydroxypropionamido, ~-hydroxybutyramido, _-nitrobenzyloxycarbamido, 5-amino-5-carboxyvaleramido, 5-(diphenylmethoxycarbonyl)-5-(acetamido)-valeramido, and 5~ nitrobenzyloxycarbonyl)-5-(2,4-di-chlorobenzamido)valeramido.
The following are illustrative of the groups .~ O
: R'-C-NH- in the above definition in which R' is ~; , a ~ - o `~ a and in whiah m is O: phenylacetamido, 4-methylphenylacet-amido, 3-ethylphenylacetamido, 4-isopropylphenylacetamido, 2-methylphenylacetamido, 4-chlorophenylacetamido, 4-nitro-phenylacetamido, 4-bromophenylacetamido, 2,4-dichloro-phenylacetamido, 3-bromophenylacetamido, 4-fluorophenyl~
acetamido, 2-fluorophenylacetamido, 3,4-dihydroxyphenyl-acetamido, 4-hydroxyphenylacetamido, 3 hydroxyphenylace~-amido, 2,6-dimethoxyphenylacetamido, 3-methoxyphenyl-acetamido, 4-isopropoxyphenylacetamido, 3-ethoxyphenyl-acetamido, 4-methoxyphenylacetamido, 3,~-dimethoxyphenyl-acetamido, 4-t-butoxyphenylacetamido, 2-aminomethylphenyl-acetamido, 4-aminomethylphenylacetamidol 3-_-butoxyphenyl-: 30 acetamidG, 3-chloro-4-methylphenylacetamido, and 3-nitro-phenylacetamido. When, in the above ~ormula, m = 1 and Z represents -0-, illustrative groups include the fol-lowin~: phenoxyacetamido, 4-methylphenoxyacetamido, 3-ethylphenoxyacetamido, 4-isopropylphenoxyacetamido, 2-methylphenoxyacetamido, 4-chlorophenoxyacetamido,

4-nitrophenoxyacetamido, 4-bromophenoxyacetamido, 2,4-di-chlorophenoxyacetamido, 3-bromophenoxyacetamido, 4-fluorophenoxyacetamido, 2-fluorophenoxyacetamido, 3,4-di-hydroxyphenoxyacetamido, 4-hydroxyphenoxyacetamido, 3-hy-droxyphenoxyacetamido, 2,6-dimethoxyphenoxyacetamido, 3-ethoxyphenoxyacetamido, 4-methoxyphenoxyacetamido, 3,4-dimethoxyphenoxyacetamido, 4-t-butoxyphenoxyacetamido, 2-_-butoxyphenoxyacetamido, 3-chloro 4-methylphenoxyacet-amido, 3-nitrophenoxyacetamido, 3-hydroxy-4-methylphenoxy-acetamido, 2-chlorophenoxyacetamidol 3-hydroxy-4-methyl-phenoxyacetamido, 2-chlorophenoxyacetamido, 4-isopropoxy-phenoxyacetamido, 2-aminomethylphenoxyacetamido, and 4-aminomethylphenoxyacetamido. When, in the foregoing formula, m = 1 and Z represents -S-, illustrative groups in-clude the ~ollowing: phenylmercaptoacetamido, 4-methyl-phenylmercaptoacetamido, 3-ethylphenylmercaptoacetamido, 4-isopropylphenylmercaptoacetamido, 2-methylphenylmer-captoacetamido, 4-chlorophenylmercaptoacetamido, 4-nitro-phenylmercaptoacetamido, 4-bromophenylmercaptoacetamido, 2,4-dichlorophenylmercaptoacetamido, 3-bromophenylmer-captoacetamido, 4-fluorophenylmercaptoacetamido, 2-~luoro~
: ~ phenylmercaptoacetamido, 3,4-dihydroxyphenylmercaptoacet-amido, 4-hydroxyphenylmercaptoacetamido, 3-hydroxyphenyl-mercaptoacetamido, 2,6-dimethoxyphenylmercaptoacetamido, 3-ethoxyphenylmercaptoacetamido, 4-methoxyphenylmercapto-: X-4273A ~9- :

:......... . . . ~ . . , ~ , , ' ', . ' acetamido, 3,4-dimethylphenylmercaptoacetamido, 4-t= butoxy-phenylmercaptoacetamido, 3-n-butoxyphenylmercaptoacet-.. amido, 3-chloro-4-methylphenylmercaptoacetamido, 3-nitro-phenylmercaptoa`cetamido, 3,4-dimethylphenylmercaptoacet-amido, 3,4-dichlorophenylmercaptoacetamido, 2,5-dichloro-phenylmercaptoacetamido, 3-fluoro-4-chlorophenylmercapto-acetamido, 3-chloro~4-fluorophenylmercaptoacetamido, . 2,6-difluorophenylmercaptoacetamido, 3-fluorophenylmercap-toacetamido, 2-aminomethylphenylmercaptoacetamido, and 4-aminomethylphenylmercaptoacetamido.
When R' represents a group of the formula P CH-O Q
illustrative groups having the overall formula R'-C-NH-include the mandelamido group of the formu~a ~ .
a ~
CH-C-NH
a ' ~--~ OH
~' ~
the o-formyl and O-acetyl derivatives thereof represented . 20~ by the general formula O
a~=-\ 11 .

" ~ ~ a ~--o~ I
T
~ in which T is hydrogen or methyl; the a~carboxyphenyl-acetamido group represented by the formula .~ .
a ~e a '~--~ COOH

- - X-4273A -10- :.
, : :

j ~ -- ~

the a-sulfophenylacetamido group represented by the formula ~ CH-G-NH-the a-aminophenylacetamido group represented by the formula a a '~*--o~ I

or the a-(substituted-amino~phenylacetamido group represented by the formula o~ ~CI l~ C-~NH
NHY

.
in which Y is benzyloxycarbonyl, t-butyloxycarbonyl, O O R'l'O
.. .. . ...................................... .
; -C-NH-C-NH2, or ---C - N---C - V in which V is, for example, ~ -: NH

phenyl, halophenyl, furyl, monomethylamino, dimethylamlno, monoethylamino, diethylamino, methylethylamino, n-propyl-amino, di-n-propylamino, di-isopropylamino, phenylamino, or diphenylamlno. Moreover, :in any of the above, R''' can be hydrogen or Cl~C3 alkyl, specifically methyl, ethyl, n-propyl or isopropyl. R''' and V can, together with the group to which they are a-ttached, form a heterocycle .: :

:: . ' such that Y has the structure O OIl 11 -- C -- N ~ C ~ N R' ' ' ' (CH ) in which n is 2 or 3 and R'''' is hydrogen, methanesulfonyl, or Cl-C3 alkyl. Also included are those a-substituted 2-thienylacetamido, 3-thienylacetamido, and l-tetrazyl-acetamido groups in which, in the above formulae, the phenylgroup is replaced by a 2-thienyl, a 3-thienyl, or a l-tetrazyl riny.
Illustrative of the foregoing acetamido groups are 4-methylmandelamido, 4-hydroxymandelamido, 3-hydroxy-mandelamido, 4-methoxymandelamido, 3-bromomandelamido, mandelamido, 4-chloromandelamido, 3-methyl-4-fluoroman-delamldo, 2-fluoromandelamido, 4-fluoromandelamido, 4-isopropylmandelamido, 3,4-dimethyl-O-formylmandelamido, 4-chloro-O-formylmandelamido, 3-i~opropoxy-O-formylmandel-amido, 3-bromo-O-~ormylmandelamido, O-formylmandelamido, 3,4-dimethoxy-O-formylmandelamido, O-acetylmandelamido, : 4-hydroxy-O-acetylmandelamido, a-hydroxy-2-thienylacetamido, a-hydroxy-3-thlenylacetamido, a formyloxy-2-thienylacetamido, . .
a-acetoxy-2-thienylacetamido, a-formyloxy-3-thienylacet-~amido, a-acetoxy-3-thienylacetamido, a-hydroxy-l-tetra- -:

zylacetamido, a-formyloxy-l-tetrazylacetamido, a-acetoxy-l-tetrazylacetamido, a-carboxyphenylacetamido, a-carboxy-4-methylphenylacetamido, a-carboxy-4-hydroxyphenylacetamido, a-carboxy-3-hydrox~yphenylacetamido, a-carboxy-4--methoxy-ph nylacetamido, a-carboxy-3-bromophenylacetamido, a-car-, boxy-4-chlorophenylacetamido, a-carboxy-3-methyl 4-fluorophenylacetamido, a-carboxy-2-~luorophenylacetamido, a-carboxy-4-~luorophenylacetamido, a-carboxy-4-isopropyl-phenylacetamido, a-carboxy-3,~-dimethylphenylacetamido, a-carboxy-3-isopropoxyphenylacetamido, a-carboxy-3,4-- dimethoxyphenylacetamido, a-carboxy-2-thienylacetamido, a-carboxy-3-thienylacetamido, a-carboxy-1-tetrazylacetamido, a-sulfophenylacetamido, a-sulfo-4-methylphenylacetamido, a-sulfo-4-hydroxyphenylacetamido, a-sulfo-3-hydroxyphenyl-acetamido, a-sulfo-4-methoxyphenylacetamido, a-sulfo-3-bromophenylacetamido, -sul~o-4-chlorophenylacetamido, a-sulfo-3-methyl-4-fluorophenylacetamido, a-sulfo-2-fluoro-phenylacetamido, a-sul~o-4-fluorophenylacetamido, a-sulfo-4-isopropylphenylacetamido, a-sul~o-3,4-dimethyl-phenylace-tamido, a-sulfo-3-isopropoxyphenylacetamido, a-sul fo-3,~-dimethoxyphenylac~tamido, a-sulfo- 2-thienyl-acetamido, a-sulfo-3-thienylacetamido, a-sulfo-l-te~ra-zylacetamido, a-aminophenylacetamido, a-amino-4~methyl-phenylacetamido, a-amino-4-hydroxyphenylacetamido, a-amino-3-hydroxyphenylacetamido, a-amino-4-methoxyphenylacetamido, a-amino-3-bromophenylacetamido, a-amino-4-chlorophenyl-acetamido, a-amino-3-chloro-4-hydroxyphenylacetamido, a-amino~-fluorophenylacetamido, a-amino-4-fluorophenyl-acetamldo, a-amino-4-isopropylphenylacetamido, a-amino-3,4-dimethylphenylacetamido, a-amino-3-isopropoxyphenyl-acetamido, a-amino-3,4-dimethoxyphenylacetamido, a-amino-2-~ thienylacetamido, a-amino-3-thienylacetamido, a-amino-l-; : tetrazylacetamido, a-(3-guanyl-1-ureido)-phenylacetamido, ~ a-(3-methylaminocarbonyl~l-ureido)phenylacetamido, : X-~273A -13-.

a-(3-dimethylaminocarbonyl-3-methyl-1-ureido)phenylacet-amido, a-[N-(imidazolidine-2-one-1-ylcarbonyl)amino~phenyl-acetamido, a-[N (3-methylimidazolidine-2-one-1-ylcarbonyl)~
amino]phenylacetamido; a-[N-(3-methanesulfonylimidazolidine-2-one-1-ylcarbonyl)aminoJphenylacetamido; a-[N-(hexa-hydropyrimidine-2-one-1-ylcarbonyl)amino]phenylacetamido;
a-[N-(3-methylhexahydropyrimidine-2-one-1-ylcarbonyl)-amino]phenylacetamido; a-[N-(3-methanesulfonylhexahydro-pyrimidine-2-one-1-ylcarbonyl)amino]phenylacetamido;
a-(3-phenylacetaminocarbonyl-3-propyl-1-ureido)phenylace-tamido, and a-(3-di-_-propylaminocarbonyl-1-ureido)phenyl-acetamido.

O
"
Illustrative of the group R'-C-NH- in the above deflnition in which R' is R''-CH2- are the following:
2-thienylacetamido, 3-thienylacetamido, 2-furylacetamido, oxazyl-2-acetamido, thiazyl-2~acetamido, tetrazyl-l-acet~
amido, benzotriazolylacetamido, 1,3,4-thiadiazolyl-2 -thioacetamido, 1,2,5-thiadiazolyl-3-thioacetamido, 1,3,4-oxadiazolyl-2-thioaGetamido, pyridylthioacetamido, 4-(cyano)-1,2,3-triazolyl-1-acetamldo, and 3-(cyano)-1,2,4-triazolyl-l-acetamido.
preferred group of cephalosporins of formula I
are ropresented by th- following formula III

. .

~-4273A -14-~ ~ ( ) rn Z I t I ~` ~OH
a ' ~--o ~ --N\~ ~CH _S~

III OOR

in which Rl, R2, a, a', Z and m have the same meanings ~s : defined above. Illustrative of these preferred compounds presented in the form of their free acid are the following:
7-phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-phenoxyacetamido-3-(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl.-3-cephem-4-carboxylic acid, ;~ 7-(4-hydroxyphenylacetamido)-3-(5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, ~.
7-(4-chlorophenoxyacetamido)-3-(4-_-propyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-car-- boxylic acid, 7-(4-methoxyphenoxyacetamido)-3-(4-methyl-: 5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl- ....
3-cephem-4-carboxylic acid, 7-(2,S-dichlorophenylthioacet-n; amido)-3-(4-isopropyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-tria-: zin-3-ylthio~methyl-3-cephem-4-carboxylic acid, and 7-phenyl-' thioacetamido-3-~4-n-butyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4 triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
: ` :
I : Another preferred group of cephalosporins of formula I are those represented by the followi~y : formula IV

~-~ X-4273A -15-::: :

:~ .

R c~l2 ~N I--T/ `I ~ I
~C~I-S--~

~OOR

in which R' ' represents 2-thienyl, 3-thieny]., 2-furyl, or l-tetrazyl, and Rl and R2 have the same meaning as defined above. Illustrative of the foregoing compounds represented by the formula IV and presented as their free acid are the following: 7-(2-thienylacetamido)-3-(4-methyl-

5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-y].thio)methyl-3-cephem-4~carboxylic acid, 7-(2-furylacetamido)-3-(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthiomethyl)-3-céphem-4-carboxyl1c acid, 7-(3-thienylacetamido)-3-(4-sec-buty1-5-oxo-6--~ ~ hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthio)methyl-3-cephem-~i~ 4-carboxyl LC acid, 7-(l-tetrazylacetamido)-3-(4-methyl-5-oxo 6-~20 hydroxy-4,5-dihydro-l,2,4-triazin~3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-~l-tetrazylacetamido)-3-(4-ethyl-5-oxo-~-.
- :
hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, and ` `~ 7-(2-thienylacetamido)-3~(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-l,2,4-triazin-3-ylthio)methyl-3-cephem-4-car-boxyl1c acid.
A further preferred group of cephalosporins of formula I are those represented by the following .~

formula V

- O H
lll / ~ ~ /OH
Q ~ C~2-S~ o OOR
~ R
V :
wherein P represents l-tetrazyl, phenyl, or a substituted phenyl group as defined herein and Q is hydroxy, formyloxy, acetoxy, carboxy, or amino. Illustrative of the foregoing compounds repxesented by formula V and presented as their free acid are the following:
7-mandelamido-3-(4-methyI-5-oxo-6-hydroxy-4,5- ; .
dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-car-boxylic acid, 7-(4-chloromandelamido)-3-(5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3 ylthio)methyl-3-cephem-4-car-boxylic acid, ., .
7-(4-hydroxymandelamido)-3-(4-ethyl-5-oxo-6-20 hydroxy-4~5-dihydro-1~2~4-triazin-3-ylthio)methyl-3~cephem-4-carboxylic acid, 7-(4 methoxymandelamido)-3-(4 n-propyl-5-oxo-6-hydroxy-4,5~dihydro-1,2,4-triazinY3-ylthio~methyl-3-cephem-4-carboxylic acid, 7- [a- (hydroxy)-1-tetrazylacetamido]-3-(4-methyl-5-oxo-6 hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-(a-formyloxyphenylacetamido)-3-(4-ethyl-5-oxo-., -

6-hydroxy-4,5Ydihydro l,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, X-4273~ -17-.; . . . .

2,.~

7-(a-acetoxyphenylacetamido)-3-(4-isobutyl-5-oxo-6-hydroxy-4,S-dihydro-1,2,4~triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-(a-carboxyphenylac~tamido)-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1~2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-[a-~carboxy)-4-hydroxyphenylacetamido]-3-(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)-methyl-3-cephem-4-carboxylic acid, 7-[-(carboxy) 4-chlorophenylacetamido~-3 (4-n-propyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)-methyl-3-cephem-4-carboxylic acid, 7-[a-(carboxy)-1-tetrazylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-(a-aminophenylacetamido)-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-~: cephem-4-carboxylic acid, ; 7-(a-aminophenylacetamido)-3-(4-ethyl-5-oxo-6-20 hydroxy-4,5-dlhydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-[a-(amino)-1-tetrazylacetamido]-3-(4-methyl-S-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-:~ ~ cephem-4-carboxylic acid, ; 7-[~-(amino)-4-hydroxyphenylacetamido]-3-(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin~3-ylthio)-methyl-3-cephem~4-carboxylic acid, and 7-[a-(amino)-4-chlorophenylacetamido]-3-(4-ethyl-5~oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4~carboxylic acid.

.

Another preferred group of cephalosporins offormula 1 are those represented by the following formula VI
2N I _ t ~ /OH
0~ / :
OOR
2 R, VI
These structures are highly useful as intermediate~ in preparing other compounds of this invention. They are readily convertible to other compounds by routine acylation procedures by which the free amino substituent in the 7-position is acylated with any of the other herein defined acyl substituents. Illustrative o~ these compounds represented by formula VI and presented as their free acid are the following:
7-amino-3-(5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, ~ 7-amino-3-(4-methyl~5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-amino-3-(4-ethyl-5-oxo-6-hydroxy-4,~5-dihydro-1,2,4-triazin-3-ylthLo)methyl-3-cephem-4-carboxyli~o~ acid, 7-amino-3,~4-n-propyl-5-oxo-6-hydroxy~4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, , 7-amino-3-(4-i~opropyl-5-oxo-6 hydroxy-4,5-dihydro-1,2,4-tria~in-3-ylthio)methyl-3-cephem-4-carboxylic acid, 7-amino-3~(4-n-butyl-5-oxo-6-hydroxy-4,5-di-hydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, X-4273A ~ -19-.. . .

:: .', , .. . : ... , .. i :.,, ",, ,,,",. . :: .: , ,. ", ". , .
. . ... .. . . . . . .. . . . . .

7-amino-3-(4-sec-butyl-S-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-car-boxylic acid, and 7-amino-3-(4-isobutyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxy-lic acid.
The compounds of formula I can be prepared by well recognized cephalosporin prPparative techniques.
They are readily available by displacement of an acetoxy function present in the 3-position o~ a 3-acetoxymethyl cephalosporin compound. Thus, any of the cephalosporins of formula I can be prepared from the corresponding 3-acetoxy-methyl cephalosporins by the well recognized displacement reactlon. Thus, a typical sequence for preparing any of the compounds of formula I from readily available 7-amino-cephalosporanic acid (7-ACA) can be as follows:

.

~, :

. -~ X-4273A -20-.~

.. . . . .
..
, . . . .

3~

~N~I-t I
- N \ ~ ~CHzO~G-CH

OOH

displacernent ~; !

H~N~ c~ s~
COOH R
pro~ection of C carboxyl , ~ 4 (if desired) functionalization (if desired) . : ' .
S ~ ~ /OH
c~lzs~ o ~, ~ ~ ~OOR R1 , :
: 20 In accordance with the above scheme,~the acetoxy function of 7-ACA is displac~d with the deined tria~inyl-thio derivative. The resulting product is the corresponding ~1 7-amino cephalosporin, a compound o formula I. This .
compound can be converted by means of well recognized acyla- :
tion techniques to any of the defined 7-acylamido cephalos-porins o ormula I~
The above sequence can be altered such that the 7~ACA is first acylated in the 7-position ko p.roduce the .
corresponding 7-acylamidocephalosporanic acid or e~ter 0 thereof~ The resulting product, if an ester, is then . . :
~ X-4273A ~ -21-: ' . . .
.

~ ` ~

cleaved to xemove the C~ ester prot~cting group, and the re-sulting acid i5 then subjected to the displacement reaction to produce the compound of formula I. An~ of the~e procedure~
used to accomplish these conversions are well recognized in the art.
The particular compound which i~ employed to displace the acetoxy function in the 3-position is a 3-mercapto-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine un-substituted in the 4-position or suitably ~ubstituted in the 4-position with a lower alkyl group as herein defined.
These 3-mercapto-1,2,4-triazines are prepared by ~ m~thod described in Pesson et al., Bulletin de la Societe Chemique de France, (1970), pages 1590-1599. In accordance with the method described in this publication, the 1,2,4-tria-zine is obtained by reaction of a thiosemicarbazide with diethyl oxalate in the presence of sodium ethoxide. The ~equence is as follows:
O O
R -NH~ NH- NH + (2) H

H
~f ~ HS~ I
S~ \N~/ \N

: R1 R, In the oregoing ~e~uence, Rl i~ hydrogsn or lo~er alkyl as herein dsfined. A~ noted, the product exist~ in tauto-meric forms. The tautomer aspect is detalled in Pes60n ' -: . . : '' ' ' ~ f~

et al., Bulletin de la Societe Chimi~ue de France (1970) - pagQs 1599-1606.
The aforementioned displacement reaction employed in preparing compounds of formula I compxises reacting the corresponding 3-acetoxymethyl compound preferably in a polar medium with the appropriate 3-mercapto-1,2,4 triazine derivative so as to displace the acetoxy group. The desired 3-substituted cephalosporin derivative is then recovered.
This reaction may conveniently be effected by maintaining the reactants in solution at a temperature such as, ~or example, from about 15C. to about 100C. and until the desired derivative is obtained in optimum yield. The reactants are advantageously employed in a ratio of about 1 molar equivalent of the 3-acetoxymethyl cephalosporin to from about one to about ten molar equivalents of the 3-mercapto-1,2,4-triazine. Preferably, the reaction is carried out using the molar equivalent amounts of these reactants or a moderate excess of the triazine compound.
The pH of the reaction solution is advantageously maintained at from about 5.0 to about 8Ø
The reaction appears to proce~d by a polar or ionic mechanism. It is preferred therefore to employ a polar medium for the reaction. This facilitates the ready ongoing of the desired reaction. Water may be employed as ~ the polar medium and, indeed, is preferred. Any other - typical and well recognized polar solvents can be employed as well. It is al50 possible to employ as supporting medium for the reaction an excess of the displacing 3-mercapto-1,2,4-triazine derivative.
` ' .

The resulting reaction product can be ~eparated from the reaction mixture by a variety of methods including crystallization, ionophoresis, paper chromatography, and chromatography on ion~exchange resins.
The,displacement reaction can be applied either to 7-amino- or to a 7-acylamido cephalosporanic acid. When 7-ACA is employed, the resulting product can then be acylated.
The acylation of the 7-amino group of cephalosporins is a well-known reaction and can be accomplished by reacting the cephalosporin with an acid halide or mixed anhydride repre-sentative of the desired acyl function. The particular method of acylation is not important to this invention.
The compounds of formula I are themselves antibiotically active and/or are intermediates to an~i-biotically active compounds. Those compounds which par-ticularly exhibit antibiotic activity are those,in which R2 is hydrogen or an alkali metal cation. Compounds in which R2 is other than hydrogen or an alkali metal cation are readily converted thereto by cleavage techniques hereinbefore ~0 described.
In the Table following, the minimum inhibitory concentrations (MIC) in micrograms per milliliter (mcg/ml) of compounds of formula I against a gram-negative organism, Esoherichia coli, and against a grarn-positive organism, penicillin-resi5tant Sta~ l occus aureus, are provided.
The MIC values were deterrnined by the Gradient Plate technique described in Bryson and Szybalski, Sclence, 116, ~5 (195~)-Table Antibiotic Activi~y ~N~~ OH
_N,f ~CH~S--~/ ~o OOH R

Minimum Inhibitory Conc. (mcg/ml~
R Rl E Coli S. aureus ClH.H- 3 15 H-CO- CH312.2 0.2 2,5-dichloro-phenylthio-acetyl CH320.5 0.6 2,5-dichloro-phenylthio-acetyl C2H5 22O5 0.7 a-hydroxyphenyl-acetyl CH34.0 0~7 a-hydroxyphenyl-acetyl (Na salt) CH3 4.5 0.9 a-formyloxyphenyl-acetyl CH33.5 4.0 a-(t-butyloxy-carbonylamino)-phenylacetyl CH3130 0.5 a-aminophenyl-acetyl CH35.3 2.0 2-thienyIacetyl CH31.5 0.3 2-thienylacetyI C2H53~0 0 5 4-aminomethyl-phenylacetyl CH33.0 0.5 a-[3-(4-chloro-benzoyl)-3-methyl-1-ureido]-phenylacetyl CH34.5 4.0 .

. .

%~

Table Antibiotic Activity cont'd.
Minimum Inhibitory Conc. (mcg/ml) R Rl_E. Coli S. aureus a-(3-furoyl-1-ureido)-phenylacetyl CH38.3 7.0 a-[3-(2-chloro-benzoyll-3~
methyl-l-ureido]phenyl-acetyl CH35.7 5.5 lH-tQtrazole-acetyl CH30.8 0.5 ~ a-amino-4-hydroxy-: phenylacetyl CH33.0 4.0 a-(3-methylamino-carbonyl-3-methyl-l~ureido~-phenylacetyl CH315.5 3.0 a-(imidazolidine-2-one-1-yl-car-bonylamino)-: phenylacetyl CH319.0 2.0 : a-(3-methanesul-:- fonylimidazol-idine-2-one-1 ylcarbonyl-amino)phenyl acetyl CH39.5 5.0 a-(amino)thien-: 2-ylacetyl CH36.5 6.0 a-(3-methyl-3-methylamino-` ~ carbonyl-l-ureido)thien-2-ylacetyl CH314.2 1.0 .~ , Table Antibiotic Ac.tivity cont'd.
Minimum Inhibitory Conc. (mc R R E. Coli S. aureus .
a-(3-methanesul-fonylimidazol-idine-2-one-l- .
ylcarbonylamino)-thien-2-yl-acetyl . CH3 4.7 l.0 a-[3-(2-chloroben-zoyl)-3-methyl-: l~ureido3-4-hydroxyphenyl- ~ -acetyl CH3 ll.l 0.7 u-(3-methanesul-fonylimida~oli-dine-2-one-l-yl-carbonylamino)-4-hydroxyphenyl-: acetyl CH3 0.6, 2.5 a-(3:-methylamino-carbonyl-3-methyl-ureido)-4-hydroxyphenyl- ~
-- acetyl CH3 4.4 2~2 .
, ~-(imidazolidine 2-one-l-ylcar-bonylamino)-4-hydroxyphenyl-acetyl C~13 5.6 2.l ~.
- ' .
.
; ' .
:

~ .

The preparation of the compounds of this in-vention is illustrated by the following examples~

Preparation of 3-Mercapto-4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine To a 22 liter (l.) flask containing 12.5 1. of anhydrous ethanol maintained in a nitrogen atmosphere were slowly added 230 g. of sodium. The mixture was maintained at room temperature overnight, a slow, but constant, stream of nitrogen being admitted to the flask.

The mixture then was heated to 50C., and 1050 g. (lO mole) of 4-methylthiosemicarbazide were added. The temperature of the mixture fell to 40C. To the mixture wexe then added 1530 g. of diethyl oxalate through a funnel. Addition was at a rate sufficient for the reaction mixture to attain a temperature of rom about 60C. to about 65C. The temp-erature of the resulting mixture rose to 65C. The mix-ture was refluxed with stixring for four hours du~ing which time a precipitate formedO The precipitate changed in appearance during the period of reflux. Upon discontinuing heating and stirring, the white solid gradually settled to the bottom of the flask leaving a clear light yellow supernatant liquid. The mixture was allow d to stand overnight. The bulk of the clear supernatant wa~ removed by suction, and the white crystalline product in the form of its sodium salt was removed from the residual mixture by filtration and washed with dry ethanol.
The product was dissolved in water, and the pH
of the solution was adju~ted to pH 5.5. A small amount of material was iltered of~ and discarded. The pH o~ the ,, -: . . : , . . .

3~

filtrate then was adjusted to 1.5, cooled, and filtered to obtain 211.5 g. of product, m.p. 212-216C.
Example 1 To 36 ml. of water were added with stirring 1.44 g. of 3-mercapto-4-methyl-5 oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine and 8.5 ml. of 1 N sodium hydroxide.
The pH of the resulting mixture was 8.3, and the pH was lowered to 706 by addition of dilute acid. 7-a-(t-Butoxy-carbonylamino)phenylacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid (4.5 g., 9 mmole.) was added to th~ mixturewith stirring, and the pH of the resulting mixture adjusted to 6.9 as the cephem compound slowly went into solution.
The mixture was stirred at 55C. After 1.75 hours, the pH
of the mixture was 6.2, and the pH was adjusted upward to :~ 7.3 by addition of dilute sodium hydroxide. ~fter 17.S .
hours, the pH of the mixture was 6.1 and was adjusted to 6.95 by addition of dilute sodium hydroxide. The mixture . was heated a total of 21 hours. The mixture was co~led ~ in ice, filtered, and the filtrat~ was adjusted to p~ 1.5.
A 501id precipitated. The solid was filtered, washed with dilute acid (~H 1.5), and air dried to give a light buff colored powder (3.60 g.). The powder was dissolved in 15 ml.
o~ a 2:1 mixture of water and methanol at pH 6.5. The mix-ture was rotary evaporated to a small volume (about 3 ml.) :~ with isopropyl alcohol being added to avoid frothing toward the end of the rotary evaporation. The residual solution was placed on a "Sephadex G-10*" (54 g.) column and eluted with water, the first fraction being 25 ml. and all ~lcceeding fractions about 15 ml. Fractions 5-8 were com~.ined, and the pH of the mixture was adjusted to 1~5 by addition of dilute X~4273A -29~

*Trademark for a particular form of insoluble adsorbent material : - composed of highly cross-linked dextran to which are attached by : . ether 1inkages functional ionic groups.

; ~ !

.

, ~f~g hydrochloric acid. The precipitated product was collected by filtration, washed with dilute acid ~pH 1.5), and air dried to give 2.19 g. of 7-a-(t-butoxycarbonylamino)phenyl-acetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid as a buff powder.
NMR DMSO d6: 9.18 (d, lH, 7~-NH); 7.3 ~bm, 5H, C6H5); 5.71 (q, lH, 7a-H); 4.05 (m, 2H, 3-CH2); 3O55 (m, 2H, 2-CH2); 3.27 (s, 3H, triazine N-CH3); and 1.36 ppm (b, 9H, t-C~Hg).
Example 2 The pxoduct from Example 1 (404 mg. ? was stirred in 4.5 ml. of acetonitrile; however, complete solution was not achieved. ~-Toluenesulfonic acid (300 mg.) was added, and within 5 minutes solutio~ was complete, and a solid began to separate. After 1.5 hours, water (0.5 ml.) was added, and the pH of the mixture was adjusted to 5.0 by addition of saturated aqueous ammonium carbonate solution.
A major portion of the acetonitrile was removed by rotary evaporation, and isopropyl alcohol was added to the stirred residue until some precipitation became evident. The pre-cipitate was removed by filtration through filter aid. Some tendency to further precipitation was noted upon passage of the solution through the filter aid. The filtrate was stirred and diluted with isopropyl alcohol to give further precipitate which was collected by filtration, washed with a mixture of isopropyl alcohol and water, and air dried to give a buff colored powder (0.13 g.). This powder was mixed with similar material from a larger scale (2.0 g.) reaction, the combined material weighing 1.23 g. This ., -, ' ., ' :

material was stirred in the cold in a mixture of 30 ml.
of water and 30 ml. of methanol. The pH of the mixture was slowly adjusted to 7.0 by addition of lN aqueous sodium hydroxide. A small amount of insoluble material was removed by filtration. The filtrate was concentrated to about 5 ml. by rotaxy evaporation, isopropyl alcohol being used in t~e later stages to prevent frothing. The residual solution was placed on a column of Sephadex G-10 (28g., 1.5 cm.) in water, and 25 ml. fractions were col-' ~ 10 lected, water being used as eluant. Fractions 3-9 were combined. The pH of the combined fractions was adjusted to pH 3.5, and the precipitated product was collected by filtration. The solid was washed with water which had been acidified to pH 3.6 by addition of HCl~ and air-dried to give 620 mg. of 7-(a-amino)phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4 t 5-dihydro-1,2,4-triazin-3-ylthio)methyl-3 cephem-4-carboxylic acid as a light brown powder.
NMR, TFA d1: 7.58 (b, 5H, C6H5); 5.96 (d, lH, 7a-H); 5.57 (s, lH, C6H5-CH); 5.21 (d, lH, 6~-H); 4.43 (q, 2H, 3-CH2); 3.77 (b, 2H, 2-CH2); and 3.58 ppm (s, 3H, triazine N~CH3).
; Example 3 To 45 ml. of water were added 2.39 g. of 3-mercapto-4-methyl-S-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine (15 mmole.). The mixture, havirlg a pH of 2.8, was stirred, and the pH was adju~ted to 7.25 by addition of 13.4 ml. of lN sodium hydroxide. 7-Mandelamido-3-acetoxymethyl-3~cephem-4-carboxylic acid (6.79 g., 15 mmole.) ~; was added, and the pH of the resulting mixture was 3.2. The pH was adjusted to 7.2 by careful addition of 14.8 ml. of lN sodium hydroxide. The resulting mixture was heated at 55C. for 4.25 hours during which the pH of the mixture became 5.85. The pH was increased to 7.17 by addition of 0.7 ml. of lN sodium hydroxide. Heating was continued for 14.25 hours during which time the pH of the mixture became 5.99. The mixtur~ was then adjusted to pH 7.09 by addition of 1.45 ml. of lN sodium hydroxide. After heating for a total of 20 hours, the mixture was cooled in an ice bath, diluted to about 120 ml. by addition of water, and the pH
was adjusted to 1.5 by addition with rapid stirring of lN
hydrochloric acid. Th~ resulting precipitate was collected by filtration, washed with dilute hydrochloric acid (pH 1~5), and air dried to give an off-white powder (5.28 g.).
This material was stirred in water (40 ml.) during which time the pH of the mixture was adjusted to 6.7 by addition of lN sodium hydroxide. The resulting solution was concentrated by addition of isopropyl alcohol and rotary ~evaporation until the isopropyl alcohol was removed~ The final volume of the mixture was about 10 ml. The mixture i ~ ~ was placed on a column of Sephadex G-10 (110 g., 2.5 cm., in water). The column was eluted with water, and fractions of 15-16 ml. each were collected. Four fractions or combinations of fractions were prepared, specifically, fractions 5 6, fractions 7-8, fraction 9, and fractions 10-12. Each was stirred in the cold, and the pH of each was adjusted to l.S by addition of lN hydrochloric acid.
A precipitate formed in each and each was collected by fil-tration, washed with dilute acid (pH 1.5), and air dried to give off~white powders (fractions 5-6--1.26 g.), (fractions 7-8 -~ 0.84 g.), (fraction 9 -- 0.31 g.), and fractions X-4273A -32~

.. . .

3~

10-12 -- 0.41 g.). Thin-layer chromatography (TLC~ showed each product to be identical, and all were combined to give 2.82 g. of an off-white powder.
This material was dissolved in water, and the solution was acidified to pH 1.5. The precipitate was filtered and air dried to give an off-white powder. The powder was dissolved in 25 ml. of tetrahydrofuran, and isopropyl alcohol was added with stirring until a total volume of 200 ml. was obtained. The mixture was filtered.
To the filtrate were added 50 ml. of isopropyl alcohol. The mixture was stirred and filtered. The filtrate was evapo-rated to dryness, and the residue was dissolved in water at pH 7. The solution was acidified to pH 1.5, filtered and air dried to obtain 7-mandelamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-~dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid as the desired product.
NMR, DMSO d6: 8.69 (d, lH, 7~~NH); 7.42 (m, 5H, C6H5); 5.75 (q~ lH, 7a-H); 5.15 (s, lH, CHOH); 4.13 (m, 2H, 3-CH2); and 3.31 (s, 3H, triazine N-CH3).
Example 4 To 20 ml. of water were added 3.46 g~ of 7-formamido-3-acetoxymethyl-3-cephem-4-carboxylic acid (12.0 mmole) and 2.0 g. of 3-mercapto-4-methyl 5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine (12.55 mmole). The resulting mix~
ture was stirred, and lN sodium hydroxide was added gradually until the pH remained at a constant 7Ø The resulting mixture was then stirred at about 55C. for 26 hours.
The resulting solution was concentrated to 20 ml.
and acidified to pH 1.2 by addition with cooling o~ 3N
hydrochloric acid. The resulting precipitate was filtered .~ :

and immediately placed into a bell jar to dr~ under vacuwn.The dried ma-terial was ground in a mortar and pestle ~2.75 g.), and was triturated three times, each with 150 ml.
of boiling isopropyl alcohol. The isopropyl alcohol solution was evaporated to dryness and the residue was triturated twice with 30 ml. of ethyl acetate. The insoluble material was filtered, washed with ethyl acetate, and dried to give 1.56 g. of 7-formamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6-D2O: 8.24 (s, lHt OC-H); 5.79 (d, lH~ 7a-H~i 5.14 (d, lH, 6a-H); 3.73 (b, 2H, 2-CH2):
and 3.40 ppm (s, 3H, triazine N-CH3)o Example 5 The product from Example 4 (0.74 g.) was stirred in 12 ml. of dry methanol, and 1.5 ml. of concentrated hydrochloric acid were added during which time complete solution occurred. After a short period of time, a white solid began to precipitate. Stirring was continued for 1.7 hours, and the mixture became thick with a white precipitate. The precipitate was filtered and dri~d. The product (0.346 g.) was shown by TLC to be a highly pure sample of the hydrochloride salt of 7-amino-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-txiazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6: 5.19 (q, 2H, 7a-H and 6a-H); 4.19 (m, 2H, 3~CH2~; 3.77 (b, 2H, 2-CH2); and 3.31 ppm (s, 3H, triazine N-CH3).

' Example 6 To 36 ml. of water were added 1.44 g. (9 mmole) of 3-mercapto-4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine. The pH of the resulting mixture was 2.9 and was adjusted to 7.20 by addition of 8.7 ml. of lN sodium hydroxide. To the mixture were added 3.77 g. ~9 mmolej of the sodium salt of 7-(2-thienyl)acetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid. The pH of the resulting mixture was 6.8 and was adjusted to 7.12 by addition of 2 drops of lN sodium hydroxide. The mixture was stirred at 55C. for 4.25 hours during which time the pH changed to 6.03. The pH was raised to 7.10 by addition of 0.3 ml. of lN sodium hydroxide. The mixture was heated at 55C. for an additional 14.25 hours during which time the pH changed to 6.02. The pH then wa~ increased to 7.09 by addition of 0.8 ml. of lN
sodium hydroxide, and heating was continued for 1.5 hours (total time at 55C. being 20 hours). The resulting reaction -~mixture then was stirred in ice water, and the pH was lowered to 1.5 by addition of lN hydrochloric acid. A soli~
formed and was collected by filtxation, washed with dilute acid (pH 1.5), and air dried to give a buff colored powder ~3.51 g.).
The solid was stirred in 25 ml. of water, and 10 mI.
of methanol were slowly added. The pH of the resulting mix-ture was adjusted to 6.8 by addition portionwise oX lN sodium hydroxide with intervening soni.cation of the resulting mix-ture. The resulting solution was concentrated -to about 5 ml.
by rotary evaporation, thereby removiny the methanol from . :
the mixture, and the concentrate was placed on a column o~

; 30 Sephadex G-10 (70 g., 2 cm. column in water). The column X-4273A -35_ '~

3~

was eluted with water, 10-12 ml. fractions being collected.
Fractions 3-5, fractions 6-7, and fractions 8-10 were com-~ined, and the resulting three portions were stirr2a in the cold and the pH of each was adjusted to 1.5 by addition of lN hydrochloric acid. The resulti~g three precipitates were collected by filtration, washed with dilute hydrochloric acid (pH 1.5), and air dried to give off-white powders, 2.13 g., 0.69 g., and 0.17 g., respectively. TLC of each of the products indicated that they were virtually identical. The products thereore were combined and dis-solved by stirring and boiling in a large volume (about 1200 ml.) of acetone, and the resulting solution was allowed to evaporate to give 1.943 g. of crystalline 7-(2-thienyl~-acetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6: 9.1 (d, lH, 7~-NH); 7.36 and 6.95 (m, 3H, thienyl); 5.70 (d, lH, 7a-Hj; 5.12 (d, lH, 6a-H); 4.14 (m, 2H, 3-CH2); 3.79 (bs, 2H, 7-CH2); 3.68 (rn, 2H, 2-CH2); and 3.33 ppm (s, 3H, triazine N-CH3).
Example 7 To 20 ml. of water was added 0.96 g. of 3-mer-capto-4-ethyl-5-oxo-6-hydroxy 4,5-dihydro-1,2,4-triazine.
The pH of the resulting mixture was 2.3 and was ad~usted to 6.5 by addition oE 5 ml. of lN sodium hydroxide. To the mixture were added 2.09 y. (5 mmole) o the sodium salt of 7-(2-thienyl)acetamido-3-acetoxymethyl-3-c0phem-4-carboxylic acid. The pH of the resulting mixture was 5.7 and was adjusted to 7.2 by addition of lN sodium hydroxide. The resulting mixture w~s stirred at 60C. for a total of 18 .

hours, the final pH being 5.9. The mixture was cooled in ice and stirred, diluted to about 10 ml., and the pH of the mixkure was adjusted to 1.5 by addition of lN hydro-chloric acid. The resulting solid was collected by filtra-tion, washed with dilute HCl (pH 1.5), and air dried to give an off-white powder (2.01 g.). The material was dissolved at pH 6.8 in 20 ml. of a 1:1 mixture of water and methanol.
The solution was evaporated on a rotary evaporator to a small volume (about 3 ml.), and the residual solution was B 10 placed on a Sephadex G-10 column (40 g., 1.6 cm. column).
The column was eluted with water~ the first fraction being -~
about 18 ml. and subsequent fractions about 5 ml. Fractions 4-11 were combined, and the pH was lowered to 1.5 by additlon of dilute hydrochloric acid to give 1.171 g. of solid. The solid was dissolved in about 30 ml. of a 3:1 mixture of acetone and methanol. Air was blown over the surface of the solution to evaporate the solution. The :
resultlng crystals of 7-(2-thienyl)acetamido-3-(4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid were filtered and acetone washed.
NMR, DMS0 d6-D20:~ 7.46 and 6.96 (m, 3H, thienyl); 5.67 (d, lH, 7~-H); 5.08 (d, lH, 6a-H); 4.5-3.S
(bm, 8H, 7-CH2, 2-CH2, 3-CH2 and tria-zine N-CH2); and 1.21 ppm (m, 3H, CH2-C~3)-; Example 8 To 12 ml. of water were added with stirring 531 `:
mg. (3 mmol.) of 3~mercapto-4-methyl-5-oxo-6-hydroxy-4,5-~dihydro-1,2,4-triazine. The pH of the resulting mixture was `: :
30 2.3 and was adjusted to 6.5 by addition of 2.9 ml. of lN

~-4273A -37-` ~

sodium hydroxide. 7-(2~5-Dichlorophenylthio)acetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid (1~54 y., 3 mmol.) was added with stirring and re~ultant formation of a gela-tinous mass. The mixture was heated to 60C. Solution occurred, and, while maintaining the mixture at this temp-erature, the pH was adjusted to 7.0, and the mixture was maintained thereat for 19 hours. After the first 16 hours, the mixture was thick and gelatinous, so much so that stirring had stopped. The mixture was thoroughly mixed with a spatula, and heating without stirring wa~ continued for the remaining three hours. On completion of heating, the mixture was cooled in an ice bath and a~idified to pH 1.5. Fluidity of the mixture was maintained ~y dilution to about 100 ml. by addition of water. After stirring at pH 1.5 at room temperature for 1.5 hours, the mixture was filtered~ washed with dilute HCl (pH 1.5), and air dried to give a cream-colored powder. The powder was dissolved in tetrahydrofuran (THF), filtered, and diluted with ethanol. The resulting solution was placed in an air stream by means of which solid was deposited followed by a yellow gum. The gum was removed with a spatula. The resulting concentrate was f iltered, and the product was washed with ethanol and air dried to give an off-white solid (861 mg.).
! The recrystallization procedure was repeated to give 707 mg.
of 7-(2,5-dichlorophenylthio)acetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid as an off-white powder.

' .' - . .- ' ' ' : . ' ' NMR, DMS0 d6-D20: 7.4 (bm, 4H, 2,5-dichlorophenyl); 5.70 (d, lH, 7a-~); 5.11 (d, lH, 6a-H); 4.15 (b, 2H, 3-CH2); 3.92 (h, 2Ht 7-CH2); 3.69 (b, 2H, 2-CH2); and 3.33 (s, 3H, triazine N-CH3).
Example 9 To 12 ml. o water were added with stirring 571 mg.
of 3-mercapto-4-ethyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine. The pH of the resulting mixture was 2.3 and was adjusted to 6.6 by addition of 2.9 ml. of lN sodium hydroxide~
7-(2,5-Dichlorophenylthio)acetamido-3-acetoxymethyl-3 cephem-4-carboxylic acid, sodium salt (1.54 g., 3 mmol.) was added, and the resulting mixture was treated in accordance with the procedure described in Example 8 to obtain 7-(2,5- ;-dichlorophenylthio)acetamido-3-(4-ethyl-5-oxo-6-hydroxy~
4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-car-~
boxylic acid.
NMRj DMS0 d6-D20: 7.4 (bm, 4H, 2,5-dichlorophenyl); 5.68 (d, ]H, 7a-H), 5.10 (d, lH, 6a-H); and 4-5-3~5 ppm (bm, 8H, 7-CH2, 2-C~2, 3-CH2, 3 -2) Example 10 ~ -To 12 ml. of dry THF were added 350 mg. (0.695 mmol.) of 7-(a-amino)phenylacetamido-3 (4-methyl-5-oxo-6-`
hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid followed by 917 mg. (7 mmol.) of N-tri-methylsilylacetamide. Upon completion of the solution~
: :. ~ : :
1 ml. of propylene oxide was added followed by 75 mg.

(0.75 mmol.) of N-(~chlorobenzoyl)-N-(chloroformyl)-30 ~ methylamine dlssolved in 2 ml. of dry THF. The addition ~: :
. , ~ .,.: . . , ~ . :

was made while the mixture was maintained at a ~emperature of -10C. The resulting mixture was stirred at -10C. for 10 minutes and then at room temperature for 15 minutes~
Water (1 ml.) was then added followed by 30 ml. of aqueous sodium bicarbonate solution. The mixture then was washed with 50 ml. of a 6:1 mixture of ethyl acetate and THF. The pH of the aqueous layer was lowered -to 2.0 by addition of lN
HCl in the presence of 50 ml. of a 6:1 mixture of fresh ethyl acetate and THF. The organic layer was separated and dried over magnesium sulfate. The mixture then was filtered and evaporated to dryness. The powder residue then was dis-solved in warm ethyl acetate, and the ethyl acetate solution was concentrated until it became cloudy. Isopropyl al- -cohol then was added, complete solution being achieved, and the mixture was again concentrated until the solution became cloudy. The concentrated solution was refrigerated over-night to obtain 234 mg. of 7~a-[3~(4-chlorobenzoyl)-3-methyl-l-ureido~phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3~cephem-4-carboxylic acid.
NMR, DMSO d6-D2O: 7.60 and 7.45 (two b, 9H, aromatic);
5.77 (d, lH, 7a-H); 5.63 (s, lH, 7-CH);
5.05 (d, lH, 6a-H)i 3.66 (m, 2H, 2-CH2);
3.40 ~, 3H, triazine N-CH3); and 3.17 ppm (s, 3H, CON(CH3)CO).
Example ll To 10 ml. Gf dry THF were added 379 mg. (0.75 mmol.) of 7-(a-amino)phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-3~ 4-carboxylic acid and 586 mg. (4~5 mmol.) of N-trimethyl-: . . . '; . ' ' ' ' ..

silylacetamide. The mixture was stirred at room tempera-ture For 2 hours. Upon completion of solution, 1 ml. of propylene oxide was added, and the solution was cooled to 0C~ N-(o-Chlorobenzoyl~ -N- (chloroformyl)methylamine (239 mg., 1.03 mmol.) dissolved in 5 ml. of dry THF was then added dropwise, and the reaction mixture was stirred for 30 minutes at 0C. and for one hour at room temperature.
Five ml. of water were then added, and the THF was removed in vacuo. To the residue then were added SO ml. of an aqueous sodium bicarbonate solution. The resulting mixture was washed with ethyl acetate, and the pH of the aqueous layer was lowered to 2.0 by addition of lN HCl. The acidi-fied aqueous mixture then was extracted with 100 ml. of a 6:1 mixture of ethyl acetate and THF. The organic layer was separated, dried over magnesium sulfate, filtered, and the filtrate was evaporated to dryness. The resulting foam residue was dissolved in ethyl acetate, and ether was added to precipitate 140 mg. of 7-a-[3-(2-chlorobenzoyl~-3-methyl-l-ureido]phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-20 dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, which was collected by filtration.
NMR, DMSO d6-D20: 7.54 and 7.41 (two b, 9H/ aromatic); 5.73 (d, lH, 7a-H); 5.59 (s, lH, 7-CH~; 4.07 (ml 2H, 3-CH2); 3.53 (m, 2H, 2-CH2); 3.28 (s, 3H, triazine N-CH3); and 2094 ppm (s, 3H, CON(CH3~CO).
Example 12 To 20 ml. of dry 'rHF ~ere added 350 mg. (0.695 mmol.) of 7-(a-amino)phenylacetamido-3-~4-methyl-5-oxo-6~
30 hydroxy-4,5-dihydro-1,2,4-triazin-3 ylthio)methyl-3-cephem-4-carboxylic acid followed by 786 mg. (6 mmol~) of N-trimethylsilylacetamide. The resulting mixture was stirred at room temperature for 2 hours during which time solution resulted. Furoyl-2-isocyanate (97.5 mg., 0.71 mmol.) was then added, and the reaction mixture was stirred for 1 hour at room temperature. Water (20 ml.) then was added to the solution. The mixture was reduced in volume until a cloudiness developed at which time 40 ml. of aqueous sodium bicarbonate solution were added. The solution became clear and was washed twice with 50 ml. of ethyl acetate. The aqueous layer was separated, and the pH was lowered to 2.0 by addition of lN HCl. The layer was then extracted twice with 50 ml. of sthyl acetate. The ethyl acetate `; extracts were combined, dried over magnesium sulfate, filtered and evaporated to a foam residue. The residue was dissolved in warm ethanol, and the ethanol solution was concentrated until a cloudiness developed. The thus-concentrated ethanol solution was then xefrigerated to obtain, upon filtration, 152 mg. of 7-a-13-furoyl-1-ureido)-20 phenylacetamido-3-(4 methyl-5-oxo-6-hydxoxy-4,5-dihydro-1,2,4~triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6: 8~03; 7.71 and 6.73 (each lH, furoyl); 7.46 (b, 5H, phenyl); 5.78 (bm, 2H, 7a-H and 7-CH~;
5.07 (d, lH, 6-H); 4.14 (b, 2H, 3-CH2); 3.61 (b, 2H, 2 CH2); and 3.32 ppm (s, 3H, tria-zine N-CH3).
Example 13 mixture of 381 mg. (1 mmole) of 7-(lH-tetra-zoleacetamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid, 30 200 mg. (1.2 mmole) of 3-mercapto-4-methyl-5~oxo-6-, .

, hydroxy-4,5-dihydro-1,2,4-triazine, and 1.25 mmole of sodium bicarbonate in 30 ml. of pH 7 buffer was prepared.
A small amount of solid remained insoluble in the mixture, the pH of the resulting mixture being about 6.6 The mix-ture was warmed at about 64C. During the first hour, the pH of the mixture rose to 7.5 and then progressively lowered to about 6.7. Heatiny was continued for an additional five hours during which time no further pH change occurred. The mixture then was cooled, layered with ethyl acetate, and the pH of the mixture was low~red to 2.7 by addition of 20 percent hydrochloric acid. The organic layer was separated, and the aqueous layer then was washed with additional ethyl acetate. The original ethyl acetate layer as well as the ethyl acetate washes were combi~ed, and the total mixture was dried over magnesium sulfate, filtered, and evaporated to give 100 mg. of 7-(lH-tetra-zoleacetamido) 3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, melting point 192-194C.
UV ~max 273 (Epsilon = 14,900~.
Analysis 15 15 9 6 2 Theory: C, 37.42; H, 3.14; N, 26.18 Found: C, 37.20; H, 3.26; N, 25.96.
Example 14 To 50 rnl. of dry tetrahydrofuran (THF) wexe added 1.85 ~ (5 mmole) of 7-amino~3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-tria~in-3-ylthio)methyl-3-cephem-4-carboxylic acid and 3.6 ml. (15 mrnole) of N,O-bis-tri-methylsilylacetamide. The mixture was stirred until X-4273A _43_ solution was complete. The solution then was cooled to -20OC.
Separately, 1.97 g. (5.5 mmole)-of the sodium salt of N-(~-methoxycarbonyl-1-methylethenyl)-4-tri-methylsilyloxyphenylglycine were added to a solution of 0.085 g. (0.5 mmole) of N-trimethylsilylsuccinimide in 75 ml. of dry tetrahydrofuran. To the resulting mixtur~
were added 6 drops of N,N-dimethylbenzylamine. The resulting suspension then was cooled to -15C., and 0.52 g. (5.5 mmole) of methyl chloroformate was added with stirring. The mixture was stirred for 15 minutes at -15C., and the above solution containing the cephalosporin nucleus was added. The resulting reaction mixture was stirred for two hours at -20C. and then for one hour at room temperature.
High pressure liquid chromatography of the reaction mixture indicated the presence of approximately 60 percent of the desired product and 40 percent of the cephalosporin nucleus starting material.
The reaction mixture was worked up by adding 10 ml. of methanol to the mixture. A precipitate formed and was removed by filtration. To the filtrate ~ en were added 10 ml. of water. The mixture was stirred for 15 minutes, and the resulting precipitate was filterçd. The filtrate was evaporated in v.c~o to about 40 mL.j and the concentrated mixture then was cooled overnight ~n a freezer.
A precipitate formed which was collected by filtration~
The resulting collected solid was stirred in 15 ml. of water. The pH of the mixture was adjusted to 1.1 by addition o~ 3 drops of concentrated hydrochloric acid. The acidic mixture then was stirred for five minutes, and the ~ .

, .~, ~ , ' insolubles were filtered. The pH of the filtrate was raised to 3.0 by addition of ~odium hydroxide, and the resulting mixture was stirred for 10 minutes at ice bath temperature.
A precipitate formed and was collected by filtration To the resulting filtrate was added 1 volume of isopropyl alcohol, and the mixture was evaporated ln vacuo to about 10 ml. The concentrated solution was s-tirred in an ice bath for 15 minutes, and the resulting precipitate~ 73 mg.
of 7-(a-amino 4-hydroxyphenyl)acetamido-3~(4-methyl-5-oxo-10 6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, was collected by filtration.
NMR, TFA dl: 7.29 (q, 4H, p-hydroxyphenyl); 5.86 (d, lH, 7~-H); 5.52 (s, lH, 7-CH); 4.48 (q, 2H, 3-CH2), - and 3.8-3.54 ppm (b, 5H, triazine N-CH3 and 2-CH2).
Example 15 To 25 ml. of dry THF were added 252 mg. tO.5 mmole~ of 7-(a-amino)phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-20 cephem-4-carboxylic acid and 650 mg (5 mmole) of N-tri-methylsilylacetamide. The mixture was stirred for about 3 hours after which time solution occurred. The solution then was cooled to 0C., and 76 mg. (0.5 mmole) of N,N'-di-methyl-N-(chloroformyl)urea were added. The resulting mixture was stirred for one hour at room temperature, and the mixture was evaporated in vacuo to an oil. Water (20 ml.) was added to the oil, and the pH of the resulting mixture was raised to 8.0 by addition of aqueous sodium bicarbonate. The resulting mixture was washed with ethyl acetate, and the pH of the aqueous layer was lowered to 2.0 by addition of lN hydrochloric acid. The resulting prP-cipitate was filtered and dried to obtain 118 mg. of 7-a-[3-(N-methylcarbonylamino)-3-methyl-1-ureido]phenylace-tamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-tria-zin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6-CD3OD: 7.43 (b, 5H, C6H5); 5.76 (d, lH, 7a-H); 5.61 (s, lH, C6H5-CH); 5.05 (d, lH, 6a-H); 4.16 Im, 2H, 3-CH2); 3.64 (b, 2H, 2-CH2); 3.39 (s, 3H, triazine N-CH3); 3.15 (s, 3H, CO-N(CH3)-CO); and 2.75 ppm (s, 3H, CONH(CH3)).
Example 16 To 25 ml. of dry THF were added 554 mg. (1.1 mmole) of 7-(a-amino)phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin~3-ylthio)rnethyl 3-cephem-4--carboxylic acid and 650 mg. (5 rnmole) of N-tri-methylsilylacetamide. The mixture was stirred for three hours until sclution occurred. The solution then was cooled to 0C., and 650 mg. (5 ~nole) of l-chloroformyl-imidazolidine-2 one were added. The resulting mixture was stirred for 30 minutes at 0C. and then for 1 hour at room temperature. Water (2 ml.) then was added to the mixture. The mixture then was evaporated ln vacuo to an oil~ Water (25 ml.) was added to the oil, and the pH
was raised to 7.S with sodium bicarbonate. The solution was washed with ethyl acet~te, and the aqueous layer then was acidified to pH 1.8 by ad-lition of lN hydrochloric ;~ ~ acid. The resulting solid was filtered and triturated with methanoI, and the rnethanol insolubles were filtered. The resulting mekhanol solution was slowly evaporated in vacuo to an oil. Water (25 ml.) was added to the oil, and the pH was raised to 7.5 with sodium bicarbonate. The solution was washed with ethyl acetate, and the aqueous layer then ~as acidified to pH 1. 8 by addition of lN hydrochloric acid.
The resulting solid was filtered and triturated with methanol, and the methanol insolubles were ~ilteredO The resulting methanol solution was slowly evaporated ln vacuo until precipitation resulted. The precipitate was filtered and dried to obtain 92 mg. of 7 a-(imidazolidine-2~one-1-yl-carbonylamino~phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6-D2O: 7.50 (b, 5H, C6H5); 5.75~(d, lH, 7a-H);
5.60 (s, lH, C6H5CH); 5.02 (d, lH, 6a-H);
and 3.36 ppm (s, 3H, triazine N-CH3).
Example 17 To 20 ml. of dry THF were added 554 mg. (1.1 mmole) of 7-(a-amino)phenylacetamido-~3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid and 650 mg. (5 mmole) of N-trimethyl-silylacetamide. The mixture was stirred for about three hours after which solution was complete. The solution then was~cooled to 9C., and 339 mg. (1.5 mmole) of l-chloro-formyl-3-methanesulfonylimidaæolidine-2-one were added. The reaction mixture was stirred for about 30 minutes at O~C. and then for about 1.5 hours at room temperature. Water (2 ml.) was added to the mixture. The solution then was evaporated in vacuo to an oil. Water (25 ml.) then was __ added, and the pM of the mixture was raised to 7.5 by addition of sodium bicarbonate. The solution then was ' washed with ethyl acetate, and the aqueous layer wasseparated and acidified to pH 1.8 by addition of lN hydro-chloric acid. The solid which precipitated was filtered and dried to obtain 416 mg. of 7-a-(3-methanesulfonyl-imidazolidine-2~one-1-ylcarbonylamino)phenylacetamido-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-yl thio)methyl-3-cephem-4~carboxylic acid.
NMR, DMSO d6-D2O: 7.47 (b, 5H, C6H5); 5.75 (d, lH, 7a-H);
5.71 (5, lH, C6H5C~); 5.02 (d, lH, 5a-H);

3-87 (b, 4H, N-CH2-CH2-N); and 3.37 ppm (b, 6H, triazine N-CH3 and SO2CH3).
Example 18 To 20 ml. of dry tetxahydrofuran (THF) were added 1.53 grams (10 mmole) of hydroxybenzotriazole and 2.57 yrams (10 mmole) of a-(t-butoxycarbonylamino)thien-2-yl-acetic acid. The mixture was cooled to 0C., and 2.06 grams (10 mmole) of N,N'-dicyclohexylcarbodiimida were added. The mixture was stirred with ice bath cooling for 2.75 hours. The mixture then was filtered rapidly, and the solids were washed with 10 ml. of dry THF. The fil-trate which was collected was maintained in an ice bath.
To 60 ml. of dry THF containing 9.8 grams (75 mmole) of N-trimethylsilylacetamide were added 3.73 grams (10 mmole) of 7~amino-3-(4-methyl-5-oxo-6-hydroxy-4,5-di-hydro-1,2,4-triazin 3~ylthio)methyl-3-cephem-4-carboxylic acid. The mixture was sonicated for about 1 hour during which time almost complete solution occurred. The resulting solution then was stirred in an ice bath, and, after 15 - minutes, was added rapidly to the above-prepared filtrate containing the acylating agent. The resulting mixture was stirred with ice bath cooling for 30 minutes and then at 32C. for three hours. The resulting brown solution was poured rapidly into stirred ice water. Ether was added, and the pH of the aqueous phase was adjusted to 8.2. The phases were separated, and the water layer was washed with a further volume of ether. The aqueous phase then was subjected to rotary evaporation to remove residual ether.
Ice was added to the resulting aqueous solution, and the mixture was stirred rapidly while the pH was adjusted to 1.8 by addition of lN hydrochloric acid. The resulting mixture was filtered, and the solid was washed with dilute hydrochloric acid (pH 1.8), air-dried, and dried in vacuo to give 4O16 grams of 7-[a-(t-butoxycarbonylamino)thien-2-ylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro~l/2,4-triazin-3-ylthio)methyl~3-cephem-4-carboxylic acid as a pale, yellow-brown solid.
NMR, TFA dl: 7O97~ 7.48 and 7.17 (m, 3H, thienyl); 5.86 (b, 2H, 7a-H and 7-CH); S.21 (b, lH, 6a-H); 4.44 (q, 2H, 3-CH~); and 3.7-3.5 ppm (b, 5H, 2-CH2 and triazine N-CH3).
Example 19 The product from Example 18 (1.~0 grams) was placed in a flask e~uipped with a stirrer bar. The flask then was cooled in an ice-acetone bath. To the flask then were added rapidly 60 ml. of trifluoroacetic acid which had first been cooled in an ice-acetone bath. The resulting mixture was stirred for 15 minutes during which time complete solution occurred. Thin-layer chromatography (TLC) of the mixture indicated thak the reaction was complete. The reaction mixture then was rotary evaporated to a gum.
Ethyl acetate (60 ml.) was added to the gum, and the mixture was sonicated, resulting in a powder which was collected by filtration, washed with ethyl acetate, and air dried to give 1.55 grams of a light brown powder. To the powder was added a mixture of 75 ml. of water and 10 ml. of ethanol. The xesulting mixture was sonicated, and the pH was adjusted to 1.4. The mixture was filtered, and the pH of the filtrate was adjusted to 3.7. The resulting mixture was again filtered, and the solid which was collected was washed with dilute acid (pH 3.8) which was added to the filtrate.
Isopropyl alcohol then was added to the filtrate and the total was rotary evaporated to a small volume. Additional isopropyl alcohol was added to the residue, and the mixture was filtered. The collected solid was washed with a 1:1 mixture of water and isopropyl alcohol maintained at a pH
of 3.8. The resulting solid was dried to obtain 280 mg.
of 7-[a-(amino)thien-2-ylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3 cephem-4-carboxylic acid.
; NMR~ DMSO d6-D2O: 7.59, 7.25 and 7.08 (m, 3H, thienyl); 5.66 (d, lH, 7a-H), 5.38 (s, lH, 7-CH); 4.99 (d, lH, 6~-H); and 3.28 ppm (bs, 3H, triazine N-CH3).
Example 20 To 24 ml. of dry tetxahydrofuran were added 408 mg. (0.8 mmole) of the product from Example 19 and 917 mg.
(7 mmole) of N-trimethylsilylacetamide. The solution was ; complete wikhin 10 minutes, and, after 15 minutes, the solution was placed in an ice bath. Propylene oxide ~1.6 ml.) and sodium bicarbonate (65 mg.) were added followed ~ 30 by 145 mg. (0.96 mmole; 1.2 mole equivalent) of N-chloro--~ X-4273A -50-~,,, .

-formyl-N,N'-dimethylurea. The resulting mixture was removed from the ice bath and stored at room temperature for 20 minutes. The mixture then ~as rotary evaporated to a small volume (about 10 ml.), and ice water was added. The re-sulting suspension then was stirred, and the pH was adjusted to 6.5. The resulting solution was washed with 2 volumes of ether, and the aqueous phase then was rotary evaporated until the ether was removed. The aqueous mixture then was acidified to pH 1.7, and the resulting solid was collected b~ filtration and partially dried on the filter. The damp product then was dried in vacuo to give 96 mg. of 7-~a-(3-methyl-3-methylaminocarbonyl-1-ureido)thien-2-ylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid as a pale yellow-brown powder.
NMR, DMSO d6-D2O: 7.43, 7.10 and 7.01 (m, 3H, thienyl);
5.79 (s, lH, 7-CH); 5.70 (d, lH, 7~-H);
5.05 (d, lH, 6a-H); 3.65 (m, 2H, 2-CH2);
3O27 (s, 3H, triazine N-CH31; 3.08 (s, 3H, CO-N(CH3)-CO); and 2.67 ppm (s, 3H, CONH(CH3)).
Example 21 Employing the same procedure as described in Example 20, but replacing the N-chloroformyl-N,N'-dimethyl-urea with 217 mg. (0.96 mmole) of 1-chloroformyl-3-methane~
sulfonylimidazolidine-2-one gave 242 mg. of 7-[u-(3-methanesul~onylimidazolidine~2-one-1-ylcarbonylamino)thien-2-ylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.

- -NMR, DMS0 d60 9.52 (d, lH, NH); 8.72 (d, lH, NH); 7.45, 7.10 and 6.99 (m, 3H, thienyl~; 5.88 (d, lH, 7-CH); 5.74 (q, lH, 7a-H); 5.08 (d, lH, 6a-H), 4.08 (m, 2H, 3-CH2), 3.78 (b, 4H, N-C~2-CH2-N), 3.60 (m, 2H, 2~CH2); 3.34 and 3.28 ppm (two s, 6H triazine, N-CH3 and S02CH3).
Example 22 To 15 ml. of dry THF were added 317 mg. (0.5 mmole) of the trifluoroacetate salt of 7 (a-amino-4-hydroxyphenyl-acetamido)-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid. N-Tri-methylsilylacetamide (0.59 grams) was added to the resulting suspension. The mixture was stirred for two hours at room temperature after which solution resulted. The mixture then was cooled to 0C., and 50 mg. of triethylamine and 1 ml. of propylene oxide were added. To the mixture then were added 123 mg. (0.5 mmole~ of N-(o-chlorobenzoyl)-N-(chloroformyl)-me~hylamine. The resulting solution then was stirred at room temperature for 1.5 hours after which the reaction mix-ture was filt~red. Water (1 ml.~ was added to the filtrate;
however, no precipitation occurred. The mixture then was evaporated in vacuc to about 10 ml., and 50 ml. of ethyl acetate were added followed by S0 ml. of water. The pH of the mixture was raised to 7.5 by addition of sodium bicar-bonate. The ethyl acetate layer then was separated rom the~aqueous layer. Fresh ethyl acetate (50 ml.) and THF
(15 ml.) were added to the aqueous layer, and the pH of the aqueous layer was lowered to 2.5 by addition of lN hydro-chloric acid. The organic layer was separated from the aqueousj dried over magnesium sulfate, and filtered. The X-4273A ~ -52-'. ~ , ~ .

fil~rate then was evaporated in vacuo to about 10 ml., and20 ml. of ether were added. The mixture then was filtered to obtain 150 mg. of 7-[a-[3-(2-chlorobenzoyl)-3-methyl-1-ureido]-4-hydroxyphenylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-- 4-carboxylic acid.
NMR, DMSO d6: 7.35 (b, 4H, o-chlorobenzoyl); 6.96 (q, 4H, p-hydroxyphenyl); 5.83 (q, lH, 7a-H); 5.47 (d, lH, 7-CH); 4.99 (d, lH, 6~-H); 4.13 (m, 2H, 3-CH2); 3.56 (m, 2H, 2-CH2); and 3.3 ppm (b, 6H, triazine N-CH3 and CO-N(CH3)-CO).
Example 23 To 25 ml. of dry THF containing 1.18 grams (9 mmole) of N-trimethylsilylacetamide were added 634 mg. (1.0 mmole) of the trifluoroacetate salt of 7-(a-amino-4~hydroxy-phenylacetamido)-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
Solution occurred within 15 minutes of completlon of ~he addition of the cephalosporin compound. The resulting solu-. ! .
tion then was cooled to 0C. in ice-acetone, and 84 mgO
(1 mmole) of sodium bicarbonate were added followed by 1 ml.
of propylene oxide. To the resulting mixture then were added 226 mg. (1 mmole) of l-chloroformyl-3-methanesulfonylimida-zolidine-2-one. The reaction mixture was warmed to room temperature and stirred at room temperature for 1.5 hours.
The reaction mixture then was filtered, and 1 ml. of water -was added to the filtrate. No precipitation occurred. The mixture then was evaporated ln vacuo to about 10 ml., and 100 ml. of a 6:1 mixture of ethyl acetate and THF along with 50 ml. of water were added. The pH of the mixture wa~

~ X-4273A _53 : :

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

raised to 7.0 by addition of sodium bicarbonate. The aqueous layer then was separated from the organic layer, and the pH of the aqueous layer was lowered to 7.0 ~y addition of lN hydrochloric acid. The mixture then was filtered, and the collected solid was washed with isopropyl alcohol and dried to obtain 382 mg. of 7-[a~(3-methanesulfonylimidazoli-dine-2-one-1-ylcarbonylamino)-4-hydroxyphenylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)-methyl-3-cephem-4-carboxylic acid.
10 NMR, TFA dl- 7.27 (q, 4H, p-hydroxyphenyl): 5.90 (d, lH, 7a-H); 5.73 (s, lH, 7-CH); 5.19 (d, lH, 6a-H);
4.46 (q, 2H, 3-CH2); 4.08 (b, 4H, N-CH2-CH2~N);
3.7-3.5 (b, 5H, 2-CH2 and triazine N-CH3); and 3.44 ppm (s, 3H, SO2CH3).
Example 24 Using the procedure of Example 22 on a 1 mmole scale, the cephalosporin was reacted with 151 mg. (1 mmole) of N-chloroformyl-N,N'-dimethylurea in the presence of sodlum bicarbonate instead of the triethylamine used in 20 Example 22 to obtain 154 mg. of 7-[a-(3-methylaminocarbonyl-3-methyl-1-ureido)-4-hydroxyphenylacetamido]-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, DMSO d6-D2O: 7.08 (q, 4H, p-hydroxyphenyl); 5.72 (d, lH, 7a H); 5.41 (s, lH, 7-CH); 5.03 (d, lH, 6a-H); 4~1 (m, 3-CH2); 3.36 (s, 3H, triazine N-CH3); 3.13 (s, 3H, CO-N (CH3)-CO); and 2.73 ppm (s, 3H, CONH(CE~3)).
;.

' 2~
Example 25 Employing the procedure of Example 24, but em-ploying l-chloroformylimidazolidine-2-one as acylating agent, there was obtained 120 mg. of 7~[a-(imidazolidine-2-one-1-ylcarbonylamino)-4-hydroxyphenylacetamido] 3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid.
NMR, ~MSO d6-D2O: 7.13 (q, 4H, p-hydroxyphenyl); 5.79 (d, lH, 7~-H); 5.51 (s, lH, 7-CH); 5.06 (dt lH, 6~-H); and 3.39 ppm (s, 3H, tria-zine N-CH3)-, ,;
.~' ' .

:

:

' . ' : ; ' ~ X-4273A _55_

Claims (4)

The embodiments of the invention for which an ex-clusive property or privilege is claimed are as follows:

1. A process for preparing cephalosporin com-pounds of the formula I

I

in which R1 is hydrogen or lower alkyl: R2 is hydrogen, an alkali metal cation, or a readily removable ester forming group; and R is hydrogen or the group which comprises reacting a 3-acetoxymethylcephalosporin compound of formula II
II .

wherein R and R2 are as defined above, with a triazinyl-thio derivative of the formula wherein R1 is as defined above; and optionally acylating the compound so obtained.

2. Cephalosporin compounds of the formula I
wherein R, R1 and R2 are as defined in claim 1, when pre-pared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A process for preparing 7-amino-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem 4-carboxylic acid which comprises reacting 7-amino-3 acetoxymethyl-3-cephem-4-carboxylic acid with 3-mercapto-4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazine.

4. 7-Amino-3-(4-methyl-5-oxo-6-hydroxy-4,5-dihydro-1,2,4-triazin-3-ylthio)methyl-3-cephem-4-carboxylic acid, when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.