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Patent 1100129 Summary

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(12) Patent: (11) CA 1100129
(21) Application Number: 232136
(54) English Title: CEPHALOSPORIN COMPOUNDS
(54) French Title: TRADUCTION NON-DISPONIBLE
Status: Expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 260/100
(51) International Patent Classification (IPC):
  • C07D 501/16 (2006.01)
  • A61K 31/545 (2006.01)
  • C07D 501/00 (2006.01)
  • C07D 501/04 (2006.01)
  • C07D 501/18 (2006.01)
  • C07D 501/36 (2006.01)
  • C07D 501/48 (2006.01)
  • C07D 501/60 (2006.01)
  • C07D 501/44 (2006.01)
  • C07D 501/46 (2006.01)
(72) Inventors :
  • LUNN, WILLIAM H.W. (United States of America)
(73) Owners :
  • ELI LILLY AND COMPANY (United States of America)
(71) Applicants :
(74) Agent: GOWLING LAFLEUR HENDERSON LLP
(74) Associate agent:
(45) Issued: 1981-04-28
(22) Filed Date: 1975-07-24
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
583,924 United States of America 1975-06-10
494,148 United States of America 1974-08-02

Abstracts

English 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.

X-4273A


Claims

Note: Claims are shown in the official language in which they were submitted.


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

Image 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 Image which comprises
reacting a 3-acetoxymethylcephalosporin compound of
formula II
Image II .

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

Image
wherein R1 is as defined above; and
optionally acylating the compound so obtained.
56


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.

57

Description

Note: Descriptions are shown in the official language in which they were submitted.



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

X-4273A -2-


~ .
",~
- , ' .


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-

X-4273A -3-


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.
X-4273A -4-

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,
X-4273A -6-


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-


X-4273A -7-


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-
X-4273A -8-


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
X-4273A -11-


.: :


:: . '


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-

X-4273A -12-




,


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
IV 2
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



X-4273A -16-
.~


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.
X-4273A -18-


.


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
20 S


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

X-4273A -22-



' -: . . : '' ' '

~ 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.
` ' .

X-4273A -23- -


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~)-




X-4273A -24-


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
X-4273A -25-


.

. .

%~

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

.~ ,


X 4273A -26-



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

~.
- '
.
.
; ' .
:




X-4273A -27-


~ .


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




X-4273A -28-



,,
-: . . : , . . .

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

X-4273A -30-



., -, ' ., ' :


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
X-4273A -31-

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
X-4273A -33-




.~ :


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).

'
X-4273A -34-


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
X-4273A -36-

.



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.




X-4273A -38-


' .' - . .- ' ' ' : . ' '

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

X-4273A -39-


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



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-

X--4273A -40-




: . . . '; . ' ' ' ' ..



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-
X-4273A -41-


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-
X-4273A -42-



, .

,



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
X-4273A -44-


~ .


,
.~, ~ , '


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
X-4273A -45- -


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
X-4273A -46-


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
X-4273A -47-

'


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

X-4273A -48-


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
X-4273A -49-


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.




X-4273A -51-
- -


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)).
;.


X-4273A -54-



'

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_

Representative Drawing

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 1981-04-28
(22) Filed 1975-07-24
(45) Issued 1981-04-28
Expired 1998-04-28

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1975-07-24
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
ELI LILLY AND COMPANY
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Drawings 1994-03-14 1 19
Claims 1994-03-14 2 46
Abstract 1994-03-14 1 21
Cover Page 1994-03-14 1 19
Description 1994-03-14 54 2,222