Preparation of cephalosporin compounds

The invention relates to a novel method for the preparation of a 7.beta.-acylamido-3-bromomethylceph-3-em-4-carboxylic acid-1-oxide compound by brominating a 7.beta.-acylamido-3-methylceph-3-em-4-carboxylic acid-1-oxide compound.

This invention is concerned with a process for the transformation of 
cephalosporin compounds. 
The compounds referred to in this specification are generally named with 
reference to cepham (see J.A.C.S. 1962, 84, 3400 and J. Chem. Soc., 1965, 
5031). The term `cephem` refers to the basic cepham structure with one 
double bond. 
In U.S. Pat. Specification No. 3,275,626 there is described a general 
method for preparing antibiotic substances, including cephalosporins, 
which comprises heating a so-called penicillin sulphoxide under acid 
conditions to a temperature of from about 100.degree. to about 175.degree. 
C. By means of this process esters of 6.beta.-acylamidopenicillanic acid 
1-oxides can be converted into analogous esters of 
7.beta.-acylamido-3-methylceph-3-em-4-carboxylic acids. By suitable choice 
of reaction conditions the cephalosporin analogues can be obtained in high 
yields. Although some of these cephalosporin analogues may have potent 
antibacterial properties it is desirable to be able to transform the 
resulting cephalosporin analogues, which contain a 3-methyl group, into 
related compounds containing a substituted 3-methyl group. Cephalosporin 
compounds containing 3-methyl groups may also be obtained by fully 
synthetic methods and it would also enable one to transfer these in like 
manner. 
Attempts which we have made to halogenate esters of 
7.beta.-acylamido-3-methylceph-3-em-4-carboxylic acids have met with 
little success. We have, however, found that bromination of compounds of 
the general formula: 
##STR1## 
(where R.sup.1 is a protected amino group and R.sup.2 is hydrogen or a 
carboxyl blocking group e.g. the residue of an ester-forming alcohol or 
phenol R.sup.2 OH) yields compounds of general formula: 
##STR2## 
The 1-oxide group in formulae I and II and in subsequent formulae 
preferably has the .beta.-configuration. 
R.sup.1 is conveniently an acylamido group, especially that of a penicillin 
obtained by a fermentation process e.g. phenylacetamido or 
phenoxyacetamido since compounds of formula I may be readily derived from 
penicillins as explained above. The group R.sup.1 may advantageously be a 
formamido group; this group is substantially stable under the bromination 
conditions and can be readily cleaved subsequently to enable another acyl 
group to be introduced. 
The acylamido group may not be the desired group in the end product but 
this can be obviated by subsequent transformations described below. It is 
not necessary that the initial acylamido group should be completely inert 
since it may be cleaved by these subsequent transformations. 
CARBOXYL BLOCKING GROUPS 
We prefer to use the cephalosporin compounds as an ester with an alcohol or 
phenol which may readily be split off, e.g. by hydrolysis or reduction, at 
a later stage of the reaction sequence. Esters are in general more soluble 
than the free acids in solvents for bromination reactions and the 
bromination proceeds more readily. If desired, the cephalosporin compound 
(I) may be employed as the free 4--COOH compound or salt thereof. 
Alcohol and phenol residues which may readily be split off include those 
containing electron-attracting substituents for example sulpho groups and 
esterified carboxyl groups; these groups may be subsequently split off by 
alkaline reagents. Benzyl and o-benzyloxyphenoxy ester groups may be 
removed by hydrogenolysis. A preferred method of removal involves cleavage 
by acid and groups which may be removed by acid include adamantyl, 
t-butyl, benzyl residues such as anisyl and the residues of alkanols 
containing electron donors in the .alpha.-position such as acyloxy, 
alkoxy, benzoyloxy, substituted benzoyloxy, halogen, alkylthio, phenyl, 
alkoxy-phenyl or aromatic heterocyclic. It should be appreciated that some 
of these groups may be subject to concomitant bromination. 
Alcohol residues which may be readily split off subsequently by reduction 
are those of a 2,2,2-trihalogenoethanol, e.g. 2,2,2-trichloroethanol, 
p-nitrobenzyl alcohol or 4-pyridylmethanol. 2,2,2-Trihalogenoethyl groups 
may conveniently be removed by zinc/acetic acid, zinc/formic acid, 
zinc/lower alcohol or zinc/pyridine or by chromous reagents; p-nitrobenzyl 
groups may conveniently be removed by hydrogenolysis. 
Where the esterifying group is subsequently removed by an acid catalysed 
reaction, this may be effected by using formic acid or trifluoroacetic 
acid (e.g. in conjunction with anisole) or alternatively by using 
hydrochloric acid (e.g. in admixture with acetic acid). 
We particularly prefer to use those compounds having a 
2,2,2-trichloroethoxycarbonyl or a t-butoxycarbonyl group as the ester 
group in the bromination process according to the invention. 
Other ester groups which can readily be converted to carboxy groups include 
silyloxycarbonyl groups. 
Although silyloxycarbonyl groups may be formed by reacting the carboxyl 
group with a silanol in some cases it may be more convenient to react the 
carboxyl group with a derivative of a silanol e.g. the corresponding 
chloride or amine. Thus silyloxycarbonyl derivatives are formed with 
tetravalent silicon moieties, and the silylating agent conveniently is a 
halosilane or a silazane of the formula R.sup.4.sub.3 SiX; R.sup.4.sub.2 
SiX.sub.2 ; R.sup.4.sub.3 Si.NR.sup.4.sub.2 ; R.sup.4.sub.3 
Si.NH.SiR.sup.4.sub.3 ; R.sup.4.sub.3 Si.NH.COR.sup.4 ; R.sup.4.sub.3 
Si.NH.CO.NH.SiR.sup.4.sub.3 ; R.sup.4 NH.CO.NR.sup.4.SiR.sup.4.sub.3 ; or 
R.sup.4 C(OSiR.sup.4.sub.3): NSiR.sup.4.sub.3 where X is a halogen and the 
various groups R.sup.4, which can be the same or different, represents 
hydrogen atoms or alkyl, e.g. methyl, ethyl, n-propyl, iso-propyl; aryl, 
e.g. phenyl; or aralkyl e.g. benzyl groups. Some of these compounds may 
not be particularly stable under the reaction conditions where R.sup.4 is 
H for all R.sup.4 groups. It is generally preferred that the R.sup.4 
groups should be hydrocarbon groups and preferably the hydrocarbon group 
should be methyl or phenyl as, for example, in hexamethyldisilazane, 
(Me.sub.3 Si).sub.2 NH. Examples of suitable silylating agents are 
trimethyl chlorosilane, hexamethyldisilazane, triethyl chlorosilane, 
methyl trichlorosilane, dimethyl dichlorosilane, triethyl bromosilane, 
tri-n-propyl chlorosilane, bromomethyl dimethyl chlorosilane, tri-n-butyl 
chlorosilane, methyl diethyl chlorosilane, dimethyl ethyl chlorosilane, 
phenyl dimethyl bromosilane, benzyl methyl ethyl chlorosilane, phenyl 
ethyl methyl chlorosilane, triphenyl chlorosilane, tri-o-tolyl 
chlorosilane, tri-p-dimethylaminophenyl chlorosilane, N-ethyl 
triethylsilylamine, hexaethyldisilazane, triphenyl silylamine, 
tri-n-propyl silylamine, tetraethyl dimethyl disilazane, tetramethyl 
diethyl disilazane, tetramethyl diphenyl disilazane, hexaphenyldisilazane 
and hexa-p-tolyl disilazane. 
When preparing compounds of the formula I having silyloxycarbonyl groups on 
a commercial scale it may be advantageous to employ silyl chlorides such 
as, for example, Me.sub.3 SiCl, or Me.sub.2 SiCl.sub.2 in conjunction with 
a base such as, for example, diethylamine, triethylamine, dimethylaniline, 
quinoline, lutidine or pyridine. 
An advantage accruing from the use of compounds of the formula I wherein 
the ester is a silyloxycarbonyl group in the process according to the 
invention is that the esterifying group is removed under mild conditions 
and hence tends to be removed during one of the isolation or subsequent 
reaction stages. 
The siloxycarbonyl group is easily converted to a carboxy group by exposing 
the derivative to an excess of a compound(s) containing active hydrogen, 
e.g. water, acidified or basified water, alcohols and phenols. 
PREATION OF 1-OXIDE STARTING MATERIALS 
Compounds of formula I may be prepared by oxidation of a compound of the 
general formula: 
##STR3## 
where R.sup.1 and R.sup.2 have the above defined meanings and the dotted 
line between the 2-, 3- and 4-position indicates that the compound may be 
a .DELTA..sup.2 or .DELTA..sup.3 compound or a mixture thereof. The 
oxidation may be carried out as described by Cocker et al (J. Chem. Soc. 
1965, 5015). The cephalosporin compound (III) is mixed with the oxidising 
agent in an amount such that approximately one atom of active oxygen is 
present per atom of dihydrothiazine sulphur. The oxidising agent should 
preferably be one that results in preferential formation of the 
1.beta.-oxide or a mixture of 1.alpha.- and 1.beta.- oxides wherein the 
1.beta.-oxide predominates. Suitable oxidising agents include metaperiodic 
acid, peracetic acid, permonophthalic acid and m-chloroperbenzoic acid. 
Care should be taken to avoid using an excess of oxidising agent which 
would result in the formation of the 1,1-dioxide. 
Compounds of the formula (I) may also be prepared by the oxidation of a 
ceph-2-em compound to form a ceph-3-em 1-oxide as described in Dutch 
published Patent Application No. 6910830. 
The 1-oxide is preferably formed at a temperature below +10.degree. C. to 
minimise sulphone formation. 
The 1-oxide may be formed in solution in an organic solvent and then 
brominated in the resulting solution after purification. Suitable solvents 
are described below under BROMINATION in particular the chlorinated 
hydrocarbons. 
The 1-oxide may be formed from a compound of formula (III) where R.sup.2 
.dbd.H and the resulting acid 1-oxide esterified. Alternatively, a 
preferred ester of formula (III) may be used, for the oxidation step. 
BROMINATION 
The bromination of the compounds of the general formula I may be effected 
by any convenient system capable of generating bromine atoms such as 
bromine itself, or a bromine transfer agent e.g. an N-bromoamide or an 
N-bromoimide. The N-amide or N-bromoimide may include a cyclic system, the 
amide or imide linkage forming part of the cyclic system; examples of such 
N-bromoamides include caprolactam and examples of such N-bromoimides 
include the 1,3-dibromo-5,5-diloweralkyl hydantoins e.g. 
1,3-dibromo-5,5-dimethylhydantoin; 1,3-dibromo-5-ethyl-5-methylhydantoin; 
1,3-dibromo-5-isopropyl-5-methylhydantoin, N-bromosuccinimide, 
N-bromophthalimide etc. Other useful N-bromoamides include N-bromo lower 
alkanoamides e.g. N-bromoacetamide. Another useful brominating agent is 
1,3,5-tribromo-1,2,4-triazole. By reason of their availability, 
particularly preferred brominating agents include N-bromosuccinimide and 
5,5-dimethyl-1,3-dibromohydantoin. 
The various brominating agents require initiation in order to generate 
bromine atoms and suitable initiating systems include free-radical 
initiators such as azo compounds e.g. azobisisobutyronitrile, peroxides 
e.g. benzoyl peroxide, irradiation by ultra violet or visible light 
sources e.g. mercury arcs or tungsten lamps, or by .gamma.-rays emitted by 
Co.sup.60 sources. 
The brominating agent may be added as such or in suspension or solution in 
a suitable solvent i.e. a solvent which solubilizes the starting material 
and which is substantially inert under the conditions of the reaction e.g. 
a hydrocarbon such as benzene or a halogenated hydrocarbon particularly a 
chlorinated hydrocarbon e.g. chloroform, methylene chloride, 
1,2-dichloroethane etc. The brominating agent is added to a solution or 
suspension of the cephalosporin compound (I) in a suitable solvent e.g. a 
halogenated hydrocarbon such as methylene chloride, chloroform, 
1,2-dichloroethane or chlorobenzene or a hydrocarbon such as benzene. The 
bromination may be effected at temperatures ranging from -80.degree. to 
+150.degree. C. e.g. from -20.degree. to +150.degree. C. preferably from 
-40.degree. to +85.degree. C. advantageously from -20.degree. to 
+85.degree. C. The course of the bromination may be followed by 
measurement of the consumption of brominating agent and by thin-layer 
chromatography. The course of the reaction may also be followed by 
monitoring the ultra violet absorption spectrum or optical rotation. 
The bromination is preferably carried out using N-bromosuccinimide or a 
dibromohydantoin as the brominating agent initiated by ultra violet 
irradiation at a low temperature e.g. from -20.degree. to +10.degree. C. 
The addition of small amounts e.g. up to 5% by volume of water or an 
aqueous solution or suspension of a weak base such as an alkali metal or 
alkaline earth metal salt of a weak acid e.g. sodium bicarbonate, sodium 
carbonate, sodium acetate or calcium carbonate has been found to assist 
the bromination reaction. In this way the times of initiation and reaction 
may be reduced and/or the yield of 3-bromomethyl compound may be 
increased. The aqueous solution or suspension of the weak base is 
preferably at a pH of 7 to 11. 
The bromination may be effected under an inert atmosphere. 
The bromination reaction according to the invention lends itself to 
continuous flow techniques. 
The 3-bromomethyl compounds obtained may be converted to other 3-halomethyl 
compounds viz 3-chloro or 3-iodomethyl compounds, e.g. by reaction with a 
suitable alkali metal halide, for the conversion of bromomethyl compounds 
into other halomethyl compounds. Such reactions may occur during 
operations involving sources of other halide ions. 
After completion of the reaction, the 3-bromomethyl compound may then be 
isolated. An advantageous procedure is to wash the resulting reaction 
mixture with water to remove by-product imide or amide resulting from the 
brominating agent which is then isolated, for example by concentration of 
the solution followed by crystallization of the product or by 
chromatography. The aqueous solution containing the imide or amide may 
then be treated with bromine after the addition of alkali to regenerate 
the brominating agent. 
Compounds of general formula: 
##STR4## 
where R.sup.1 and R.sup.2 have the above defined meanings, and X is 
bromine, chlorine or iodine are new compounds and are a feature of the 
invention. Important compounds of formula (IV) include 
2,2,2-trichloroethyl 3-bromomethyl-7.beta.-phenylacetamidoceph-3-em-4-carb 
oxylate, 1.beta.-oxide; 2,2,2-trichloroethyl 
3-bromomethyl-7.beta.-formamido-ceph-3-em-4-carboxylate, 1.beta.-oxide; 
t-butyl 3-bromomethyl-7.beta.-formamidoceph-3-em-4-carboxylate, 
1.beta.-oxide; 2,2,2-trichloroethyl 
3-bromomethyl-7.beta.-phenoxyacetamidoceph-3-em-4-carboxylate, 
1.beta.-oxide; 2,2,2trichloroethyl 3-bromomethyl-7.beta.-[D - 2 - 
(2,2,2-trichloroethoxycarbonylamino) 
2-phenylacetamido]ceph-3-em-4-carboxylate, 1.beta.-oxide; p-methoxybenzyl 
3-bromomethyl-7.beta.-phenylacetamidoceph-3-em-4-carboxylate, 
1.beta.-oxide; t-butyl 
3-bromomethyl-7.beta.-phenylacetamidoceph-3-em-4-carboxylate, 
1.beta.-oxide; 2,2,2-trichloroethyl 
3-bromomethyl-7.beta.-(2,2,2-trichloroethoxycarbonylamino)ceph-3-em-4-carb 
oxylate, 1.beta.-oxide; 
t-butyl-3-bromomethyl-7.beta.-phenoxyacetamidoceph-3-em-4-carboxylate, 
1.beta.-oxide and 2,2,2 -trichloroethyl 
3-bromomethyl-7.beta.-(DL-2-bromo-2-phenylacetamido)ceph-3-em-4-carboxylat 
e, 1.beta.-oxide. 
In addition to bromination in the 3-methyl group, other bromination 
reactions may take place. One may thus form as by-products compounds of 
formula: 
##STR5## 
(wherein R.sup.1 and R.sup.2 have the above defined meanings and Z is 
hydrogen or bromine). Compounds of formula V where Z.dbd.H may be reduced 
e.g. with Zn/acid to re-form the compound of formula I which may then be 
re-brominated to yield the desired compound of formula II. Bromination may 
also occur in the group R.sup.1 e.g. in the case where R.sup.1 = 
phenylacetamido to yield 2-bromo-2-phenylacetamido-3-bromomethyl 
compounds. 
ACYL GROUPS 
The group R.sup.1 in the above formulae may represent a wide variety of 
acylamido groups which may contain 1-20 carbon atoms. Specific acyl groups 
are illustrated in the accompanying list which is not intended to be 
exhaustive: 
(i) R.sup.u C.sub.n H.sub.2n CO-- where R.sup.u is aryl (carbocyclic or 
heterocyclic), cycloalkyl, substituted aryl, substituted cycloalkyl, 
cyclohexadienyl, or a non-aromatic or mesoionic group, and n is an integer 
from 1-4. Examples of this group include phenylacetyl; substituted 
phenylacetyl e.g. fluorophenylacetyl, nitrophenylacetyl, 
aminophenylacetyl, acetoxyphenylacetyl, methoxyphenylacetyl, 
methylphenylacetyl, or hydroxyphenylacetyl; N,N-bis (2-chloroethyl) 
aminophenylporpionyl; thien-2- and -3-ylacetyl; 4-isoxazolyl and 
substituted 4-isoxazolylacetyl; pyridylacetyl; tetrazolylacetyl or a 
sydnoneacetyl group. The substituted 4-isoxazolyl group may be a 
3-aryl-5-methyl isoxazol-4-yl group, the aryl group being e.g. phenyl or 
halophenyl e.g. chloro- or bromo- phenyl. An acyl group of this type is 
3-o-chlorophenyl-5-methyl isoxazol-4acetyl. 
(ii) C.sub.n H.sub.2n+1 CO- where n is an integer from 1-7. The alkyl group 
may be straight or branched and, if desired, may be interrupted by an 
oxygen or sulphur atom or substituted by e.g. a cyano group, a carboxy 
group, an alkoxycarbonyl group, a hydroxy group or a carboxycarbonyl group 
(--CO.COOH). Examples of such groups include cyanoacetyl, hexanoyl, 
heptanoyl, octanoyl and butylthioacetyl. 
(iii) C.sub.n H.sub.2n-1 CO- where n is an integer from 2-7. The alkenyl 
group may be straight or branched and, if desired, may be interrupted by 
an oxygen or a sulphur atom. An example of such a group is 
allylthioacetyl. 
(iv) 
##STR6## 
where R.sup.u has the meaning defined under (i) and in addition may be 
benzyl, and R.sup.v and R.sup.w which may be the same or different each 
represent hydrogen, phenyl, benzyl, phenethyl or lower alkyl. Examples of 
such groups include phenoxyacetyl, 2-phenoxy-2-phenylacetyl, 
phenoxypropionyl, 2-phenoxybutyryl benzyloxycarbonyl, 2-phenoxypropionyl, 
2-phenoxybutyryl, methylthiophenoxyacetyl. 
(v) 
##STR7## 
where R.sup.u has the meaning defined under (i) and, in addition, may be 
benzyl and R.sup.v and R.sup.w have the meanings defined under (iv). 
Examples of such groups include S-phenylthioacetyl, 
S-chlorophenylthioacetyl, S-fluorophenylthioacetyl, pyridylthioacetyl, and 
S-benzylthioacetyl. 
(vi) R.sup.u Z(CH.sub.2).sub.m CO- where R.sup.u has the meaning defined 
under (i) and, in addition, may be benzyl, Z is an oxygen or sulphur atom 
and m is an integer from 2-5. An example of such a group is 
S-benzylthiopropionyl. 
(vii) R.sup.u CO- where R.sup.u has the meaning defined under (i). Examples 
of such groups include benzoyl, substituted benzoyl (e.g. aminobenzoyl), 
4-isoxazolyl- and substituted 4-isoxazolylcarbonyl, cyclopentanecarbonyl, 
sydnonecarbonyl, naphthoyl and substituted naphthoyl (e.g. 
2-ethoxynaphthoyl) quinoxalinylcarbonyl and substituted 
quinoxalinylcarbonyl (e.g. 3-carboxy-2-quinoxalinylcarbonyl). Other 
possible substituents for benzoyl include alkyl, alkoxy, phenyl, carboxy, 
alkylamido, cycloalkylamido, allylamido, phenyl(lower)alkyl amido, 
morpholinocarbonyl, pyrrolidinocarbonyl, piperidinocarbonyl, 
tetrahydropyridino, furfurylamido or N-alkyl-N-anilino, or derivatives 
thereof, and such substitutents may be in the 2- or 2- and 6-positions. 
Examples of such substituted benzoyl groups are 2,6-dimethoxybenzoyl, 
2-methylamidobenzoyl and 2-carboxybenzoyl. Where the group R.sup.u 
represents a substituted 4-isoxazolyl group, the substituents may be as 
set out above under (i). Examples of such 4-isoxazolyl groups are 
3-phenyl-5-methyl-isoxazol-4yl carbonyl, 
3o-chlorophenyl-5-methyl-isoxazol-4-yl carbonyl and 
3-(2,6-dichlorophenyl)-5-methyl-isoxazol-4-yl carbonyl. 
(viii) 
##STR8## 
where R.sup.u has the meaning defined under (i) and X is amino, 
substituted amino (e.g. acylamido or a group obtained by reacting the 
.alpha.-aminoacylamido group of the 7-side chain with an aldehyde or 
ketone e.g. acetone, methylethylketone or ethyl acetoacetate), hydroxy, 
carboxy, esterified carboxy, triazolyl, tetrazolyl, cyano, halogeno, 
acyloxy (e.g. formyloxy or lower alkanoyloxy) or etherified hydroxy group. 
Examples of such acyl groups are .alpha.-aminophenylacetyl, and 
.alpha.-carboxyphenyl-acetyl. 
(ix) 
##STR9## 
where R.sup.x, R.sup.y and R.sup.z which may be the same or different may 
each represent lower alkyl, phenyl or substituted phenyl or R.sup.x 
represents hydrogen. An example of such an acyl group is triphenylcarbonyl 
(x) 
##STR10## 
where R.sup.u has the meaning defined under (i) and in addition may be 
hydrogen, lower alkyl or halogen substituted lower alkyl, and Y represents 
oxygen or sulphur. An example of such a group is Cl(CH.sub.2).sub.2 NHCO. 
(xi) 
##STR11## 
where X has the meaning defined under (viii) above and n is an integer of 
from 1 to 4. An example of such an acyl group is 
1-aminocyclohexanecarbonyl. 
(xii) Amino acyl, for example R.sup.w CH(NH.sub.2).(CH.sub.2).sub.n CO-- 
where n is an integer from 1-10, or NH.sub.2. C.sub.n H.sub.2n 
Ar(CH.sub.2).sub.m CO, where m is zero or an integer from 1-10, and n is 
0, 1 or 2, R.sup.w is a hydrogen atom or an alkyl, aralkyl or carboxy 
group or a group as defined under R.sup.u above, and Ar is an arylene 
group, e.g. p-phenylene or 1,4-naphthylene. Examples of such groups are 
disclosed in British Patent Specification No. 1,054,806. A group of this 
type is the p-aminophenylacetyl group. Other acyl groups of this type 
include those, e.g. .delta.-aminodipoyl, derived from naturally occurring 
amino acids and derivatives thereof e.g. N-benzoyl .gamma.-aminodipoyl. 
(xiii) Substituted glyoxylyl groups of the formula R.sup.y.CO.CO- where 
R.sup.y is an aliphatic, araliphatic or aromatic group, e.g. a thienyl 
group, a phenyl group, or a mono-, di- or tri- substituted phenyl group, 
the substituents being, for example, one or more halogen atoms (F, Cl, Br, 
or I), methoxy groups, methyl groups or amino groups, or a fused benzene 
ring. Included in this group are also the .alpha.-carbonyl derivatives of 
the above substituted glyoxylyl groups, formed for example with 
hydroxylamine, semicarbazide, thiosemicarbazide, isoniazide or hydrazine. 
(xiv) Formyl. 
(xv) Hydrocarbyloxycarbonyl and substituted hydrocarbyloxy groups (wherein 
the 7-amino group forms part of a urethane), in particular lower 
alkoxycarbonyl groups (such as methoxycarbonyl, ethoxycarbonyl and, most 
preferably, t-butoxycarbonyl groups); halo lower alkoxycarbonyl groups 
e.g. 2,2,2-trichloroethoxycarbonyl; aralkoxycarbonyl groups such as 
benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, diphenylmethoxycarbonyl and 
4-nitrobenzyloxycarbonyl groups. Cycloalkoxycarbonyl groups are also 
advantageous, especially the adamantyloxycarbonyl group. 
(xvi) Haloformyl e.g. chloroformyl. 
DISPLACEMENT WITH NUCLEOPHILES 
Compounds of general formula IV may, for example, be reacted with an alkali 
metal salt of a lower aliphatic acid e.g. potassium acetate, for example 
by heating in acetone, or in the cold with N,N-dimethylformamide, to yield 
a compound of the general formula: 
##STR12## 
where R.sup.1 and R.sup.2 have the above defined meanings and R.sup.3 COOH 
is a lower aliphatic acid. The use of an alkali metal salt of a lower 
aliphatic acid is however only an example of a range of compounds having a 
nucleophilic atom, e.g. nitrogen, carbon, sulphur or oxygen which may be 
employed under conditions which are effective to displace the group X by 
the nucleophile. Reactions of this type may have advantages over 
conventional nucleophilic reactions involving acetates in cephalosporin C 
type compounds. Nucleophilic substances which may be used have been widely 
described in earlier literature and patents pertaining to cephalosporin 
chemistry. Examples of such nucleophiles include: 
NITROGEN NUCLEOPHILES 
Examples of nitrogen nucleophiles include tertiary aliphatic, aromatic, 
araliphatic and cyclic amines including trialkylamines, for example, 
triethylamine, pyridine bases such as pyridine and alkyl pyridines; 
heterocyclic amines having more than one heteroatom, at least one 
heteroatom being nitrogen, such as pyrimidines, purines, pyridazines, 
pyrazines, pyrazoles, imidazoles, triazoles and thiazoles. 
Thus the term "nitrogen nucleophile" includes compounds of the following 
formulae: 
EQU NR.sup.a R.sup.b R.sup.c (a) 
in which R.sup.a, R.sup.b and R.sup.c, which may be the same or different 
are hydrogen atoms or substituted or unsubstituted e.g. lower alkyl 
aliphatic, araliphatic e.g. benzyl or aromatic e.g. phenyl, groups; any 
two together with the nitrogen atom if desired forming a heterocyclic ring 
which may be interrupted by one or more further hetero-atoms. Examples of 
such compounds include N-methylaniline, piperidine morpholine, etc. 
##STR13## 
in which n is 0 or an integer from 1 to 5 and R.sup.d, which when n is 
from 2 to 5, may be the same or different, is an aliphatic, e.g. lower 
alkyl such as methyl, ethyl, n-propyl, iso-propyl etc; an aryl e.g. 
phenyl; an araliphatic, e.g. phenyl lower alkyl such as benzyl, 
phenylethyl etc; or an alkoxymethyl e.g. methoxymethyl, ethoxymethyl, 
n-propoxymethyl, iso-propoxymethyl etc; or acyloxymethyl e.g. 
alkanoyloxymethyl such as acetoxymethyl; formyl; carbamoyl; acyloxy e.g. 
alkanoyloxy such as acetoxy; esterified carboxyl; alkoxy e.g. methoxy, 
ethoxy, n-propoxy, iso-propoxy etc; aryloxy e.g. phenoxy; aralkoxy e.g. 
benzyloxy; alkylthio e.g. methylthio, ethylthio; arylthio; aralkylthio; 
cyano; hydroxy; N-monoloweralkylcarbamoyl e.g. N-methylcarbamoyl, 
N-ethylcarbamoyl etc; N,N-diloweralkylcarbamoyl e.g. 
N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl etc; N-(hydroxyloweralkyl) 
carbamoyl e.g. N-(hydroxymethylcarbamoyl, N-(hydroxyethyl)carbamoyl etc; 
or carbamoylloweralkyl e.g. carbamoylmethyl, carbamoylethyl etc. group. 
in which R.sup.d is as defined in (b) and m is 0 or an integer from 1 to 
4, 
##STR14## 
in which R.sup.d and m are as defined in (c), 
##STR15## 
in which R.sup.d and m are as defined in (c), 
##STR16## 
in which R.sup.d is as defined in (b), p is 0 or an integer from 1 to 3, 
and R.sup.e is an aliphatic, araliphatic, aryl, or acyl radical or a 
hydrogen atom. 
##STR17## 
in which R.sup.d, R.sup.e and p are as defined in (f), 
##STR18## 
in which R.sup.d, R.sup.e and p are as defined in (f), 
##STR19## 
in which R.sup.d and R.sup.e are as defined in (f) and q is 0, 1 or 2, 
##STR20## 
in which R.sup.d and q are as defined in (i) 
##STR21## 
in which R.sup.d and p are as defined in (f), and in which R.sup.d and p 
are as defined in (f). 
(m) azides e.g. alkali metal azides. 
CARBON NUCLEOPHILES 
Examples of "carbon nucleophiles" include inorganic cyanides, pyrroles and 
substituted pyrroles, e.g. indoles, and compounds giving stabilised 
carbanions, for example, acetylenes and compounds having .beta.-diketone 
groups, for example, acetoacetic and malonic esters and 
cyclohexane-1,3-diones or enamines, ynamines or enols. 
Thus the term "carbon nucleophile" includes compounds of the following 
formulae: 
EQU M.sup.v+ (CN).sub.v.sup.- (a') 
in which M is a metal cation, preferably an alkali metal or alkaline earth 
metal cation or a quaternary ammonium ion, and v is the valency of the 
cation. 
##STR22## 
in which R.sup.j is an aliphatic, araliphatic or aryl group or an 
esterified carboxy, acyloxy or acyl group, p is 0 or an integer from 1 to 
3, and R.sup.k is an alkyl, aralkyl, or aryl group or a hydrogen atom, at 
least one of the .beta.-positions being unsubstituted, 
##STR23## 
in which R.sup.j and R.sup.k are as defined in (b')and n is 0 or an 
integer from 1 to 5, the 3-position being unsubstituted, (d') 
EQU (R.sup.g -- C.vertline.C.sup.-).sub.v M.sup.v+ 
in which R.sup.g is an aliphatic, araliphatic or aryl group or a hydrogen 
atom, and M and v are as defined in (a') 
##STR24## 
in which the groups R.sup.h, which may be the same or different, are 
hydrogen atoms or alkyl, aralkyl or aryl groups and R.sup.m is an alkyl, 
aralkyl, aryl, alkoxy, aralkoxy or aryloxy group. 
##STR25## 
where R is an electron donating group or atom and n is 0 or an integer of 
from 1 to 5. 
SULPHUR NUCLEOPHILES 
Examples of "sulphur nucleophiles" include thiourea and aliphatic, 
aromatic, araliphatic, alicyclic and heterocyclic substituted thioureas; 
dithiocarbamates; aromatic, aliphatic and cyclic thioamides, for example 
thioacetamide and thiosemicarbazide; thiosulphates; thiols; thiophenols; 
thioacids, e.g. thiobenzoic acid or thiopicolinic acid; and dithioacids. 
Thus the term "sulphur nucleophile" includes compounds of the formulae: 
EQU R.sup.1 R.sup.2 N -- CS -- NR.sup.3 R.sup.4 ( a") 
in which R.sup.1, R.sup.2, R.sup.3 and R.sup.4, which may be the same or 
different may each represent a hydrogen atom, an aliphatic e.g. lower 
alkyl such as methyl, ethyl, n-propyl etc. group; an alicyclic e.g. 
cyclohexyl, cyclopentyl etc. group; an aromatic e.g. phenyl, naphthyl etc. 
group; an araliphatic e.g. benzyl group; or a heterocyclic group; or 
R.sup.1 and R.sup.3 together form a divalent group. R.sup.2 or R.sup.4 may 
alternatively be a group --NR.sup.1 R.sup.3 where R.sup.1 and R.sup.3 are 
as defined above. 
EQU HR.sup.q.CS.NR.sup.r R.sup.s (b") 
in which R.sup.q is a straight or branched chain aliphatic or araliphatic 
group, and R.sup.r and R.sup.s, which may be the same or different, may 
each represent a hydrogen atom or an aliphatic group e.g. lower alkyl such 
as methyl, ethyl, n-propyl etc. group; an araliphatic e.g. benzyl group; 
an acyl e.g. a lower alkanoyl such as acetyl etc. group or an aryl e.g. 
phenyl, naphthyl etc. group. 
##STR26## 
in which M is a metal cation, preferably an alkali or alkaline earth metal 
cation, or a quaternary ammonium ion, and v is the valency of the cation. 
EQU R.sup.1.S(O).sub.n H (d") 
in which R.sup.1 has the above defind meaning, e.g. lower alkyl, and n is 
0, 1 or 2. A preferred class of nucleophiles falling within the above 
formula (d") are those having the general formula 
EQU R.sup.a SH 
in which R.sup.a is an aliphatic e.g. lower alkyl e.g. methyl, ethyl, 
n-propyl etc.; araliphatic, e.g. phenyl lower alkyl e.g. benzyl, 
phenylethyl etc. or substituted phenyl lowe alkyl; alicylic e.g. 
cycloalkyl e.g. cyclopentyl or cyclohexyl; aromatic e.g. phenyl or 
substituted phenyl or heterocyclic group e.g. 
5-methyl-1,3,4-thiadiazol-2-yl. 
OXYGEN NUCLEOPHILES 
Examples of oxygen nucleophiles include water, alcohols, for example 
alkanols such as methanol, ethanol, propanol and butanol and lower 
alkanoic acids. Water furnishes both H.sub.2 O: and OH.sup.- and is thus a 
competitor nucleophile in any reaction occurring in aqueous medium. 
The term "oxygen nucleophile" thus includes compounds of the following 
formula: 
EQU R.sup.t (CO).sub.n OH 
in which the group R.sup.t may be lower alkyl (e.g. methyl, ethyl, 
n-propyl, isopropyl, n-butyl, isobutyl etc.); lower alkenyl (e.g. allyl); 
lower alkynyl (e.g. propynyl, etc.); lower cycloalkyl (e.g. cyclopentyl, 
cyclohexyl, etc); lower cycloalkyl lower alkyl (e.g. cyclopentylmethyl, 
cyclohexylethyl etc.); aryl (e.g. phenyl or naphthyl); aryl lower alkyl 
(e.g. benzyl); heterocyclic; heterocyclic lower alkyl (e.g. furfuryl) or 
any of these groups substituted by, for example, one or more of lower 
alkoxy (methoxy, ethoxy, etc.), lower alkylthio (methylthio, ethylthio, 
etc), halogen (chlorine, bromine, iodine or fluorine), lower alkyl 
(methyl, ethyl etc), nitro, hydroxy, acyloxy, carboxy, carbalkoxy, lower 
alkylcarbonyl, lower alkylsulphonyl, lower alkoxysulphonyl, aino, lower 
alkylamino or acylamino groups; and n is 0 or 1. Where the oxygen 
nucleophile is an acid this will generally be employed as a salt with an 
inorganic or organic base. Such salts include alkali metal e.g. sodium or 
potassium or trialkylammonium e.g. triethylammonium. 
Reactions with nucleophiles, particularly oxygen nucleophiles, may be 
facilitated by the presence of a salt of mercury, silver or gold, 
preferably mercury. We particularly prefer to use mercuric (Hg.sup.++) 
salts. The efficacy of the reaction is also dependent on other factors 
including the nature of the anion of the salt, the type of cations which 
it produces in aqueous solution and the solubility of the salt in the 
reaction medium which, whenever the nucleophile is a lower alkanol, is 
conveniently the latter itself. 
The metal salt is advantageously one of the formula HgD.sub.2 or HgD which 
furnishes Hg.sup.++ and/or HgD.sup.+ cations, preferably the former, in 
aqueous solution, D.sup.- being a weakly nucleophilic anion; a like-acting 
salt Hg.sub.n E.sub.2 of mercury with a di- or polyvalent anion where E is 
an n-valent anion, n being 2 or greater, or a salt of the formula Ag.sub.m 
F where F is an m-valent anion of a weakly nucleophilic nature and m is 1 
or greater. 
The anion of the salt should be substantially non-oxidising to compound 
(IV) under the conditions of the reaction and should preferably be an 
anion of a strong acid, i.e. an acid having a pKa value in aqueous 
solution of less than 2, to facilitate formation of the desired cations. 
Nucleophilic properties in the anion may complete with those in the chosen 
nucleophile; therefore it is desirable that the anion having a 
nucleophilic constant less than that of the acetate ion for conventional 
one-step nucleophilic displacement in aqueous media at a tetrahedral 
carbon centre (see, for example, Hine's "Physical Organic Chemistry" 
McGraw-Hill, 1962 pp 159-161). Mercuric salts with anions of nucleophilic 
constant less than acetate generally promote fast reactions of the 
required type. Mercuric and silver salts with the attributes described 
above include the perchlorate, nitrate, trifluoroacetate and 
tetrafluoroborate. Mercurous perchlorate also possesses the desired 
properties. 
The metal salt will normally be used in an amount at least equivalent to 
the compound of formula (IV). 
REACTION CONDITIONS FOR THE DISPLACEMENT OF X BY THE NUCLEOPHILE 
The displacement of X in compounds of formula (IV) by the nucleophile may 
conveniently be effected by maintaining the reactants in solution or 
suspension at a moderate temperature, e.g. from -40.degree. to 
+120.degree. C. such as from 0.degree. to +120.degree. C. preferably from 
-20 to +35.degree. C. e.g. from 0 to +35.degree. C. advantageously at 
ambient temperature. Reactions are usually complete for the replacement of 
bromine by pyridine in N,N-dimethylformamide in about 2 hours at 
20.degree. C. and in correspondingly longer times at lower temperatures or 
correspondingly shorter times at higher temperatures. 
The nucleophile displacement reactions may be facilitated by the addition 
of an acid acceptor such as an organic base which promotes the formation 
of the nucleophile anion in the form of a salt. Suitable organic bases 
include tri (lower alkyl)amines e.g. triethylamine. However, reactions 
with tertiary nitrogen nucleophiles in general do not require an acid 
acceptor. 
The reaction is advantageously effected using from one to ten molar 
equivalents of incoming nucleophile. The pH value of the reaction solution 
under aqueous conditions is advantageously maintained within the limits 5 
- 8. When working under non-aqueous conditions, the reaction medium should 
be neither extremely basic nor extremely acidic. 
Organic solvents such as dioxan, ethyl acetate, formamide, 
N,N-dimethylformamide or acetone may be employed. The organic solvents may 
be used in the presence or absence of water. In certain cases the 
nucleophile itself may be the solvent e.g. when the nucleophile is 
pyridine or a lower alcohol. 
Organic media whic may be used include lower alkanoic acid nitriles e.g. 
acetonitrile or propionitrile; halogenated hydrocarbons e.g. methylene 
chloride, chloroform, ethylene dichloride or perchloroethylene; 
hydrocarbons e.g. benzene; cyclic ethers e.g. dioxan or tetrahydrofuran; 
amides of the general formula R.sup.5.CO.NR.sup.6 R.sup.7 where R.sup.5 is 
a hydrogen atom or an alkyl group containing 1 to 5 carbon atoms and 
R.sup.6 and R.sup. 7, which may be the same or different, are each a 
hydrogen atom or an alkyl group containing 1 to 5 carbon atoms, or, 
alternatively R.sup.6 and R.sup.7 together form a divalent aliphatic group 
which, together with the adjacent nitrogen atom, forms a heterocyclic 
ring. Examples of amides of this type are N,N-dimethylformamide, 
N,N-diethylformamide, N,N-dimethylacetamide, formamide and 
N-methylformamide. Other solvents which may be used include N-lower alkyl 
pyrrolidones e.g. N-methylpyrrolidone and di-lower alkyl sulphoxides, e.g. 
dimethylsulphoxide. 
The reaction medium need not be liquid at room temperature. Solids, e.g. 
acetamide, may be used so long as they are liquid at the reaction 
temperature. 
The reaction product may be separated from the reaction mixture, which may 
contain, for example, unchanged cephalosporin and other substances, by a 
variety of processes including recrystallization, ionophoresis, paper 
chromatogrphy or by chromatography on ion-exchange resins. 
After the introduction of the desired nucleophilic group the 1-sulphinyl 
group may be reduced by any convenient means. This may, for example, be 
effected by reduction of the corresponding acyloxysulphonium or 
alkyloxysulphonium salt prepared in situ by reaction with e.g. acetyl 
chloride in the case of an acetoxy-sulphonium salt, reduction being 
effected by, for example, sodium dithionite or by iodide ion as in a 
solution of potassium iodide in a water miscible solvent e.g. acetic acid, 
tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The 
reaction may be effected at a temperature of -20.degree. to +50.degree. C. 
Alternatively, reduction of the 1-sulphinyl group may be effected by 
phosphorus trichloride or tribromide in solvents such as methylene 
chloride, dimethylformamide or tetrahydrofuran, preferably at a temprature 
of -20.degree. to +50.degree. C. 
It will be observed from the foregoing that the .DELTA..sup.3 unsaturation 
has not been subject to isomerisation throughout the sequence of reactions 
described. This is an important feature of the invention. 
Alternatively, the compound of general formula (IV) may be first reduced in 
like manner to form a compound of general formula: 
##STR27## 
wherein R.sup.1, R.sup.2 and X have the above defined meanings, which may 
then be reacted with a nucleophile as directed above. This step may 
however give rise to .DELTA..sup.3 .fwdarw..DELTA..sup.2 isomerisation. 
Where the group R.sup.2 is the residue of an alcohol or phenol this may be 
removed at any convenient stage of the synthesis by the methods indicated 
above. Protection of the 4-carboxyl may be required in some of the stages 
and the exact point of removal will depend on this factor. Should the 
group R.sup.2 be removed during a specific reaction it may be necessary to 
re-esterify the carboxyl group if protection is subsequently necessary. 
When the group R.sup.2 is removed after the introduction of a nitrogen 
nucleophile, particularly a nitrogen nucleophile of (b) above, the 
resulting compound may conveniently be recovered as a quaternary ammonium 
salt e.g. the hydronitrate. The resulting salt may then be converted to 
the free betaine by the methods described in British Pat. Nos. 1,101,561 
or 1,101,562. 
Where at any stage the product of a 7.beta.-acylamido compound not having 
the desired acyl group, the 7.beta.-acylamido compound may be N-deacylated 
to yield the corresonding 7.beta.-amino compound and the latter acylated 
with an appropriate acylating reagent. 
Suitable methods of N-deacylating cephalosporin derivatives having 
7.beta.-acylamido groups are described in British Pat. Nos. 1,041,985 and 
1,119,806; Belgian Pat. No. 719,712 and in South African Pat. 
Specifications Nos. 68/5048 and 68/5327. Another method of N-deacylation 
which may be used is acid catalysis. For example, N-deformylation of a 
7.beta.-formamido group may be effected with a mineral acid at a 
temperature of minus 15.degree. to +100.degree. C. preferably +15 to 
40.degree. C. A convenient reagent for the N-deformylation is concentraed 
hydrochloric acid in methanol, dioxan or tetrahydrofuran. Alternativey 
N-deformylation may be effected with the aid of a Lewis acid in a lower 
alkanol or a lower alkane diol, under substantially anhydrous conditions. 
N-deformylation under such substantially anhydrous conditions may be 
effected at a temperature of from -40.degree. to +100.degree. C. 
advantageously at from -20.degree. to +70.degree. C. 
The 7.beta.-amino compound may be separated as an insoluble salt e.g. a 
hydrochloride or a hydrogen p-toluene sulphonate or it may be precipitated 
by adjustment of the pH (e.g. to an isoelectric point), if necessary by 
extraction with a suitable solvent. The 7.beta.-amino compound may then be 
reacylated. Reacylation can then be effected with the acylating agent of 
choice. A wide variety of acylating agents for use in the cephalosporin 
field have been described in the literature. 
When the 7.beta.-acylamido group contains an amino group it will be 
necessary to protect this during the various reaction stages. The 
protecting group is conveniently one which can be removed by hydrolysis 
without affecting the rest of the molecule, especially the lactam and 
7.beta.-amido linkages. The amine protecting group and the esterifying 
group at the 4--COOH position can be removed using the same reagent. An 
advantageous procedure is to remove both groups at the last stage in the 
sequence. Protected amine groups include urethane, arylmethyl (e.g. 
trityl) amino, or sulphenylamino types. Such groups can in general be 
removed by one or more reagents selected from dilute mineral acids e.g. 
dilute hydrochloric acid, concentrated organic acids, e.g. concentrated 
acetic acid, trifluoroacetic acid, and liquid hydrogen bromide at very low 
temperatures, e.g. -80.degree. C. A convenient protecting group is the 
t-butoxycarbonyl group, which is radily removed by hydrolysis with dilute 
mineral acid, e.g. dilute hydrochloric acid, or preferably with a strong 
organic acid (e.g. formic acid or trifluoroacetic acid) e.g. at a 
temperature of 0-40.degree. C., preferably at room temperature 
(15-25.degree. C.). Another convenient protecting group is the 
2,2,2-trichloroethoxycarbonyl group which may be split off by an agent 
such as zinc/acetic acid, zinc/formic acid, zinc/lower alcohols or 
zinc/pyridine. 
According to one embodiment of the invention the reactions described above 
may be proceeded with via the following sequence of steps wherein R.sup.1 
and R.sup.2 have the above defined meanings, R.sup.10 CO is the final acyl 
group and Y is the residue of the nucleophile. 
##STR28## 
After the formation of the --CH.sub.2 Br group, the bromine may be 
exchanged for iodine or chlorine to yield the analogous --CH.sub.2 I or 
CH.sub.2 Cl compounds. The latter may then be subjected to the 
nucleophilic reaction, similar considerations also applying to the 
reaction schemes set out below. 
An advantageous embodiment of the process according to the invention 
involves the following sequence of reactions: 
##STR29## 
wherein X, R.sup. 2, R.sup.d and n have the above defined meanings, and 
R.sup.10 CO is the final acyl group. 
The above sequence is advantageous for a number of reasons: (1) step A 
involving the bromination of the 3-methyl group, followed if desired by 
halogen transfer, can be effected without substantial attack of the 
formamido group which might be the case with R.sup.10 CO e.g. when it is 
2-thienylacetyl; (2) step B involving N-deformylation can be effected 
readily; (3) step C involving the introduction of the final acyl group 
R.sup.10 CO is effected before step D which might otherwise be 
disadvantageous and (4) retention of the oxide group until step D is 
complete minimises .DELTA..sup.3 .fwdarw..DELTA..sup.2 isomerisation which 
would otherwise tend to occur in the presence of the pyridine nucleophile. 
By proceeding in this manner we are able to arrive at a satisfactory route 
to the compounds of general formula (VIII) defined below. 
Another advantageous embodiment of the process according to the invention 
involves the following sequence of reaction steps: 
##STR30## 
The sequence with steps A, B.sup.1 and D.sup.1 affords similar advantages 
to that described for the earlier sequence with steps A, B and D. 
The present invention provides a significant, alternative route to those 
already known for preparing compounds of formula (VIII) below. 
Compounds of the general formula 
##STR31## 
where R.sup.10 CO is an acyl group and Y is the residue of a nucleophile 
and their salts in general possess antibacterial activity. 
Important members of formula (VIII) which may be prepared by the process 
according to the invention include cephaloram, cephalothin, cephaloridine, 
cefazolin, cephaloglycine, 
7.beta.-(D-2-amino-2-phenylacetamido)-3-methylthiomethylceph-3-em-4-carbox 
ylic acid, 
7.beta.-(D-2-amino-2-phenylacetamido)-3-methoxymethylceph-3-em-4-carboxyli 
c acid and 
N-[7.beta.-(2-thienylacetamido)ceph-3-em-3-ylmethyl]-4-(N-hydroxymethylcar 
bamoyl)pyridinium-4-carboxylate. 
In order that the invention may be well understood the following 
Preparations and Examples are given by way of illustration only. In the 
Preparations and Examples, unless otherwise stated, 
(1) ultra-violet (UV) spectra were measured onsolutions in ethanol, 
(2) infra-red (IR) spectra were measured on mulls in Nujol, 
(3) proton magnetic resonance (PMR) spectra were determined at 60 or 100 
MHz as 5-10% solutions in dimethylsulphoxide-d.sub.6. 
The signs of coupling constants (J) are not assigned. Signals are assigned 
as singlets (s), doublets (d), double doublets (dd), triplets (t), 
AB-quartets (AB-q) or multiplets (m). 
(4) optical rotations were determined at 19.degree. to 30.degree. at 
concentrations in the range 0.8 to 1.2% as solutions in 
dimethylsulphoxide, 
(5) solutions were dried over anhydrous magnesium sulphate, 
(6) light petroleum was a fraction, b.p. 60.degree.-80.degree., 
(7) all grades of kieselgel G were supplied by Merck AG, Darmstadt, 
Germany, 
(8) methylene chloride was dried by passage through basic alumina (Woelm, 
activity I); N,N-dimethylformamide was dried by distillation over acidic 
alumina (Woelm, activity I). 
(9) paper electrophoresis was performed on Whatman No. 3 MM paper at 30 
v/cm in pH 1.9 buffer consisting of formic acid (16.7 ml, 98%), acetic 
acid (84 ml), acetone (105 ml) and water (495 ml) or where indicated with 
a dilution of this buffer to pH 2.2 with four volumes of water. Spots were 
located by visual examination with a Hanovia "Chromatolite" ultraviolet 
lamp. R.sub.c values represent movement with respect to 
N-[7.beta.-(2-thienylacetamido) ceph-3-em-3-ylmethyl] 
pyridinium-4-carboxylate (cephaloridine), R.sub.c 1.00, as standard; 
vitamin B.sub.12 served as an uncharged marker.