Process for the preparation of 3-hydroxymethyl cephalosporins

A process for the preparation of a 3-hydroxymethyl cephalosporin compound which comprises selectively hydrolyzing a 3-formyloxymethyl cephalosporin compound under acidic conditions in the presence of a protic solvent whereby a 3-hydroxymethyl cephalosporin is produced.

This invention relates to the preparation of cephalosporin compounds and is 
particularly concerned with the preparation of 3-hydroxymethyl 
cephalosporins. 
The cephalosporin compounds referred to in this specification are generally 
named with reference to "cepham" (J. Am. Chem. Soc. 1962, 84, 3400). The 
term "cephem" refers to the cepham structure with one double bond. 
3-Hydroxymethyl cephalosporin compounds are valuable intermediates in the 
synthesis of a range of cephalosporin antibiotics possessing substituted 
methyl groups at the 3-position by virtue of the chemical reactivity of 
the hydroxyl group and the consequent ease with which the hydroxymethyl 
group may be converted to a desired 3-(substituted methyl) group. In 
particular they are of value in the manufacture of 3-carbamoyloxymethyl 
and 3-(N-substituted)carbamoyloxymethyl cephalosporin antibiotics. 
The preparation of 3-hydroxymethyl cephalosporin compounds has attracted a 
great deal of attention not only because of the value of such compounds 
but also because of the difficulty in making them. 
Thus, hydrolysis of 3-acyloxymethylceph-3-em-4-carboxylic acids and their 
esters to their 3-hydroxymethyl analogues by chemical methods has proved 
to be generally impractical. Base catalysed hydrolysis may lead to opening 
of the .beta.-lactam ring and acid catalysed hydrolysis may be accompanied 
by rapid and substantially irreversible lactonisation involving reaction 
of the 3-hydroxymethyl and 4-carboxy groups. 
In British Pat. No. 1,474,519 we have described how esterases obtained from 
yeast microorganisms of the genus Rhodotorula and mutants thereof promote 
hydrolysis of 3-acyloxymethylceph-3-em-4-carboxylic acids to their 
3-hydroxymethyl analogues in good yield. However this enzymatic hydrolysis 
is only effective with cephalosporin compounds having a free carboxy group 
in the 4-position whereas it is frequently desired to prepare 
3-hydroxymethyl cephalosporin compounds possessing a carboxylate ester 
group in the 4-position. 
There is thus a need for alternative sources and methods of obtaining 
3-hydroxymethyl cephalosporin compounds. 
We have now found that 3-formyloxymethyl cephalosporin compounds may be 
converted to 3-hydroxymethyl cephalosporin compounds in good yield. This 
finding is important because 3-formyloxymethyl cephalosporins may in turn 
be derived in a multi-stage process from penicillin compounds. 
Consequently one is afforded not only a new method of making 
3-hydroxymethyl cephalosporin compounds but also an alternative source to 
existing methods relying on fermentation produced 3-acetoxymethyl 
cephalosporin compounds. 
This multi-stage process may be shown schematically as follows: 
##STR1## 
where R is a blocked amino group, preferably a C.sub.1 -C.sub.20 acylamido 
groups, Z is &gt;S or &gt;S.fwdarw.O, R.sup.1 is a carboxyl blocking group and 
the dotted line bridging the 2-, 3- and 4-positions indicates that the 
compound may be a ceph-2-em or ceph-3-em compound). 
If Z = &gt;S.fwdarw.O the end-product is obtained as a ceph-3-em compound for 
direct use in subsequent reactions since the multi-stage process does not 
produce any substantial quantity of ceph-2-em isomers. If Z = S, the 
.DELTA..sup.3 product may be accompanied by some of its .DELTA..sup.2 
isomer. In this event, or if the use of .DELTA..sup.2 intermediates is 
desired, .DELTA..sup.3 compounds may subsequently be produced by standard 
methods of sulphoxidation and reduction. In general, therefore, Z is 
preferably &gt;S.fwdarw.O. 
According to one embodiment of the invention there is provided a process 
for the preparation of a 3-hydroxymethyl cephalosporin compound which 
comprises selectively hydrolysing a 3-formyloxmethyl cephalosporin 
compound under acidic conditions in association with a protic solvent, 
preferably in the presence of an inert diluent, and advantageously at a 
temperature within the range of 0.degree.-50.degree. C., preferably 
15.degree.-40.degree. C. 
Any functional groups in the 3-formyloxymethyl cephalosporin may be 
protected during the hydrolysis reaction e.g. as is indicated in the above 
schematic representation of the multi-stage process. 
The hydrolysis is advantageously effected using an acid having a pKa of 
less than 4, such as sulphuric, phosphoric, perchloric, hydrochloric, 
trifluoroacetic, p-toluene sulphonic, hydrobromic, nitric, formic or 
oxalic acid or an alkylphosphoric acid (which may, for example, be formed 
in situ by reaction of phosphorus pentoxide and an alkanol). The acid is 
preferably employed in a sufficient amount to maintain the pH of the 
reaction mixture at less than 4, advantageously at a pH of 1 to 3. 
The protic solvent may be water or a lower alkanol. Preferably water is 
employed. 
Any inert organic diluent used is preferably miscible with the protic 
solvent(s) and is preferably one in which the 3-formyloxy methyl 
cephalosporin is soluble. The diluent is conveniently an organic diluent, 
preferably dimethyl formamide but examples of other diluents which may be 
employed include tetrahydrofuran, dioxan, acetone, methanol, 
dimethylacetamide, dichloromethane, chloroform, industrial methylated 
spirits, ethylene glycol and ethyl acetate as well as mixtures of two or 
more thereof. 
The hydrolysis is advantageously effected at room temperature using an 
aqueous hydrochloric acid/dimethylformamide system. 
The reaction may be monitored by thin layer chromatography and the 
3-hydroxymethyl cephalosporin product of formula V may be isolated for 
example by extraction or precipitation. Purification may be effected for 
example by crystallisation, by precipitation or by column chromatography 
on a suitable adsorbent e.g. silica gel of 60-200 mesh size. 
The compounds of general formula (IV), i.e. 3-formyloxymethyl starting 
compounds, may be prepared in an analogous manner to that described in 
British Pat. No. 1326531, the halogen of a 3-halomethyl cephalosporin 
being replaced by a formate anion in a nucleophilic displacement reaction. 
Sources of the formate anion may be inorganic or organic formate salts e.g. 
sodium formate or triethylammonium formate, and the displacement may be 
carried out in an inert solvent system e.g. acetone or tetrahydrofuran, if 
necessary with added water to aid the solubility of any ionic reagents. 
Heavy metals, such as silver and mercury, may also be present to promote 
the reaction. 
The source of the formate group may also be a substituted formamide e.g. 
N,N-dimethylformamide, which may react directly with the halomethyl 
compound if desired in the presence of a catalyst such as a mild base 
(e.g. NaHCO.sub.3) or an inorganic salt (e.g. Na.sub.2 SO.sub.3). When the 
3-formyloxymethyl cephalosporin is prepared in this way, it may be 
hydrolysed in situ in accordance with this invention, for example, by 
adding an aqueous acid. 
According to a preferred embodiment of the present invention we provide a 
process for the preparation of a 3-hydroxymethyl cephalosporin compound of 
formula 
##STR2## 
(wherein R is a blocked amino group, preferably a C.sub.1 -C.sub.20 
acylamido group, Z is &gt;S or &gt;S.fwdarw.O, R.sup.1 is a carboxyl blocking 
group and the dotted line bridging the 2-, 3- and 4-positions indicates 
that the compound may be a ceph-2-em or ceph-3-em compound) in which a 
3-formyloxymethyl cephalosporin compound of formula 
##STR3## 
(wherein R, Z, R.sup.1 and the dotted line are as defined above) is 
selectively hydrolysed, whereafter, if desired in each instance, any of 
the following reactions are carried out: 
(i) conversion of a .DELTA..sup.2 isomer into the corresponding 
.DELTA..sup.3 isomer 
(ii) reduction of a compound in which Z is &gt;S.fwdarw.O to form a compound 
in which Z is &gt;S; and 
(iii) removal of any carboxyl blocking groups. 
The acyl moiety of R may be selected from the extensive lists of such acyl 
groups in the penicillin and cephalosporin literature. 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, 
cycloalkadienyl, or a non-aromatic heterocyclic or mesoionic group and n 
is 0 or an integer from 1-4. Examples of this group include phenylacetyl; 
thien-2- and -3-ylacetyl, 3- and 4-isoxazolylacetyl both substituted or 
unsubstituted; pyridylacetyl, tetrazolylacetyl or a sydnoneacetyl group. 
Where n is other than 0, especially when n is 1, the .alpha.-carbon atom 
of the acyl group may be substituted by, for example, a hydroxy, 
esterified hydroxy (e.g. lower alkanoyloxy such as acetoxy), blocked amino 
(e.g. amino substituted by any of the blocking groups specified 
hereinafter), hydroxyimino, acyloxyimino (e.g. lower alkanoyloxyimino such 
as acetoxyimino or halo-substituted lower alkanoyloxyimino such as mono- 
or dichloroacetoxyimino) or etherified oxyimino (e.g. lower alkoxyimino 
such as methoxyimino or t-butoxyimino, lower cycloalkyloxyimino such as 
cyclopentyloxyimino or aryloxyimino such as phenoxyimino) group; examples 
of .alpha.-substituted acyl groups of this type include 
2-hydroxy-2-phenylacetyl, N-blocked 2-amino-2-phenylacetyl and 
2-(fur-2-yl)-2-hydroxyiminoacetyl. 
(ii) C.sub.n H.sub.2n+1 CO-- where n is 0 or an integer from 1-7. The alkyl 
group may be straight or branched and may be substituted by e.g. a cyano 
group, a carboxy group, an alkoxycarbonyl group, a hydroxy group, a 
blocked amino group or a carboxycarbonyl group (--CO.COOH) or any such 
group in which the functional group is blocked. Examples of such groups 
include formyl, glutaroyl, and N-blocked (e.g. N-ethoxycarbonyl) 
R-5-amino-5-carboxypentanoyl. 
(iii) 
##STR4## 
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 and Z is an 
oxygen or sulphur atom. Examples of this group include phenoxyacetyl or 
pyridylthioacetyl. 
The carboxyl blocking group R.sup.1 is desirably a group which may readily 
be split off at a suitable stage. It may, however, be convenient in some 
instances to employ biologically acceptable, metabolically labile carboxyl 
blocking groups and retain these in the final product. 
Suitable carboxyl blocking groups are well known in the art, a list of 
representative blocked carboxyl groups being included in Belgian Patent 
No. 783,449. Preferred blocked carboxyl groups include aryl lower 
alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl 
groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such 
as 2,2,2-trichloroethoxycarbonyl. The carboxyl blocking group may 
subsequently be removed by any of the appropriate methods disclosed in the 
literature; for instance by acid catalysis (e.g. with anhydrous 
trifluoroacetic acid) or by reduction (e.g. by hydrogenation) or by 
enzymically-catalysed hydrolyses. 
In order that the invention may be well understood the following examples 
are given by way of illustration. 
All temperatures are quoted in .degree. C. Melting points were measured in 
capillary tubes, the values are uncorrected. 
Petrol is petroleum b.p. 40.degree. to 60.degree.. 
T.l.c. is thin-layer chromatography using pre-coated plates (Merck 
F.sub.254, 0.25mm thick coating) which were examined under ultraviolet 
light at 254nm and were developed with iodine.

EXAMPLE 1 
(a) 
Diphenylmethyl(1S,6R,7R)-3-Bromomethyl-7-phenoxyacetamidoceph-3-em-4-carbo 
xylate, 1-oxide 
A cooled (-9.degree.) solution of diphenylmethyl 
(1S,6R,7R)-3-methyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 1-oxide 
(9.70mmole) in dichloroethane (250ml) was stirred with 
1,3-dibromo-5,5-dimethylhydantoin (2.08g, 7.28mmole) under a nitrogen 
atomsphere. 
The mixture was irradiated with ultra-violet light (from a 125 watt Hanovia 
mercury arc lamp) at -9.degree. for 1 hour, filtered, washed with 3% 
sodium bicarbonate solution (2.times.100ml) and water (50ml), then dried 
and concentrated giving the crude title compound (2.87g) as a solid, 
[.alpha.].sub.D - 53.degree. (c 0.94, DMSO), .lambda..sub.max (EtOH) 
268.5nm (.epsilon. 9,255), 274.5 (.epsilon. 9,930) with inflection at 
264(.epsilon. 8,045) and 280nm (.epsilon. 9,260) respectively. 
Chromatography of the filtrate afforded a further crop of the title 
compound (348mg). 
(b) Diphenylmethyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate 
1-oxide 
A suspension of sodium sulphite heptahydrate (103mg, 0.41mmole) in N,N 
dimethylformamide (1ml) was treated with urea (100mg) but the sodium 
sulphite did not dissolve appreciably. 
Diphenylmethyl(1S,6R,7R)-3-bromoethyl-7-phenoxyacetamidoceph-3-em-4-carboxy 
late, 1-oxide (244mg, 0.40mmole) was added to the above mixture which was 
stirred at 20.degree. for 24 hours and allowed to stand at 20.degree. for 
a further 21/2 days by which time t.l.c. showed complete reaction. 
The stirred solution (containing very little residue) was diluted with 
water (20ml) and the mixture was stirred at 20.degree. for 30 minutes, the 
resultant brown precipitate was filtered off and washed with water and 
dried in vacuo to give the crude title compound (151mg), m.p. 122 to 
140.degree. [.alpha.].sub.D.sup.22 +36.degree. (c 0.64, CHCL.sub.3), 
.lambda..sub.max (EtOH) 269nm (E .sub.1cm.sup.1% 138, .epsilon. 7,940) and 
274.5nm (E.sub.1cm.sup.1% 122, .epsilon. 7,020) with an inflection at 
266nm (E.sub.1cm.sup.1% 132, .epsilon.7,600). 
(c) Diphenylmethyl 
(1S,6R,7R)-3-hydroxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide 
To a warmed (&lt;30.degree.) stirred suspension of diphenylmethyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide (400mg, 0.7mmole) in methanol (40ml) was added perchloric acid 
(60%; 0.1ml, 0.9mmole). The mixture was stirred at 25.degree. for 45 
minutes then dichloromethane (12ml) was added and the solution was stirred 
at 25.degree. for 5 minutes then partitioned between dichloromethane and 
water. The organic layer was separated and washed with water and dried 
(sodium sulphate) and evaporated in vacuo to a brown residue (382 mg) 
which, on trituration with ether-petrol (1:2) gave the crude title 
compound (312mg), as an amorphous orange solid, m.p. 92 to 150.degree. C., 
[.alpha.].sub.D.sup.22 +36.degree.(CHCL.sub.3, c 0.49). 
A sample of the crude title compound (150mg) was subjected to column 
chromatography on silica-gel (Crosfield 60 to 200 mesh, 10g); elution was 
with ethyl acetate and 15ml fractions were collected. Fractions 2 to 7 
were combined and evaporated in vacuo to an off-white solid (80mg) which, 
on trituration with ether, afforded the title compound (52mg) as an 
off-white solid, m.p. 181 to 184.degree. [.alpha.].sub.D.sup.22 + 
19.5.degree. (CHCl.sub.3, c 0.41) .lambda..sub.max (EtOH) 263nm 
(E.sub.1cm.sup.1% 158, .epsilon.8,650) with inflections at 266nm 
(E.sub.1cm.sup.1% 156, .epsilon. 8,500) and 274mn (E.sub.1cm.sup.1% 116, 
.epsilon. 6,350) [Found C, 62.6; H, 4.6; N,5.4. C.sub.29 H.sub.26 N.sub.2 
O.sub.7 S 1/2H.sub.2 O requires C, 62.7; H 4.9; N, 5.05%]. 
EXAMPLE 2 
(a) t-Butyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide 
t-Butyl (1S,6R,7R)-3-bromomethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide (500mg, 1mmole) in acetone (20ml) was treated with formic acid 
(0.25ml) and triethylamine (0.5ml) and the reaction solution was stirred 
at 30.degree. to 40.degree. for 4 hours, and then left to stand at 
20.degree. for 18 hours. The solution was concentrated by evaporation and 
then partitioned between ethyl acetate and water. The organic layer was 
washed with water, and dried over sodium sulphate and evaporated to a 
brown oil (450mg). The oil in ethyl acetate (.about.10ml) was filtered and 
the filtrate was added slowly to well stirred petrol (.about.120ml). The 
precipitate was filtered off and dried in vacuo to give the title compound 
(338mg) as a pale brown solid. [.alpha.]D + 45.degree. (CHCl.sub.3, c 
0.51%), .lambda.inf266nm (.epsilon.9250), .lambda.max 268.5nm 
(.epsilon.9530), .lambda.max 273nm (.epsilon.8460) (ethanol) [Found: C, 
54.25; H, 5.2; N, 5.7; S, 6.9. C.sub.21 H.sub.24 N.sub.2 O.sub.8 S 
requires C, 54.3; H, 5.2; N, 6.1; S, 6.9%]. cl (b) t-Butyl 
(1S,6R,7R)-3-hydroxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide 
t-Butyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide (50mg, 0.11mmole) in methanol (5ml) was treated with 2N 
hydrochloric acid (0.025ml,0.05mmole) and the solution was stirred at 
24.degree. for 2 hours. Propylene oxide (0.1ml) was then added, and the 
mixture was stirred at 24.degree. for 3 minutes and then partitioned 
between ethyl acetate and water. The organic layer was washed with water 
and dried over sodium sulphate and evaporated to an oil (47mg, 100%). A 
solution of the oil in ethyl acetate (2ml) was added slowly to stirred 
petrol (100ml) and the white precipitate was filtered off and dried in 
vacuo to give the title compound (31mg, 66%) as a white solid, 
[.alpha.].sub.D + 56.degree. (CHCl.sub.3,c 0.21%), &gt; 90% pure by t.l.c. 
and n.m.r. 
EXAMPLE 3 
Diphenylmethyl 
(1S,6R,7R)-3-hydroxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide 
Diphenylmethyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide (50mg, 0.087mmole) was suspended in methanol (5ml) and 2N 
hydrochloric acid (0.02ml, 0.04mmole) was added. The suspension was 
stirred at 27.degree. for 3.5 hours, during which time it became a 
homogeneous solution of apparent pH ca 2-3. Propylene oxide (0.1 ml) was 
added, and after two minutes the solution was partitioned between ethyl 
acetate and water. The organic layer was washed with water and dried over 
anhydrous sodium sulphate and evaporated to an oily foam (49mg). A 
solution of the foam in ethyl acetate (2ml) was added to stirred 
40.degree.-60.degree. petroleum ether (100ml) and the precipitate was 
filtered off and dried in vacuo to give the slightly impure title compound 
(28mg) as an off-white solid, [.alpha.].sub.D.sup.22 + 37.degree. 
(CHCl.sub.3, c 0.21%). The product appeared (by t.l.c. and n.m.r.) to 
contain about 10 to 20% of lactone impurity. 
EXAMPLE 4 
Diphenylmethyl (1S,6R,7R)-3- 
hydroxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 1-oxide 
(i) variation of acid catalyst 
Diphenylmethyl 
(1S,6R,7R)-3-formyloxymethyl-7-phenoxyacetamidoceph-3-em-4-carboxylate, 
1-oxide (25mg) in methanol (5ml) was treated with one or two molar 
equivalents (see Table below) of the specified acid at 30.degree.. The 
reaction was judged to be complete when there was only a trace, if any, of 
the 3-formyloxymethyl starting material by thin-layer chromatography 
(t.l.c.), and the ratio of products in the final reaction mixture was 
determined by the t.l.c. (silica plates eluted with ethyl acetate). The 
approximate R.sub.f values of the components were: 3-formyloxymethyl 
starting material: 0.65; title compound: 0.55; and corresponding lactone: 
0.1. The apparent pH of the reaction mixture was determined by spotting an 
aliquot on to moistened universal pH indicator paper. 
TABLE 
______________________________________ 
Amount of 
Molar lactone in 
equiva- Time for final Apparent 
lents of 
Complete reaction 
pH of 
Acid used acid reaction mixture reaction 
______________________________________ 
20% aq. HNO.sub.3 
1 2 hr trace 2 
phosphorus 
pentoxide 
P.sub.2 O.sub.5 
1 2 hr .about. 10% 
2.5 
trichloro- 
acetic acid 
1 19 hr .about. 10% 
3.5 
CCl.sub.3 CO.sub.2 H 
H.sub.3 PO.sub.4 
(orthophos- 
2 48 hr trace 3 - 4 
phoric acid) 
oxalic acid 
(CO.sub.2 H).sub.2 . 2H.sub.2 O 
2 4 days trace 3 - 4 
______________________________________ 
(ii) Variation of solvent 
The same conditions were used as in (i) above except that the specified 
solvent was used in place of methanol; the results are set out in the 
Table below: 
__________________________________________________________________________ 
Molar 
Time for 
Amount of 
lactone in 
Amount of eq. of 
complete 
final App. pH of 
Solvent used 
solvent 
Acid used 
acid 
reaction 
mixture 
reaction 
__________________________________________________________________________ 
1,2-ethanediol 
5 ml 2N hydrochloric 
1 71/2 hr 
.about. 20% 
.about. 3 
acid 
acetone 1 ml " 2 5 hr 
.about. 10% 
1.5 
tetrahydrofuran 
1 ml " 4 4 hr 
.about. 10% 
.about. 1 
N,N-dimethyl- 
1 ml " 4 24 hr 
trace 3 
formamide 
__________________________________________________________________________ 
(iii) Variation of reaction conditions 
The above 3-formyloxymethyl starting material was dissolved in the 
specified solvent or solvent mixture and treated with the specified acid, 
as indicated in the Table below. When t.l.c. indicated complete reation 
[as in (i) above] the product was isolated by the work-up described, and 
its purity was assessed by t.l.c. [as in (i) above]. The n.m.r. spectrum 
of each product agreed with the spectrum of the authentic material, plus 
an amount of lactone roughly in accord with the amount observed by t.l.c. 
The optical rotation of an impure sample of the lactone in chloroform 
(+94.degree.) was considerably higher than that of the authentic 
3-hydroxymethyl compound (+19.5.degree.). The purity of each product could 
therefore be estimated from its rotation, and this estimate was consistent 
with those from the other methods mentioned above. The details of the 
reactions are summarised in the Table below. 
__________________________________________________________________________ 
3-formyl- Molar 
oxymethyl equiv. 
pH of 
Reac- Isolated 
% Lactone 
Compound of reac- 
tion Work- 
Isolated 
Prod. [.alpha.].sup.22 
.sub.D 
(mg) Solvent (ml) 
Acid (ml) 
acid 
tion time up Prod. Wt. 
Yield 
t.l.c. 
(CHCl.sub.3) 
__________________________________________________________________________ 
50 Acetone (0.5) 
2N 
Methanol (1.0) 
hydrochloric 
2.3 1.5 13/4 hr 
A 35mg 74% &lt;10% + 20.degree. 
acid (0.1ml) 
50 Dichlorometh- 
2N 
ane (0.5) 
Methanol (1.0) 
hydrochloric 
0.6 .about.2 
3 hr B .32mg 67% .about.10% 
+ 32.degree. 
acid (0.025 ml) 
50 Acetone (0.5) 
2N 
Methanol (1.0) 
Sulphuric acid 
0.3 2 to 3 
5 hr 40m 
C 32mg 67% .about.10% 
+ 32.degree. 
(0.025ml) 
200 Methylated 
spirit (Indus- 
trial) (10.0) 
6N 
Dichlorometh- 
hydrochloric 
0.9 .about.2.5 
41/2 hr 
D 177mg 93% 10-20% 
+ 36.degree. 
ane (3.0) 
acid (0.05ml) 
100 N,N-Dimethyl- 
2N 
4 2 to 3 6 hr A 71mg 75% &lt;5% + 23.degree. 
formamide 
hydro 
(2.0) chloric acid 
(0.36 ml) 
200 N,N-Dimethyl 
2N 0.55 
3 24 hr 
E 164 mg 
86% &lt;5% + 23.degree. 
formamide 
hydro- 
(1.0) chloric acid 
(0.1ml) 
__________________________________________________________________________ 
NOTES TO TABLE 
WORK UP 
A. Propylene oxide (in excess) (0.1 to 0.4 ml) was added and the mixture 
was stirred for 5 minutes and then partitioned between ethyl acetate (ca 
75 ml) and water (ca 75 ml). The organic layer was washed with water (2 
.times. ca 50 ml) and dried over anhydrous sodium sulphate and filtered 
and evaporated to ca 3 to 5 ml. This solution was added dropwise to 
stirred 40.degree.-60.degree. petroleum ether (ca 150 ml). The resulting 
precipitate was filtered off and dried in vacuo. 
B. As above for A but dichloromethane was used in place of ethyl acetate. 
C. As above for A but no propylene oxide was added. 
D. Saturated aqueous sodium hydrogen carbonate solution (0.3 ml) was added 
and the resulting mixture was concentrated to about 0.75 of its volume. 
This mixture was partitioned between ethyl acetate (75 ml) and water (75 
ml). The organic layer was washed with water (2 .times. 75 ml) and dried 
over anhydrous sodium sulphate and filtered and then evaporated to a foam. 
E. Propylene oxide (0.05 ml) was added and the mixture was stirred for 3 
minutes. Water (20 ml) was added and the resulting solid was filtered off, 
washed with water (2 .times. 10 ml), dried on the sinter for 5 minutes and 
then dried in vacuo. 
EXAMPLE 5 
t-Butyl 
(6R,7R)-3-Hydroxymethyl-7-(2-thienylacetamido)-ceph-2-em-4-carboxylate 
t-Butyl 
(6R,7R)-3-formyloxymethyl-7-(2-thienylacetamido)ceph-2-em-4-carboxylate 
(50 mg.; 0.115 mmole), warm methanol (2 ml) and 2N hydrochloric acid 
(0.025 ml) were stirred together at 25.degree. C. at an apparent pH of 2. 
T.l.c. after 2 hours 20 minutes showed a virtually complete reaction. 
Propylene oxide (0.1 ml) was added and the mixture was stirred at 
25.degree. C. for 5 minutes. The mixture was partitioned between ethyl 
acetate (40 ml) and water (40 ml). The aqueous layer was extracted with 
ethyl acetate (20 ml) and the combined organic layers were washed with 
water (2 .times. 20 ml) and dried over anhydrous sodium sulphate and 
filtered and evaporated to a gum. This gum was dissolved in ethyl acetate 
(2 ml) and added dropwise to stirred 40 to 60.degree. petroleum ether (100 
ml) to give a pale yellow solid (23mg.; 50%). T.l.c. after work up 
indicated approximately 20% lactone. .tau. (d.sup.6 DMSO) 0.81 (d, J 9Hz; 
NH), 2.6, 3.0 (mult.; thienyls), 3.5 (broad s; .DELTA..sup.2 2-H), 4.51 
(d,d J 9, 5Hz; 7-H), 4.80 (d, J 5Hz; 6-H), 4.92 (broad s; --CH.sub.2 OH), 
5.10 (broad s; 4-H), 5.95 (mult; 3 CH.sub.2), 6.20 (s; 
##STR5## 
and 8.55 (s; tBu). 
EXAMPLE 6 
Diphenylmethyl 
(1S,6R,7R)-7-Formamido-3-hydroxymethylceph-3-em-4-carboxylate, 1-oxide 
(a) Diphenylmethyl 
(1S,6R,7R)-7-formamido-3-formyloxymethylceph-3-em-4-carboxylate, 1-oxide 
(100mg) in N,N-dimethylformamide (2 ml) was treated with 2N hydrochloric 
acid (0.05 ml) and the solution was stirred at 25.degree. for 22 hours, 
during which time the apparent pH of the reaction rose from about 3.0 to 
about 3.5. More 2N hydrochloric acid (0.02 ml) was added, and the solution 
was stirred for 6 hours and then left to stand (at 25.degree.) for 60 
hours. Propylene oxide (0.07 ml) was added and the solution was stirred at 
25.degree. for 5 minutes, and it was then partitioned between ethyl 
acetate (50 ml) and water (50 ml). The organic layer was washed with water 
(50 ml) and dried over sodium sulphate and filtered and evaporated to a 
gum which was suspended in ethyl acetate (8 ml) and added slowly to 
stirred 40.degree. to 60.degree. petroleum ether (100 ml). The flocculent 
precipitate was filtered off and dried in vacuo to give the title compound 
(47 mg) as a pale brown solid. 
T.l.c. (elution with ethyl acetate/acetic acid 9/1 or with 
chloroform/methanol/acetic acid 90/16/20) and n.m.r. showed the product to 
contain some unchanged formate ester (.about.10%), but no discernable 
amount of lactone: .tau. (DMSO-d.sub.6) 1.63 (d, J 9, NH), 1.81 (s, HCO), 
ca 2.3 to b 2.9 (m, Ph.sub.2), 3.08 (s, CHPh.sub.2), 3.99 (dd, J 5.9, 
7-H), 4.85 (m, OH), 5.02 (d, J5, 7-H), 5.57 and 5.82 (ABqm, J14, 
3-CH.sub.2), 5.98 and 6.38 (ABq, J 18, 2-H.sub.2) 
(b) The procedure described in (a) above was repeated except that the 
product was isolated (after the 60 hour reaction period) by adding water 
(20 ml) and filtering off and drying (in vacuo) the precipitate. This 
yielded the title compound (49 mg) as a buff-coloured solid. T.l.c. and 
n.m.r. showed the product to contain some unchanged formate ester (&lt;10%), 
but no discernable amount of lactone: the n.m.r. resembled that of the 
product from part (a) above. 
(c) Diphenylmethyl 
(1S,6R,7R)-7-formamido-3-formyloxymethylceph-3-em-4-carboxylate, 1-oxide 
(127 mg, 0.25 mmole) was suspended in methanol (5ml) and perchloric acid 
(60%, 0.015 ml, 0.13 mmole) was added. The suspension was stirred at 
30.degree. for 75 minutes, but no reaction appeared to occur (by t.l.c.). 
Chloroform (5ml) was added and the mixture (still not all in solution) was 
stirred at 30.degree. for 4 hour, by which time a solution had formed. The 
solution was partitioned between chloroform and water, and the organic 
layer was washed with water and dried over sodium sulphate and filtered 
and evaporated to a foam (80mg) which was triturated with ether to give 
the title compound (51 mg) as a light brown solid. 
T.l.c. and n.m.r. showed the product to contain small amounts of the 
formate ester (10-20%) and the lactone (&lt;5%): .nu..sub.max (CHBr.sub.3) ca 
3600 (OH), 3400 (NH), 1798 (.beta.-lactam), 1725 (CO.sub.2 R), 1692 and 
1500 (CONH) and 1042 cm.sup.-1 (sulphoxide). The NMR spectrum resembled 
that in preparation (a) above. 
Preparation 1 
Diphenylmethyl (1S, 6R, 7R)-7-Formamido-3-formyloxymethylceph-3 
em-4-carboxylate, 1-oxide 
Diphenylmethyl (1S, 6R, 
7R)-3-Bromomethyl-7-formamidoceph-3-em-4-carboxylate, 1-oxide (503 mg - 1 
mmole) was suspended in acetone (10 ml) and to the vigorously stirred 
mixture was added formic acid (0.4 ml; ca 10 mmole) and triethylamine (0.8 
ml; ca 6 mmole). The stirred mixture was warmed to 45.degree. and then 
allowed to cool to room temperature (30.degree.) over 1 hour. (A thick 
buff coloured precipitate was formed over 1/2 hour). The mixture was 
stirred at 30.degree. for a further 11/2 hours, when t.l.c. showed a 
complete reaction, and then it was slowly diluted with water (20 ml). The 
fine pale-brown precipitate was filtered off, washed with water and dried 
in vacuo to give the title compound (361 L mg) m.p. 198.degree. to 
200.degree. (decomp.) [.alpha.].sub.D +34.degree. (DMSO, c 
0.87%).lambda.max (chloroform) 269.5 mm (.epsilon.8,650 ), .nu.max 
(CHBr.sub.3) 3320, 3280 (NH), 1788 (.beta.-lactam), 1720,1710(shoulder) 
(ester, OCHO), 1660, 1655 (shoulder) and 1535 cm.sup.-1 (amide). 
Preparation 2 
t-Butyl (6R, 7R)-3-Formyloxymethyl-7-(2-thienylacetamido) 
ceph-2-em-4-carboxylate 
t-Butyl (6R, 7R)-3-Bromomethyl-7-(2-thienylacetamido) 
ceph-2-em-4-carboxylate (592 mg; 1.25 mmole), acetone (25 ml), 98-100% 
formic acid (0.25 ml; 6.25 mmole; 5.0 molar equivalents) and triethylamine 
(0.5 ml; 3.75 mmole; 3.0 molar equivalents) were stirred together at 
25.degree. C. under nitrogen. Acetone (10ml) was added, but a suspension 
was still formed. The mixture was stirred for 221/2 hours when t.l.c. 
(ethyl acetate:toluene 1:1) showed a complete reaction. The mixture was 
concentrated to 1/2 it's volume and poured into swirling water (125 ml). 
The resulting precipitate was extracted into ethyl acetate (1 .times. 75 
ml; 1 .times. 20 ml). The yellow organic layer was washed with water (1 
.times. 75 ml) and dried over anhydrous sodium sulphate and filtered. The 
filtrate was stirred with charcoal for 1/2 hour. The mixture was filtered 
through kieselguhr, the cake washed with ethyl acetate, and then the 
filtrate was evaporated to a pale yellow gum, the title compound (242mg) 
[.alpha.].sub.D.sup.21 + 264.degree. (CHCl.sub.3 ; c 0.53%), .lambda.max 
(ethanol) 237 nm (.epsilon.14,600), .nu.max (CHBr.sub.3) 3400 (NH), 1770 
(.beta.-lactam), 1720 (ester) 1675 and 1500 cm.sup.-1 (amide).