Formyl derivatives of hydrazinopenicillins

Formyl derivatives of hydrazinopenicillins of the formula ##STR1## wherein R is thienyl or phenyl, R' is hydrogen, formyl or alkyl containing from 1 to 3 carbon atoms and R" is hydrogen or formyl, provided that at least one of R' and R" is a formyl group, the carbon atom with the asterisk indicating a center of asymmetry of the molecule, and pharmaceutically acceptable salts and esters thereof, as mixture or as separated epimers. These compounds have antibacterial activity against Gram-negative and Gram-positive bacteria and are prepared by a process wherein the corresponding hydrazinopenicillin is reacted under anhydrous conditions with acetoformic anhydride to give the desired compounds.

The present invention is concerned with new penicillins and more 
particularly with formyl derivatives of hydrazinopenicillins. 
In our previous patent application No. 31,558 A of Nov. 10, 1972 new 
hydrazino derivatives of penicillins of the general formula 
##STR2## 
wherein R is hydrogen or alkyl containing up to 6 carbon atoms, as well as 
its non-toxic salts, either as a mixture or as separated epimers, were 
described. 
We have now found and this is one of the objects of the present invention, 
that by replacing the hydrogen atoms of the nitrogen in position 1 and/or 
2 of the hydrazino group in compounds of formula Ia and in its carboxylic 
derivatives, new penicillins displaying interesting antibacterial activity 
are obtained. 
Formyl derivatives of hydrazinopenicillins I, in which the phenyl group is 
substituted with a heterocyclic group, are a further object of the present 
invention. 
Compounds belonging to the class having the general formula 
##STR3## 
wherein R is phenyl or thienyl, R' is hydrogen, alkyl containing 1 to 3 
carbon atoms as, formyl and R" is hydrogen or formyl provided that at 
least one of R' and R" be a formyl group, the carbon atom having the 
asterisk indicating a center of asymmetry of the molecule, in the form of 
mixture or separated epimers, are prepared and hereinafter described. 
The invention further includes suitable pharmaceutically acceptable esters 
and salts of penicillane derivatives of formula I. 
The new formylhydrazino penicillins of formula I are prepared starting from 
hydrazino penicillins of the formula 
##STR4## 
wherein R is phenyl or thienyl, R'a is hydrogen or alkyl having from 1 to 
3 carbon atoms and R'" is hydrogen or a pharmaceutically acceptable, such 
as pivaloyloxymethyl and 1-(ethoxycarbonyloxy) ethyl, the carbon atom 
having the asterisk indicating a center of asymmetry of the molecule, in 
the form of a mixture or separated epimers, by reaction under anhydrous 
conditions with mixed acetoformic anhydride. The formylating reaction is 
carried out in an organic solvent suitable for the reagent materials, such 
as for example a tertiary organic base or an aprotic, optionally 
halogenated solvent. 
Compounds of the present invention can also be prepared by standard 
procedures known in the art such as for example by reacting 
6.beta.-amino-penicillanic acid optionally esterified with a suitable 
derivative, already formylated, at the substituted .alpha.-hydrazino 
acetic acid. 
The compounds of the present invention display interesting anti-bacterial 
activity against Gram-positive and Gram-negative bacteria. The minimum 
inhibiting concentration (MIC) in vitro of the compounds of the invention 
expressed in .gamma./ml are listed in the following Table. 
The following Examples are given for the purpose of illustrating the 
present invention without limiting it. 
__________________________________________________________________________ 
(R) 6.beta.-[.alpha.-(2-formyl-1-methyl 
(R) 6.beta.-[.alpha.-(1.2-diformyl- 
(R) 6.beta.-[.alpha.-(2-formylhydr 
a- 
hydrazino)phenylacetamido] 
hydrazino)phenylacetami- 
zino)phenylacetamido]pe- 
Microorganisms 
penicillanic acid 
do]penicillanic acid 
nicillanic acid 
__________________________________________________________________________ 
Staphylococcus aureus 
0.19 0.19 0.048 
Staphylococcus aureus* 
3.12 6.25 1.56 
Streptococcus pyogenes 
0.048 0.097 &lt;0.012 
Streptococcus pneumoniae 
0.024 0.024 0.012 
Streptococcus foecalis 
3.12 6.25 1.56 
Sarcina lutea 
0.012 0.024 &lt;0.012 
Escherichia coli 
1.56 3.12 0.78 
Shigella dysenteriae 
1.56 3.12 0.78 
Salmonella typhi 
3.12 6.25 1.56 
Salmonella typhymurium 
12.5 25 3.12 
Salmonella enteritidis 
25 25 6.25 
Neisseria meningitidis 
0.012 0.097 &lt;0.012 
Pseudomonas aeruginosa 
&gt;200 &gt;200 200 
__________________________________________________________________________ 
*Penicillinase producing strain

EXAMPLE 1 
Pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
A solution consisting of 3 g 
R(-).alpha.-[1-(p.nitrobenzyloxycarbonyl)hydrazino]phenylacetic acid in 20 
ml formic acid stirred at 4.degree. C. is treated dropwise with 10 ml 
acetoformic anhydride under stirring for 10 minutes and then for a further 
hour at ambient temperature. The product is evaporated to dryness and the 
residue is taken up several times with diethyl ether until there is 
obtained a solid compound which is triturated with diethyl ether, taken up 
under vacuum to give 3.06 g 
R(-).alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacetic 
acid melting at 158.degree.-161.degree. C. (with decomposition); 
[.alpha.].sub.D =-82.47.degree. C. (c=1 in tetrahydrofuran). 
0.78 grams of dicyclohexylcarbodiimide are added to a solution of 1.20 g 
pivaloyloxymethyl ester of 6.beta.-aminopenicillanic acid in 5 ml 
tetrahydrofuran, while stirring at 4.degree. C., and then dropwise, 1.4 g 
R(-).alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacetic 
acid diluted in 8 ml tetrahydrofuran are added. Stirring is continued at 
4.degree. C. for a further 10 minutes and then at ambient temperature for 
30 minutes. The insoluble residue is filtered off, evaporated to dryness, 
taken up with 30 ml methylene chloride, washed twice with 10 ml solution 
of 5% sodium bicarbonate and 10 ml water; it is then made anhydrous and 
evaporated to dryness. The residue is triturated with diisopropyl ether 
and purified by chromatography (silicagel, diethyl ether 100%): 1.93 g 
pivaloyloxymethyl ester of (R) 
6.beta.-{.alpha.-[1-(p.nitrobenzyloxycarbonyl-2-formylhydrazino)phenylacet 
amido]}penicillanic acid are obtained melting at 76.degree.-78.5.degree. C. 
(with decomposition); [.alpha.].sub.D =+55.5.degree. (c=1 in 
tetrahydrofuran). 
To a solution consisting of 1.2 g pivaloyloxymethyl ester of (R) 
6.beta.-{.alpha.-[1-(p.nitrobenzyloxycarbonyl-2-formylhydrazino)phenylacet 
amido]}penicillanic acid in 10 ml of 95% ethyl alcohol and 3 ml water are 
added, under stirring, 1.8 g of 5% Palladium/charcoal. Hydrogen is bubbled 
in for 20 hours. It is filtered over celite, evaporated to dryness and 
taken up several times with ether until a solid is obtained. The product 
is then triturated with diisopropyl ether and taken up under vacuum. It is 
purified by chromatography (silicagel, hexane/ethyl acetate 1:1) to obtain 
0.20 g pivaloyloxymethyl ester of R 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
melting at 69.3.degree.-74.8.degree. C. (with decomposition). 
EXAMPLE 2 
(R,S) 6.beta.-[.alpha.-(1-formylhydrazino)phenylacetamido]penicillanic acid 
A solution consisting of 1.86 ml formic acid in 7.5 ml diethyl ether, 
cooled to -15.degree. C. is treated with a solution of 3.43 g 
dicyclohexylcarbodiimide in 10 ml diethyl ether and stirred for 30 
minutes. To the so obtained mixture is added a suspension in methylene 
chloride of (R,S) .alpha.-(2-benzyloxycarbonylhydrazino)phenylacetic acid 
trimethylsilylester obtained by refluxing the free acid with 
hexamethyldisilazane in acetonitrile and stirring at 0.degree. C. for 2 
hours. Fifty milliliters of cold water are added thereto and the pH 
adjusted to 9. The solid residue is filtered off and the layers are 
separated. The aqueous layer, acidified to pH 1.5, is extracted three 
times with 50 ml methylene chloride, the organic extracts collected 
together and washed with 20 ml water, made anhydrous and evaporated to 
dryness to give 2.64 g (R,S) 
.alpha.-(1-formyl-2-benzyloxycarbonylhydrazino)phenylacetic acid in the 
oily form from which the corresponding sodium salt is obtained melting at 
148.degree.-150.degree. C. (with decomposition). To a solution of 10 g of 
the sodium salt of (R,S) 
.alpha.-(1-formyl-2-benzyloxycarbonylhydrazino)phenylacetic acid in 85 ml 
acetone cooled to -40.degree. C., are added 2.95 ml ethyl chloroformiate 
and then the mixture is stirred for 30 minutes. An aqueous-acetonic 
solution (30 ml) containing 5.85 g of the triethylamine salt of 
6.beta.-aminopenicillanic acid is added thereto and the mixture is stirred 
at -25.degree. C. for 45 minutes. Then 50 ml cold water and 150 ml 
methylene chloride are added to the mixture and this is acidified to pH 2. 
The layers are then separated and the organic one is washed with 50 ml 
water, made anhydrous and evaporated to dryness. The residue is triturated 
with cyclohexane and there are obtained 13 g (R,S) 
6.beta.-[.alpha.-(1-formyl-2-benzyloxycarbonylhydrazino)phenylacetamido]pe 
nicillanic acid melting at 98.degree.-100.degree. C. (with decomposition). 
To a solution of 1.16 g sodium bicarbonate in 37.5 ml water are added 7.3 g 
(R,S) 
6.beta.-[.alpha.-(1-formyl-2-benzyloxycarbonylhydrazino)phenylacetamido]pe 
nicillanic acid and 5.25 g 10% palladium over charcoal. Hydrogen is bubbled 
in for 2 hours, then the catalyst is filtered off and the filtrate is 
stratified with 130 ml methylene chloride, cooled to 0.degree. C. and 
acidified to pH 2. The layers are separated, the organic one is extracted 
twice with 20 ml methylene chloride and the organic extracts collected 
together are washed three times with 30 ml water, made anhydrous and 
evaporated to dryness. 2.15 grams (R,S) 
6.beta.-[.alpha.-(1-formylhydrazino)-phenylacetamido]penicillanic acid are 
obtained, melting at 156.degree.-160.degree. C. (with decomposition). 
EXAMPLE 3 
(R) 6.beta.-[.alpha.-(2-Formylhydrazino)phenylacetamido]penicillanic acid 
To a solution of 2.26 g p.nitrobenzyl ester of 6.beta.-aminopenicillanic 
acid in 10 ml tetrahydrofuran stirred at 4.degree. C. are added 1.33 g 
dicyclohexylcarbodiimide. Then 2.40 g 
R(-).alpha.-[1-(p.nitrobenzyloxycarbonyl-2-formylhydrazino)]phenylacetic 
acid dissolved in 20 ml tetrahydrofuran (obtained in a manner similar to 
that described in Example 1) are added thereto. The mixture is further 
stirred at 4.degree. C. for 10 minutes, and at ambient temperature for 3 
hours, then the residue is removed by filtration and the filtrate 
evaporated to dryness, and taken up several times with diethyl ether until 
a solid is obtained. It is dissolved in 50 ml ethyl acetate, washed three 
times with 10 ml of 5% sodium bicarbonate solution, made anhydrous and 
evaporated to dryness. It is purified by chromatography (silicagel, 
hexane/ethyl acetate 3:7) to give 3.42 g p.nitrobenzyl ester of 
(R)6.beta.-{.alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]}pheny 
lacetamido penicillanic acid (4b) melting at 101.5.degree.-104.7.degree. C. 
(with decomposition); [.alpha.].sub.D =+74.47.degree. C. (c=1 in 
tetrahydrofuran). 
To a solution of 2.20 g p.nitrobenzyl ester of (R) 
6.beta.-{.alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]}phenylac 
etamido penicillanic acid in 30 ml tetrahydrofuran and 14 ml water are 
added, under stirring, 4.50 g 5% palladium/calcium carbonate. Hydrogen is 
bubbled in for 20 hours, it is filtered over celite and evaporated to 
dryness. The residue is triturated with ether, collected under vacuum and 
dissolved in 5.5 ml tetrahydrofuran and 1 ml water. Fifty milliliters of 
isopropyl alcohol are added to the solution stirred at 4.degree. C., 
stirring is further continued for 10 minutes, then the solid residue is 
collected under vacuum and 1.09 g (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid as 
calcium salt are obtained, melting at 200.degree. C. (with decomposition). 
EXAMPLE 4 
1-(Ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)]phenylacetamido penicillanic acid 
To a solution of 1.21 g 1-(ethoxycarbonyloxy)ethyl ester of 
6.beta.-aminopenicillanic acid in 5 ml tetrahydrofuran, cooled to 
4.degree. C., 0.78 g dicyclohexylcarbodiimide are added and then treated 
dropwise with a solution of 1.40 g R(-) 
.alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacetic acid 
(prepared in a manner similar to that described in Example 1). The product 
is stirred at ambient temperature for 30 minutes. The solid is filtered 
off and the filtrate is evaporated to dryness. The residue is dissolved in 
30 ml methylene chloride, washed twice with 5% sodium bicarbonate solution 
and with 10 ml water, then made anhydrous and evaporated to dryness. The 
residue is purified by chromatography (silicagel, ether) and 1.6 g 
1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-.alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacet 
amido penicillanic acid are obtained, melting at 89.3.degree.-97.3.degree. 
C. 
To a solution of 1.20 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-.alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacet 
amido penicillanic acid in 10 ml 95% ethyl alcohol and 3 ml water are added 
1.80 g 5% palladium over calcium carbonate, and hydrogen is bubbled in for 
20 hours. The catalyst is filtered off, the mixture evaporated to dryness, 
then the residue purified by chromatography (silicagel, hexane/ethyl 
acetate 1:1) to give 0.43 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid, 
melting at 58.6.degree.-64.3.degree. C.; [.alpha.].sub.D =+111.3.degree. 
(c=1 in chloroform). 
EXAMPLE 5 
Pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)phenylacetamido]penicillanic acid 
Three milliliters of acetoformic anhydride are added dropwise to a solution 
of 1 g R(-).alpha.-hydrazinophenyl acetic acid in 3 ml formic acid, while 
stirring at 4.degree. C., and stirring is continued for a further 10 
minutes, then for a further 30 minutes at ambient temperature. The product 
is evaporated to dryness and taken up with diethyl ether and the solid 
obtained collected under vacuum to give 0.94 g 
R(-).alpha.-(1.2-diformylhydrazino)phenylacetic acid melting at 
141.7.degree.-155.4.degree. C. (with decomposition); [.alpha.].sub.D 
=-222.5.degree. C. (c=1 in tetrahydrofuran). 
To a solution of 2.97 g pivaloyloxymethyl ester of 
6.beta.-amminopenicillanic acid in 10 ml tetrahydrofuran stirred at 
4.degree. C., 1.85 g dicyclohexylcarbodiimide are added, and 2 g 
R(-).alpha.-(1.2-diformylhydrazino)phenylacetic acid dissolved in 30 ml 
tetrahydrofuran are added dropwise thereto. Stirring is continued for 10 
minutes at 4.degree. C. and then for a further 2 hours at ambient 
temperature. The product is then filtered, the filtrate dried and the 
residue triturated with diisopropylether, collected under vacuum and 
chromatographed (silicagel, hexane/ethyl acetate 3:7) to give 2.4 g 
pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)phenylacetamido]penicillanic acid 
melting at 91.7.degree.-93.1.degree. C.; [.alpha.].sub.D =+96.42.degree. 
C. (c=1 in tetrahydrofuran). 
EXAMPLE 6 
Pivaloyloxymethyl ester of 1 g (R,S) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)-2-thienylacetamido]penicillanic 
acid 
A solution of 1 g (RS) .alpha.-hydrazino-2-thienylacetic acid in 3 ml 
formic acid is stirred at 4.degree. C. and treated dropwise with a 
solution of 3 ml acetoformic anhydride. Stirring is continued for 20 
minutes at the same temperature and for a further 30 minutes at ambient 
temperature, then the product is evaporated to dryness, taken up with 
diethyl ether and the so obtained solid is triturated with ethyl ether 
collected under vacuum, to give 1.26 g (RS) 
.alpha.-(1.2-diformylhydrazino)-2-thienylacetic acid melting at 
168.degree.-170.degree. C. (with decomposition). 
To a solution of 1.1 g pivaloyloxymethyl ester of 6.beta.-aminopenicillanic 
acid in 3 ml tetrahydrofuran stirred at ambient temperature are added 686 
mg dicyclohexylcarbodiimide and dropwise 760 mg (RS) 
.alpha.-(1.2-diformylhydrazino)-2-thienylacetic acid dissolved in 20 ml 
tetrahydrofuran. The mixture is stirred for one hour and a half, the 
residue is removed, the filtrate evaporated to dryness and the residue 
triturated with diisopropylether, collected under vacuum and purified by 
chromatography (silicagel, hexane/ethyl acetate 3:7) to obtain 1.25 g 
(69%) pivaloyloxymethyl ester of (RS) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)-2-thienylacetamido]penicillanic 
acid melting at 67.4.degree. C.; [.alpha.].sub.D =+147.2.degree. C. (c=1 
in tetrahydrofuran). 
EXAMPLE 7 
Pivaloyloxymethyl of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid 
To a solution of 5 g (R) .alpha.-(1-methylhydrazino)phenylacetic acid in 10 
ml formic acid, kept under stirring at 4.degree. C., 15 ml acetoformic 
anhydride are added dropwise, and the mixture is stirred at 4.degree. C. 
for 15 minutes and then an hour and a half at ambient temperature. The 
product is evaporated to dryness and the oily residue taken up with 30 ml 
methylene chloride and 30 ml water, then adjusted to pH 7.2, the aqueous 
layer is washed with methylene chloride, stratified with 50 ml methylene 
chloride, adjusted to pH 2 and extracted with methylene chloride. The 
organic layers are collected, made anhydrous, evaporated to dryness and 
triturated with diethyl ether to give a solid residue, which is collected 
under vacuum, consisting of 3.46 g (R) 
.alpha.-(2-formyl-1-methylhydrazino) phenylacetic acid melting at 
129.degree.-131.degree. C.; [.alpha.].sub.D =104.8.degree. C. (c=1 in 0.1 
N sodium hydroxide). 
To a solution of 0.50 g (R) 
.alpha.-(2-formyl-1-methylhydrazino)phenylacetic acid and 0.34 ml 
triethylamine in 5 ml tetrahydrofuran at -50.degree. C., 0.25 ml ethyl 
chloroformiate are added, the mixture is stirred at -20.degree. C. for 30 
minutes, then a solution of 0.72 g pivaloyloxymethyl ester of 
6.beta.-aminopenicillanic acid in 8 ml tetrahydrofuran are added thereto. 
The mixture is stirred at 0.degree. C. for 1 hour, thereafter 20 ml 
methylene chloride and 20 ml water are added. The layers are separated and 
the organic one is washed twice with 10 ml solution of 5% sodium 
bicarbonate and 10 ml water. The product is made anhydrous and evaporated 
to dryness, the residue is purified through chromatography (silicagel, 
hexane/ethyl acetate 10:2) to give 0.30 g pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid melting at 147.degree.-154.3.degree. C. (with decomposition); 
[.alpha. ].sub.D =+129.4.degree. C. (c=0.5 in chloroform). 
EXAMPLE 8 
1-(Ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid 
In a manner analogous to that described above, using 
1-(ethoxycarbonyloxy)ethyl ester of 6.beta.-aminopenicillanic acid, 
1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid is obtained, melting at 71.3.degree.-77.4.degree. C. (with 
decomposition); [.alpha.].sub.D =131.6.degree. C. (c=0.5 in chloroform). 
EXAMPLE 9 
(R) 6.beta.-[.alpha.-(1.2-Diformylhydrazino)phenylacetamido]penicillanic 
acid 
A solution of 0.5 g R 
6.beta.-(.alpha.-hydrazinophenylacetamido)penicillanic acid in 6 ml 
pyridine, stirred at 4.degree. C., is treated dropwise with 1.5 ml 
acetoformic anhydride solution and kept under stirring for a further 5 
hours. Then it is washed three times with 10 ml hexane and the oily 
residue is triturated with 30 ml diethyl ether and collected under vacuum 
to give 0.38 g (R) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)phenylacetamido]penicillanic acid 
melting at 137.degree.-177.degree. C. (with decomposition). 
The corresponding sodium salt, obtained by treatment with a solution of 
2-ethylhexanoate sodium in isopropyl alcohol, melts at 180.degree. C. 
(with decomposition). 
EXAMPLE 10 
(R) 
6.beta.-[.alpha.-(2-Formyl-1-methylhydrazino)phenylacetamido]penicillanic 
acid 
A solution of 2 g (R) 
6.beta.-[.alpha.-(1-methylhydrazino)phenylacetamido]penicillanic acid in 
15 ml pyridine, under stirring at 4.degree. C., is treated dropwise with 6 
ml acetoformic anhydride and kept under stirring for a further 30 minutes 
at the same temperature. The mixture is washed with hexane until there is 
obtained a thick oily product, which is triturated with 50 ml diethyl 
ether three times, and under vacuum 1.5 g (R) 
6.beta.-[.alpha.-(2-formyl-1-methylhydrazino)phenylacetamido]penicillanic 
acid is separated, melting at 157.degree.-158.1.degree. C. (with 
decomposition); [.alpha.].sub.D =+201.51.degree. C. (c=1 in 
tetrahydrofuran). 
EXAMPLE 11 
(R) 6.beta.-[.alpha.-(2-Formylhydrazino)phenylacetamido]penicillanic acid 
To a solution consisting of 5 g (R) 
6.beta.-(.alpha.-hydrazinophenylacetamido)penicillanic acid and 5.4 ml 
triethylamine in 100 ml methylene chloride cooled to -20.degree. C. are 
added dropwise 2.3 ml acetoformic anhydride solution in 20 ml methylene 
chloride. The mixture is stirred at -20.degree. C. for 30 minutes, then at 
ambient temperature for 1 hour. It is evaporated to dryness and the 
residue is diluted with 80 ml of 3.5% sodium bicarbonate. It is stratified 
with 40 ml ethyl acetate and the layers are separated: the aqueous layer, 
adjusted to pH 2 with 10% hydrochloric acid, is again stratified with 100 
ml ethyl acetate. The layers are separated and the organic one, made 
anhydrous, is concentrated, the solid removed by filtration, and the clear 
filtrate is evaporated to dryness. The residue, triturated with diethyl 
ether, gives 2.6 (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
melting at 145.5.degree.-147.8.degree. C. (with decomposition); [.alpha. 
].sub.D =+133.8.degree. C. (c=1 in tetrahydrofuran). 
EXAMPLE 12 
Pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid 
To a solution of 3.3 g pivaloyloxymethyl ester of 6.beta.-aminopenicillanic 
acid in 30 ml methylene chloride cooled to 0.degree. C. are added 13 ml 
propylene oxide, while stirring, and then to -30.degree. C. 2.3 g chloride 
hydrochloride of (R) .alpha.-(1-methylhydrazino)phenylacetic acid are 
added, and the product is kept under stirring for 30 minutes at 
-20.degree. C. and for a further 2 hours at 0.degree. C. Then, it is 
evaporated to dryness, the residue is triturated with diisopropyl ether, 
and 4.2 g pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1-methylhydrazino)phenylacetamido]penicillanic acid 
hydrochloride are obtained, melting at 88.degree.-90.degree. C. (with 
decomposition); [.alpha.].sub.D =+140.53.degree. C. (c=1 in methyl 
alcohol). 
A solution of 4 g pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1-methylhydrazino)phenylacetamido]penicillanic acid in 
25 ml anhydrous pyridine is cooled to 4.degree. C., and 4.2 ml acetoformic 
anhydride are added dropwise thereto. The product is kept under stirring 
at 4.degree. C. for 30 minutes, then evaporated to dryness and the residue 
dissolved in 100 ml ethyl acetate and washed with water. The organic layer 
is made anhydrous and evaporated to dryness, then the residue purified by 
chromatography (silicagel, hexane:ethyl acetate, 10:2) gives 2.50 g 
pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid melting at 147.degree.-154.3.degree. C. (with decomposition); 
[.alpha.].sub.D =+129.4.degree. C. (c=0.5 in chloroform). 
EXAMPLE 13 
1-(Ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-1-formylhydrazino)phenylacetamido]penicillanic 
acid 
Twenty-four milliliters of propylene oxide are added to a solution of 6 g 
1-(ethoxycarbonyloxy)ethyl ester of 6.beta.-aminopenicillanic acid in 60 
ml methylene chloride; the mixture is cooled to -30.degree. C. and while 
stirring 4.25 g chloride hydrochloride of (R) 
.alpha.-(1-methylhydrazino)phenylacetic acid are added thereto. The 
temperature is maintained at -30.degree. C. for 30 minutes, then increased 
to ambient temperature and the product is kept under stirring for a 
further hour. The mixture is evaporated to dryness, the residue is 
triturated with diethyl ether, treated with sodium bicarbonate and 7 g 
1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methylhydrazino)phenylacetamido]penicillanic acid 
melting at 45.6.degree.-47.degree. C. (with decomposition); 
[.alpha.].sub.D =+76.7.degree. C. (c=0.5 in chloroform). 
Ten milliliters of acetoformic anhydride are added dropwise to a solution 
of 5 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methylhydrazino)phenylacetamido]penicillanic acid in 
40 ml anhydrous pyridine, cooled to 4.degree. C. and kept under stirring 
for 30 minutes. To that mixture 150 ml ethyl acetate are added, it is 
washed four times with 100 ml water, the organic layer, after separation, 
is made anhydrous and evaporated to dryness. The residue is purified by 
chromatography (silicagel, ethyl acetate/hexane, 9:1) to give 2.20 g 
1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid melting at 71.3.degree.-77.4.degree. C. (with decomposition); 
[.alpha.].sub.D =+131.6.degree. C. (c=0.5 in chloroform). 
EXAMPLE 14 
1-(Ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid 
To a solution of 0.80 g (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid in 5 ml dimethylsulphoxide are added 0.28 ml triethylamine and 0.30 g 
.alpha.-chlorodiethylcarbonate. The mixture is stirred for 16 hours, and 
20 ml ethyl acetate are added thereto. The product is washed four times 
with 20 ml water, the organic layer is separated, thereafter made 
anhydrous and evaporated to dryness. The residue is recrystallized from 
isopropyl alcohol:water and 0.15 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(1-methyl-2-formylhydrazino)phenylacetamido]penicillanic 
acid exactly like the acid of the previous Example is obtained. 
EXAMPLE 15 
1-(Ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
To a solution of 13.28 g 1-(ethoxycarbonyloxy)ethyl ester of 
6.beta.-aminopenicillanic acid in 150 ml methylene chloride are added 40 
ml propylene oxide; the product is cooled to -30.degree. C., 8.85 g 
chloride hydrochloride of (R) .alpha.-hydrazinophenylacetic acid are added 
to the mixture, and it is kept under stirring for 1 hour at the same 
temperature. By evaporating to dryness and triturating the oily residue 
with diethyl ether, there are obtained 20 g 1-(ethoxycarbonyloxy)ethyl 
ester of (R) 6.beta.-(.alpha.-hydrazinophenylacetamido)penicillanic acid 
hydrochloride melting at 108.4.degree.-109.6.degree. C.; [.alpha.].sub.D 
=+109.9.degree. C. (c=1 in chloroform). 
To a solution of 15 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-(.alpha.-hydrazinophenylacetamido)penicillanic acid in 150 ml 
pyridine cooled to 0.degree. C., are slowly added 4.95 ml acetoformic 
anhydride in 30 ml chloroform. The mixture is stirred for 30 minutes at 
0.degree. C., for a further hour at ambient temperature, then 500 ml ethyl 
acetate are added thereto and it is washed four times with 100 ml water. 
The organic layer is made anhydrous and evaporated to dryness, the residue 
is taken up with petroleum ether until a gummy product is obtained, which 
is then purified by chromatography (silicagel, hexane:ethyl acetate, 1:1) 
to give 2.35 g 1-(ethoxycarbonyloxy)ethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
melting at 58.6.degree.-64.3.degree. C.; [.alpha.].sub.D =+111.3.degree. 
C. (c=1 in chloroform). 
EXAMPLE 16 
Pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
Operating in a manner analogous to that previously described, starting from 
pivaloyloxymethyl ester of 6.beta.-aminopenicillanic acid, 
pivaloyloxymethyl ester of (R) 
6.beta.-(.alpha.-hydrazinophenylacetamido)penicillanic acid is at first 
obtained and then pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid is 
obtained, melting at 69.3.degree.-74.8.degree. C. (with decomposition). 
EXAMPLE 17 
(R) 6.beta.-[.alpha.-(2-Formylhydrazino)phenylacetamido]penicillanic acid 
To a solution of 6.57 g 
R(-).alpha.-[1-(p.nitrobenzyloxycarbonyl)-2-formylhydrazino]phenylacetic 
acid (prepared as previously described) in 60 ml water, while stirring is 
added a solution of 0.95 g sodium carbonate, to the so obtained mixture 
are added 10 g of 5% palladium/calcium carbonate and hydrogen is bubbled 
in for 13 hours. The reaction mixture is filtered over celite, washed with 
chloroform, adjusted to pH 2 and concentrated until a precipitate is 
obtained, which is collected under vacuum in order to obtain 1.39 g 
R(-).alpha.-(2-formylhydrazino)phenylacetic acid. The aqueous mother 
liquors, after evaporating to dryness, are extracted with tetrahydrofuran; 
the organic layer, after removal of the solvent, is triturated with 
diethyl ether. The solid is collected under vacuum and 0.85 g R(-) 
.alpha.-(2-formylhydrazino)phenylacetic acid is obtained, melting at 
136.9.degree. C.; [.alpha.].sub.D =169.degree. C. (c=1 in 
tetrahydrofuran). 
To a solution of 3.4 g p.nitrobenzyl ester of 6.beta.-aminopenicillanic 
acid in 20 ml tetrahydrofuran is added, while stirring at 4.degree. C., a 
solution of 2 g dicyclohexylcarbodiimide, and 1.88 g 
R(-).alpha.-(2-formylhydrazino)phenylacetic acid dissolved in 30 ml 
tetrahydrofuran are added dropwise to the mixture. Stirring is continued 
at 4.degree. C. for 1 hour and then for a further 5 hours at ambient 
temperature. The insoluble is filtered off, the filtrate is evaporated to 
dryness, purified by chromatography (silicagel, hexane/ethyl acetate, 3:7) 
and 1.72 g p.nitrobenzyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid are 
obtained, melting at 84.9.degree.-86.9.degree. C.; [.alpha.].sub.D 
=+127.degree. C. (c=1 in tetrahydrofuran). 
To a solution of 0.5 g p.nitrobenzyl ester of (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid in 8 
ml tetrahydrofuran and 4 ml water are added 0.35 g 10% palladium over 
charcoal while stirring, and hydrogen is bubbled in for 7 hours. It is 
filtered over celite washing with tetrahydrofuran and 5% aqueous sodium 
bicarbonate. The clear aqueous liquors are washed five times with 10 ml 
ethyl acetate and adjusted to pH 2. The product is evaporated to dryness, 
the residue is taken up with tetrahydrofuran, filtered and evaporated to 
dryness. The oily residue is triturated with diethyl ether and collected 
in a vacuum to give 0.2 g (R) 
6.beta.-[.alpha.-(2-formylhydrazino)phenylacetamido]penicillanic acid 
melting at 145.5.degree.-147.8.degree. C. (with decomposition); 
[.alpha.].sub.D =+117.6.degree. C. (c=1 in tetrahydrofuran). 
EXAMPLES 18, 19 
Operating in a manner similar to that described in Example 12, using (RS) 
.alpha.-hydrazinothienylacetic acid and (R) .alpha.-hydrazinophenylacetic 
acid, pivaloyloxymethyl ester of (R,S) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)-2-thienylacetamido]penicillanic 
acid and pivaloyloxymethyl ester of (R) 
6.beta.-[.alpha.-(1.2-diformylhydrazino)phenylacetamido]penicillanic acid 
are obtained respectively, identical to that obtained in Examples 6 and 5.