Synthesis and activity of cephalosporins containing an oxyiminomethylene functionality in the ortho-position of a phenyl- or phenoxyacetic acid C-7 side chain substituent

Article abstract of DOI:10.1016/S0014-827X(98)00112-8

A new series of cephalosporins having in the C-7 side chain a phenyl- or a phenoxyacetamido group bearing an oxyiminomethyl function in the ortho-position of the aromatic ring was prepared. Their in vitro activity was tested against both Gram+ and Gram- s

Full text of DOI:10.1016/S0014-827X(98)00112-8

Il Farmaco 53 (1998) 709–717
Synthesis and activity of cephalosporins containing an
oxyiminomethylene functionality in the ortho-position of a
phenyl- or phenoxyacetic acid C-7 side chain substituent
Domenico Albanese a, Dario Landini a, Mario Leone b, Michele Penso a,*,
Maurizio Zenoni b
a Dipartimento di Chimica Organica e Industriale and Centro CNR, Via Golgi 19, I-20133 Milan, Italy
b ACS-Dobfar, Via Addetta 8, I-20067 Tribiano, Italy
Abstract
A new series of cephalosporins having in the C-7 side chain a phenyl- or a phenoxyacetamido group bearing an oxyiminomethyl
function in the ortho-position of the aromatic ring was prepared. Their in vitro activity was tested against both Gram+ and
Gram− strains. © 1998 Elsevier Science S.A. All rights reserved.
Keywords: b-Lactam antibiotics; Oxyiminomethylene cephalosporins; Oxyacetamido cephalosporins; Phase transfer catalysis
peculiar presence in the C-7 side chain of a phenyl- (1)
or a phenoxyacetamido (2) group bearing an oxyimi-
nomethyl function in the ortho-position of the aromatic
ring. The ortho polar moiety could enhance the activity
of these compounds, like e.g. the ortho-aminomethyl
group does in ceforanide.
Products 1 and 2 were tested in vitro against numer-
ous bacterial strains and their activity was comparable
with many of the commercial 2-aminothiazole cepha-
losporins and those bearing an a-oxyiminoacetamido
group at the C-7 side chain.
1. Introduction
As is well known, cephalosporin antibiotics exert
their intrinsic activity by inhibiting the biosynthesis of
cell wall of many bacteria species [1–3]. Natural or
acquired resistance to cephalosporins is due to several
factors, but very often can be imputed to the genera-
tion, by the microorganism, of a b-lactamase enzyme
[4]. Among the structural modifications of the
cephalosporanic moiety that confer good stability to
b-lactamases, the introduction in the C-7 side chain of
an E-oxyimino group and/or an aromatic or heteroaro-
matic substituent bearing an ortho-positioned polar
group, are the more frequently used. Several marketed
or studied cephalosporins [5] have one (e.g. for the
oxime derivatives: cefuroxime [6–10], cefuroxime axetil
[11,12]; e.g. for an o-polar group: ceforanide [13–15])
or both these features (e.g. cefixime [16–18], cefotaxime
[19–22], ceftizoxime [23–26], cefmenoxime [27,28], cef-
triaxone [29–32], ceftazidime [33–36], cefpirome
[37,38], cefepime [39,40], etc. [41–44]).
2. Chemistry
2.1. Preparation of cephalosporins 1 and 2
The cephalosporins 1 and 2 can be considered to
consist of a cephalosporanic nucleus 3 and a substituted
phenylacetic (4) or phenoxyacetic (5) acid (Scheme 1).
The condensation of these two units leads to products 1
and 2 in good yields (55–78%) and is stereoconserva-
tive, i.e. no racemization of the stereogenic centres (C-6
and C-7) occurs. The formation of the oxyiminomethyl
group from the corresponding aldehyde is more conve-
niently performed before the condensation stage and
proceeds in a stereoselective fashion affording the E-
In the present work we describe the preparation of a
series of new cephalosporins 1 and 2 (Fig. 1) having the
* Corresponding author. Tel.: +39-02-70638399; fax: +39-02-
2364369.
E-mail address: michele@iumchz.chimorg.unimi.it (M. Penso)
0014-827X/98/$ - see front matter © 1998 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(98)00112-8

710
D. Albanese et al. / Il Farmaco 53 (1998) 709–717
Fig. 1. Prepared cephalosporins 1 and 2.
oxime. The synthesis of 1 and 2 was realized by reacting
the corresponding substituted cephalosporanic acid 3
with bis-trimethylsilylacetamide (BSA) to produce the
intermediate 7-silylamido-4-silyl ester (6) (Scheme 2).
At the same time, the phenylacetic (4) or phenoxyacetic
(5) acid was transformed into the corresponding mixed
anhydride 7 or 8 by alkylation with ethyl chlorofor-
mate. Finally, addition of 6 to the solution containing
7 or 8, followed by hydrolysis with water, gave the
corresponding cephalosporins 1 and 2. When R is
CH2COOBut, the solvolysis is conducted with formic
acid to release the carboxylic acid from its t-butyl ester.
2.2. Preparation of the substituted phenyl- or
phenoxyacetic acid deri6ati6es 4 and 5
The preparations of the intermediate oxyimino con-
taining acetic acids 4 and 5 were realized by using
known synthetic procedures, both under homogeneous
and phase transfer catalysis (PTC) conditions. Oximes
4a,5a (Scheme 3) were prepared by reacting the corre-
sponding ortho-formyl phenyl- or phenoxy acetic acid 9
or 10 with hydroxylamine hydrochloride. These oximes
were simultaneously O-alkylated and esterified by reac-
tion with an excess of methyl iodide in the presence of

D. Albanese et al. / Il Farmaco 53 (1998) 709–717
711
Scheme 1. Structural constituents of cephalosporins 1 and 2.
Scheme 2. Synthesis of cephalosporins 1 and 2.
solid, anhydrous potassium carbonate under solid–liq-
3. Experimental
uid PTC (SL-PTC) conditions to give 11 or 12, that
were directly saponified to methoximes 4b or 5b with
aqueous NaOH under liquid–liquid PTC (LL-PTC).
In a similar way, oximes 5c and 5d (Scheme 4) were
generated by tandem SL-PTC bis-alkylation of 5a with
t-butyl chloroacetate or chloroacetamide and LL-PTC
hydrolysis of the resulting intermediate 13 or 14.
2,4-Dihydroxybenzaldehyde (15) was chosen for the
synthesis of methoximes 5e and 5f (Scheme 5). The
tetrahydropyranyl (THP) ether 16, prepared by a stan-
dard procedure [45], was alkylated in good yields with
ethyl bromoacetate under SL-PTC conditions and the
resulting ester 17 was saponified with NaOH (LL-PTC)
and O-deprotected to 18 by acid hydrolysis. Finally, the
aldehyde 18 was transformed into the oxime 5e with
O-methylhydroxylamine in methanol in 42% overall
yield from aldehyde 15. The bis-alkylation of 5e with
chloroacetamide (SL-PTC) followed by saponification
(LL-PTC) gave the oxime 5f.
As shown in Scheme 6, the oxime 5g was prepared
from 2-hydroxy-5-nitrobenzaldehyde (19) through O-
alkylation with chloroacetic acid in the presence of
aqueous NaOH followed by reaction of the resulting
(2-formylaryl)oxyacetic acid (20) with hydroxylamine
hydrochloride. Oxime 5h was generated by reduction of
5g with sulfidric acid in aqueous ammonia to the amino
derivative 21 which was subjected to the alkylation–
hydrolysis protocol previously described.
3.1. Chemistry
Melting points were determined on a Bu¨chi 535
apparatus and are corrected. NMR spectra were
recorded on a Bruker AC 300 spectrometer; TMS was
used as external reference; l are in ppm and J are in
Hz. IR spectra were recorded on a FT-IR 1725 X
Perkin–Elmer spectrometer and frequency values are in
cm−1. Reagent-grade commercially available reagents
and solvents were used and were dried, when required,
before use. All the cephalosporanic nuclei 3 are known
in the literature [5].
3.1.1. Representati6e procedure for the synthesis of
cephalosporins 1,2. Preparation of 7-[(E)-(2-methoxy-
iminomethylphenoxy)acetamido]-3-acetoxy-3-cephem-
4-carboxylic acid (2a)
(E)-2-(Methoxyiminomethylphenoxy)-acetic acid (5b)
(5 g, 24 mmol) was suspended in 50 ml of
dichloromethane and triethylamine (3.3 ml) was added
dropwise. When the dissolution of 5b was complete, the
solution was cooled to −60°C, N-methylmorpholine
(50 ml) was added followed by ethyl chloroformate (2.3
ml, 24 mmol). The reaction mixture was stirred at
−30°C for 1 h to form the intermediate ‘mixed anhy-
dride’ 8. Separately, a mixture of 7-aminocephalospo-
ranic acid (3a) (7-ACA) (8.4 g, 31 mmol) and BSA (8.3

712
D. Albanese et al. / Il Farmaco 53 (1998) 709–717
Scheme 3. Preparation of oximes 4a,b and 5a,b.
Scheme 4. Preparation of oximes 5c,d by PTC alkylation/hydrolysis of 5a.
ml, 34 mmol) in 70 ml of anhydrous dichloromethane
was refluxed until complete dissolution of 7-ACA, then
it was cooled to −10°C. The solution was added
dropwise to the solution of 8 at 0°C and stirred at this
temperature for 3 h. The reaction mixture was then
washed with ice cold water (2×30 ml), 1N HCl (100
ml) (to remove the unreacted 7-ACA) and water (2×30
ml). The organic phase was evaporated under vacuum
until a solid residue was obtained that was dissolved in
200 ml of anhydrous iso-propanol. A solution of
sodium hexanoate (4 g) in 100 ml of iso-propanol was
added at 30°C and the mixture was left to crystallize at
30°C for 2 h, cooled with an ice/salt bath and filtered.
After drying, 9.1 g (78%) of product 2a (as sodium salt)
were obtained. The same procedure was applied to the
synthesis of products 1a,b and 2b,d–h,j. In the prepara-
tion of products 2c,i the t-butyl ester function was
solvolyzed in the last step of the synthesis by taking up
the crude of the condensation in absolute formic acid
(35 ml) instead of iso-propanol. The acid solution was
stirred for 4 h and then diluted with toluene (100 ml)
and formic acid was eliminated by azeotropic distilla-
tion under vacuum. The solid residue was dissolved in
iso-propanol (50 ml) and sodium hexanoate (7.5 g) in
iso-propanol (100 ml) was added. The mixture was left
to crystallize overnight.
The physical and spectroscopic characteristics of
products 1 and 2 are reported in Table 1.
3.1.2. (E)-(2-Hydroxyiminomethylphenyl)-acetic acid
(4a)
Hydroxylamine hydrochloride (5 g, 72 mmol) and
sodium acetate (20 g, 0.24 mol) were added to a solu-
tion of (2-formylphenyl)acetic acid (9) (5 g, 30 mmol) in
50 ml of water. The reaction mixture was refluxed for
10 min, cooled to 20°C, the pH adjusted at 2 with
diluted HCl and left to crystallize. The product 4a is
filtered, washed with water and dried under vacuum at
1
50°C. Yield 91%. M.p. 147°C. H NMR (DMSO-d6) l
13.20 (bs, 1H), 11.27 (s, 1H), 8.27 (s, 1H), 7.68–7.26
(m, 4H), 3.78 (s, 2H). IR (KBr) 3250, 1760, 1600, 1550
cm−1. Anal. Calc. for C9H9NO3: C, 60.33; H, 5.06; N,
7.82. Found: C, 60.25; H, 5.30; N, 7.73%.
3.1.3. (E)-(2-Hydroxyiminomethylphenoxy)-acetic acid
(5a)
5a was prepared following the same procedure used
for 4a. Yield 90%. M.p. 140°C (lit. [46], 138°C).

D. Albanese et al. / Il Farmaco 53 (1998) 709–717
713
Scheme 5. Preparation of oximes 5e,f from 2,4-dihydroxybenzaldehyde (15).
3.1.4. (E)-(2-Methoxyiminomethylphenyl)-acetic acid
(4b)
9.30 (bs, 1H), 8.17 (s, 1H), 7.43–6.90 (m, 4H), 4.70 (s,
2H), 3.99 (s, 3H). IR (KBr) 3050–2577, 1969, 1740,
1713, 1609, 1601, 1495 cm−1. Anal. Calc. for C9H9NO4:
C, 57.41; H, 5.30; N, 6.70. Found: C, 57.31; H, 5.42; N,
6.64%.
A solution of acid 4a (1.8 g, 10 mmol), MeI (1.5 ml,
24 mmol) and triethylbenzylammonium chloride
(TEBA) (114 mg, 0.5 mmol) in anhydrous acetonitrile
(20 ml) was stirred at 80°C for 6 h in the presence of
anhydrous potassium carbonate (5 g, 36 mmol). After
this period the reaction mixture was cooled to 20°C and
stirred overnight at this temperature. The salts were
filtered and the filtrate was evaporated under vacuum.
The crude methyl ester 11 was dissolved in
dichloromethane (50 ml) and tetrabutylammonium bro-
mide (200 mg, 0.6 mmol) and 15% aqueous NaOH (50
ml) were added to this solution. The heterogeneous
mixture was stirred at 20°C for 12 h, then the aqueous
phase was separated, acidified to pH 2 with diluted HCl
and left to crystallize overnight at 4°C. The product 4b
is filtered, washed with water and dried under vacuum
3.1.6. (E)-(2-t-Butoxycarbonylmethyleneoxyimino-
methylphenoxy)-acetic acid (5c)
A heterogeneous mixture of a solution of 5a (2.93 g,
15 mmol), t-butyl chloroacetate (5 ml, 35 mmol) and
TEBA (228 mg, 1 mmol) in anhydrous acetonitrile (20
ml) and of anhydrous potassium carbonate (5.5 g, 4
mmol) was stirred at 80°C for 20 h. After usual work-
up the ester function was hydrolyzed under LL-PTC
conditions as previously described for 4b, and 5c was
obtained after crystallization from water. Yield 65%.
M.p. 128–130°C. 1H NMR (DMSO-d6) l 13.10 (bs,
1H), 8.51 (s, 1H), 7.63–7.00 (m, 4H), 4.78 (s, 2H), 4.61
(s, 2H), 1.43 (s, 9H). IR (KBr) 3020, 2990, 2900, 1740,
1610, 1600 cm−1. Anal. Calc. for C15H19NO6: C, 58.25;
H, 6.19; N, 4.53. Found: C, 58.09; H, 6.11; N, 4.62%.
1
at 50°C. Yield 65%. M.p. 120°C. H NMR (CDCl3) l
9.22 (bs, 1H), 8.22 (s, 1H), 7.58–7.26 (m, 4H), 3.94 (s,
3H), 3.89 (s, 2H). IR (KBr) 3000–2500, 1780, 1608,
1600 cm−1. Anal. Calc. for C10H11NO3: C, 62.17; H,
5.74; N, 7.25. Found: C, 62.12; H, 5.91; N, 7.10%.
3.1.7. (E)-(2-Aminocarbonylmethyleneoxyiminomethyl-
phenoxy)-acetic acid (5d)
3.1.5. (E)-(2-Methoxyiminomethylphenoxy)-acetic acid
(5b)
5d was prepared starting from 5a (2.93 g, 15 mmol),
chloroacetamide (3.27 g, 35 mmol), TEBA (228 mg, 1
mmol), acetonitrile (20 ml) and potassium carbonate
(5.5 g, 40 mmol), with a similar procedure to that used
5b was prepared with the same procedure used for
1
4b. Yield 67%. M.p. 122–124°C. H NMR (CDCl3) l

714
D. Albanese et al. / Il Farmaco 53 (1998) 709–717
Scheme 6. Preparation of oximes 5g,h from 2-hydroxy-5-nitrobenzaldehyde (19).
Table 1
Isolated yields, melting points and spectroscopical data of cephalosporins 1 and 2
Com-
pound
Yield (%)
M.p. (°C)
1H NMR l (ppm), J (Hz)a; IR (C=O, b-lactam) (cm−1 b
)
1a
77
180 (dec.)
12.40 (bs, 1H, COOH), 8.68 (d, 1H, J=8), 8.52 (s, 1H), 7.70–7.20 (m, 4H), 5.53 (dd, 1H, J=8, 5),
5.00 (d, 1H, J=5), 4.95 and 4.72 (ABq, 2H, J=15), 3.90 (s, 3H), 3.75 (s, 2H), 3.51 and 3.27 (ABq,
2H, J=13), 2.00 (s, 3H); 1767; acid form
1b
2a
75
78
120 (dec.)
175 (dec.)
9.10 (d, 1H, J=8), 8.45 (s, 1H), 7.70–7.20 (m, 4H), 3.85 (s, 3H), 3.70 (s, 2H), 3.60 and 3.30 (ABq,
2H, J=13); 1759; sodium salt
9.00 (d, 1H, J=8), 8.45 (s, 1H), 7.70–7.00 (m, 4H), 5.55 (dd, 1H, J=8, 5), 5.00 (d, 1H, J=5),
5.00–4.75 (ABq, 2H, J=15), 4.72 (s, 2H), 3.90 (s, 3H), 3.45 and 3.15 (ABq, 2H, J=13), 2.00 (s,
2H); 1767; sodium salt
2b
2c
2d
2e
2f
75
59
61
62
76
60
71
57
55
147 (dec.)
205
11.27 (s, 1H), 9.10 (d, 1H, J=8), 8.27 (s, 1H), 7.68–7.26 (m, 4H), 5.65 (dd, 1H, J=8, 5), 5.05 (d,
1H, J=5), 4.70 (s, 2H), 4.35 and 4.25 (ABq, 2H, J=13), 3.90 (s, 3H), 3.60 and 3.30 (ABq, 2H,
J=14); 1756; sodium salt
9.20 (d, 1H, J=8), 8.50 (s, 1H), 7.65–7.00 (m, 4H), 5.71 (dd, 1H, J=8, 5), 5.12 (d, 1H, J=5), 4.78
(s, 2H), 4.65 (s, 2H), 4.42 and 4.31 (ABq, 2H, J=13), 3.90 (s, 3H), 3.75 and 3.60 (ABq, 2H,
J=15); 1760; disodium salt
12.50 (bs, 1H), 9.20 (d, 1H, J=8), 8.16 (s, 1H), 7.65–7.30 (m, 4H), 7.00–6.90 (m, 2H), 5.72 (dd, 1H,
J=8, 5), 5.13 (d, 1H, J=5), 4.70 (s, 2H), 4.45 (s, 2H), 4.41 and 4.34 (ABq, 2H, J=14), 3.95 (s,
3H), 3.83 and 3.62 (ABq, 2H, J=15); 1770; acid form
8.95 (d, 1H, J=8), 8.45 (s, 1H), 7.72–6.95 (m, 4H), 5.50 (dd, 1H, J=8, 5), 4.95 (d, 1H, J=5), 4.70
(s, 2H), 4.35 (t, 2H, J=6), 4.41 and 4.20 (ABq, 2H, J=13), 3.91 (s, 3H), 3.90 and 3.35 (ABq, 2H,
J=15), 2.71 (t, 2H, J=6), 2.15 (s, 6H); 1763; sodium salt
13.10 (bs, 1H), 9.92 (s, 1H), 9.23 (d, 1H, J=9), 8.33 (s, 1H), 7.52–6.31 (m, 3H), 5.70 (dd, 1H, J=9,
5), 5.15 (d, 1H, J=5), 4.67 (s, 2H), 4.42 and 4.23 (ABq, 2H, J=13), 3.90 (s, 3H), 3.83 (s, 3H), 3.81
and 3.64 (ABq, 2H, J=15); 1761; acid form
9.17 (d, 1H, J=8), 8.32 (s, 1H), 7.51–6.30 (m, 3H+2H), 5.32 (dd, 1H, J=8, 5), 5.11 (d, 1H, J=5),
4.73 (s, 2H), 4.50 (s, 2H), 4.40 and 4.20 (ABq, 2H, J=14), 3.92 (s, 3H), 3.82 (s, 3H), 3.81 and 3.63
(ABq, 2H, J=12); 1755; sodium salt
13.21 (bs, 1H), 11.70 (s, 1H), 9.05 (d, 1H, J=8), 8.46–8.21 (m, 3H), 7.24 (d, 1H, J=6), 5.61 (dd,
1H, J=8, 5), 5.14 (d, 1H, J=5), 4.96 (s, 2H), 4.45 and 4.26 (ABq, 2H, J=15), 3.51 and 3.37
(ABq, 2H, J=13), 2.65 (s, 3H); 1758; acid form
13.00 (bs, 1H), 9.00 (d, 1H, J=9), 8.17 (s, 1H), 7.22–6.70 (m, 3H), 5.70 (dd, 1H, J=9, 5), 5.15 (d,
1H, J=5), 4.73 (s, 2H), 4.64 (s, 2H), 4.52 and 4.40 (ABq, 2H, J=15), 3.95 (s, 3H), 3.50 (bs, 3H),
3.72 and 3.64 (ABq, 2H, J=13); 1701; inner salt
110 (dec.)
110 (dec.)
189
2g
2h
2i
136 (dec.)
195
180
2j
115
13.02 (bs, 1H), 9.00 (d, 1H, J=8), 8.17 (s, 1H), 7.22–6.67 (m, 3H), 5.70 (dd, 1H, J=8, 5), 5.17 (d,
1H, J=5), 4.70 (s, 2H), 4.51 and 4.43 (ABq, 2H, J=15), 3.92 (s, 3H), 3.72 and 3.64 (ABq, 2H,
J=13), 3.45 (bs, 3H), 2.72 (s, 3H); 1765; inner salt
a In DMSO-d6.
b KBr pellets.

D. Albanese et al. / Il Farmaco 53 (1998) 709–717
715
Table 2
In vitro MICs of cephalosporins 1b,2b–j against some Gram+ strains
Compound
MICs (mg/ml)a
S. a.
S. a.
S. e.
S. e.
S. f.
ATCC 6538
F2 ISF 3
HCF B.
CHPL A2
LEP Br
1b
2b
2c
2d
2e
2f
2g
2h
2i
0.031
0.063
0.25
0.063
0.063
0.031
0.016
0.031
0.25
32
32
128
32
120
8
2
16
8
0.125
0.25
1
0.125
0.25
0.031
0.031
0.063
0.25
0.031
2
2
8
128
4
1
0.5
1
0.5
8
32
128
128
0.402
120
0.25
0.102
32
0.25
0.25
2j
0.016
8
a Determined by a standard dilution method. MICs, minimal inhibitory concentrations; S. a., Staphylococcus aureus; S. e., Staphylococcus
epidermidis; S. f., Streptococcus fecalis.
Table 3
In vitro MICs of cephalosporins 1b,2b–j against some Gram− strains
Compound
MICs (mg/ml)a
E. c.
E. c.
E. c. R
P. 6.
K. p.
S. e.
ATCC 8739
ISF 432
ISF 10
CNUR 6
ATCC 10031
1b
2b
2c
2d
2e
2f
2g
2h
2i
32
4
16
4
2
2
0.25
8
2
4
4
4
4
4
100
100
100
100
100
100
32
100
16
32
100
100
100
100
100
100
100
100
100
100
4
2
4
4
4
2
0.25
8
2
\128
128
6
128
128
16
16
\128
2
2
0.5
16
4
2j
4
4
6
6
a Determined by a standard dilution method. MICs, minimal inhibitory concentrations; E. c., Escherichia coli; E. c. R, Escherichia coli
R+TEM; P. 6., Proteus 6ulgaris; K. p., Klebsiella pneumoniae; S. e., Shighella enteridis.
for 5c. Acid 5d was isolated as a solid after crystalliza-
tion. Yield 43%. M.p. 179–182°C. H NMR (DMSO-
mmol) and potassium carbonate (158 mg, 1.1 mmol)
were added. This mixture was stirred at 80°C for 2 h,
cooled at 20°C, diluted with 50 ml of ethyl ether and
filtered through Celite. The filtrate was evaporated un-
der vacuum and the crude was flash chromatographed
(silica gel 230–400 mesh) using ethyl ether/petroleum
1
d6) l 13.10 (bs, 1H), 8.55 (s, 1H), 7.65–7.37 (m, 2H),
7.35 (bs, 1H), 7.27 (bs, 1H), 7.25–7.00 (m, 2H), 4.78 (s,
2H), 4.56 (s, 2H). IR (KBr) 3990, 3250, 2900–2600,
1710, 1650, 1585 cm−1. Anal. Calc. for C11H12N2O5: C,
52.38; H, 4.80; N, 11.11. Found: C, 52.25; H, 4.88; N,
11.00%.
1
ether (1:2) as eluant. Yield 80%. M.p. 65°C. H NMR
(CDCl3), l 10.39 (s, 1H), 7.83 (d, 1H, J=9), 6.76 (dd,
1H, J=9, 3) 6.50 (d, 1H, J=3), 5.48 (t, 1H, J=3),
4.70 (s, 2H), 4.26 (q, 2H, J=7), 3.87–3.75 (m, 1H),
3.65–3.52 (m, 1H), 1.95–1.56 (m, 6H), 1.19 (t, 3H,
J=7). Anal. Calc. for C16H20O6: C, 62.33; H, 6.54.
Found: C, 62.30; H, 6.48%.
3.1.8. Preparation of the tetrahydropyranyl ether i.e.
17
A mixture of 2,4-dihydroxybenzaldehyde (15) (138
mg, 1.01 mmol), 3,4-dihydro-2H-pyran (340 mg, 4
mmol) and pyridinium p-toluenesulfonate (PPTS) (25
mg) in dichloromethane (2 ml) was stirred under anhy-
drous atmosphere at 20°C for 24 h. The solvent was
evaporated under vacuum, the crude of the reaction
was dissolved in anhydrous acetonitrile (1 ml) and ethyl
bromoacetate (184 mg, 1.1 mmol), TEBA (23 mg, 0.1
3.1.9. (E)-(4-Hydroxy-2-methoxyiminomethylphenoxy)-
acetic acid (5e)
The ethyl ester 17 (244 mg, 0.80 mmol) was hy-
drolyzed through the LL-PTC procedure used for 4b.
The crystallization at pH 2 hydrolyzed the tetrahy-

716
D. Albanese et al. / Il Farmaco 53 (1998) 709–717
dropyranyl ether function and gave the crude (2-
formyl-4-hydroxyphenoxy)acetic acid 18 (150 mg, 0.76
mmol). To a heterogeneous mixture of 18 and sodium
hydrogencarbonate (305 mg, 3.0 mmol) in MeOH (2
ml) O-methylhydroxylamine hydrochloride (30%
aqueous solution) (115 ml, 1.52 mmol) was added drop-
wise, keeping the pH in the range 3–4. After decol-
oration with charcoal, the product was crystallized
twice with water in order to obtain the desired pure
E-isomer 5e as white crystals. Yield 42% (overall from
15). M.p. 120°C. 1H NMR (DMSO-d6) l 13.07 (bs,
1H), 9.96 (s, 1H), 8.28 (s, 1H), 7.59 (d, 1H, J=9), 6.41
(dd, 1H, J=9, 3), 6.32 (d, 1H, J=3), 4.67 (s, 2H), 3.80
mmol) was stirred at 40°C for 5 h. The excess hydrogen
sulfide was removed under vacuum at 50°C. The reac-
tion mixture was cooled at 20°C, the sulfur produced
during the reduction crystallized and was filtered off.
The pH was adjusted to 4 to crystallize the product 21
that was filtered, redissolved in an aqueous solution
buffered at pH 8, decolorized over charcoal and recrys-
tallized with hydrochloric acid at pH 3. The product 21
was isolated as a clear crystalline powder. Yield 59%.
M.p. 187°C. 1H NMR (CDCl3) l 11.70 (bs, 1H), 8.17 (s,
1H), 7.10–6.68 (m, 3H), 4.70 (s, 2H), 3.50 (bs, 3H). IR
(KBr) 3500–3050, 1850, 1650, 1505 cm−1. Anal. Calc.
for C9H10N2O4: C, 51.43; H, 4.80; N, 13.33. Found: C,
51.30; H, 4.90; N, 13.21%.
(s, 3H). IR (KBr) 3300–3200, 1705, 1620, 1600 cm−1
.
Anal. Calc. for C10H11NO5: C, 53.33; H, 4.92; N, 6.22.
Found: C, 53.25; H, 4.81; N, 6.15%.
3.1.14. (E)-(4-Amino-2-t-butoxycarbonylmethyleneoxy-
iminomethylphenoxy)-acetic acid (5h)
3.1.10. (E)-(5-Aminocarbonylmethyleneoxy-2-methoxy-
iminomethylphenoxy)-acetic acid (5f)
Acid 5h was prepared according to the procedure
described for the synthesis of 5c starting from 21 (420
mg, 2 mmol), t-butyl chloroacetate (0.7 ml, 5 mmol),
TEBA (20 mg), anhydrous potassium carbonate (1 g,
7.2 mmol) and anhydrous acetonitrile (2 ml). After
usual work-up the ester function was hydrolyzed under
LL-PTC conditions as in 5c. Crude 5h was obtained as
a solid after crystallization from water and was used as
such in the condensation reactions with the cephalospo-
ranic nuclei.
The product 5f was prepared from 5e by alkylation
(SL-PTC)–hydrolysis (LL-PTC) as reported for the
synthesis of 5d (Section 3.1.7). Yield 35%. M.p. 183–
1
185°C. H NMR (DMSO-d6) l 13.10 (bs, 1H), 8.27 (s,
1H), 7.51 (d, 1H, J=9), 7.35–7.28 (bs, 2H), 6.42 (dd,
1H, J=9, 3), 6.33 (d, 1H, J=3), 4.70 (s, 2H), 3.80 (s,
3H). IR (KBr) 3300–3200, 1705, 1620, 1600 cm−1
.
Anal. Calc. for C12H14N2O6: C, 51.06; H, 5.00; N, 9.93.
Found: C, 51.21; H, 5.15; N, 9.86%.
3.2. Microbiology
3.1.11. (E)-(2-Formyl-4-nitrophenoxy)-acetic acid (20)
A solution of 2-hydroxy-5-nitrobenzaldehyde (19)
(2.5 g, 15 mmol), chloroacetic acid (1.41 g, 15 mmol),
NaOH (1.2 g, 30 mmol) in H2O (40 ml) was refluxed
under stirring for 3 h. After cooling the reaction mixture
was acidified to pH 3 with 32% HCl and left to
crystallize overnight at 20°C. Yield 70%. M.p. 190°C
(lit. [47] 190–192°C).
Cephalosporins 1b,2b–j were tested for in vitro an-
tibacterial activity against some Gram+ (Staphylo-
coccus aureus ATCC 6538, S. aureus F2 ISF 3, Staphy-
lococcus epidermidis HCF BersetC, S. epidermidis CHPL
A2, Streptococcus fecalis LEP Br) and some Gram−
(Escherichia coli ATCC 8739, E. coli ISF 432, E. coli
R+ TEM ISF 10, Proteus 6ulgaris CNUR 6, Klebsiella
pneumoniae ATCC 10031, Shighella enteridis) bacterial
strains. Minimal inhibitory concentrations (MICs) were
determined by an Agar dilution technique using
Mu¨ller–Hinton 2 agar (bioMerieux). The inocolum
used was 104 CFU per spot. The cephalosporins 1 and
2 were diluted in the test medium in the range 128–
0.016 mg/ml.
3.1.12. (E)-(2-Hydroxyiminomethyl-4-nitrophenoxy)-
acetic acid (5g)
The oxime 5g was prepared starting from aldehyde 20
(2.36 g, 10.4 mmol), hydroxylamine hydrochloride (1.67
g, 24.1 mmol) and sodium acetate (6.9 g, 84 mmol) in 20
ml of water by using the procedure reported for 4a
(Section 3.1.2) and was isolated after crystallization
from water. Yield 89%. M.p. 193°C. 1H NMR (DMSO-
d6) l 13.40 (bs, 1H), 11.72 (s, 1H), 8.46 (d, 1H, J=3),
8.34 (s, 1H), 8.22 (dd, 1H, J=9, 3), 7.42 (d, 1H, J=9),
4.97 (s, 2H). IR (KBr) 3600, 3490, 3250–2900, 1720,
1610, 1508 cm−1. Anal. Calc. for C9H8N2O6: C, 45.01;
H, 3.36; N, 11.66. Found: C, 44.92; H, 3.44; N, 11.50%.
4. Results and discussion
Even if the MICs values found for the cephalosporins
1 and 2 (Tables 2 and 3) represent an initial screening,
they give an indication of the potentiality of these new
b-lactam antibiotics.
In general, these products showed a good activity
against Gram+ microorganism (Table 2) and part of
the Gram− tested (Table 3), whereas they resulted less
or no active against E. coli R+ TEM, P. 6ulgaris and
3.1.13. (E)-(4-Amino-2-hydroxyiminomethylphenoxy)-
acetic acid (21)
A solution of 5g (1.32 g, 5.5 mmol) and hydrogen
sulfide (0.6 g, 17.6 mmol) in 6N ammonia (6.5 ml, 39

D. Albanese et al. / Il Farmaco 53 (1998) 709–717
717
[15] Drugs Today 20 (1984) 615.
S. enteridis. Among the tested products, cephalosporin
2g, that contains an oxyiminomethyl and an oxy-
acetamido group in the C-7 side chain benzene ring,
[16] Drugs Future 8 (1983) 682.
[17] Drugs Future 13 (1988) 790.
[18] Drugs 38 (1989) 524.
[19] Drugs Future 3 (1978) 905.
[20] Drugs Future 5 (1980) 519.
[21] Drugs Today 17 (1981) 16.
[22] M. Ochiai, O. Aki, A. Morimoto, T. Okada, K. Kawakita, Y.
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[24] Drugs Future 8 (1983) 445.
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a (1-methyl-1,2,3,4-tetrazol-5-yl)thio-
methyl group at the C-3 position, seems to be the more
active against both Gram+ and Gram− strains.
In conclusion, the introduction of an oxyimino-
methylene function at the ortho-position of the aro-
matic ring generated a new class of cephalosporins with
an interesting antibacterial activity, that could stimulate
the search for further useful structural modifications.
[27] Drugs Today 19 (1983) 335.
[28] Drugs Future 5 (1980) 146.
[29] Drugs Future 6 (1981) 132.
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