Synthesis and antimicrobial properties of cephalosporin derivatives substituted on the C(7) nitrogen with arylmethyloxyimino or arylmethyloxyamino alkanoyl groups

Article abstract of DOI:10.1016/S0014-827X(99)00017-8

Some 7-aminocephalosporanic acid (7-ACA) derivatives substituted on the C(7) nitrogen with 2-(arylmethyloxyimino)propionyl (3a-f), 2-(arylmethyloxyamino)propionyl (4a-d) and (arylmethyloxyamino)acetyl (2a-d) moieties were synthesized by reaction of the ap

Full text of DOI:10.1016/S0014-827X(99)00017-8

Il Farmaco 54 (1999) 224–231
Synthesis and antimicrobial properties of cephalosporin derivatives
substituted on the C(7) nitrogen with arylmethyloxyimino or
arylmethyloxyamino alkanoyl groups
Daniela Gentili a, Marco Macchia a, Elisabetta Menchini a, Susanna Nencetti a,*,
Elisabetta Orlandini a, Armando Rossello a, Giampietro Broccali b, Donatella Limonta b
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
b Laboratorio B.T. Biotecnica S.R.L., Via G. Ferrari 21, 20047 Saronno (Va), Italy
Received 20 July 1998; accepted 10 February 1999
Abstract
Some 7-aminocephalosporanic acid (7-ACA) derivatives substituted on the C(7) nitrogen with 2-(arylmethyloxyimino)propionyl
(3a–f), 2-(arylmethyloxyamino)propionyl (4a–d) and (arylmethyloxyamino)acetyl (2a–d) moieties were synthesized by reaction of
the appropriate acylating agents with 7-ACA protected as a t-butyl ester, followed by removal of the t-butyl protecting group.
The new compounds, tested in vitro for their antimicrobial activity against Gram-positive and Gram-negative bacteria, proved to
possess a modest activity directed only against Gram-positive microorganisms. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: b-lactams; Cephalosporins; Antimicrobial activity
1. Introduction
cleus, we previously described compounds 1a–g [11], in
which this side-chain contains an aryl-substituted
By now, it is widely accepted that the antimicrobial
properties of cephalosporinic b-lactam antibiotics and
their stability to acids and resistance to enzyme inacti-
vation, depend on various factors, of which, one of the
most important is the chemical nature of the amidic
substituent linked to the C(7) carbon of the b-lactam
nucleus [1–5].
In the majority of cephalosporins of therapeutic in-
terest, this amidic side-chain is substituted by an aryl or
aromatic heterocycle, together with another more or
less complex moiety, which, in many cases, contains an
oximethereal group or a protonatable aminic moiety
[6].
[(methyloxy)imino]methyl
moiety
(CH2ONꢀC,
MOIMM). This oximethereal group was chosen on the
basis of the fact that, in the field of b-adrenergic
blocking drugs [13,14], it acted as a valid bioisoster of
aryls, and therefore might also be able to effectively
replace the aromatic moiety usually present on the
amidic side-chains of the most active cephalosporins.
As compounds 1a–g show a modest activity directed
only against Gram-positive microorganisms, we
thought it of interest to verify whether an increase in
the polarity of the amidic side-chain might improve the
antimicrobial properties of these types of compounds.
Consequently, compounds 2a–d were synthesized, in
which the oximethereal moiety of compounds 1a–g is
replaced by the more polar hydroxylaminoethereal por-
tion. This moiety, in addition to those present in some
b-lactam antibiotics of clinical interest, possesses the
basic characteristics needed to widen the activity spec-
trum and gives to the new compounds a stability to
acids. Furthermore, in order to examine whether the
antimicrobial activity of 2-arylmethyloxyimino (1a–g)
and 2-arylmethyloxyamino (2a–d) compounds could be
In a series of studies [7–12] in the field of cephalo-
sporinic b-lactam antibiotics aiming at investigating the
effects on the antimicrobial properties induced by cer-
tain structural modifications on the amidic side-chain
linked to the C(7) carbon of the cephalosporanic nu-
* Corresponding author. Tel.: +39-050-500-209; fax: +39-050-
40517.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00017-8

D. Gentili et al. / Il Farmaco 54 (1999) 224–231
225
influenced by an increase in the steric bulk of the
amidic side-chain, compounds 3a–f and 4a–d were also
prepared, in which a methyl substituent is present on
the oxime carbon of 1 or on the corresponding carbon
of the saturated compounds 2.
iminoacids 8a–f. Reduction of 7a–d with borane-
trimethylamine complex in anhydrous 8 N ethanolic
hydrochloric acid afforded the aminoesters 9a–d which
were hydrolyzed with THF–H2O NaOH to form the
corresponding aminoacids 11a–d.
The b-lactam derivatives 3a–f, 2a–d and 4a–d were
prepared as indicated in Scheme 2. Treatment of imi-
noacids 8a–f and aminoacids 10a–d and 11a–d with
7-ACA, protected as a t-butyl ester, in the presence of
[N-(3-dimethylaminopropyl)N-ethyl carbodiimide hy-
drochloride] (EDCI) as the coupling agent, afforded the
corresponding b-lactam esters 12a–f, 13a–d and 14a–d,
which were purified by column chromatography and
then hydrolyzed to the free acids 3a–f, 2a–d and 4a–d,
using trifluoroacetic acid and anisole.
In view of the presence of the new chiral center at the
level of the amidic side-chain, both the b-lactam esters
14a–d and the corresponding acids 4a–d, should be
mixtures of two diastereoisomers, these proved to be
inseparable by means of the usual separation methods.
The configuration around the oximic double bond of
esters 7 was assigned by 1H NMR study of the shielding
induced by an anisotropic solvent (C6D6) on one of
these esters (7a) in the unprotonated and protonated
forms. For this compound, in agreement with findings
for methylketone oxime ethers which present a syn
relationship between the oximic oxygen and the methyl
group [16], the protonation of the oximic nitrogen
determines a shift of the signal of the methyl protons to
a higher field, ranging from 0.14 to 0.26 ppm, depend-
ing on the quantity of acid added to the oximic com-
pound 7a (see Section 4). The very similar chemical
shift value of the methyl signal for 7a and for its
analogs 7b–f in the same experimental conditions,
made it possible to also assign the same type of configu-
ration (E) to 7b–f. The E configuration around the
iminic double bond of acids 8a–f, of b-lactam esters
12a–f and of the final compounds 3a–f was assigned
2. Chemistry
The N-(arylmethyloxy)iminoacids 8a–f and the N-
(arylmethyloxy)aminoacids 10a–d and 11a–d to be
used as 7-aminocephalosporanic acid (7-ACA) acylat-
ing agents in the synthesis of the b-lactam compounds
3a–f, 2a–d and 4a–d, were prepared as outlined in
Scheme 1. For acids 10a–d the synthetic route followed
the one previously described [15], consisting of the
reaction of 5a–d with glyoxylic acid and subsequent
reduction of the resulting oximic acids 6a–d.
As regards acids 8a–f and 11a–d, the reaction of the
appropriate O-(arylmethyl)hydroxylamines (5a–f) with
ethylpyruvate afforded the corresponding ethyl esters of
the (E)-N-(arylmethyloxy)-2-iminopropionic acids (7a–
f) as the only configurational isomer. Hydrolysis of
7a–f with ethanolic NaOH yielded the corresponding
Scheme 1.

226
D. Gentili et al. / Il Farmaco 54 (1999) 224–231
Scheme 2.
on the basis of the observation that the value of the
chemical shift of the signal of the methyl protons of
these compounds is practically the same for the starting
E oximes 7a–f, acids 8a–f, b-lactam esters 12a–f and
the corresponding acids 3a–f.
2-arylmethyloxyamino ones of type 2, respectively, in
the insertion of a methyl group on the MOIMM of 1
and on the corresponding carbon of the hydroxyl-
aminoethereal portion of 2, exhibited an antimicrobial
profile similar to that of 1 and 2, i.e. a modest activity
towards some Staphylococcus strains, and a complete
inactivity towards Gram-negative bacteria.
Compounds 2 were synthesized in order to verify
whether the substitution of the oximethereal portion of
1 with the more polar hydroxylaminoethereal moiety
which also possesses basic characteristics, might have a
positive effect on the activity of these compounds, while
compounds 3 and 4 were prepared with the aim of
testing the effects on the antimicrobial activity of the
insertion of a methyl group on the oximic or hydroxyl-
aminic portions of 1 and 2, respectively.
The results obtained showed that all three types of
cephalosporinic derivatives synthesized (2–4) present
similar antimicrobial characteristics to those of the type
1 compounds previously studied.
3. Results and discussion
Compounds 2a–d, 3a–f and 4a–d were tested on 14
bacterial strains of Gram-positive and Gram-negative
microorganisms; the results of these tests are expressed
as MIC (minimum inhibitory concentration) values and
are shown in Table 1, together with those obtained on
the same strains for 1b [11], one of the most active
previously described compounds, cephaloram,
cephalosporin antibiotic active against Gram-positive
bacteria [17], and ceftazidime, wide-range
a
a
cephalosporin antibiotic, particularly active against
Gram-negative bacteria [18].
It may thus be concluded that the types of structural
modifications which, starting from the structure of
compound 1, lead to compounds 2, 3 and 4, do not
induce any improvement in the activity, and should
not, therefore, be able to substantially modify any of
the molecular parameters involved in the definition of
the antimicrobial properties of those types of cephalo-
sporinic derivatives.
The 2-arylmethyloxyamino cephalosporins 2a–d, like
the previously reported 2-arylmethyloxyimino analogs
1a–g, showed a low activity directed only towards two
Staphylococcus epidermidis strains, and appeared to be
completely inactive against the tested Gram-negative
bacteria at concentrations lower than 128 mg/ml.
Compounds 3a–f and 4a–d, which differ from the
2-arylmethyloxyimino cephalosporins of type 1 and the

Table 1
Antimicrobial activity (MIC a, mg/ml) of cephalosporins 1b, 2a–d, 3a–f and 4a–d against Gram-positive and Gram-negative bacteria
b
Microorganisms
CFL
CFT c
1b
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
Gram positive
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus epidermidis
Enterococco faecalis
d
e
/
0.036
4
0.036
8
128
2
8
32
128
8
\128
\128
2
32
\128
\128
\128
4
64
\128
64
\128
1
32
\128
\128
\128
4
64
\128
\128
32
16
\128
\128
\128
\128
32
16
\128
\128
\128
8
16
64
\128
\128
32
32
\128
\128
\128
8
32
\128
\128
\128
64
2
2
\128
64
2
1
\128
8
8
64
2
2
32
1
1
16
f
4
4
64
g
16
16
h
\128
\128
\128
\128
\128
\128
5
Gram negative i
(
Geometric averages
\128
0.469
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
\128
a The in vitro antibacterial activities were evaluated by a two-fold serial dilution method with a multiinocular device (see Ref. [19]).
2
b Cephaloram.
c Ceftazidime.
231
d S.a. MPR 5.
e S.a. ATCC 6538.
f S.e. HCF Berset C.
g S.e. CPLH A2.
h E.f. LEP Br.
i Strain tested: Escherichia coli ATCC 8739, Escherichia coli ISF 432, Enterobacter cloacae OMNFI 153, Proteus 6ulgaris CUNR 6, Pro6idencia stuardi CUNR 5, Klebsiella pneumoniae ATCC10031, Shighella enteritidis,
Pseudomonas aeruginosa CNUR 4, Pseudomonas aeruginosa ATCC 9027.

228
D. Gentili et al. / Il Farmaco 54 (1999) 224–231
1
4. Experimental
4.1. Chemistry
4H); 7c (75%): IR w 1726 cm−1; H NMR (CDCl3) l
1.36 (t, 3H, J=7 Hz), 2.11 (s, 3H), 4.36 (q, 2H, J=7
Hz), 5.30 (s, 2H), 7.26–7.56 (m, 4H); 7d (69%): IR w
1726 cm−1; H NMR (CDCl3) l 1.35 (t, 3H, J=7 Hz),
1
Melting points were determined on a Kofler hot-
stage apparatus and are uncorrected. IR spectra, for
comparison of compounds, were recorded on an FTIR
Mattson 1000 Unicam spectrometer as Nujol mulls in
the case of solid substances or as liquid film in the case
of liquids. 1H NMR spectra were recorded with a
Varian CFT20 instrument operating at 80 MHz in ca.
3% CDCl3 or DMSO-d6 solutions. The proton mag-
netic resonance assignments were established on the
basis of the expected chemical shifts and the multiplic-
ity of the signals. Evaporations were made in vacuo
(rotating evaporator). Analytical TLCs were carried out
on 0.25 mm layer silica gel plates (Merck F254).
Column chromatography was carried out on 70–230
mesh silica gel. MgSO4 was always used as a drying
agent. Elemental analyses were performed in our ana-
lytical laboratory and agreed with theoretical values to
within 90.4%.
2.07 (s, 3H), 4.35 (q, 2H, J=7 Hz), 5.3 (s, 2H), 7.37
1
(m, 4H); 7e (81%): IR w 1726 cm−1; H NMR (CDCl3)
l 1.36 (t, 3H, J=7 Hz), 2.13 (s, 3H), 3.86 (s, 3H), 4.36
(q, 2H, J=7 Hz), 5.46 (s, 2H), 6.86–7.73 (m, 4H); 7f
1
(85%): IR w 1726 cm−1; H NMR (CDCl3) l 1.35 (t,
3H, J=7 Hz), 2.09 (s, 3H), 3.80 (s, 3H), 4.32 (q, 2H,
J=7 Hz), 5.28 (s, 2H), 6.81–7.30 (m, 4H).
1H NMR study of the shielding effect induced by an
anisotropic solvent on 7a was carried out with a 5%
solution (w/w) in C6D6 (0.5 ml) by adding increasing
amounts (50, 100 and 1000 ml) of CF3CO2D. The
spectrum was recorded for the free base and after each
addition of acid. The chemical shifts of the methyl
proton signals were 1.97 ppm for the free base and 1.83,
1.76 and 1.71 ppm for the protonated form, after the
addition of 50, 100 and 1000 ml.
An analogous experiment carried out in the same
conditions, using the solvent CDCl3 instead of C6D6,
did not reveal any appreciable effect on the chemical
shift of the same methyl group.
4.1.1. N-(Arylmethyloxy)glycines (10a–d)
These compounds were prepared following the syn-
thetic route previously described [15]. Treatment of the
appropriate O-(arylmethyl)hydroxylamine hydrochlo-
ride (5a–d) (0.041 mmol) with glyoxylic acid (0.049
mmol) in acetonitrile for 12 h at room temperature
(r.t.) afforded the (E)-N-(arylmethyloxy)iminoacetic
acids (6a–d), which by reduction with the borane–tri-
ethylamine complex in EtOH in the presence of 10%
aqueous HCl, yielded, after the usual work-up, the
aminoacids 10a–d.
4.1.3. (E)-N-(Arylmethyloxy)-2-iminopropionic acid
deri6ati6es (8a–f)
A 2 N NaOH ethanolic solution (73.2 ml) was added
to a solution of the appropriate ethyl esters (7a–f) (22.6
mmol) in EtOH (4 ml). After stirring at 40°C for 30
min, the reaction mixture was evaporated at reduced
pressure and the resulting residue was diluted with H2O
and washed with Et2O. The aqueous phase was aci-
dified with 10% aqueous HCl to pH$4 and extracted
with AcOEt. Evaporation of the washed (H2O) organic
extracts gave the appropriate acids as solids which were
purified by crystallization from hexane to yield exclu-
sively the E-isomers 8a–f.
4.1.2. Ethyl esters of (E)-N-(arylmethyloxy)-2-imino-
propionic acids (7a–f)
A
solution of the appropriate O-(arylmethyl)-
hydroxylamine hydrochloride (5a–f) (0.092 mol) and
ethylpyruvate (0.092 mol) in anhydrous EtOH (74 ml)
was treated dropwise, while stirring, with a solution of
AcONa (0.138 mol) in anhydrous EtOH (74 ml). The
resulting mixture was stirred at r.t. for 24 h and then
evaporated at reduced pressure. The residue was added
to H2O and extracted with Et2O. The organic phase
was separated, washed (5% aqueous HCl, 10% aqueous
NaHCO3 and H2O) and evaporated to dryness to yield
a crude oily residue which was subjected to column
chromatography on silica gel, eluting with a 4:1 hex-
ane–AcOEt mixture to give 7 as only one of the two
possible E/Z isomers (1H NMR and GLC).
1
8a (58%): m.p. 83–85°C; IR w 1703 cm−1; H NMR
(CDCl3) l 2.10 (s, 3H), 3.60–4.03 (br, 1H, D2O ex-
changeable), 5.37 (s, 2H), 7.45 (s, 5H); 8b (44%): m.p.
1
98–99°C; IR w 1703 cm−1; H NMR (CDCl3–DMSO-
d6) l 2.04 (s, 3H), 5.26 (s, 2H), 7.34 (m, 4H); 8c (68%):
1
m.p. 104–105°C; IR w 1749 cm−1; H NMR (CDCl3–
DMSO-d6) l 2.13 (s, 3H), 5.73 (s, 2H), 8.00 (m, 4H); 8d
1
(75%): m.p. 110–111°C; IR w 1703 cm−1; H NMR
(CDCl3) l 2.13 (s, 3H), 5.35 (s, 2H), 5.83–6.10 (br, 1H,
D2O exchangeable), 7.47 (m, 4H); 8e (54%): m.p. 75–
1
76°C; IR w 1703 cm−1; H NMR (CDCl3) l 2.07 (s,
1
7a (85%): IR w 1724 cm−1; H NMR (CDCl3) l 1.37
3H), 3.83 (s, 3H), 5.34 (s, 2H), 6.80–7.35 (m, 4H), 8.90
(br, 1H, D2O exchangeable); 8f (97%): m.p. 80–81°C;
(t, 3H, J=7 Hz), 2.07 (s, 3H), 4.30 (q, 2H, J=7 Hz),
1
IR w 1711 cm−1; H NMR (CDCl3) l 2.07 (s, 3H), 3.83
5.33 (s, 2H), 7.43 (s, 5H); 7b (88%): IR w 1726 cm−1
;
1H NMR (CDCl3) l 1.30 (t, 3H, J=7 Hz), 2.08 (s,
3H), 4.33 (q, 2H, J=7 Hz), 5.43 (s, 2H), 7.16–7.66 (m,
(s, 3H), 5.34 (s, 2H), 6.80–7.35 (m, 4H), 8.90 (br, 1H,
D2O exchangeable).

D. Gentili et al. / Il Farmaco 54 (1999) 224–231
229
4.1.4. Ethyl esters of N-(arylmethyloxy)alanines (9a–d)
4.1.6. t-Butyl esters of the 7i-{(E)-[N-(arylmethyl-
oxy)imino]-2-propionamido}-3-(acetoxymethyl)-3-
cephem-4-carboxylic acid deri6ati6es (12a–f)
Ethanolic hydrochloric acid (8 N, 109 ml) was added
dropwise to a stirred mixture (0°C) of borane–
trimethylamine complex (21.9 mmol) and the appropri-
ate oximether (7a–d) (14.6 mmol). Stirring was
continued for 20 h, then the solvent was evaporated
and the residue was dissolved in CH2Cl2 supplemented
with solid NaHCO3. After stirring for several hours, the
suspension was filtered and the solvent was evaporated.
The residue was filtered through a silica gel column
eluting with a hexane/AcOEt (4:1) mixture. Evapora-
tion of the final fraction yielded pure 9a–d as oils
(GLC).
A stirred solution of the appropriate acid (8a–f)
(0.763 mmol) and the t-butyl ester of 7-ACA (0.738
mmol) in anhydrous CH2Cl2 (10 ml) was cooled at 0°C
and then treated portionwise with EDCI (0.738 mmol).
After stirring for 24 h at r.t. the reaction mixture was
washed (5% aqueous HCl, saturated aqueous NaHCO3
and brine), filtered and evaporated to give an oil which
was purified by silica gel column chromatography (hex-
ane/2-pentanone 2:1). Evaporation of the middle frac-
tions yielded 12a–f as a vitreous product.
9a [20] (69%): IR w 3269, 1742 cm−1 1H NMR
;
1
12a (67%): H NMR (CDCl3) l 1.55 (s, 9H), 2.03 (s,
(CDCl3) l 1.03–1.50 (m, 6H), 3.40–3.96 (m, 1H), 4.26
(q, 2H, J=7 Hz), 4.76 (s, 2H), 6.05 (br, 1H, J=9 Hz,
D2O exchangeable), 7.40 (s, 5H); 9b (73%): IR w 3269,
3H), 2.07 (s, 3H), 3.30 and 3.55 (2d, 2H, J=18.1 Hz),
4.97 (d, 1H, J=4.8 Hz), 5.82 (dd, 1H, J=4.2 and 9.0
1
Hz); 12b (55%): H NMR (CDCl3) l 1.55 (s, 9H), 2.09
1
1742 cm−1; H NMR (CDCl3) l 1.10–1.50 (m, 6H),
(s, 6H), 3.29 and 3.63 (2d, 2H, J=18.6 Hz), 4.79 (d,
1H, J=12.6 Hz), 5.01 (d, 1H, J=4.2 Hz), 5.11 (d, 1H,
J=12.6 Hz), 5.33 (s, 2H), 5.86 (dd, 1H, J=4.2 and 9.0
3.53–4.00 (m, 1H), 4.26 (q, 2H, J=7 Hz), 4.90 (s, 2H),
5.93–6.38 (br, 1H, D2O exchangeable), 7.16–7.60 (m,
4H); 9c (83%): IR w 3269, 1742 cm−1 1H NMR
;
1
Hz), 7.31 (m, 5H); 12c (61%): H NMR (CDCl3) l 1.55
(CDCl3) l 1.10–1.50 (m, 6H), 3.42–3.97 (m, 1H), 4.25
(q, 2H, J=7 Hz), 4.70 (s, 2H), 6.03 (br, 1H, J=8 Hz,
D2O exchangeable), 7.26–7.53 (m, 4H); 9d (86%): IR w
(s, 9H), 2.06 (s, 3H), 2.08 (s, 3H), 3.29 and 3.65 (2d,
2H, J=18.6 Hz), 4.79 (d, 1H, J=12.6 Hz), 5.0 (d, 1H,
J=4.8 Hz), 5.10 (d, 1H, J=12.6 Hz), 5.17 (s, 2H), 5.86
(dd, 1H, J=4.8 and 9.6 Hz), 7.27 (m, 5H); 12d (70%):
1H NMR (CDCl3) l 1.54 (s, 9H), 2.03 (s, 3H), 2.08 (s,
3H), 3.29 and 3.65 (2d, 2H, J=18.6 Hz), 4.79 (d, 1H,
J=12.6 Hz), 5.0 (d, 1H, J=4.8 Hz), 5.10 (d, 1H,
J=12.6 Hz), 5.17 (s, 2H), 5.86 (dd, 1H, J=4.8 and 9.6
1
3269, 1742 cm−1; H NMR (CDCl3) l 1.10–1.50 (m,
6H), 3.4–4.03 (m, 1H), 4.25 (q, 2H, J=7 Hz), 4.70 (s,
2H), 6.02 (br, 1H, J=9 Hz, D2O exchangeable), 7.37
(m, 4H).
1
Hz), 7.27 (m, 5H); 12e (55%): H NMR (CDCl3) l 1.54
4.1.5. N-(Arylmethyloxy)alanines (11a–d)
(s, 9H), 2.03 (s, 3H), 2.08 (s, 3H), 3.30 and 3.60 (2d,
2H, J=17.5 Hz), 3.82 (s, 3H), 4.78 (d, 1H, J=12.6
Hz), 4.96 (d, 1H, J=4.8 Hz), 5.07 (d, 1H, J=12.6 Hz),
5.24 (s, 2H), 5.82 (dd, 1H, J=4.8 and 8.8 Hz), 6.80–
A stirred THF/H2O (7:3) solution (10 ml) of the
appropriate ester (9a–d) (1.37 mmol) was cooled at 0°C
and then treated dropwise with 1 N aqueous NaOH
(1.37 mmol). At the end of the reaction, monitored by
TLC (AcOEt/hexane 3:2), the THF was evaporated and
the aqueous solution was acidified to pH$3–4 with
aqueous H3PO4 and extracted with AcOEt. The organic
phase was evaporated to dryness to give the appropri-
ate alanine derivatives (11 a–d) as a solid residue which
was purified by crystallization from hexane.
1
7.41 (m, 5H); 12f (65%): H NMR (CDCl3) l 1.54 (s,
9H), 2.04 (s, 3H), 2.08 (s, 3H), 3.30 and 3.59 (2d, 2H,
J=17.6 Hz), 3.79 (s, 3H), 4.78 (d, 1H, J=12.8 Hz),
4.97 (d, 1H, J=5.6 Hz), 5.07 (d, 1H, J=12.8 Hz), 5.15
(s, 2H), 5.82 (dd, 1H, J=5.6 and 8.8 Hz), 6.86–7.35
(m, 5H).
11a (61%): m.p. 121–122°C (lit. [21] m.p. 123–
1
124°C); IR w 3236, 1719 cm−1, H NMR (CDCl3) l
4.1.7. 7i-{(E)-[N-(Arylmethyloxy)imino]-2-propion-
amido}-3-(acetoxymethyl)-3-cephem-4-carboxylic acid
deri6ati6es (3a–f)
1.27 (m, 3H, J=7 Hz), 3.56–4.03 (m, 1H, J=7 Hz),
4.82 (s, 2H), 7.43 (m, 5H), 8.68 (br, 2H, D2O exchange-
able); 11b (77%): m.p. 130–131°C; IR w 3236, 1719
Trifluoroacetic acid (1.3 ml) was added dropwise to a
stirred and cooled (0°C) solution of the appropriate
ester (12a–f) (0.29 mmol) in anisole (0.15 ml) and
anhydrous CH2Cl2 (1 ml). After stirring at 0°C for 5 h,
the solution was concentrated at reduced pressure, di-
luted with AcOEt and extracted with 10% aqueous
NaHCO3. The aqueous phase was cooled at 0°C, aci-
dified at pH$3 with 10% aqueous HCl and then
extracted with AcOEt. Evaporation of the washed
(H2O) organic extracts gave the pure acids 3a–f as
oils.
cm−1 1H NMR (CDCl3) l 1.26 (d, 3H, J=7 Hz),
,
3.50–4.03 (m, 1H, J=7 Hz), 4.90 (s, 2H), 7.23–8.05
(m, 6H); 11c (60%): m.p. 119–120°C; IR w 3236, 1719
cm−1 1H NMR (CDCl3) l 1.24 (d, 3H, J=7 Hz),
,
3.50–4.00 (m, 1H, J=7 Hz), 4.77 (s, 2H), 7.20–7.56
(m, 4H), 7.63–8.10 (br, 2H, D2O exchangeable); 11d
(50%): m.p. 129–130°C; IR w 3236, 1719 cm−1 1H
,
NMR (CDCl3) l 1.24 (d, 3H, J=7 Hz), 3.47–3.93 (m,
1H, J=7 Hz), 4.73 (s, 2H), 7.38 (m, 4H), 8.08–8.58
(br, 2H, D2O exchangeable).

230
D. Gentili et al. / Il Farmaco 54 (1999) 224–231
1
3a (82%): H NMR (CDCl3) l 2.03 (s, 3H), 2.07 (s,
3H), 3.34 and 3.60 (2d, 2H, J=18.4 Hz), 5.18 (s, 2H),
5.84 (dd, 1H, J=4.2 and 9.6 Hz), 7.20–7.70 (m, 5H);
NMR (CDCl3) 1.26 (m, 3H), 1.54 (s, 9H), 2.07 (s,
3H), 3.25 and 3.62 (2d, 2H, J=18.4 Hz), 3.70 (m, H),
4.65–5.25 (m, 6H), 5.82 (m, H), 7.05–7.60 (m, 5H);
1
1
3b (82%): H NMR (CDCl3) l 2.03 (s, 3H), 2.08 (s,
14c (43%): H NMR (CDCl3) 1.25 (m, 1H), 1.55 (s,
3H), 3.34 and 3.72 (2d, 2H, J=17.7 Hz), 4.80 (d, 1H,
J=12.7 Hz), 5.02 (d, 1H, J=9.3 Hz), 5.28 (d, 1H,
J=12.7 Hz), 5.31 (s, 2H), 5.84 (dd, 1H, J=4.9 and
9.3), 7.20–7.60 (m, 5H); 3c (87%): H NMR (CDCl3)
l 2.06 (s, 3H), 2.09 (s, 3H), 3.40 and 3.62 (2d, 2H,
9H), 2.08 (s, 3H), 3.10–3.90 (m, 3H), 4.50–5.75 (m,
6H), 5.80 (m, H), 7.00–7.70 (m, 5H); 14d (31%): H
NMR (CDCl3) 1.24 (m, 3H), 1.55 (s, 9H), 2.08 (s,
3H), 3.15–3.80 (m, 3H), 4.40–5.20 (m, 6H), 5.82 (m,
H), 7.00–7.55 (m, 5H).
1
1
J=17.7 Hz), 4.85–5.38 (m, 5H), 5.84 (dd, 1H, J=5.0
1
and 9.6 Hz), 7.00–7.55 (m, 5H); 3d (98%): H NMR
4.1.9. 7i-{[N-(Arylmethyloxy)amino]acetamido} (2a–d)
and 7i-{[N-(arylmethyloxy)amino]-2-propionamido}-
3-(acetoxymethyl)-3-cephem-4-carboxylic acid
deri6ati6es (4a-d)
(CDCl3) l 2.03 (s, 3H), 2.09 (s, 3H), 3.35 and 3.60
(2d, 2H, J=18.4 Hz), 5.15 (s, 2H), 5.45–6.10 (m, 6H),
7.20–7.80 (m, 5H); 3e (60%): 1H NMR (CDCl3–
DMSO-d6) l 2.04 (s, 3H), 2.09 (s, 3H), 3.79 (s, 3H),
5.01 (d, 1H, J=4.9 Hz), 5.25 (s, 2H), 5.85 (dd, 1H,
Trifluoroacetic acid (1.15 ml) was added dropwise to
a stirred and cooled (0°C) solution of the appropriate
ester (13a–d or 14a–d) (0.81 mmol) in a mixture of
anisole (0.18 ml) and anhydrous CH2Cl2 (1 ml). After
12 h at the same temperature, the reaction mixture
was evaporated and the oily residue was dissolved in
AcOEt and extracted with 10% aqueous NaHCO3.
The aqueous phase was cooled to 0°C, acidified at
pH$2.5 with 10% aqueous HCl and extracted with
AcOEt. Evaporation of the washed (H2O) organic ex-
tract gave an amorphous solid which was purified by
trituration to yield 2a–d (CHCl3/hexane) and 4a–d
(Et2O).
1
J=4.9 and 9.4 Hz), 6.80–7.60 (m, 5H); 3f (93%): H
NMR (CDCl3) l 1.98 (s, 3H), 2.02 (s, 3H), 3.33 and
3.52 (2d, 2H, J=18.5 Hz), 3.73 (s, 3H), 5.11 (s, 2H),
5.81 (dd, 1H, J=4.9 and 9.6 Hz), 6.70–7.30 (m, 4H).
4.1.8. t-Butyl esters of 7i-{[N-(arylmethyloxy)amino]-
acetamido} (13a–d) and of 7i-{[N-(arylmethyloxy)-
amino]propionamido}-3-(acetoxymethyl)-3-cephem-
4-carboxylic acid deri6ati6es (14a–d)
A stirred and cooled (0°C) solution of the appropri-
ate acid (9a–d or 10a–d) (0.55 mmol) and the t-butyl
ester of 7-ACA (0.55 mmol) in anhydrous CH2Cl2 (10
ml) was treated portionwise with EDCI (0.55 mmol).
The mixture was stirred at 20°C for 12 h, diluted with
CH2Cl2, washed with aqueous NaHCO3 and brine,
filtered and evaporated to give a semisolid which, after
purification by column chromatography (hexane–
AcOEt 1:1), yielded pure 13a–d or 14a–d as oils.
1
2a (65%): H NMR (CDCl3) 2.07 (s, 3H), 3.31 and
3.60 (2d, 2H, J=17.6 Hz), 3.58 (m, 2H), 4.14 (br, 1H,
D2O exchangeable), 4.73 (s, 2H), 4.80–5.35 (m, 3H),
5.87 (m, H), 7.00–7.65 (m, 5H), 7.86 (d, 1H, J=9.6
1
Hz, D2O exchangeable); 2b (88%): H NMR (CDCl3)
2.07 (s, 3H), 3.17–3.85 (m, 4H), 4.65–5.25 (m, 5H),
5.65 (m, 1H, D2O exchangeable), 5.80 (m, H), 7.10–
7.50 (m, 5H); 2c (78%): 1H NMR (CDCl3) 2.07 (s,
3H), 3.29 (d, 1H, J=17.6 Hz), 3.59 (m, 2H), 3.60 (d,
1H, J=17.6 Hz), 4.69 (s, 2H), 4.80–5.42 (m, 3H),
5.85 (dd, 1H, J=4.8 and 9.6 Hz), 7.10–7.45 (m, 4H),
7.84 (d, 1H, J=9.6 Hz, D2O exchangeable); 2d (88%):
1H NMR (CDCl3) 2.07 (s, 3H), 3.31–3.80 (m, 4H),
3.55–3.95 (m, 2H), 4.70 (s, 2H), 4.91–5.30 (m, 5H),
5.52 (br, 1H, D2O exchangeable), 5.86 (dd, 1H, J=4.8
and 9.6 Hz), 7.20–7.45 (m, 5H), 7.67 (d, 1H, J=9.6
1
13a (20%): H NMR (CDCl3) 1.55 (s, 9H), 2.08 (s,
3H), 3.30 (d, 1H, J=17.6 Hz), 3.58 (m, 2H), 3.60 (d,
1H, J=17.6 Hz), 4.75 (s, 2H), 4.78 (d, 1H, J=12.8
Hz), 4.98 (d, 1H, J=5.6 Hz), 5.06 (d, 1H, J=12.8
Hz), 5.87 (dd, 1H, J=5.6 and 9.6 Hz), 7.0–7.80 (m,
6H); 13b (15%): H NMR (CDCl3) 1.55 (s, 9H), 2.08
(s, 3H), 3.30 and 3.58 (2d, 2H, J=17.6 Hz), 3.62 (m,
2H), 4.55–5.55 (m, 5H), 5.87 (dd, 1H, J=4.8 and 9.6
Hz), 7.00–7.55 (m, 5H); 13c (22%): H NMR (CDCl3)
1.48 (s, 9H), 2.08 (s, 3H), 3.32 and 3.59 (2d, 2H,
J=17.6 Hz), 3.60 (m, 2H), 4.71 (s, 2H), 4.79 (d, 1H,
J=12.8 Hz), 4.97 (d, 1H, J=4.8 Hz), 5.09 (d, 1H,
J=12.8 Hz), 5.87 (dd, 1H, J=4.8 and 8.8 Hz), 7.10–
1
1
1
Hz); 4a (24%): H NMR (CDCl3) 1.24 (m, 3H), 2.06
(s, 3H), 3.20–3.65 (m, 3H), 4.70–5.15 (m, 5H), 5.65–
1
6.00 (m, 2H), 7.15–7.55 (m, 6H); 4b (22%): H NMR
(CDCl3, DMSO-d6) 1.26 (m, 3H), 2.08 (s, 3H), 3.25
1
7.80 (m, 5H); 13d (32%): H NMR (CDCl3) 1.54 (s,
and 3.70 (m, 3H), 4.50–6.20 (m, 6H), 6.80–7.55 (m,
1
9H), 2.07 (s, 3H), 3.30 and 3.58 (2d, 2H, J=17.6 Hz),
3.57 (m, 2H), 4.70 (s, 2H), 4.90 (d, 1H, J=4.8 Hz),
4.78 (d, 1H, J=13.6 Hz), 4.96 (d, 1H, J=4.8 Hz),
5.05 (d, 1H, J=13.6 Hz), 5.85 (dd, 1H, J=5.6 and
9.6 Hz), 7.15–7.45 (m, 4H); 14a (42%): 1H NMR
(CDCl3) 1.25 (m, 3H), 1.55 (s, 9H), 2.08 (s, 3H),
3.10–3.85 (m, 3H), 4.65–5.20 (m, 6H), 5.83 (dd, 1H,
5H); 4c (86%): H NMR (CDCl3–DMSO-d6) 1.29 (m,
3H), 2.08 (s, 3H), 3.15 and 3.80 (m, 3H), 3.65–3.90
(m, H), 4.50–5.20 (s, 5H), 5.80 (m, 2H), 7.15–7.40 (m,
1
5H); 4d (41%): H NMR (CDCl3–DMSO-d6) 1.23 (m,
3H), 2.07 (s, 3H), 3.30 (d, 1H, J=17.6 Hz), 3.45–4.00
(m, 2H), 4.66 (s, 2H), 4.73–5.20 (m, 3H), 5.80 (dd,
1H, J=4.8 and 9.6 Hz), 7.20–7.40 (m, 4H), 7.78 (d,
1H, J=9.6 Hz).
1
J=4.8 Hz, 8.8 Hz), 7.10–7.70 (m, 6H); 14b (42%): H

D. Gentili et al. / Il Farmaco 54 (1999) 224–231
231
[11] M. Macchia, E. Menchini, E. Orlandini, A. Rossello, G. Broc-
cali, M. Visconti, Synthesis and antimicrobial activity of 7b-[N-
(arylmethyloxyimino)acetamido]cephalosporanic acid derivati-
ves, Farmaco 50 (1995) 713.
References
[1] K.E. Price, Structure activity relationships of semisynthetic peni-
cillins (supplement), in: D. Perlman (Ed.), Structure Activity
Relationships Among the Semisynthetic Antibiotics, vol. 12,
Academic Press, New York, 1977, pp. 61–68.
[2] M.L. Sassiver, A. Lewis, Structure activity relationships of
semisynthetic cephalosporins. I. The first generation of com-
pounds, in: D. Perlman (Ed.), Structure Activity Relationships
Among the Semisynthetic Antibiotics, vol. 12, Academic Press,
New York, 1977, pp. 87–160.
[3] J.A. Webber, W.J. Wheeler, Antimicrobial and pharmacokinetic
properties of newer penicillins and cephalosporins, in: R.B.
Morin, M. Gorman (Eds.), Chemistry and Biology of b-Lactam
Antibiotics, vol. I, Academic Press, New York, 1982, pp. 371–
436.
[4] D.M. Boyd, Theoretical and physicochemical studies on b-lac-
tam antibiotics, in: R.B. Morin, M. Gorman (Eds.), Chemistry
and Biology of b-Lactam Antibiotics, vol. I, Academic Press,
New York, 1982, pp. 437–545.
[5] C.M. Cimarusti, Dependence of b-lactamase stability on sub-
structure within b-lactam antibiotics, J. Med. Chem. 27 (1984)
247.
[6] W. Durckheimer, F. Adam, C. Fisher, R. Kirrstetter, in: B.
Testa (Ed.), Advances in Drug Research, vol. 17, Academic
Press, New York, 1988, pp. 61–234.
[7] A. Balsamo, B. Macchia, F. Macchia, A. Rossello, R. Giani, G.
Pifferi, M. Pinza, G. Broccali, Synthesis and antibacterial activi-
ties of new (a-hydrazinobenzyl)cephalosporins, J. Med. Chem.
26 (1983) 1648.
[8] A. Balsamo, G. Broccali, A. Lapucci, B. Macchia, F. Macchia,
E. Orlandini, A. Rossello, Synthesis and antimicrobial properties
[12] M. Macchia, E. Menchini, E. Orlandini, A. Rossello, G. Broc-
cali, M. Visconti, Synthesis and antimicrobial activity of 7b-(S)-
and
7b-[(R)-3-(methylenaminoxy)-2-methylpropionamido]sub-
stituted cephalosporanic acid derivatives, Farmaco 51 (1996)
283.
[13] B. Macchia, A. Balsamo, M.C. Breschi, G. Chiellini, M. Mac-
chia, A. Martinelli, C. Martini, S. Nencetti, A. Rossello, R.
Scatizzi, The (methyloxymino)methyl moiety as bioisoster of
aryl. A novel class of completely aliphatic b-adrenergic receptor
antagonist, J. Med. Chem. 37 (1994) 1518.
[14] A. Balsamo, M.C. Breschi, G. Chiellini, L. Favero, M. Macchia,
A. Martinelli, C. Martini, A. Rossello, R. Scatizzi, Synthesis and
b-adrenergic properties of (E)-N-[3-(alkylamino)-2-hydroxy-
propylidene](methyloxy)amines substituted with an aromatic
group on their [(methyloxy)imino]methyl moiety (MOIMM): an
investigation into the biopharmacological effects of an aryl
substitution in the class of MOIMM b-blocking drugs, Eur. J.
Med. Chem. 30 (1995) 743.
[15] A. Balsamo, M.S. Belfiore, M. Macchia, C. Martini, S. Nencetti,
E. Orlandini, A. Rossello, Synthesis and aldose reductase in-
hibitory activity of N-(arylsulfonyl)- and N-(aroyl)-N-(aryl-
methyloxy)glycines, Eur. J. Med. Chem. 29 (1994) 787.
[16] B.L. Fox, J.E. Reboulet, Assignment of ketoxime stereochem-
istry by a nuclear magnetic resonance method, J. Org. Chem. 35
(1970) 4234.
[17] M.L. Sassiver, A. Lewis, R.G. Shepperd, in: G.L. Hobby (Ed.),
Antimicrobial Agent and Chemotherapy-1968, Williams and
Wilkins, Baltimore MD, 1969, pp. 101–105.
[18] C.E. Newall, Recent Advances in the Chemistry of b-lactam
Antibiotics, Third International Symposium, The Royal Society
of Chemistry, 1984, p. 4.
[19] E. Steers, E.L. Foltz, B.S. Graves, Antimicrobial Chemother. 9
(1959) 307.
[20] M.W. Tijhuis, J.D.M. Herscheid, H.C.J. Ottenheijm, A practical
synthesis of N-hydroxy- a-amino acid derivatives, Synthesis 11
(1980) 890.
[21] T. Kolasa, A. Chimiak, O-Protected derivatives of N-hydroxy-
amino acids, Tetrahedron 30 (1974) 3591–3595.
of
substituted
b-aminoxypropionyl
penicillins
and
cephalosporins, J. Med. Chem. 32 (1989) 1398.
[9] A. Balsamo, B. Macchia, A. Martinelli, E. Orlandini, A.
Rossello, F. Macchia, G. Broccali, P. Domiano, Synthesis and
antimicrobial properties of substituted 3-aminoxy-(E)-2-
methoxyiminopropionyl penicillins and cephalosporins, Eur. J.
Med. Chem. 25 (1990) 227.
[10] A. Balsamo, B. Macchia, E. Orlandini, A. Rossello, F. Macchia,
G. Broccali, W. Fonio, Synthesis and antimicrobial properties of
substituted 3-aminoxypropionyl and 3-aminoxy-(E)-2-methoxy-
iminopropionyl monobactams, Farmaco 45 (1990) 879.
.