Synthesis and inhibitory activity towards human leukocyte elastase of new 7α-methoxy and 7α-chloro (2-acyloxymethyl) cephem derivatives

Article abstract of DOI:10.1016/S0223-5234(01)01211-9

Some new cephem derivatives of types 4 and 5, viewed as analogues of type I esters in which the atomic sequence of the C-2 ester group is formally inverted, were synthesised and tested in vitro for their inhibitory activity towards human leukocyte elastase and porcine pancreatic elastase. An examination of the inhibition data obtained for the new type 4 and 5 derivatives, while exhibiting a considerable reduction in their activity against porcine pancreatic elastase, indicated that these compounds still maintain an appreciable inhibitory activity against human leukocyte elastase. On this basis the new type of C-2 substitution appears to contribute to the research of new, potentially interesting, cephalosporinic human leukocyte elastase inhibitors.

Full text of DOI:10.1016/S0223-5234(01)01211-9

Eur. J. Med. Chem. 36 (2001) 185–193
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01211-9/FLA
Preliminary communication
Synthesis and inhibitory activity towards human leukocyte elastase of new
7a-methoxy and 7a-chloro (2-acyloxymethyl) cephem derivatives
Aldo Balsamoa, Giovanni Cercignanib, Daniela Gentilia, Annalina Lapuccia, Marco Macchiaa,
Elisabetta Orlandinia, Simona Rapposellia, Armando Rosselloa*
aDipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Universita` degli Studi di Pisa,Via Bonanno, 6, 56100 Pisa, Italy
bLaboratorio di Biochimica, Dipartimento di Fisiologia e Biochimica, Universita` degli Studi di Pisa, Via S. Maria, 55,
56100 Pisa, Italy
Received 1 August 2000; accepted 11 December 2000
Abstract – Some new cephem derivatives of types 4 and 5, viewed as analogues of type I esters in which the atomic sequence of the
C-2 ester group is formally inverted, were synthesised and tested in vitro for their inhibitory activity towards human leukocyte
elastase and porcine pancreatic elastase. An examination of the inhibition data obtained for the new type 4 and 5 derivatives, while
exhibiting a considerable reduction in their activity against porcine pancreatic elastase, indicated that these compounds still maintain
an appreciable inhibitory activity against human leukocyte elastase. On this basis the new type of C-2 substitution appears to
´
contribute to the research of new, potentially interesting, cephalosporinic human leukocyte elastase inhibitors. © 2001 Editions
scientifiques et me´dicales Elsevier SAS
elastase inhibitor / HLE inhibitor / cephalosporin derivative / C-2 hydroxymethyl cephem derivative
1. Introduction
Cephalosporins of type I (figure 1) were the first
b-lactam derivatives studied for their HLE inhibitory
Human leukocyte elastase (HLE) is a serine
protease [1–11] whose hyperexpession may lead to the
degradation of the connective tissue associated with
certain pulmonary, vascular and neoplastic patholo-
gies [12–16]. Therefore, compounds capable of in-
hibiting HLE may restore the balance between the
free enzyme and endogenous antiproteinases, and
thus may have considerable therapeutic potential.
activity, and their inhibition mechanism has been
analysed using chemical [23, 24], crystallographic [25]
and kinetic [26] methods.
Molecular modelling studies on a cephem sulphone
of type I have indicated the importance of the C-2
substituent, which appears to be involved in the bind-
ing process with the S1%-S2% sites of HLE [27]. The
character of this enzyme, an endopeptidase, offers the
possibility of changing the chemical nature of the C-2
In the past decade, HLE inhibitors obtained from
suitably derivatized b-lactam compounds such as
substituent on the cephem nucleus, where the C-2
carboxyl may be substituted with other groups with-
cephalosporins [17], penicillins [18], monocyclic b-lac-
tams [19, 20] and unconventional b-lactams [21, 22],
out affecting the HLE binding process [21, 28]. There-
fore, attention has focused on compounds obtained
by modifying the substituents in the C-2, C-3, C-4
and C-7 positions of the cephem nucleus, with the aim
of improving their inhibitory activity and enzyme
specificity. These efforts have produced some new
b-lactam HLE inhibitors such as the cephem com-
pounds II [28], and the 2-spirocyclopropyl cephems
have been proposed for the therapeutic treatment of
patients affected by chronic bronchitis and cystic
fibrosis. Among these b-lactam compounds studied,
cephalosporins and synthetic b-lactams have been the
most promising.
* Correspondence and reprints.
E-mail address: aros@farm.unipi.it (A. Rossello).

186
A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
III [22], which, in spite of their structural differences,
do not appear to differ substantially from I, as re-
gards the reactivity of the b-lactam carbonyl involved
in the initial step of the sequence of the enzymatic
reactions, which consisted in the attack on the same
carbonyl of the serine hydroxy group present in the
catalytic site of this enzyme. However, for these com-
pounds, the high reactivity of the b-lactam is likely to
have a negative influence on their stability.
A major differentiation in the reactivity of the
b-lactam carbonyl of new cephem inhibitors from
that of derivatives I–III may be obtained by introduc-
ing into the C-2 position of the cephem nucleus sub-
stituents able to exert electronic effects different from
those exerted by the carboxylic or carbonylic groups
present in the type I–III structures. Normally in the
cephem derivatives, the reactivity of the b-lactam
nucleus may be considered to be linked to its low
degree of amidic resonance shown in the d form
(figure 2). Substituents on C-2 possessing high elec-
tron-withdrawing characteristics due to their conju-
gating effects, such as the carboxylic or carbonylic
groups, reduce this amidic resonance by increasing
the possibility of the enolic resonance form b, thus
consequently improving the reactivity of the b-lactam
Figure 2.
ring to the nucleophile attack of the enzyme [29]. On
the contrary, the presence of substituents devoid of
any conjugating electron-withdrawing effect in C-2
does not allow the contribution of the resonance form
b; as a consequence, the b-lactam maintains a certain
degree of amidic resonance, resulting in a lower level
of electrophilic reactivity of the b-lactam itself, and a
higher level of nucleophilicity of the C-2 C-3 double
bond of the dihydrothiazine ring.
Our previous results showed that the substitution
of the C-2 carboxyl group of natural cephalosporins 1
(figure 3) with the hydroxymethyl group of com-
pound 2 or the arylacyloxymethyl one of compounds
3 [30, 31] increases the nucleophile reactivity of the
C-2 C-3 double bond of the cephalosporin system,
thus indicating a smaller contribution of form b to the
resonance and therefore a greater weight of the less
reactive form d.
On the basis of these results, we decided to transfer
the same type of substituent to the C-2 carbon atom
of type I and II inhibitors, in order to evaluate the
effects of this structural modification, potentially ca-
pable modulating the b-lactam system reactivity, on
the anti-HLE properties of the cephalosporin
derivatives.
This work describes the synthesis and the in vitro
inhibitory activity towards HLE and porcine pancre-
atic elastase (PPE) of a series of new cephem esters of
types 4 (4a–c) and 5 (5a,b), in which the esterified C-2
carboxylic group of the cephalosporins of type I is
replaced by a hydroxymethyl group esterified by aro-
matic or aliphatic acyls. The esters were chosen so as
to have a C-2 side chain sterically similar to that of
Figure 1.

A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
187
anhydrous DCM, in the presence of triethylamine
(TEA) as the base and 2,6-dimethylaminopyridine
(DMAP) as the catalyst. Imides 10a–c, without isola-
tion, were treated with N,N-diethylethylendiamine in
anhydrous DCM to give the crude N-7-t-(BOC)-
amides 11a–c, which without any purification were
transformed into the desired 7b-amino-3-cephem es-
ters 12a–c by reaction with trifluoroacetic acid (TFA)
in anhydrous DCM at room temperature. 12a–c were
purified by flash chromatography and then trans-
formed into the 7-diazoketons 13a–c (figure 5) by
treatment with an aqueous sodium nitrite solution in
the presence of 2 N sulphuric acid at 0 °C. For the
preparation of the 7a-methoxy cephem esters 14a–c,
the appropriate 7-diazoketon 13a–c was treated with
rhodium acetate dimer (Rh2OAc4) in methanol at
0 °C. For the preparation of the 7a-chlorine deriva-
tives 15a,b, a crude mixture of 13a,b was treated with
concentrated hydrogen chloride in EtOH at room
temperature for 20 s. The crude 7a-methoxy and
7a-chlorine derivatives, 14a–c and 15a,b, were
purified by flash chromatography on silica gel and
then oxidised to the sulfones 4a–c and 5a,b with
m-chloroperoxybenzoic acid (MCPBA) in anhydrous
DCM. Pure 4a–c and 5a,b were obtained by flash
chromatography on silica gel of the crude products.
Figure 3.
the more interesting cephalosporin esters of type I
(the benzyl and the t-butyl one [27]). Compounds 4
and 5 may therefore be viewed as analogues of type I
drugs in which the atomic sequence of the ester group
is formally inverted. The substituents on the C-7
carbon atom of 4 (the methoxy group) or 5 (the
chlorine atom) were selected because they are present
on the same carbon of the most interesting com-
pounds of type I [32].
3. Results and discussion
The new cephem esters 4a–c and 5a,b were evalu-
ated for their ability to inhibit HLE- and PPE-
catalysed
hydrolysis
of
the
substrate
N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.
Table I shows the results obtained with these enzymes
expressed as concentration-independent second-order
rate constant kinact/KI [20]. Table I also shows the
results obtained in the same tests with the cephem
sulphone 16 [17], used as a reference type I drug. All
the tested cephem esters 4a–c and 5a,b were found to
possess a moderate inhibitory activity against HLE,
with kinact/KI values ranging from 1 540 of 4a to 7 600
M–1s–1 of 5b which proved to be the most active in
the series. On the contrary, in the case of PPE, the
compounds tested showed a poor inhibitory activity,
with kinact/KI values ranging from 240 of 4a to 1 800
M–1s–1 of 4c. In the same tests, the reference drug 16
showed a kinact/KI value of 25 000 M–1s–1 against HLE
and 131 000 M–1s–1 against PPE.
2. Chemistry
The cephem esters 4a–c and 5b–c were synthesised
as shown in figure 4. The starting cephaloram 6 was
transformed into the acid chloride 7 by treatment
with oxalyl chloride (ClCO)2 and catalytic dimethyl-
formamide (DMF) in dichloromethane (DCM), and
then, without the isolation of 7, into the alcohol 8 by
reduction of 7 with LiAl(t-BuO)3H in THF at 0 °C.
Reaction of alcohol 8 with the appropriate acid in
anhydrous DCM, using 1-ethyl-3-[3-(dimethy-
lamino)propyl]carbodiimide hydrochloride (EDCI) as
the condensing agent gave the esters 9a–c which were
converted into the N-7-t-Boc-imides 10a–c by treat-
ment with di-tert-butyl dicarbonate [t-(BOC)2O] in

188
A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
Figure 4. Conditions: i: (ClCO)2, DCM, cat. DMF, 0 °C; ii: LiAl(t-BuO)3H, THF, 0 °C; iii: RCO2H, EDCL, cat. DMAP, DCM,
−5 to 0 °C; iv: (t-BOC)2O, TEA, cat. DMAP, DCM, 0 °C; v: N,N-diethylethylenediamine, DCM, room temperature; vi: TFA,
DCM, room temperature.
An examination of these results indicates that the
newly synthesised cephem sulfones 4a–c and 5a,b
maintain a still appreciable inhibitory activity against
HLE, with the most active derivative in this series (the
p-fluorobenzoate 5b, substituted on the C-7 carbon
with an a-chlorine atom) which is only 3 times less
active than the reference drug 16; on the contrary, the
activity against PPE is considerably reduced, with the
most active compound 4c which is 73 times less active
than 16.
In conclusion, the results obtained with the new
cephem derivatives of types 4 and 5 indicate that the
substitution of the more electron-withdrawing car-
boxy group, linked to the C-2 double bond of type I
drugs, with the less electron-withdrawing hydroxyl
group esterified with aliphatic or aromatic acyls of 4
and 5 seems to reduce the ability of the new com-
pounds to interact with PPE considerably. On the
contrary, this type of substitution does not substan-
tially hinder the inhibition process towards HLE if
compared with the reference type I drug (16). There-
fore, this new type of C-2 substitution, which does not
have a too significant effect on the anti-HLE activity
of the new compounds, appears to be able to con-
tribute to the development of a new class of poten-
tially interesting cephalosporinic HLE inhibitors.
4. Experimental protocols
4.1. Chemistry
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. Infrared (IR) spectra for
comparison of compounds were recorded on a Mattson
1000 FTIR spectrometer. Nuclear magnetic resonance
(1H-NMR) spectra were recorded on a Varian CFT-20
(80 MHz) or on a Bruker AC-200 (200 MHz) in a ca.
2% solution of CDCl3 for all compounds. The proton
magnetic resonance assignments were established on the
basis of the expected chemical shifts and the multiplicity
of the signals. Analytical thin layer chromatography
(TLC) was carried out on 0.25-mm layer silica gel plates
containing a fluorescent indicator, and spots were de-

A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
189
4.1.1. N-((6R)-3-acetoxymethyl-2-hydroxymethyl-8-
oxo-(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-7t-yl)-2-
phenyl-2-yl-acetamide 8
A solution of acid 6 (13.0 mmol, 5.0 g), in anhydrous
DCM (76 mL) and anhydrous DMF (0.19 mL), cooled
at 0 °C, was treated dropwise with a solution of (ClCO)2
(34.2 mmol, 4.3 g) in anhydrous DCM (6.7 mL). After
stirring at 0 °C for 1 h, the reaction mixture was evapo-
rated at 0–5 °C and the crude residue was washed with
benzene to remove the excess of (ClCO)2.
LiAl(t-BuO)3H (30.7 mmol, 7.3 g) was added portion-
wise over a period of 20 min to the residue, dissolved in
anhydrous THF (133 mL) and cooled at 0 °C. The
reaction mixture was stirred at the same temperature for
another 30 min and then poured into 2 N aqueous HCl
at pH 2.8, extracted with AcOEt, washed with 5%
aqueous NaHCO3 and brine and dried. The organic
phase was evaporated to give a crude residue (20 g)
which was purified by flash chromatography on silica
gel, eluting with 4:1 AcOEt/toluene to obtain the pure
alcohol 8 as an oil (1.7 g, 35%). IR (CHCl3): w 1778
1
cm–1; H-NMR: l 1.97 (s; 3H); 3.11 and 3.45 (2d; 2H,
J=17.8 Hz); 3.56 (s; 2H); 3.60–4.80 (m; 4H); 4.88 (d;
Table I. Inhibition of HLE and PPE by 4a–c, 5a,b and
reference drug 16.
Figure 5. Conditions: i: NaNO2,
2 N H2SO4, 0 °C; ii:
Rh2OAc4, MeOH, 0 °C; iii: HCl, abs. EtOH, 0 °C; iv:
MCPBA, DCM, 0 °C.
tected under UV light (254 nm). Flash chromatography
or preparative medium pressure liquid chromatography
(MPLC) were carried out through glass columns con-
taining 40–63 mm silica gel (Macherey-Nagel Silica Gel
60) or reversed phase of octadecyl silica gel (C18
Polygosil 60-4063 Macherey-Nagel). The MPLCs were
performed using a chromatographic apparatus consist-
ing of a Buchi 681 pump, a Knauer differential refrac-
tometer detector, and a Philips PM 8220 pen recorder.
Solvents and reagents were obtained from commercial
vendors in the appropriate grade and were used without
further purification unless otherwise indicated. Elemen-
tal analyses were carried out by our analytical labora-
tory and were consistent with theoretical values to
within 0.4%.
Compd.
R
X
HLEa
PPEa
inact/KI9S.D.b
k
inact/KI
k
9S.D.b
(M–1 s–1)
(M–1 s–1)
4a
4b
4c
5a
5b
16
Ph
MeO
1 5409125
1 6009145
4 0009410
1 6009170
7 6009910
240915
p-F-Ph MeO
CMe3
Ph
350930
1 8009145
432935
MeO
Cl
p-F-Ph Cl
580950
25 00092 250 131 00097 860
a
Substrate: N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanil-
ide.
b
S.D.: standard deviation.

190
A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
1H, J=4.8 Hz); 5.67 (dd; 1H, J=4.8 and 8.9 Hz); 6.23
(d; 1H, J=8.9 Hz, D2O exchangeable); 7.24 (brs; 5H).
Anal. C18H20N2O5S (C, H, N).
stirred and cooled at 0 °C. After stirring for 1 h at room
temperature, the reaction mixture was washed with
cooled 1 N HCl (2×25 mL) and brine (2×50 mL), dried
and evaporated to give the crude imide 10a–c. The
residue was dissolved in anhydrous DCM (300 mL) and
N,N-diethylethylendiamine (10.8 mmol) was added to
the resulting solution. After stirring for 48 h at room
temperature, the reaction mixture was washed with
cooled 1 N HCl (2×25 mL) and brine (2×25 mL), dried
and evaporated to give the crude t-BOC-amide 11a–c,
which was dissolved in anhydrous DCM (182 mL). TFA
(50 mL) was added to the resulting solution and the
mixture was stirred for 20 min at room temperature and
then evaporated. The residue, dissolved in CHCl3, was
poured into ice and 5% NaHCO3 (100 mL), cooling at
0 °C and keeping it in these conditions for 1 h. The
mixture was then extracted with AcOEt (3×50 mL),
washed with brine (2×50 mL), dried and evaporated to
give a crude residue which was purified by flash chro-
matography on silica gel or used for the next reaction.
(6R) - 3 - acetoxymethyl - 2 - benzoyloxymethyl - 8-
oxo-(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-7t-amine
12a. The residue was purified by flash chromatography
on silica gel eluting with a 9:1 AcOEt/hexane mixture to
obtain the pure amine 12a as an oil (30%).
4.1.2. General procedure for esters 9a–c
A solution of alcohol 8 (3.0 mmol, 2.5 g) in anhy-
drous DCM (26 mL), stirred and cooled at −5 °C, was
treated with the appropriate carboxylic acid (5.3 mmol)
in the presence of EDCI (10.6 mmol) and DMAP (2.6
mmol). The solution was stirred at 0 °C for 3 h and then
washed with cooled solutions of 10% HCl, 5% NaHCO3
and H2O. Evaporation of the dried and filtered extracts
gave the crude esters 9a–c, which were purified as
follows. 9a was purified by flash chromatography on
silica gel, eluting with a 2:1 hexane/AcOEt mixture and
was obtained it as an oil (42%); N-((6R)-3-ace-
toxymethyl-2-benzoyloxymethyl-8-oxo-(6rH)-5-thia-1-
aza-bicyclo[4.2.0]oct-2-en-7t-yl)-2-phenyl-2-yl-aceta-
mide 9a. m.p.: 60 °C (benzene). IR (CHCl3): w 1775
1
cm–1; H-NMR (CDCl3): l 2.02 (s; 3H); 3.20 and 3.48
(2d; 2H, J=17.7 Hz); 3.64 (s; 2H); 4.58 (d; 1H, J=12.8
Hz); 4.89 (d; 1H, J=4.8 Hz); 4.90 (d; 1H, J=12.8 Hz);
5.12 and 5.40 (2d; 2H, J=12.6 Hz); 5.70 (dd; 1H,
J=4.8 and 9.0 Hz); 6.02 (d; 1H, J=9.6 Hz, D2O
exchangeable); 7.06–8.07 (m; 10H). Anal. C25H24N2O6S
(C, H, N). Crude 9b was purified by trituration with
petroleum ether (40–60 °C) to give pure 9b as an oil
1
IR (CHCl3): w 1772 cm–1; H-NMR (CDCl3): l 1.86
(brs; 2H); 2.05 (s; 3H); 3.35 and 3.55 (2d; 2H, J=17.6
Hz); 4.40–5.50 (m; 6H); 7.30–8.20 (brs; 5H). Anal.
C17H18N2O5S (C, H, N).
(70%);
N-((6R)-3-acetoxymethyl-2-(4-fluorobenzoyl)-
oxymethyl-8-oxo-(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-
2-en-7t-yl)-2-phenyl-2-yl-acetamide 9b. IR (CHCl3): w
(6R)-3-acetoxymethyl-2-(4-fluorobenzoyl)oxymethyl-
8 - oxo - (6rH) - 5 - thia - 1 - aza - bicyclo[4.2.0]oct - 2 - en - 7t-
amine 12b. IR (CDCl3): w 1773 cm–1; 1H-NMR (CDCl3):
l 1.89 (brs; 2H); 2.10 (s; 3H); 3.39 and 3.60 (2d; 2H,
J=17.6 Hz); 4.65–5.50 (m; 6H); 7.01–7.25 and 7.98–
8.18 (m; 4H). Anal. C17H17FN2O5S (C, H, N).
(6R) - 3 - acetoxymethyl - 2 - pivaloyloxymethyl - 8 - oxo-
(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-7t-amine 12c.
1
1773 cm–1; H-NMR (CDCl3): l 2.06 (s; 3H); 3.20 and
3.55 (2d; 2H, J=17.6 Hz); 3.67 (s; 2H); 4.68 and 4.96
(2d; 2H, J=12.8 Hz); 4.97 (d; 1H, J=4.8 Hz); 5.25 and
5.42 (2d; 2H, J=8.8 Hz); 5.79 (dd; 1H, J=4.8 and 8.8
Hz); 6.38 (d; 2H, J=8.8 Hz, D2O exchangeable); 6.99–
7.35 and 7.94–8.27 (2m; 9H). Anal. C25H23FN2O6S (C,
H, N). 9c was directly obtained as a pure oil (90%);
N-((6R)-3-acetoxymethyl-2-pivaloyloxymethyl-8-oxo-
(6rH) - 5 - thia - 1 - aza - bicyclo[4.2.0]oct - 2 - en - 7t - yl) - 2-
phenyl-2-yl-acetamide 9c. IR (CHCl3): w 1773 cm–1;
1H-NMR (CDCl3): l 1.77 (s; 9H); 2.07 (s; 3H); 3.16 and
3.63 (2d; 2H, J=17.9 Hz); 3.89 (s; 2H); 4.76 (d; 1H,
J=4.8 Hz); 5.03 (m; 2H); 5.82 (dd; 1H, J=4.8 and 9.4
Hz); 6.59 (d; 1H, J=9.4 Hz, D2O exchangeable); 7.32
(brs; 5H). Anal. C23H28N2O6S (C, H, N).
1
IR (CDCl3): w 1770 cm–1; H-NMR (CDCl3): l 1.19 (s;
9H); 1.89 (brs; 2H, D2O exchangeable); 2.09 (s; 3H);
3.16 and 3.40 (2d; 2H, J=17.8 Hz); 3.60–5.16(m; 8H).
Anal. C15H22N2O5S (C, H, N).
4.1.4. General procedure for the synthesis of the
7h-methoxy cephem derivatives 14a–c
A solution of NaNO2 (0.56 mmol) in H2O (4.1 mL)
was added to a solution of the appropriate amine 12a–c
(0.55 mmol) in DCM (4.1 mL). The resulting mixture
was cooled in an ice bath and stirred vigorously, then 2
N aqueous H2SO4 (0.42 mL) was added dropwise over
30 min. Stirring was continued for 1 h in the same
conditions and the reaction was monitored by IR spec-
4.1.3. General procedure for amines 12a–c
t-(BOC)2O (15.6 mmol), DMAP (9.2 mmol) and TEA
(8.9 mmol) were added to a solution of the appropriate
ester 9a–c (8.9 mmol) in anhydrous DCM (190 mL),

A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
191
troscopy controlling the appearance of the diazo group
characteristic stretching-band at 2 100 cm–1.
troscopy controlling the appearance of the diazo group
characteristic stretching-band at 2 100 cm–1.
The two phases were separated and the aqueous
medium was extracted with more DCM (5 mL). The
combined organic layers were washed with brine (10
mL) and dried over anhydrous Na2SO4, previously dried
in muffle. The resulting anhydrous DCM solution of the
diazo derivative 13a–c, stirred and cooled at 0 °C, was
diluted with anhydrous MeOH (4.3 mL) and treated
with Rh2OAc4 (4.05×10–3 mmol). After stirring in these
conditions for 90 min, the mixture was filtered through
silica gel, concentrated, and dried in vacuo to give a
crude residue which was purified by flash chromatogra-
phy on silica gel eluting with a 2:1 hexane/AcOEt
mixture.
The two phases were separated and the aqueous
medium was extracted with more DCM (2.5 mL). The
combined organic layers were washed with brine (5 mL)
and dried over anhydrous Na2SO4, previously dried in
muffle. The resulting anhydrous DCM solution of the
diazoderivative 13a,b, stirred and cooled at 0 °C, was
diluted with absolute EtOH (0.65 mL) and concentrated
aqueous HCl (0.026 mL). After stirring at room temper-
ature for 20 s, the reaction mixture was poured into a 1
M KH2PO4 solution (15 mL) and extracted with DCM
(5 mL). The organic phase was washed with brine (10
mL), dried and evaporated to obtain 15a,b as crude
residues.
(6R)-3-acetoxymethyl-8-oxo-(6rH)-5-thia-1-aza-bi-
cyclo[4.2.0]oct-2-en-7c-methyloxy-2-benzoyloxy methyl-
(6R)-3-acetoxymethyl-8-oxo-(6rH)-5-thia-1-aza-bi-
cyclo[4.2.0]oct-2-en-7c-chloro-2-benzoyloxymethylester
15a. The crude residue was purified by flash chromatog-
raphy on silica gel eluting with 2:1 hexane/AcOEt, ob-
taining 15b as an oil (32%). IR (CDCl3): w 1780 cm–1;
1H-NMR (CDCl3): l 2.04 (s; 3H); 3.39 and 3.59 (dd;
2H, J=18.0 Hz); 4.73 (d; 1H, J=12.7 Hz); 4.76 (d; 1H,
J=2.0 Hz); 4.78 (d; 1H, J=2.0 Hz); 4.99 (d; 1H,
J=12.7 Hz); 5.30 and 5.49 (dd; 2H, J=13.2 Hz);
1
ester 13a. IR (CDCl3): w 1780 cm–1; H-NMR (CDCl3):
l 2.04 (s; 3H); 3.33 (d; 1H, J=17.7 Hz); 3.55 (s; 3H);
3.59 (d; 1H, J=17.7 Hz); 4.56 (d; 1H, J=1.9 Hz); 4.69
(d; 1H, J=1.9 Hz); 4.73 and 4.99 (dd; 2H, J=12.6 Hz);
5.31 and 5.49 (dd; 2H, J=12.9 Hz); 7.30–7.60 and
8.05–8.12 (2m; 5H). Anal. C18H19NO6S (C, H, N).
(6R)-3-acetoxymethyl-8-oxo-(6rH)-5-thia-1-aza-bi-
cyclo[4.2.0]oct-2-en-7c-methyloxy-2-(4-fluorobenzoyl)-
7.40–7.60
and
8.00–8.12
(2m;
5H).
Anal.
1
oxymethylester 13b. IR (CDCl3): w 1780 cm–1; H-NMR
C17H16ClNO5S (C, H, N). (6R)-3-acetoxymethyl-8-oxo-
(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-7c-chloro-2-
(4-fluorobenzoyl)oxymethylester 15b. The residue was ex-
clusively composed of the desired compound (90%). IR
(CDCl3): w 1780 cm–1; 1H-NMR (CDCl3): l 2.03 (s;
3H); 3.30 and 3.62 (dd; 2H, J=18.0 Hz); 4.76 and 4.92
(2d; 2H, J=12.7 Hz); 5.22 and 5.45 (2d; 2H, J=13.2
Hz); 6.90–7.30 and 7.82–8.20 (2m; 4H). Anal.
C17H17ClFNO5S (C, H, N).
(CDCl3): l 2.08 (s; 3H); 3.32 (d; 1H, J=18.2 Hz); 3.59
(s; 3H); 3.68 (d; 1H, J=18.2 Hz); 4.57 (d; 1H, J=2.0
Hz); 4.72 (d; 1H, J=2.0 Hz); 4.74 and 5.01 (dd; 2H,
J=12.8 Hz); 5.32 and 5.55 (dd; 2H, J=12.8 Hz);
6.99–7.32 and 7.95–8.22 (2m; 4H). Anal. C18H18FNO6S
(C, H, N).
(6R)-3-acetoxymethyl-8-oxo-(6rH)-5-thia-1-aza-bi-
cyclo[4.2.0]oct-2-en-7c-methyloxy-2-pivaloyloxymethyl-
1
ester 13c. IR (CDCl3): w 1780 cm–1; H-NMR (CDCl3):
l 1.19 (s; 9H); 2.05 (s; 3H); 3.18 (d; 1H, J=18.9 Hz);
3.54 (s; 3H); 3.56 (d; 1H, J=18.0 Hz); 4.51 (d; 1H,
J=2.0 Hz); 4.56 (d; 1H, J=12.6 Hz); 4.66 (d; 1H,
J=2.0 Hz); 4.83 (d; 1H, J=12.6 Hz). Anal.
C16H23NO6S (C, H, N).
4.1.6. General procedure for the synthesis of sulphones
4a–c and 5a,b
Solid MCPBA (0.65 mmol, 70% pure) and NaHCO3
(0.65 mmol) were added to a stirred solution, cooled at
0 °C, of the appropriate sulphur derivative 14a–c, 15a,b
(0.26 mmol) in anhydrous DCM (5.3 mL). After stirring
in these conditions for 2 h, the mixture was diluted with
DCM (5 mL) and washed with cooled 10% aqueous
Na2S2O3 (2×10 mL), 5% aqueous NaHCO3 (2×10 mL)
and brine. The extracts were then dried and evaporated
in vacuo to give a crude residue, which was purified by
flash chromatography on silica gel, eluting with a hex-
ane/AcOEt 2:1 mixture.
4.1.5. General procedure for the synthesis of 7h-chloro
cephem derivatives 15a,b
A solution of NaNO2 (0.35 mmol) in H2O (2.5 mL)
was added to a solution of the appropriate amine 12a,b
(0.32 mmol) in DCM (2.5 mL). The resulting mixture
was cooled in an ice bath and stirred vigorously, then 2
N aqueous H2SO4 (0.28 mL) was added dropwise over
30 min. Stirring was continued for 1 h in the same
conditions and the reaction was monitored by IR spec-
(6R) - 3 - acetoxymethyl - 2 - benzoyloxymethyl - 8 - oxo-
(6rH)-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-7c-methyloxy-

192
A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
5,5-dioxide 4a. IR (CHCl3): w 1796 cm–1; 1H-NMR
(CDCl3): l 2.06 (s; 3H); 3.55 (s; 3H); 3.73 and 3.99 (2d;
2H, J=17.9 Hz); 4.61 (d; 1H, J=13.0 Hz); 4.71 (d; 1H,
J=1.9 Hz); 4.98 (d; 1H, J=13.0 Hz); 5.19 (d; 1H,
J=1.9 Hz); 5.26 and 5.44 (2d; 2H, J=13.2 Hz); 7.40–
7.65 and 7.95–8.20 (2m; 5H). Anal. C18H19NO8S (C, H,
N).
(6R)-3-acetoxymethyl-2-((4-fluorobenzoyl)oxymethyl)-
8-oxo-(6rH)-5-thia-1-aza-bicyclo[4.2.0]-oct-2-en-7c-
methyloxy-5,5-dioxide 4b. IR (CHCl3): n 1796 cm–1;
1H-NMR (CDCl3): d 2.06 (s; 3H); 3.55 (s; 3H); 3.73 and
3.99 (2d; 2H, J=18.0 Hz); 4.61 (d; 1H, J=13.0 Hz);
4.69 (d; 1H, J=1.6 Hz); 5.25 and 5.42 (2d; 2H, J=13.3
Hz); 7.01–7.19 and 8.0–8.12 (2m; 4H). Anal.
C18H18FNO8S (C, H, N).
troaniline was monitored from the increase in ab-
sorbance at 390 nm (m for MAAPVNA=500 M–1 cm–1;
m for p-nitroaniline=13 000 M–1 cm–1) with a Beckman
DU-7 UV–visible spectrophotometer thermostated at
37 °C [34]. The KM values of HLE and PPE for
MAAPVNA at 37 °C were evaluated and found to be
0.12 and 0.43 mM, respectively; the latter value is
strongly at variance with that reported at 25 °C [33, 35].
A typical inhibition assay (carried out in a final volume
of 500 mL of the buffer containing 4% DMSO) was run
as follows: HLE (about 5 milliunits; one milliunit re-
leases one nmol of p-nitroaniline per min from
MAAPVNA) was added to a reaction mixture contain-
ing MAAPVNA (0.2–0.6 mM), and the increase in
absorbance was recorded for 3 min (time course A); one
of the inhibitors dissolved in <2 mL of DMSO was
subsequently added to a final concentration ranging
from 0.1 to 20 mM and the absorbance was further
recorded up to 30 min (time course B). As a reference,
Fig. 11 in [20] can be used. Control assays, in which the
inhibitor was omitted, ran linearly in time up to an
absorbance of 1.0 to 1.1 AU, before the slope started to
change due to substrate consumption; assays in which
the inhibitor was added readily showed a time-depen-
dent decline in the slope, and the absorbance reading
levelled off between 0.5 and 1.0 AU at 30 min, depend-
ing on the inhibitor concentration and potency.
The first-order decay in HLE or PPE activity was
evaluated by taking the slope value every 60 s over time
course B and dividing it by that of time course A. The
natural log of this ratio was plotted as a function of
time, the negative slope of this plot yielding a kobs value
(apparent first-order rate constant). The inactivation
time course of the enzyme in the presence of both
inhibitor and substrate strictly obeyed pseudo-first-order
kinetics up to a residual activity lower than 10% (i.e.,
over more than 2 ln units). Since kobs values did not
increase linearly with [I], 1/kobs values obtained at the
same substrate concentration and at varying concentra-
tions of a single inhibitor were plotted versus 1/[I]
(double-reciprocal plot). The reciprocal of the slope of
this plot yielded
(6R) - 3 - acetoxymethyl - 2 - pivaloyloxymethyl - 8 - oxo-
(6rH)-5-thia-1-aza-bicyclo[4.2.0]-oct-2-en-7c-methyl-
1
oxy-5,5-dioxide 4c. IR (CHCl3): n 1796 cm–1; H-NMR
(CDCl3): d 1.21 (s; 9H); 2.09 (s; 3H); 3.57 (s; 3H); 3.71
and 3.98 (2d; 2H, J=18.4 Hz); 4.54 (d; 1H, J=13.0
Hz); 4.66 (d; 1H, J=1.8 Hz); 4.86 (d; 1H, J=13.0 Hz);
5.01–5.10 (2d; 2H, J=13.3 Hz); 5.17 (d; 1H, J=1.8
Hz). Anal. C16H23NO8S (C, H, N).
(6R) - 3 - acetoxymethyl - 2 - benzoyloxymethyl - 8 - oxo-
(6rH)-5-thia-1-aza-bicyclo[4.2.0]-oct-2-en-7c-chloro-5,5
-dioxide 5a. IR (CHCl3): n 1812 cm–1; 1H-NMR
(CDCl3): d 2.07 (s; 3H); 3.79 and 4.02 (2d; 2H, J=18.1
Hz); 4.62 (d; 1H, J=13.1 Hz); 4.80 (d; 1H, J=1.9 Hz);
4.98 (d; 1H, J=13.1 Hz); 5.24 (d; 1H, J=13.5 Hz); 5.36
(d; 1H, J=1.9 Hz); 5.45 (d; 1H, J=13.5 Hz); 7.40–7.65
and 7.98–8.19 (2m; 5H). Anal. C17H16ClNO7S (C, H,
N). (6R) - 3 - acetoxymethyl - 2 - ((4 - fluorobenzoyl)oxy-
methyl)-8-oxo-(6rH)-5-thia-1-aza-bicyclo[4.2.0]-oct-2-
en-7c-chloro-5,5-dioxide 5b. IR (CHCl3): n 1812 cm–1;
1H-NMR (CDCl3): d 2.12 (s; 3H); 3.78 and 4.09 (2d;
2H, J=18.50 Hz); 4.65 (d; 1H, J=12.7 Hz); 4.82 (d;
1H, J=2.0 Hz); 4.99 (d; 1H, J=12.7 Hz); 5.25 (d; 1H,
J=13.2 Hz); 5.38 (d; 1H, J=2.0 Hz); 5.48 (d; 1H,
J=13.2 Hz). Anal. C17H15ClFNO7S (C, H, N).
4.2. HLE and PPE inhibition
kinact
KI(1+[S]/KM)
The activities of HLE (EC 3.4.21.37, Calbiochem) and
PPE (EC 3.4.21.36, Sigma Chemical Company) were
assayed spectrophotometrically, using N-methoxysuc-
cinyl-Ala-Ala-Pro-Val-p-nitroanilide (MAAPVNA) as
the substrate [33] in 0.05 M sodium phosphate buffer,
pH 7.8. Both substrate and inhibitor solutions were
prepared in DMSO. The hydrolytic release of p-ni-
,
from which the concentration-independent second-order
rate constant kinact/KI was obtained, using the appropri-
ate KM value (for HLE or PPE) in computing the factor
(1+[S]/KM). Similar values of this parameter were ob-

A. Balsamo et al. / European Journal of Medicinal Chemistry 36 (2001) 185–193
193
Osinga D.G., Peterson L.B., Williams D.T., Metzger J.M.,
Bonney R.J., Humes J.L., Pacholok S.P., Hanlon W.A., Opas E.,
Stolk J., Davies P., Proc. Natl. Acad. Sci. USA 90 (1993)
8727–8731.
tained for the same inhibitor at different substrate con-
centrations, in agreement with a competitive mechanism
of inhibition [20].
[20] Macchia B., Gentili D., Macchia M., Mamone F., Martinelli A.,
Orlandini E., Rossello A., Cercignani G., Pierotti R., Allegretti
M., Asti C., Caselli G., Eur. J. Med. Chem. 35 (2000) 53–67.
Acknowledgements
[21] Alpegiani M., Bissolino P., Corigli R., Rizzo V., Perrone E.,
Bioorg. Med. Chem. Lett. 5 (1995) 687–690.
This work was supported in part by a grant from the
consiglio Nazionale delle Ricerche and the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica.
[22] Maiti S.N., Czajkowski D.P., Narender A.V., Reddy A.V.,
Spevak P., Kaleta J., Micetich G., Bioorg. Med. Chem. Lett. 7
(1996) 823–826.
[23] Alpegiani M., Bissolino P., Borghi D., Rizzo V., Perrone E.,
Bioorg. Med. Chem. Lett. 3 (1993) 2259–2264.
[24] Rizzo V., Borghi D., Sacchi N., Alpegiani M., Perrone E., Bioorg.
Med. Chem. Lett. 3 (1993) 2265–2270.
References
[25] Navia M.A., Springer J.P., Lin T.Y., Williams H.R., Firestone
R.A., Pisano J.M., Doherty J.B., Finke P.E., Hoogsteen K.,
Nature 327 (1987) 79–82.
[1] Travis J., Dumbin A., Potempa J., Watorek W., Kurdowska A.,
Ann. NY Acad. Sci. 624 (1991) 81–86.
[26] Knight W.B., Maycock A.L., Greene B.G., Ashe B.M., Gale P.,
Weston H., Finke P.E., Hagmann W.K., Shah S.K., Doherty J.B.,
Biochem. 31 (1992) 4980–4986.
[2] Janoff A., Scherer J., J. Exp. Med. 128 (1968) 1137–1155.
[3] Dal Nogare A.R., Toews G.B., Pierce A.K., Am. Rev. Respir.
Dis. 135 (1987) 671–675.
[27] Finke P.E., Ashe B.M., Knight W.B., Maycock A.L., Navia
M.A., Shah S.K., Tompson K.R., Underwood D., Weston H.,
Zimmerman M., Doherty J.B., J. Med. Chem. 33 (1990) 2522–
2528.
[4] McDonald J.A., Kelly D.G., J. Biol. Chem. 255 (1987) 8848–
8858.
[5] McDonald J.A., Baum B.J., Rosemberg D.M., Kelman J.A., Brin
S.C., Crystal R.G., Lab. Invest. 40 (1979) 350–357.
[28] Alpegiani M., Bissolino P., Corigli R., Del Nero S., Perrone E.,
Rizzo V., Sacchi N., Cassinelli G., Franceschi G., J. Med. Chem.
37 (1994) 4003–4019.
[6] Brown G.M., Donaldson K., Thorax 43 (1988) 132–139.
[7] Starkey P.M., Acta Biol. Med. Ger. 36 (1977) 1549–1554.
[29] Alpegiani M., Bissolino P., Borghi D., Corigli R., Del Nero S.,
Perrone E., Razzano G., Rizzo V., Bioorg. Med. Chem. Lett. 2
(1992) 1127–1132.
[8] Gadek J.E., Fells G.A., Wright D.G., Crystal R.G., Biochem.
Biophys. Res. Commun. 98 (1980) 1815–1822.
[9] Mainard C.L., Dixit S.N., Kang A.H., J. Biol. Chem. 225 (1980)
5435–5441.
[30] Balsamo A., Domiano P., Macchia B., Macchia F., Orlandini E.,
Rossello A., J. Chem. Res. (S) (1984) 226–227.
[10] Janoff A., Blondin J., Proc. Soc. Exp. Biol. Med. 135 (1970)
302–306.
[31] Balsamo A., Benvenuti M., Lapucci A., Macchia B., Nencetti S.,
Macchia F., Rossello A., Domiano P., Dradi E., J. Org. Chem.
56 (1991) 2148–2153.
[11] Itoh Y., Nagase H., J. Biol. Chem. 270 (1995) 16518–16521.
[12] Snider G.L., Ciccolella D.E., Morris S.M., Ann. NY Acad. Sci.
624 (1991) 45–59.
[32] Doherty J.B., Ashe B.M., Barker P.L., Blacklock T.J., Butcher
J.W., Chandler G.O., Dahlgren M.E., Davies Dorn C.P., Finke
P.E., Firestone R.A., Hagman K.W., Halgren T., Knight W.B.,
Maycock A.L., Navia M.A., O’Grady L., Pisano J.M., Shah S.K.,
Tompson K.R., Weston H., Zimmerman M., J. Med. Chem. 33
(1990) 2513–2521.
[13] Merritt T.A., Cochrane C.G., Holcomb K., J. Clin. Invest. 72
(1983) 656–666.
[14] Jackson A.H., Hill S.L., Afford S.C., J. Resp. Dis. 65 (1984)
114–124.
[33] Nakajima K., Powers J.C., Ashe B.M., Zimmerman M., J. Biol.
Chem. 254 (1979) 4027–4032.
[15] Stockley R.A., Hill S.L., Burnett D., Ann. NY Acad. Sci. 624
(1991) 257–266.
[34] Shah S.K., Dorn C.P., Finke P.E., Hale J.J., Hagmann W.K.,
Brause K.A., Chandler G.O., Kissinger A.L., Ashe B.M., Weston
H., Knight W.B., Maycock A.L., Dellea P.S., Fletcher D.S., Hand
K.M., Mumford R.A., Underwood D.J., Doherty J.B., J. Med.
Chem. 35 (1992) 3745–3754.
[16] Bjornland K., Buo L., Scott H., Konttinen Y., Johansen H.,
Aasen A.O., Int. J. Oncol. 12 (1998) 535–540.
[17] Finche P.E., Shah S.K., Ashe B.M., Ban R.G., Blacklock T.J.,
Bonney R.J., Brause K.A., Chandler G.O., Cotton M., Davies
P., Dellea P.S., Dorn C.P., Fletcher D.S., O’Grady L.A., Munford
R.A., Osinga D.G., Sohar P., Tompson K.R., Weston H.,
Doherty J.B., J. Med. Chem. 35 (1992) 3731–3744.
[35] The KM value of MAAPVNA for PPE was experimentally
determined both under our assay conditions (37 °C, 0.05 M
sodium phosphate buffer, pH 7.8) and under those of Nakajima
et al. [33], always getting a value in the 0.4–0.6 mM range. We
never obtained a value as high as that reported in [33] (6 mM),
and therefore KM=0.4 mM (the value obtained under our assay
conditions) was used for the correct evaluation of the inhibition
parameters against PPE of the compounds under study in this
work. The KM value of MAAPVNA for HLE, experimentally
determined both under our assay conditions and under those of
Nakajima et al. [33], was always found to be in agreement with
the value reported by these authors.
[18] Tompson K.R., Finche P.E., Shah S.K., Ashe B.M., Dahlgren
M.E., Maycock A.L., Doherty J.B., Bioorg. Med. Chem. Lett.
3 (1993) 2283–2288.
[19] Doherty J.B., Shah S.K., Finche P.E., Dorn C.P., Hagman K.W.,
Hale J.J., Kissinger A.L., Tompson K.R., Brause K.A., Chandler
G.O., Knight W.B., Maycock A.L., Ashe B.M., Weston H., Gale
P., Munford R.A., Andersen O.F., Williams H.R., Nolan T.E.,
Frankenfield D. L., Underwood D., Vyas K.P., Kari P.H.,
Dahlgreen M.E., Mao T., Fletcher D.S., Dellea P.S., Hand K.M.,
.