Carnosine analogues containing NO-donor substructures: Synthesis, physico-chemical characterization and preliminary pharmacological profile

Article abstract of DOI:10.1016/j.ejmech.2012.04.032

The synthesis, physico-chemical, and biological characterisation of a short series of carnosine amides bearing NO-donor nitrooxy functionalities are described. The NO-donor carnosine analogues and their des-NO derivatives display carnosine-like properties

Full text of DOI:10.1016/j.ejmech.2012.04.032

European Journal of Medicinal Chemistry 54 (2012) 103e112
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Original article
Carnosine analogues containing NO-donor substructures: Synthesis,
physico-chemical characterization and preliminary pharmacological profile
Massimo Bertinaria a, Barbara Rolando a, Marta Giorgis a, Gabriele Montanaro a, Elisabetta Marini a,
,
a
Massimo Collino a, Elisa Benetti a, Pier Giuseppe Daniele b, Roberta Fruttero a , Alberto Gasco
*
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P. Giuria 9, I-10125 Torino, Italy
b Dipartimento di Chimica, Università degli Studi di Torino, Via P. Giuria 7, I-10125 Torino, Italy
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< We synthesized and evaluated car-
nosine nitrooxy derivatives as anti-
oxidant and organ protective agents.
< New compounds showed multifac-
torial antioxidant activity in vitro.
< Both NO and des-NO carnosine
derivatives protect from ischaemia/
reperfusion injury in vivo.
< One model NO-donor carnosine
derivative was able to lower TNF-
a
in vivo.
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, physico-chemical, and biological characterisation of a short series of carnosine amides
bearing NO-donor nitrooxy functionalities are described. The NO-donor carnosine analogues and their
des-NO derivatives display carnosine-like properties, differing from the lead for their high serum
stability. The newly-synthesised compounds are able to complex Cu2þ ions at physiological pH, dis-
playing significant copper ion sequestering ability, and protect LDL from oxidation catalysed by Cu2þ
ions. All products show moderately-potent HNE quenching activity. The NO-donor compounds 7cef
relaxed rat aorta strips via an NO-dependent mechanism. In vivo evaluation of organ protection in
a model of cerebral ischaemia/reperfusion injury, using the selected NO-donor 7e and its des-NO
analogue 7a, showed that both derivatives protect from hypoxia-induced brain damage, at lower
Received 16 January 2012
Received in revised form
4 April 2012
Accepted 24 April 2012
Available online 2 May 2012
Keywords:
Carnosine
Carnosine amides
Cerebral ischaemia
Nitric oxide
concentrations than carnosine; 7e also decreased serum TNF-a levels. This class of NO-donor carnosine
amides is worthy of further study as potential tools for treating a wide range of chronic vascular and
neurodegenerative diseases in which NO-bioavailability is reduced.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Nitrooxy derivatives
1. Introduction
belonging to the family of metalloproteases [2]. At present, the
physiological role of carnosine remains unclear. In vitro, it triggers
Carnosine 1 is a histidine-containing dipeptide (
b
-alanyl-
L
-hys-
a variety of biological actions [3]. In particular, it exerts protection
against oxidative stress, being a scavenger of reactive oxygen (ROS)
and nitrogen (RNS) species, which are responsible for oxidative
damage of cellular lipids, enzymes, proteins and DNA [1,4e6]. In
tidine) (Chart 1) which inhumansisprincipallypresent inmuscle and
nerve tissues [1]. Itisrapidlycleaved into its constituents, in bothcells
and serum, under the action of carnosinases, a group of dipeptidases
addition, carnosine can trap toxic electrophilic
a,b- unsaturated
aldehydes (e.g., (E) 4-hydroxy-trans-2,3-nonenal (HNE) and malon-
dialdehyde) deriving from the oxidation of unsaturated membrane
lipids [7,8]. It protects proteins from glycation, thus inhibiting the
* Corresponding author. Tel.: þ39 011 6707850; fax: þ39 011 6707286.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.032

104
M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
Fig. 3. Alterations in serum levels of TNF-a induced by compound 7e. Rats were
subjected to 30 min ischaemia and 24 h reperfusion (I/R). Carnosine (1) (1 g/kg),
compound 7e (500 mg/kg) or its des-NO analogue 7a (500 mg/kg) were administered
during reperfusion. Data are means ꢀ S.E.M. of five animals per group. C p < 0.05
versus 7e.
hypoxia-ischaemic injuries [13].
tion proved able to promote a strong reduction in the hippocampal
intraneuronal A accumulation, and to rescue AD and ageing-related
mithocondrial disfunction in 3xTG-AD mice [14]. Unfortunately one
limitation in employing -carnosine as a protective drug is that it is
rapidly inactivated by carnosinases. The chemical derivatization of
carnosine is a very promising approach to realize therapeutic
carnosinase-resistant molecules based on carnosine. The conjugation
of carnosine with several types of organic molecules has the main
purpose of reducing the carnosinase action on the peptide moiety,
improving the multifunctional activity, because of the potential
beneficial role of the conjugating moiety and the synergism with the
peptide properties as well. Different modifications of the carnosine
structure have been performed generating a number of multifunc-
tional drug-like molecules which were recently reviewed [15,16].
Recent work on carnosine is directed towards the development of
L-carnosine dietary supplementa-
b
L
Fig. 1. a) Relationship between antioxidant activity (expressed as Dtlag) and calculated
lipophilicity (clog D7.4oct) obtained for carnosine amide derivatives (B) [21] and for
new compound 7b (C). Calculated clog D7.4oct for designed NO-donor analogues 7cef
(:) is also reported. b) Relationship between antioxidant activity (expressed as
Dtlag)
and measured lipophilicity (log D7.4oct) obtained for all carnosine amide derivatives:
(B) compounds described in [21], (C) new reference compound 7b, (:) NO-donor
carnosine analogues 7cef.
formation of toxic advanced glycation end-products (AGEs) [8e10].
Since oxidative stress and AGEs are involved in the development and
progression of some cardiovascular and neurodegenerative diseases
[11,12], as well as in ageing, there is currently great interest in car-
nosine and its derivatives. Recently the effects of carnosine have been
extensively studied both in vivo and in vitro in different models of
oxidative driven damages, such as neurodegenerative disorders and
both
carnosine itself proved stable to carnosinase and superior to
nosine in some in vitro and in vivo pharmacological models [17,18].
The major drawback with -carnosine lies in its low bioavailability,
D- and
L-carnosine derivatives stable to carnosinase action.
D-
L-car-
D
since it does not interact with hPEPT1, a transporter responsible for
the uptake of a broad array of small peptides in the colon. This
problem was addressed with the development of D-carnosine pro-
drugs; the octyl ester derivative was evaluated in vivo proving able to
decrease hypertension, improve dislipidemia and restore renal
function in Zucker fa/fa obese rats as well as to inhibit AGEs formation
and oxidative stress [19].
Interestingly some new L-carnosine derivatives stable towards
carnosinase action were recently synthesized by converting the
carboxylic function to an amide and by conjugating the terminal
primary amino group to a sugar substructure; these compounds
showed carnosinase stability while retaining copper complexing
ability [20].
Also our group has recently described some new L-carnosine
amides bearing alkyl or arylalkyl substituent at the amide nitrogen
endowed with different lipophilicities (Chart 1, general structure I)
[21]. These products share a number of properties with carnosine,
including the ability to complex Cu2þ ions, to protect LDL from
oxidation catalyzed by Cu2þ, and HNE quenching activity. Further,
in contrast to the lead, they also display high serum stability.
NO is a pivotal signalling messenger in the cardiovascular
system. Reduced NO bioavailability and abnormalities in NO-
Fig. 2. Effects of carnosine derivatives on infarct volume induced by cerebral I/R injury.
Effects evoked by the administration of carnosine (1) (1 g/kg), compound 7e
(500 mg/kg) or its des-NO analogue 7a (500 mg/kg) on cerebral infarct volume were
evaluated at 24 h reperfusion. Data are means ꢀ S.E.M. of five animals/group. + p < 0.01
versus I/R.

M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
105
Chart 1.
dependent signalling are among the central factors in vascular
2. Results and discussion
disease, and loss of endothelium-derived NO is known to be critical
in the pathogenesis of vascular diseases, including hypertension,
atherosclerosis, and vasospasm with compromised blood flow
[22,23]. Microvascular disorders, occurring with advanced age or in
the presence of increased oxidative stress, are consequent on
endothelial dysfunction, namely on a reduced capacity of endo-
thelial cells to produce nitric oxide (NO) [24]. Furthermore, chronic
mild long-lasting cerebrovascular dysfunction, involving basal NO
deficit, significantly contributes to plaque development in Alz-
heimer’s disease (AD) [23]. However, NO’s role in the brain is by no
means fully elucidated, and both neuroprotective and neurotoxic
effects have been widely reported and reviewed [25e27]. As
a continuation of our work on NO-donor antioxidant drugs [28],
this study aimed to synthesize and characterize new NO-donor
carnosine derivatives, with high serum stability. New NO-donor
compounds (Chart 1, derivatives 7cef) were designed taking into
account the relationship between clogD 7.4 and antioxidant activity
we previously found (Fig. 1a); the determination of clogD values for
these derivatives (7c ¼ ꢁ3.12; 7d ¼ ꢁ3.18, 7e ¼ ꢁ3,19, 7f ¼ ꢁ1,72)
allowed us to anticipate their optimal hydrophilic-lipophilic
balance in order to obtain potent antioxidant molecules. The NO-
donor carnosine amides described were obtained by introducing
one or two NO-donor nitrooxy groups on the lateral chain of the
amide nitrogen of scaffolds 7a,b. The introduction of two nitrooxy
2.1. Chemistry
The NO-donor carnosine amide structures, object of this study,
were synthesized by the pathway depicted in Scheme 1. The
commercially-available Na-fluorenylmethoxycarbonyl-N-trityl-
L-
histidine (Fmoc-His(Trt)-OH) 2 was coupled with amines 3a,b or
with the appropriate NO-donor amines 3cef, using the O-benzo-
triazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate
(HBTU)/1-hydroxybenzotriazole
(HOBt)/di-isopropylethylamine
(DIPEA) protocol, to give the corresponding intermediate amides
4aef. Fmoc deprotection of these intermediates was carried out in
DMF at room temperature in the presence of piperidine, to afford
the amines 5aef. Coupling of free amines with tert-butoxycarbonyl
protected
b-alanine (Boc-b-Ala-OH) in the presence of dicyclo-
hexylcarbodiimide (DCC) afforded 6aef, and the subsequent
simultaneous Trt/Boc deprotection using 10% (v/v) CF3COOH in
CH2Cl2 furnished the target compounds 7aef.
Intermediate substituted benzylamine 3b was synthesized by
alkylation of commercially-available 8, and the subsequent LiAlH4-
mediated reduction of the cyano group. The amines bearing dini-
trooxy functionalities 3d,e were synthesized by nitration with
fuming nitric acid in CH2Cl2 following the method reported else-
where [29]. Compound 3f was obtained by reacting tosylate 11 [30]
with Boc-protected benzylaminophenol 12 [31] followed by
CF3COOH-mediated deprotection (Scheme 2).
groups at the
u,
uꢁ1 positions of the propyl chain of 7a afforded the
two diastereoisomers 7d,e.
The preliminary pharmacological profile of these products, its
comparison with that of the related des-NO analogues 7a,b,
including the results of an in vivo study of the protective effects of
7e and of 7a in a model of cerebral ischaemia/reperfusion (I/R)
injury, are also discussed.
2.2. Stability in human serum
Two isoforms of carnosinase enzyme have been characterized
thus far. The first is a cytosolic form (tissue carnosinase) while the

106
M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
Scheme 1. Preparation of derivatives 7aef. Reagents and conditions: (a) DIPEA, HBTU, HOBtcat, 10 min RT, then 3aef 1e18 h, RT (57e94%); b) Piperidine, dry DMF, 1 h, RT.
(74e100%); c) Boc-
-Ala(OH), DCC, dry CH2Cl2, 0 ꢂC to RT. 2e18 h (59e91%); d) CF3COOH 10% in CH2Cl2, 20 h, RT (34e89%).
b
second is present in the serum (serum carnosinase). It is not
known why the enzyme is present in the serum. Although the
neuroprotective effects of carnosine have been widely docu-
mented in the literature and recently confirmed by clinical studies
in patients with stroke [32], widespread medical use of carnosine
is limited by a high rate of its degradation in serum and tissues by
carnosinase, which dictates the use of high doses and repeated
administration of carnosine. Consequently, an important requisite
that analogues of carnosine should posses in order to have ther-
apeutic potential is a good degree of metabolic stability in human
serum. For this reason, the NO-donor carnosine amides 7cef were
studied for their stability in this medium at 37 ꢂC. Unlike the lead,
which is rapidly metabolized into its constituents following
pseudo-first-order kinetics (t1/2 ¼ 5 min), all products were stable
over 3 h, similarly to the related des-NO amides 7a,b. This is in
keeping with the lack of carboxylic group in their structures,
which is essential to bind a Zn2þ ion present in the active cleft of
the enzyme [33].
2.3. Dissociation constants (pKas) and lipophilicity determination
Dissociation Constants (pKas) of 7aef were determined by
potentiometric titration; the results are reported in Table 1. As
expected, all NO-donor derivatives and related des-NO analogues
show pKa values that are very close either to that of the basic
imidazole centre or of the amino group present on the lateral chain.
At physiological pH (7.4) they exist prevalently as monocharged
(92e93%) and dicharged cations (5e6%). The apparent partition
coefficient log D7.4 of the products was evaluated by the shake-flask
technique, using buffered water (pH 7.4)/n-octanol as partition pair
Scheme 2. Preparation of intermediate amines 3b, 3def. Reagents and conditions: (a) NaH, RT, dry DMF, 1 h, then 3-Bromopropane, RT, 3 h; (b) LiAlH4, dry Et2O, RT to reflux,
30 min; (c) fuming HNO3, 0 ꢂC, dry CH2Cl2, 2 h, then Ac2O, 0 ꢂC, 1 h; (d) Tert-BuOK, THF dry, RT, 15 min then reflux 3 h; (e) CF3COOH 10% in CH2Cl2, 2 h, RT.

M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
107
Table 1
Dissociation constants (pKa), formation constants (log bpqr) of copper(II) complexes and logarithm of the ratio between [Cu2þ
derivatives 7aef.
]
and [Cu2þ
]
free
for carnosine (1) and
total
log ([Cu2þ
3.81
]
tot
/ [Cu2þ
] )
free
a
a
c
N.
pKa1
pKa2
logb111
logb110
logb11-1
logb22-2
logb22-3
logb22-4
logb120
b
1
13.30
8.47
2.44
8.35
14.05
b
7a
7b
7c
7d
7e
7f
6.18
6.22
6.16
6.19
6.19
6.21
9.19
9.19
9.19
9.21
9.23
9.16
12.22
6.72
1.25
5.40
ꢁ3.09
ꢁ12.67
13.47
2.93
d
d
d
d
d
d
d
d
12.27
12.10
6.81
6.42
1.22
1.21
6.14
5.24
ꢁ2.73
ꢁ3.52
ꢁ12.68
ꢁ13.08
13.49
13.29
3.06
3.00
12.34
7.15
1.39
6.22
ꢁ2.47
ꢁ12.01
13.28
3.05
d
d
d
d
d
d
d
d
a
Determined by potentiometric titration with GLpKa apparatus; n ꢄ 4, SD < 0.03; carnosine pKa1-3 were respectively 2.60, 6.79, 9.42.
b
c
Ref. [21].
Calculated for [Cu2þ
]
¼ 2.5 ꢃ 10ꢁ6 mol Lꢁ1 and [ligand]total ¼ 1 ꢃ 10ꢁ4 mol Lꢁ1 at pH 7.4.
total
d
Not determined because of low solubility of compounds in experimental conditions.
(Table 2). As expected, the introduction of nitrooxy function(s) on
des-NO amide scaffolds slightly increases the lipophilicity.
(II) ions, quite similar to those of the related des-NO analogues, even
if their copper(II) complexes are weaker, because of the absence of
carboxylate group, than that of carnosine. The capacities of the two
diastereoisomers 7d,e to sequester Cu2þ ions are similar.
2.4. Copper complexes
Particular attention was paid to carnosine’s capacity to form
complexes with Cu2þ, in view of its role in protecting LDL against
oxidation induced by Cu2þ ions [34,35]. Complex formation
between copper (II) and nitrooxy substituted carnosine amides
7cee was thus determined, following the same pH-metric tech-
nique used to study the parent des-NO analogue 7a. The study of 7f
and of its des-NO analogue 7b was hindered by the low water
solubility of the products in the experimental conditions. The
stability constants of the complexes, expressed as bpqr ¼ [CupLqHr]/
[Cu]p[L]q[H]r for the reaction pCu þ qL þ rH ¼ CupLqHr, where L is the
carnosine derivative, and p, q, r are the stoichiometric coefficients,
the charges being omitted for simplicity. Since for ligands here
studied the dissociation of both peptide and amide hydrogens takes
place at too high pH values to allow a reliable evaluation of the
relative pKH in aqueous solution, the concentration [L] in the
expression of bpqr takes into account only the hydrogen ion disso-
ciation from protonated imidazole and amino nitrogens. If the
presence of metal ion promotes further dissociation of hydrogen ion
from peptide and amide group, in the above reaction the species Hþ
is subtracted and the index r becomes negative. So the r index is
negative if the complex is formed by hydrogen ion dissociation from
peptide and/or amide group. From the stability constants reported
in Table 1, the ratio between free and total copper (II) concentrations
2.5. Biological activity
2.5.1. Antioxidant activity
Free transition-metal ions, especially iron and copper, play
important roles in LDL peroxidation. In particular, copper ions at
mM concentrations induce generation of free radicals at the low-
density lipoprotein (LDL) surface, with consequent oxidative
modifications that are of paramount importance in the pathogen-
esis of a number of cardiovascular and neurodegenerative diseases
[36e39]. This oxidation can be prevented by using copper chela-
tors, such as carnosine and related products. In previous research
we assayed the ability of carnosine amides to control this oxidation,
by evaluating
Dlag time (Dtlag), namely the duration of the period
prior to the onset of rapid peroxidation (propagation phase)
compared to control, and the propagation rate of the oxidation (R).
D
tlag values were found to be dependent on the lipophilicity of the
products, the most hydrophilic compounds being the most active
(Fig. 1b) [21]. lag time and R values of 7cef, evaluated under the
same conditions used previously, are collected in Table 2. lag time
D
D
values are slightly higher than those of the related des-NO refer-
ence compounds 7a,b and of carnosine, and closely fit the corre-
lation reported in Fig. 1b, between measured log D7.4 and
antioxidant activity of carnosine amide derivatives. R values are
slightly lower than those of the reference compounds, and close to
that of carnosine. The propagation rates of oxidation in the
can be calculated, assuming [Cu]total
¼
2.5
ꢃ
10ꢁ6 and
[ligand]total ¼ 1 ꢃ 10ꢁ4 M. Analysis of the data shows that the NO-
donor carnosine amides display a good ability to sequester copper
Table 2
Lipophilicity, antioxidant activity, HNE-scavenging ability and vasodilator properties of carnosine (1) and derivatives 7aef.
N.
Lipophilicity
Antioxidant activity
HNE-scavenging ability
Vasodilator properties
log D7.4 a
D
tlag (min) ꢀ SEMb
R (nmol minꢁ1 mgꢁ1 LDL prot) ꢀ SEMb
HNE % scavenged ꢀ SEM 24 hc
EC50 ꢀ SEM (
m
M)d
1
nd
67 ꢀ 2
68 ꢀ 2
64 ꢀ 6
90 ꢀ 3
75 ꢀ 4
88 ꢀ 3
70 ꢀ 1
5.8 ꢀ 0.3
7.7 ꢀ 0.3
9.5 ꢀ 0.5
3.9 ꢀ 0.2
5.8 ꢀ 0.1
5.8 ꢀ 0.3
6.3 ꢀ 0.2
88.8 ꢀ 1.6
81.0 ꢀ 1.6
73.3 ꢀ 1.3
75.4 ꢀ 3.5
67.2 ꢀ 4.5
77.6 ꢀ 3.2
74.3 ꢀ 2.7
nd
nd
7a
7b
7c
7d
7e
7f
ꢁ2.72
ꢁ1.15
ꢁ2.43
ꢁ2.33
ꢁ2.19
ꢁ1.01
nd
e
169 ꢀ 30
70 ꢀ 11
3.0 ꢀ 0.5
a
Determined by the shake-flask technique; n ꢄ 6, SD < 0.06.
b
Obtained by CuSO4-induced human LDL oxidation assay in the presence of compounds at 100 mM. R values were calculated from DA234 as a function of time, using
3
¼ 29500 Mꢁ1 cmꢁ1 for conjugated lipid peroxides. For control LDL samples, R ¼ 12.1 ꢀ 0.5 nmol minꢁ1 mgꢁ1 LDL prot.
234
c
Determined in phosphate buffer (pH 7.4, 1 mM) at 37 ꢂC; HNE (50
mM), test compound (1 mM). Scavenging % was calculated from the following formula: scavenging
(%) ¼ 100 e{[ (amount of HNE left after t h in the presence of the scavenger)/(amount of HNE left after t h in the control)] x 100}.
d
The EC50 value represents the
46 ꢀ 3% tissue relaxation at the maximum concentration tested (1 mM). nd ¼ not determined.
m
M equivalents of test compounds required to relax the pre-contracted rat aorta strips by 50%.
e

108
M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
presence of the two diastereoisomers 7d,e are the same, but its
onset is slightly faster for 7d.
reinforce experimental evidence of the therapeutic potential of the
NO-donor carnosine amides described here, the most promising
derivative (compound 7e), its des-NO analogue 7a, and carnosine
(1) were tested in a well-known in vivo model of cerebral I/R injury,
as we report elsewhere [48]. In comparison with brain sections
obtained from sham-operated rats, those rats that underwent
cerebral I/R injury showed an infarct volume of 31.6 ꢀ 4.1% of the
total brain. As shown in Fig. 2, compound 1 administration (1 g/kg)
almost halved the I/R-induced infarct volume. Our results are
consistent with previous studies showing that exogenously
administered carnosine evoked a marked reduction in infarct size
when administered before the ischaemic injury either in adult and
neonatal rat models [49e51]. Carnosine has been reported to exert
protective effects on neurological symptoms and animal mortality
even when injected into the blood stream after the ischaemic event
[52]. Here for the first time we show that a marked reduction in the
mean size of the ischaemic area was also measured when two
carnosinase-stable derivatives, compound 7e and its des-NO
analogue 7a, were administered at 500 mg/kg, without any
significant difference among groups. These results demonstrate
that both of these carnosine derivatives exert protective effects
against I/R injury, at lower doses than is required for carnosine,
being about four time more active than the lead (1). In our acute
experimental model, the potential further beneficial effect of NO
release by compound 7e was blunted by the predominant role of
antioxidant activity due to the carnosine amide substructure. This
observation is in keeping with previous in vivo and in vitro studies
reporting that carnosine neuroprotective effects were almost
entirely attributable to its ability to inhibit and/or prevent oxidative
and nitrosative stress [13]. However, recently, carnosine has also
been demonstrated to reduce glutamate excitotoxicity through the
regulation of the glutamate transporter-1 [50], to protect the
bioelectric activity of nervous cells by restoring the activity of the
glutamatergic and GABA-ergic receptors [53] and to affect the
apoptotic cascade in the ischaemic brain by inhibiting mRNA
expression of apoptosis-inducing factor and caspase-3 [51]. Here
we show that the NO-donor carnosine derivative 7e may modulate
cytokine production in the ischaemic brain. In fact, when serum
2.5.2. Vasodilator properties
The ability of the target products to supply exogenous NO to
blood vessels was evaluated by assessing their capacity to induce
relaxation of endothelium-denuded rat-aorta strips, pre-contracted
with phenylephrine. All products were capable of relaxing the
contracted tissue in
a concentration-dependent manner. The
vasodilator potencies of the products, expressed as EC50 values, are
reported in Table 2. They rank in order 7f > 7e > 7d>>7c. The
vasorelaxant effect was cGMP-dependent, being blocked if exper-
iments were carried out in the presence of 1H- [1,2,4]oxadiazolo
[4,3-a]quinoxalin-1-one (ODQ), a well-known inhibitor of soluble
guanylate cyclase (sGC). Interestingly, 7e is a vasodilator about
three times more potent than its diastereoisomer 7d, indicating
a possible role for the stereoisomery in the interaction of these
compounds with the enzyme responsible for their NO production.
2.5.3. HNE quenching activity
As mentioned above, HNE is a particularly abundant reactive
aldehyde, generated during lipidic peroxidation through
b-cleavage
of hydroperoxide derivatives of -6 polyunsaturated fatty acids. It
u
is a potent bifunctional electrophile (Michael system and carbonyl
group) able to interact with the nucleophile centres of DNA and
proteins. After the first electrophile centre has reacted, the second
centre undergoes further reaction, producing cross-linking of
macromolecules. Carnosine is an efficient quencher of HNE, as well
as of its amide derivatives. In order to evaluate the HNE trapping
ability of products 7aef, they were incubated in phosphate buffer,
pH 7.4, with the aldehyde at 37 ꢂC. HNE consumption over time was
monitored by RP-HPLC [7]. The results obtained after 24 h are
summarized in Table 2. The quenching effects on HNE fell in the
range 67.2e81.0%, the value for carnosine being 88.8%. Among the
two diastereoisomers, 7e is a slightly stronger quencher than 7d.
2.5.4. Cerebral ischaemia-reperfusion injury
Reduced blood supply to an organ induces organ injury, whose
severity depends on the degree and duration of blood-flow
reduction. Reperfusion of ischaemic tissue results in further
injury. The molecular mechanisms which underlie I/R injury are
complex and not fully understood. It is certain that overproduction
of reactive oxygen metabolites during reperfusion, in particular of
the superoxide anion (Oꢁ2 .), plays a central role. Hydrogen peroxide
(H2O2) is formed from this radical by action of superoxide dis-
mutase (SOD). H2O2 is a source, through the HabereWeiss reaction,
tumour necrosis factor (TNF)-
typical marker of the excessive inflammatory response associated
with I/R injury (Fig. 3), the two-fold increase in TNF- levels evoked
a concentrations were measured, as
a
by I/R was significantly reduced by administering compound 7e; in
contrast, both des-NO analogue 7a and carnosine had no effect on
TNF-a production. TNF-a is one of the key immunomodulatory and
pro-inflammatory cytokines, which has been clearly demonstrated
to be up-regulated during cerebral I/R injury [54]. Recent evidence
_
of the highly-toxic hydroxyl radical (OH), the principal agent
also suggests that chronic exposure to high levels of TNF-a may
responsible for the cell damage induced by oxidative stress. Release
of pro-inflammatory cytokines, increased expression of endothelial
adhesion molecules and of chemotactic factors, as well as infiltra-
tion of leukocytes, also play important roles in post-ischaemic
inflammation, contributing to progression of tissue damage [40].
Several studies have clearly demonstrated that carnosine exerts
beneficial effects against I/R-induced organ damage, including the
brain [41e43]. However, its rapid and complete enzymatic hydro-
lysis by carnosinases severely limits its potential therapeutic use.
The role of NO in I/R is complex. It exerts either protective or toxic
effects, depending on the level, location, source, and environment
[44,45]. Beneficial effects in I/R are due to its numerous capabilities:
it relaxes vascular smooth muscle, protects the microvasculature,
prevents neutrophile infiltration, maintains vascular permeability,
modulates cytokine expression, as well as displaying antioxidant,
anti-platelet-activating factor, and antimicrobial properties.
Consequently, a supply of exogenous NO (NO-donors) might have
beneficial effects in acute ischaemic damage [46,47]. In order to
induce changes in both lipid and glucose metabolism, likely to exert
pro-atherogenic effects, thus promoting the development of
atherosclerotic lesions [55]. Therefore, although decreased cyto-
kine production is not essential for protection against acute cere-
bral injury, it is possible that the decreased cytokine production
evoked by compound 7e might significantly contribute to the
potential protective effects of its prolonged administration in
vascular disorders characterized by high levels of chronic inflam-
mation, such as I/R-induced organ damage, atherosclerosis and
Alzheimer’s disease.
3. Conclusions
In conclusion, we have designed and realized a new series of
carnosine amide derivatives with NO-donor properties. The results
of this study demonstrate that our compounds are a new class of
NO-donor antioxidants endowed with HNE scavenging and Cu2þ
chelating properties, able to inhibit LDL oxidation. Preliminary

M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
109
pharmacological characterization of 7e shows that these
compounds are worthy of additional study as potential tools in the
treatment of complex vascular and neurodegenerative disorders,
whose onset and progression involve oxidative stress and
decreased NO availability.
and the semisolid residue was treated with water (30 mL). The
precipitate formed was filtered off through a sintered glass funnel,
the water phase was washed with CH2Cl2 (2 ꢃ 20 mL), then with
EtOAc (2 ꢃ 20 mL) and evaporated to afford the final products 7bef.
4.2.1.1.
b
-alanyl-N-[4-(propyloxy)benzyl]-
L
-histidinamide
ditri-
4. Experimental
fluoroacetate (7b). The product was recrystallized twice from dry
MeOH/Et2O and freeze-dried to yield 7b as a white foam (67%). 1H
4.1. Instrumentation and chemicals
NMR (CD3OD): d, 8.71 (s, 1H, ImH2); 7.24 (s, 1H, ImH5); 7.13 (d, 2H,
J ¼ 8.4 Hz, ArH2’,6’); 6.84 (d, 2H, J ¼ 8.4 Hz, ArH3’,5’); 4.70 (m,1H, CH);
4.30e4.23 (m, 2H, NHCH2); 3.90 (t, 2H, J ¼ 6.3 Hz, OCH2); 3.22e3.08
(m, 4H, ImCH2, CH2NHþ3 ); 2.68e2.62 (m, 2H, COCH2); 1.80e1.73 (m,
Determination of melting points of final compounds (7bef) was
affected by their complex thermal behaviour; consequently,
melting points are not reported here. 1H and 13C-NMR spectra were
obtained on a Bruker Avance 300, at 300 and 75 MHz, respectively,
2H, CH2CH2CH3); 1.03 (t, 3H, J ¼ 7.2 Hz, CH3). 13C NMR (CD3OD):
d,
172.4; 171.9; 160; 135; 131.5; 131.2; 130; 118.4; 115.5; 70.6; 53.8;
43.7; 36.8; 32.7; 28.3; 23.7; 10.8. Anal. Calc. for C19H27N5O3 $ 2
CF3COOH $ H2O, C% 44.59, H% 5.04, N% 11.30. Found: C% 44.60, H%
4.98, N% 11.42.
or on a Bruker AC-200, at 200 and 50 MHz, respectively;
d in ppm
rel. to SiMe4 as the internal standard; coupling constants J in Hz.
13C-NMR spectra were fully decoupled. The following abbreviations
are used: s: singlet, d: doublet, dd: doublet doublet, t: triplet, qt:
quartet, m: multiplet, br: broad, Im: imidazole ring. Mass spectra
were recorded on a Finnigan-Mat TSQ-700. Flash chromatography
4.2.1.2.
acetate (7c). The product was recrystallized from dry MeOH/Et2O and
freeze-dried to yield 7c as a white foam (89%). 1H NMR (CD3OD):
d,
b
-alanyl-N-[3-(nitrooxy)propyl]-
L
-histidinamide
ditrifluoro-
(FC) was performed on BDH silica gel (particle size 40e63
mm).
When not otherwise specified, anhydrous magnesium sulphate
(MgSO4) was used as drying agent for organic phases. Analysis (C,
H, N) of the target compounds was performed by Service de
Microanalyse, Université de Genève, Genève (CH), and the results
were within ꢀ0.4% of theoretical values. Derivatives 7a [21], 3c [29],
11 [30], and 12 [31] were synthesised by the methods reported.
HNE (4-hydroxy-trans-2,3-nonenal) was prepared by acid treat-
ment (1 mM HCl) of HNE-DMA (4-hydroxy-trans-2,3-nonenal-
dimethylacetal; Sigma).
8.81 (s, 1H, ImH2); 7.35 (s, 1H, ImH5); 4.64 (m, 1H, CH); 4.48 (t, 2H,
J ¼ 6.3 Hz, CH2ONO2); 3.31e3.10 (m, 6H, ImCH2, CH2NHþ3 , CH2NH);
2.68e2.65 (m, 2H, COCH2); 1.92e1.88 (m, 2H, CH2CH2CH2). 13C NMR
(CD3OD): d, 172.44; 172.41; 135.1; 131.2; 118.4; 72.3; 53.9; 37.1; 36.8;
32.7; 28.0; 27.8. Anal. Calc. for: C12H20N6O5 $ 2 CF3COOH $ H2O, C%
33.46, H% 4.21, N% 14.63; Found C% 33.83, H% 4.14, N% 14.40.
4.2.1.3.
b-Alanyl-N-[(2S)-2,3-bis(nitrooxy)propyl]-L-histidinamide
ditrifluoroacetate (7d). The productwas recrystallized twice from dry
MeOH/Et2O and freeze-dried to yield 7d as a white foam (70%). 1H
4.2. Chemistry
NMR (CD3OD): d, 8.73 (s, 1H, ImH2); 7.33 (s, 1H, ImH5); 5.45 (m, 1H,
CHONO2); 4.88e4.58 (m, 3H, CHNH, CH2ONO2); 3.61e3.41 (m, 2H,
4.2.1. General procedure for the synthesis of derivatives 7bef
NHCH2); 3.30e3.06 (m, 4H, ImCH2, CH2NHþ3 ); 2.86e2.73 (m, 2H,
To a stirred solution of Fmoc-His-(Trt)-OH (2) (2.5 g; 4 mmol) in
dry DMF (40 mL), DIPEA (1.03 mL; 6 mmol), HBTU (2.29 g; 6 mmol)
and HOBt (0.08 g; 0.6 mmol) were added. After 10 min, the appro-
priate amine 3bef (6 mmol) was added; the reaction mixture was
stirred at RT until TLC evidenced complete consumption of starting
material (1e18 h). The solvent was removed under reduced pressure
(oil pump) and the residue taken up with CH2Cl2 (40 mL) and
washed with water (3 ꢃ 30 mL), brine (30 mL), then dried and
evaporated under reduced pressure. The residual oil was purified by
flash chromatography, eluting with CH2Cl2/MeOH 9.9/0.1 to 9.5/0.5
to afford the desired intermediate 4bef (57e94%). To a stirred
solution of the resulting intermediate (2.59 mmol) in dry DMF
(23 mL), piperidine (1.15 mL; 11.6 mmol) was added and the reaction
mixture stirred at RT for 1 h. The solvent was evaporated under
reduced pressure and the solid residue was taken up with CH2Cl2
(30 mL) and washed with water (3 ꢃ 30 mL) and brine (30 mL). The
organic phase was dried (Na2SO4) and the crude product was puri-
fied by flash chromatography, eluting with CH2Cl2/MeOH 9.8/0.2 to
8/2 to yield the desired intermediates 5bef (74e100%). To a stirred
COCH2). 13C NMR (CD3OD):
d, 173.1; 172.5; 133.5; 131.2; 118.4; 79.2;
71.2; 54; 39.1; 36.8; 32.6; 27.8. Anal. Calc. for: C12H19N7O8 $ 2 CF3COOH
C% 31.13, H% 3.43, N% 15.,88. Found: C% 31.02, H% 3.61, N% 15.94.
4.2.1.4.
b-alanyl-N-[(2R)-2,3-bis(nitrooxy)propyl]-L-histidinamide
ditrifluoroacetate (7e). The product was recrystallized twice from
dry MeOH/Et2O and freeze-dried to yield 7e as a white foam (63%).
1H NMR (CD3OD):
d, 8.73 (s,1H, ImH2); 7.34 (s,1H, ImH5); 5.52e5.47
(m, 1H, CHONO2); 4.91e4.86 (m, 1H, CHNH); 4.68e4.59 (m, 2H,
CH2ONO2); 3.74e3.68 (m, 2H, NHCH2); 3.31e3.07 (m, 4H, ImCH2,
CH2NHþ3 ); 2.7e2.64 (m, 2H, COCH2). 13C NMR (CD3OD):
d, 173.1;
172.6; 135.2; 131.2; 118.4; 79.2; 71.3; 54; 39.2; 36.9; 32.6; 27.8. Anal.
Calc. for C12H19N7O8 $ 2 CF3COOH C% 31.13, H% 3.43, N% 15.88.
Found: C% 30.91, H% 3.61, N% 15.97.
4.2.1.5.
b-alanyl-N-{4-[3-(nitrooxy)propyloxy]benzyl}-L-histidina-
mide ditrifluoroacetate (7f). The product was recrystallized twice
from dry MeOH/Et2O and freeze-dried to yield 7f as a white foam
(42%). 1H NMR (CD3OD):
d, 8.64 (s,1H, ImH2); 7.23 (s,1H, ImH5); 7.16
solution of the free amines 5bef (1.75 mmol) and Boc-
b
-Ala-OH
(d, 2H, J ¼ 8.4 Hz, ArH2’,6’); 6.87 (d, 2H, J ¼ 8.4 Hz, ArH3’,5’);
4.73e4.65 (m, 3H, CH, CH2ONO2); 4.35e4.23 (m, 2H, NHCH2); 4.06
(t, 2H, J ¼ 6 Hz, OCH2); 3.27e3.05 (m, 4H, ImCH2, CH2NHþ3 );
2.72e2.60 (m, 2H, COCH2); 2.22e2.14 (m, 2H, CH2CH2CH2). 13C NMR
(0.35 g; 1.83 mmol) in dry CH2Cl2 (30 mL) kept at 0 ꢂC, DCC (0.36 g;
1.75 mmol) was added; the ice bath was removed and the reaction
was stirred at RT for 2e18 h. The reaction mixture was cooled to 0 ꢂC.
The precipitate was filtered and washed with cold (0 ꢂC) CH2Cl2. The
liquid phase was washed with water (3 ꢃ 30 mL), brine (30 mL), then
dried and evaporated under reduced pressure to leave a white solid.
The crude material was purified by flash chromatography, eluting
with CH2Cl2/MeOH 9.8/0.2 to 9.5/0.5 to give the desired derivatives
6bef (59e91%) as white foams. The resulting products were dis-
solved in CH2Cl2 (21 mL), treated with CF3COOH (2.1 mL) and stirred
at RT for 20 h. The solvent was evaporated under reduced pressure
(CD3OD): d, 172.3; 172; 159.4; 135.2; 132; 131.6; 130.1; 118.3; 115.6;
71.6; 65.2; 54; 43.7; 36.9; 32.7; 28.6; 28.1. Anal. Calc. for C19H26N6O6
$ 2 CF3COOH $ 2H2O, C% 39.55, H% 4.62, N% 12.03. Found: C% 39.19,
H% 4.27, N% 12.08.
4.2.1.6. 4-propoxybenzenemethanamine (3b). In a flame-dried flask,
NaH 60% in mineral oil (0.80 g; 20.1 mmol) was suspended in dry
DMF (20 mL), then p-hydroxybenzonitrile (8) (2.00 g; 16.8 mmol)

110
M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
was added portionwise and the mixture was stirred for 1 h at RT.
After this time, a solution of bromopropane (4.13 g; 33.6 mmol) in
dry DMF (10 mL) was added and the reaction stirred for a further
3 h. The mixture was diluted with CH2Cl2 (150 mL), washed with
water (3 ꢃ 100 mL), dried (Na2SO4) and evaporated to afford the
desired alkylated intermediate, which was dissolved in dry Et2O
(40 mL) and added dropwise during 30 min to a stirred suspension
of LiAlH4 (0.95 g; 25.1 mmol) in dry Et2O (60 mL). When addition
was complete, the mixture was refluxed for 30 min, then cooled
(0 ꢂC) and cautiously treated with water (10 mL) and CH2Cl2
(80 mL), the precipitate formed was filtered through a sintered
funnel and the resulting mixture was washed with brine
(3 ꢃ 40 mL), dried, and evaporated to afford 3b (2.65 g; 87%) as
a pale yellow oil. The product was used immediately in the next
step without further purification.
colourless oil. 1H NMR (CDCl3):
d
, 7.23 (d, 2H, J ¼ 8.5 Hz, 2 ArH2’,6’);
6.84 (d, 2H, J ¼ 8.5 Hz, 2 ArH3’,5’); 4.66 (t, 2H, J ¼ 6.3 Hz, CH2ONO2);
4.04 (t, 2H, J ¼ 5.8, OCH2); 3.79 (m, 2H, CH2NH2); 2.24e2.15 (m, 4H,
CH2; NH2). 13C NMR (CDCl3):
d, 157.5; 135.2; 128.5; 114.5; 70; 63.6;
45.8; 27. MS CI (isobutane) (m/z): 227 [MHþ].
4.3. Determination of dissociation constants and lipophilicity
descriptor
The ionisation constants of the compounds were determined
by potentiometric titration with the GLpKa apparatus (Sirius
Analytical Instruments Ltd, Forest Row, East Sussex, UK). Ionisa-
tion constants of carnosine
1 and compounds 7aef were
obtained by aqueous titration, with at least four separate titra-
tions for each compound: different aqueous solutions (ionic
strength adjusted to 0.15 M with KCl) of the compounds (20 mL,
about 1 mM) were initially acidified to pH 1.8 with 0.5 N HCl; the
solutions were then titrated with standardized 0.5 N KOH to pH
12.2.
4.2.1.7. (2S) 2,3 (dinitrooxy)propan-1-ammonium nitrate (3d). In
a flame-dried flask equipped with a CaCl2 guard tube and an air
condenser, (R)-3-amino-1,2-propandiol (1.17 g; 12.8 mmol) was
suspended in dry CH2Cl2 (34 mL) and kept at 0 ꢂC. Fuming HNO3
(d ¼ 1.52 g/mL) (3.30 mL) was added cautiously and the mixture
stirred vigorously at 0 ꢂC for 2.5 h. Acetic anhydride was slowly added
and after 1 h the precipitate was filtered through a sintered glass
funnel, collected and dried over P2O5, KOH overnight, to afford 2.65 g
(85%) of an off-white solid. The product wasrecrystallized fromiPrOH
Calculated partition coefficients (clog D7.4) were obtained from
CLOGP (CLOGP for windows, version 4.0, BioByte Corp. Claremont,
CA, USA) according to the equation: clog D7.4 ¼ CLOGP - log
[1 þ 10(pKa ꢁpH) þ 10(pKa Ka e2pH)].
2
2ꢁp
1
The apparent partition coefficients log D7.4 were obtained by the
shake-flask procedure at pH 7.4 (phosphate buffer solutions with
ionic strength adjusted to 0.15 M with KCl); n-octanol was added to
the buffers and the two phases were mutually saturated by shaking
for 4 h. The compounds were solubilised in the buffered aqueous
phase at a concentration of about 0.1 mM and an appropriate
amount of n-octanol was added. The two phases were shaken for
about 20 min, by which time the partitioning equilibrium of solutes
was reached, and then centrifuged (10,000 rpm, 10 min). The
concentration of solute in the aqueous phase was measured by UV
spectrophotometry (UV-2501PC, Shimadzu) at 230 nm. For each
compound, at least 6 log D values were measured.
to obtain a white solid. 1H NMR (DMSO-d6): , 8.15 (s, br 3H, NHþ3 );
d
5.68e5.61 (m,1H, CHONO2); 4.98 (dd, 2H, Jgem ¼ 12.9 Hz; Jvic ¼ 3.3 Hz,
CHHONO2); 4.79 (dd, 2H, Jgem ¼ 12.9; Jvic ¼ 5.4 Hz, CHHONO2); 3.38
(dd, 2H, Jgem ¼ 14.1; Jvic ¼ 3.3 Hz, CHHNH3þ); 3.21 (dd, 2H, Jgem ¼ 14.1;
Jvic ¼ 9.3 Hz, CHHNH3þ).13C NMR (DMSO-d6):
d, 76.4; 70; 37.3, spectral
data are in agreement with those of an authentic sample [56].
4.2.1.8. (2R) 2,3 (dinitrooxy)propan-1-ammonium nitrate (3e). In
a flame-dried flask equipped with a CaCl2 guard tube and an air
condenser, kept at 0 ꢂC, dry CH2Cl2 (34 mL) and fuming HNO3
(d ¼ 1.52) (3.30 mL) were mixed cautiously; to this mixture,
powdered (R)-3-amino-1,2-propandiol (1.2 g; 13.2 mmol) was
added portionwise. The mixture was stirred vigorously at 0 ꢂC for
2 h, then acetic anhydride (3.0 mL; 31.8 mmol) was added and
the reaction stirred at 0 ꢂC for a further 45 min The resulting
precipitate was filtered through a sintered glass funnel, collected
and dried over P2O5, KOH overnight, to afford 2.60 g (81%) of an
off-white solid. The product was recrystallized from iPrOH to
4.4. Copper complexes
Complex formation, between copper(II) and carnosine (1) and
derivatives 7aef, was investigated by means of the classical pH-
metric technique with the GLpKa apparatus. For each compound,
at least three separate titrations were performed: different aqueous
solutions of the compounds and of CuCl2 equimolar (1 mM) were
initially acidified to pH 1.8 with 0.5 N HCl; the solutions were then
titrated with standardized 0.5 N KOH to pH 12.2. The measure-
ments were made under nitrogen at 25.0 ꢀ 0.1 ꢂC and ionic
strength adjusted to 0.15 M (KCl). Elaboration of experimental pH-
metric data, to calculate the values of formation constants, was
done using the BSTAC software [58].
obtain a white solid. 1H NMR (DMSO-d6): , 8.16 (s, br 3H, NHþ3 );
d
5.67e5.62 (m, 1H, CHONO2); 4.98 (dd, 2H, Jgem ¼ 12.9 Hz;
Jvic ¼ 3.3 Hz, CHHONO2); 4.79 (dd, 2H, Jgem ¼ 12.9; Jvic ¼ 5.4 Hz,
CHHONO2); 3.39e3.33 (m, 2H, CHHNH3þ); 3.21 (dd, 2H,
Jgem ¼ 14.1; Jvic ¼ 9.3 Hz, CHHNH3þ). 13C NMR (DMSO-d6):
d, 76.4;
70; 37.3, spectral data are in agreement with those of an
authentic sample [57].
4.5. Stability in human serum
4.2.1.9. 3-[4-(aminomethyl)phenoxy]propyl nitrate (3f). To a stirred
solution of 12 (1.7 g; 5.84 mmol) in dry THF (40 mL) potassium tert-
butoxide was added, and after 15 min 11 (1.37 g; 6.13 mmol) in dry
THF (15 mL) was slowly added. The reaction mixture was stirred at
60 ꢂC for 3 h, cooled and cautiously treated with water (30 mL). The
aqueous layer was extracted with CH2Cl2 (3 ꢃ 30 mL), dried
(MgSO4) and evaporated under reduced pressure to afford the
expected intermediate as a yellow oil. This oil was dissolved in
CH2Cl2 (60 mL) and CF3COOH (4.25 mL) and stirred for 2 h. The
solvent was evaporated and the residue taken up with 5% aqueous
NaHCO3, extracted with CH2Cl2 (3 ꢃ 20 mL) and dried (Na2SO4) to
leave a orange coloured oil which was purified by flash chroma-
tography, eluting with CH2Cl2/MeOH 9.9/0.1 gradient to CH2Cl2/
MeOH 8/2 to afford the title product in quantitative yield as
A solution of each compound (10 mM) in water was added to
human serum (Sigma) preheated to 37 ꢂC; the final concentration
of the compound was 0.5 mM. The solutions were incubated at
37 ꢀ 0.5 ꢂC and, at appropriate time intervals, 500
m
L of reaction
mixture were withdrawn and added to 500
mL of acetonitrile
containing 0.1% trifluoroacetic acid, in order to deproteinize the
serum. The sample was sonicated, vortexed and then centrifuged
for 10 min at 2150 g. The clear supernatant was filtered through
0.45 mm PTFE filters (Alltech, Deerfield, IL, USA) and analysed by
RP-HPLC. HPLC analyses were performed with an HP 1100 chro-
matograph system (Agilent Technologies, Palo Alto, CA, USA)
equipped with a quaternary pump (model G1311A), membrane
degasser (G1379A), diode-array detector (DAD) (model G1315B)

M. Bertinaria et al. / European Journal of Medicinal Chemistry 54 (2012) 103e112
111
integrated in the HP 1100 system. Data analysis was done using an
HP ChemStation system (Agilent Technologies, Palo Alto, CA, USA).
The analytical column was Purospher C18-endcapped
(250 4.6 mm, particle size) (Merck Darmstadt,
Germany). The mobile phase consisting of methanol/20 mM
CH3COONa pH 4.5e5 mM SDS (80/20 to 60/40 in accordance with
the polarity of the compound) and the flow-rate was 0.7 mL/min.
4.7. In vivo experimental protocols
4.7.1. Animals and surgery
a
ꢃ
5
m
m
Male Wistar rats (Harlan-Italy; Udine, Italy) weighing 210e230 g
were housed in a controlled environment at 25 ꢀ 2 ꢂC with alter-
nating 12-h light and darkcycles. They wereprovided with a Piccioni
pellet diet (n.48, Gessate Milanese, Italy) and water ad libitum. All
rats were acclimatised in our animal facility for at least 1 week prior
to experiments, and stressful stimuli were avoided. Animal care was
in compliance with Italian regulations on the protection of animals
used for experimental and other scientific purposes (D.M.116/92) as
well as with the Guide for the Care and Use of Laboratory Animals, as
adopted and promulgated by the U.S. National Institutes of Health.
The experimental protocol, approved by the Turin University Ethics
Committee, was performed as described elsewhere [48]. Briefly, rats
were anaesthetised with i.p. injection (30 mg/kg) of Zoletil 100
(mixture of tiletamine and zolazepam, Laboratoires Virbac, France),
which was supplemented as needed. Anaesthetised rats were
placed onto a thermostatically controlled heating pad, a rectal
temperature probe was inserted and body temperature was moni-
tored and maintained at 37 ꢂC. Both common carotid arteries were
exposed over a midline incision and a dissection was made between
the sternocleidomastoid and the sternohyoid muscles, parallel to
the trachea. Each carotid artery was freed from its adventitial sheath
and vagus nerve, which was carefully separated and maintained.
Ischaemia was achieved by clamping the bilateral common carotid
arteries for 30 min, using non-traumatic artery clamps (Micro
Bulldog Clamps, Harvard Apparatus Ltd., Kent, U.K.). During
ischaemia, the animals were monitored for body temperature,
respiration pattern, loss of righting reflex, unresponsiveness,
corneal reflexes, and fixed and dilated pupils. Recirculation of blood
flow was established by releasing the clips, and restoration of blood
flow in the carotid arteries was confirmed by careful observation.
Reperfusion was allowed for 24 h. Post-surgery, the animals were
kept for at least 3 h in a 37 ꢂC incubator to ensure that postoperative
recovery was satisfactory. Thereafter, they were group-housed
under temperature- and light-controlled conditions, with food
and water ad libitum. At the end of the reperfusion period, the rats
were anaesthetised and killed by decapitation, after aortic exsan-
guination; the forebrain was rapidly dissected at 0 ꢂC. Sham-
operated rats underwent identical surgical procedures, except that
no artery clamps were applied.
The injection volume was 20
mL (Rheodyne, Cotati, CA). The
column effluent was monitored at 210 and 223 nm, referenced
against a 360 nm wavelength.
4.6. Biological activity
4.6.1. LDL isolation and oxidation
Human plasma from healthy donors was provided by the Blood
Bank (A.O. San Giovanni Battista Turin) and fortified with 0.1%
EDTA. The LDL fraction was isolated by ultracentrifugation through
NaCl discontinuous gradients, and collected as the fraction floating
at a density of 1.019e1.063 g/mL. EDTA was removed by rapid
filtration through disposable desalting columns Econo-Pac 10 DG
(Bio-Rad). Filtered LDL aliquots were diluted with PBS (10 mM
phosphate buffer, pH 7.4) to give a final concentration of 50
protein/mL, and transferred to a 1-cm cuvette, either with 50
water alone, or with 50 L of the test compound solution in water at
a final concentration of 100 M. Oxidation was initiated by adding
to the LDL a suspension of CuSO4 at a final concentration of 2.5 M.
mg LDL
mL of
m
m
m
Formation of conjugated dienes was measured spectrophotomet-
rically in a Varian Cary 50 Bio spectrophotometer, equipped with
a thermostatic control (37 ꢂC) and an automatically exchangeable
multi-position cuvette holder, operating at 234 nm. The lag phase
(tlag) and propagation rate (R) were determined as described else-
where [59].
4.6.2. HNE-scavenging studies
HNE (final concentration 50 mM in 1 mM phosphate buffer, pH
7.4) was incubated with a solution of compounds 7aef, or with
carnosine (1) (final concentration 1 mM in 1 mM phosphate buffer,
pH 7.4) for different periods (up to 24 h) at 37 ꢂC. Samples for each
different incubation time were analyzed directly by HPLC to
measure HNE consumption, as described elsewhere [7]. HNE was
determined by reverse-phase HPLC, using an HP 1100 chromato-
graph system (Agilent Technologies, Palo Alto, CA, USA) previously
described. Reaction mixture (20
mL) was eluted on a Agilent Zorbax
Eclipse XDB-C18 column (150 ꢃ 4.6 mm; particle size 5
m
m). The
4.7.2. Drug treatments
mobile phase was 60% A (water/acetonitrile/formic acid; 9:1:0.01,
v/v/v) and 40% B (water/acetonitrile; 1:9, v/v) delivered at a flow-
rate of 1 mL/min. The column effluent was monitored at 223 nm.
Animals were randomly allocated into different groups (n ¼ 5
per group): Sham (the common carotid arteries were exposed but
not occluded); I/R group; carnosine group (the drug was adminis-
tered at 1 g/kg); compound 7e group (the drug was administered at
500 mg/kg) and des-NO analogue 7a group (the drug was admin-
istered at 500 mg/kg). All compounds were prepared in 5% DMSO
and injected i.v., at the beginning of reperfusion and again after 6 h.
For all experiments, concentrations of drugs were calculated such
that all animals received equal volumes of DMSO. The dose of
carnosine used was based on that previously shown to attenuate
injury in animal models of cerebral I/R injury [49].
4.6.3. Vasodilator activity assay
Thoracic aortas were isolated from male Wistar rats weighing
180e200 g. The endothelium was removed and the vessels were cut
helically: three strips were obtained from each aorta. The strips
were mounted in organ baths containing 30 mL of Krebs-
bicarbonate buffer of the following composition (mM): NaCl
111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0, NaHCO3 12, glucose
11.1 maintained at 37 ꢂC and continuously gassed with 95% O2-5%
CO2 (pH ¼ 7.4). The aortic strips were allowed to equilibrate for
4.7.3. Determination of infarct volume
90 min and then contracted with 1
response to the agonist reached
concentrationeresponse curves were determined. The effect of
M ODQ was evaluated in a separate series of experiments, in
mM
L-phenylephrine. When the
After 24 h of reperfusion, the rats were killed with an overdose of
Zoletil 100 (mixture of tiletamine and zolazepam) and decapitated.
The rats’ brains were immediately removed and placed in ice-cold
saline for 5 min. Each brain was then placed in a brain matrix and
coronal sections were cut into 2-mm slices. Brain slices were
immediately immersed in 2% 2,3,5-triphenyltetrazolium chloride
monohydrate (TTC) solution (in saline) at 37 ꢂC for 30 min, followed
by 4% paraformaldehyde solution. The infarct area and hemisphere
a
plateau, cumulative
1
m
which the inhibitor of sGC was added 5 min before contraction.
EC50 values are the mean of at least 5 determinations. Responses
were recorded by an isometric transducer connected to the MacLab
System PowerLab.

112
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