New synthesis and promising neuroprotective role in experimental ischemic stroke of ONO-1714

Article abstract of DOI:10.1016/j.ejmech.2012.05.031

In an experimental permanent stroke model, we report here the contribution of ONO-1714 to brain damage prevention. Daily drug administration, twenty-one days prior to and two days after an experimental infarct, was performed by using mini-osmotic pumps (ALZET). Infarct volumes were assessed by image analysis of sequential coronal brain 1 mm³ sections stained following the 2,3,5-triphenyltetrazolium chloride histological staining technique. Results of this study provide evidence of a significant reduction of the brain lesion size, suggesting ONO-1714 as a potential neuroprotective agent in stroke patients. ONO-1714 was prepared in our laboratory following a procedure which resulted in the supply of the desired compound in an easy and excellent yield.

Full text of DOI:10.1016/j.ejmech.2012.05.031

European Journal of Medicinal Chemistry 54 (2012) 439e446
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New synthesis and promising neuroprotective role in experimental ischemic
stroke of ONO-1714
Andrea Pozo-Rodrigálvarez b, Ana Gradillas a, Julia Serrano b, Ana Patricia Fernández b, Ricardo Martínez-
, ,
,
,1
**
Murillo b 1, Javier Pérez-Castells a
*
a Departamento de Química, Facultad de Farmacia, Universidad San Pablo CEU, Urbanización Montepríncipe, 28668 Boadilla del Monte, Madrid, Spain
b Neurovascular Research Group, Department of Molecular, Cellular and Developmental Neurobiology, Instituto Cajal (CSIC), Av. Doctor Arce 37, 28002 Madrid, Spain
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
< New synthesis of selective iNOS
inhibitor ONO-1714.
< ONO-1714 is potentially effective as
therapeutic intervention in stroke.
< Results suggest ONO-1714 is a neu-
roprotective molecule in the context
of brain ischemia.
a r t i c l e i n f o
a b s t r a c t
Article history:
In an experimental permanent stroke model, we report here the contribution of ONO-1714 to brain
damage prevention. Daily drug administration, twenty-one days prior to and two days after an
experimental infarct, was performed by using mini-osmotic pumps (ALZET). Infarct volumes were
Received 13 February 2012
Received in revised form
16 May 2012
Accepted 22 May 2012
Available online 29 May 2012
assessed by image analysis of sequential coronal brain
1
mm3 sections stained following the
2,3,5-triphenyltetrazolium chloride histological staining technique. Results of this study provide
evidence of a significant reduction of the brain lesion size, suggesting ONO-1714 as a potential
neuroprotective agent in stroke patients. ONO-1714 was prepared in our laboratory following a proce-
dure which resulted in the supply of the desired compound in an easy and excellent yield.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Focal ischemia
Infarct size
iNOS
Neuroprotection
ONO-1714
1. Introduction
pharmacological approaches to protecting the brain have been
considered [3,4]. There are many evidences that inflammation,
Stroke is one of the main causes of death and a major cause of
long-term disability in the western world. More than 80% of all
strokes are caused by cerebral ischemia [1], resulting in devastating
neurological sequelae accompanied by severe morphological and
molecular alterations [2]. Many advances in the understanding of
the mechanisms of ischemic brain injury have been done and many
among other contributing factors, plays an important role in the
outcome of ischemic stroke (IS). It is accepted that inflammatory
mediators, such as nitric oxide (NO), contribute to brain damage [4].
Since stroke is common and current drug therapies for the
management of stroke patients are limited, the progress toward the
identification of new targets and the development of more effective
new drugs has become a challenging task for prevention and early
management of patients [4,5]. Actually, more than 50 neuro-
protective agents have been evaluated in promising Phase III clin-
ical trials with disappointing results. Restoration of blood flow
needs to be achieved as quickly as possible. Yet, only intravenous
administration of the tissue plaminogen activator (tPA), a clot-
* Corresponding author. Tel.: þ34 915854714; fax: þ34 915854754.
** Corresponding author. Tel.: þ34 913724936; fax: þ34 913510496.
E-mail addresses: r.martinez@cajal.csic.es (R. Martínez-Murillo), jpercas@ceu.es
(J. Pérez-Castells).
1
These two authors contributed equally to this work as co-directors and should
be considered last authors.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.031

440
A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
dissolving agent, has been proven to be effective. However, to reap
the full benefits of tPA, there is a very short window of opportunity
for drug administration [4].
protecting group, N-PMB (N-p-methoxybenzil), that they proposed.
All efforts for the deprotection step proved to be unsuccessful, so
that we initiated a similar route shown in Scheme 1, with certain
modifications with regard to Kawanaka’s one, which resulted in the
supply of the desired compound in an easier and better overall yield.
Studies with animals and humans provided increasing knowl-
edge on the mechanisms of cell death following IS, including
excitotoxicity, calcium ion [Ca2þ] overload, free-radicals, inflam-
mation, and apoptosis [4]. In the past decade several studies have
examined the role of the vasodilator mediator NO and its synthe-
sizing enzyme system Nitric Oxide Synthase (NOS) in cerebrovas-
cular diseases, including stroke [6]. NO is an intercellular
messenger present in all vertebrates, modulating blood flow,
thrombosis, and neural activity. Basically, there are three isoforms
of the NOS: neuronal (nNOS), endothelial (eNOS), and inducible
(iNOS), each one involved in specific events in the brain. The
vascular endothelium synthesizes NO through the action of eNOS,
constitutively active, calcium dependent, that generates NO in
response to shear stress and other physiological stimuli, and iNOS,
transcriptionally regulated, calcium independent, which is
produced in response to cytokines and endotoxin signals. Acute
expression of highly reactive mediators, such as iNOS, and matrix
metalloproteinase-9 (MMP9), participates in brain damage after
stroke [6,7]. The reaction of NO with superoxide (O2ꢀ) to form the
much more powerful oxidant peroxynitrite (ONOOꢀ) is a key
element in resolving the contrasting roles of NO in physiology and
pathology [8]. When ischemia has been developed, over-
production of NO generated by the neuronal or inducible iso-
forms (nNOS, iNOS) is neurotoxic, thus contributing to brain injury
and responsible for the progression of brain damage [9]. This
probably occurs through NO-induced formation of peroxynitrite
and toxic free radicals leading to damage by lipid peroxidation [10].
NO over-expression has been also reported to stimulate the release
of the neurotransmitter glutamate, thus contributing to excitotox-
icity [11]. In striking contrast, NO derived from the eNOS isoform is
beneficial because plays a prominent role in preventing neuronal
injury by maintaining cerebral blood flow [12].
Taking together, available data define iNOS as an important
player in IS, and also that pharmacological manipulations of the NO
levels in the area of infarct by regulating iNOS activity might
prevent neuronal injury and neurological deficits [5]. It has been
found that selective iNOS inhibitors significantly reduced infarct
volume, which is in striking contrast with non-selective inhibitors
that were ineffective. ONO-1714, a cyclic amidine derivative and
selective and highly potent inhibitor of rat and human iNOS
(Ki ¼ 1.8 nM) with a 10-fold selectivity over eNOS, has appeared as
a newly developed competitive NOS inhibitor that has selective
potency for iNOS [13], but few data has been reported regarding the
neuroprotective effect of this compound [14].
ONO-1714 was prepared in our laboratory following a procedure
which resulted in the supply of the desired compound in an easy an
excellent overall yield. In vitro experiments carried out in this study
assessed the efficiency of ONO-1714 on the inhibition of NO
production in lipopolysaccharide (LPS) and interferon-gamma
2. Results and discussion
2.1. Chemistry
The new synthesis proposed in this paper required the prepa-
ration of the optically active enamide (ꢀ)-1 which synthesis is
already described in the literature [18,19]. Thus, the cyclo-
propanation of (ꢀ)-1 with CHCl3 under alkaline conditions resulted
in (ꢀ)-anti-2 (45%) (the newly introduced cyclopropane moiety and
the methyl moiety showed anti-stereochemistry) and (ꢀ)-syn-2
(12%) (the newly introduced cyclopropane moiety and the methyl
moiety showed syn-stereochemistry) respectively, as a separable
mixture by column chromatography on silica gel [20].
The mayor isomer (ꢀ)-anti-2 was reduced, to remove one
chlorine, with tin hydride resulting in an inseparable mixture
consisting of an endo-isomer, (ꢀ)-anti-cis-3 and an exo-isomer,
(ꢀ)-anti-trans-3 (2:1) (74%). The mixture obtained was then
transformed into the corresponding thioamido derivatives by
treatment with Lawesson’s reagent. We were pleased to obtain the
corresponding mixture of isomeric thiolactams, which could be
efficiently separated, by column chromatography on silica gel, to
give (ꢀ)-anti-cis-4 (48%) and (ꢀ)-anti-trans-4 (18%) [21]. In addi-
tion, easily removal of the Dmob group from (ꢀ)-anti-cis-4 with TFA
provided (ꢀ)-anti-cis-5 in 70% yield. The deprotected thioamide
was then stirred with a saturated solution of NH3 in methanol to
yield the corresponding amidine 6 which was converted into its
corresponding hydrochloride salt after acidification of the crude
product with HCleCH3OH.
The transformation of the lactam group into the thiolactam
system has revealed a convenient way to improve isomer separa-
tion and deprotection efficiency. The synthesis of amidine 6 by
deprotection of (ꢀ)-3 and afterward transformation of the amide
group into thioamide was discarded as the deprotection of
a mixture of 3 isomers only provided 35% yield of the corre-
sponding amide.
In view of this efficient optimization we have extended this
methodology to the synthesis of several compounds structurally
related to 6 with replacement for the chloro group on the cyclo-
propane ring with different substituents such as CONH2 [18], COOEt
[19], CF3 [18], dichloro, all in racemic and optically active forms.
After being transformed into their corresponding amidine hydro-
chloride salts they were biologically evaluated for their ability to
inhibit the iNOS. Replacement of the chloro group showed always
a marked reduction of iNOS inhibition [18,19].
2.2. Biological results
(INF-
g) stimulated mouse peritoneal macrophages, and on iNOS
activity assay. In addition, the effect of ONO-1714 on the patho-
physiology of stroke was explored in a model of permanent (p)
middle cerebral artery (MCA) occlusion (pMCAO) in mouse. The
pMCAO model is accepted as a pre-clinical experimental ischemic
model for the evaluation of potential neuro-protective agents in
preclinical assays [15,16]. Following this procedure, the results of
this study suggest ONO-1714 as a potential neuroprotective agent
in stroke patients.
The structure of ONO-1714 is shown in Scheme 1 and it was
obtained following a procedure which involved an adaptation of the
procedure described by Kawanaka [17], which was thwarted by our
initial inability to cleavage satisfactoriously the remaining
2.2.1. Biological in vitro results
Endogenous iNOS inhibition was spectrophotometrically
analyzed by using the Griess assay [22]. To confirm the effect of
ONO-1714 on NO production, in vitro experiments were conducted
using a LPS and INF-
model [22]. As shown in Fig. 1A, the treatment with LPS (100 ng/
mL) and INF- (100 ng/mL) markedly increased the production of
g stimulated mouse peritoneal macrophage
g
NO from the basal level following 48 h incubation. When cells were
simultaneously treated with various concentrations of ONO-1714,
100 mM, 10 mM, 1 mM and 0.1 mM, and LPS/INF-g, NO production
was significantly down-regulated in a dose-dependent manner.
Characteristically, progressive reduction of nitrite-containing in

A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
441
H
H
Cl
Cl
Cl
Cl
a
+
Dmob = 2,4-dimethoxybenzyl
N
Dmob
O
N
Dmob
O
N
Dmob
O
H
H
(-)-1
(-)-anti-2
(-)-syn-2
b
H
Cl
N
H
O
Dmob
H
H
H
H
H
H
c
(-)-anti-cis-3 + (-)-anti-trans-3
insaparable mixture (2:1)
+
Cl
Cl
N
S
N
Dmob
S
Dmob
(-)-anti-cis-4
(-)-anti-trans-4
d
H
H
e
Cl
Cl
N
S
N
NH
H
H
H
H2
Cl
(-)-anti-cis-5
6
ONO-1714
Scheme 1. Synthesis of ONO-1714. Reagents: (a) aliquat-336, CHCl3, NaOH, rt. Stereochemistry correlations determined by the NOE correlation are described in Ref. 3; (b) Ph3SnH,
AIBN, benzene, 80 ꢂC, (ꢀ)-anti-trans-3 and (ꢀ)-anti-cis-3 were obtained as an inseparable mixture (1:2); (c) (i) Lawesson’s reagent, benzene, 80 ꢂC, Stereochemistry correlations
determined by the NOE correlation are described in Ref. 4; (ii) chromatographic separation; (d) TFA, 80 ꢂC; (e) (i) MeOH sat. NH3, (ii) HCl, MeOH.
macrophage samples correlated well with ONO-1714 concentra-
tions (0.1, 1, 10, 100 M) (Fig. 1A). The IC50 of the product was
calculated at 0.82 M.
carried out in this work involved the frontal branch of the MCA,
whereas the stem of this artery remained untie. This procedure
yielded a smaller infarct size than that determined by ligature of
the arterial stem, allowing a better assessment of final infarct
volume among control and treated groups and also reducing the
sample size.
m
m
The survival of mouse activated macrophages has been deter-
mined by the colorimetric sulforhodamine B (SRB) assay, a suitable
survival test procedure for the screening of compounds for poten-
tial in therapy because it is a fast and efficient method that is
reproducible and technically advantageous. The SRB assay is an
alternative to the very laborious clonogenic assay that yields
comparable results [23]. It is noteworthy that the different
concentrations of the iNOS inhibitor employed showed prominent
survival rates (means ꢁ S.E.; 98.368 ꢁ 1.52%; 93.588 ꢁ 1.72%;
99.104 ꢁ 0.90%; 97.242 ꢁ 1.61%, respectively) (Fig. 1B). This ruled
out the possibility that ONO-1714 might act as a cytotoxic agent.
The results in this study are consistent with previous research [13].
Finally, IC50 value for inhibition of recombinant murine iNOS
NO generated oxidatively from L-arginine by NOS isoforms,
which are dependent on cofactor binding and dimerization to
become active [24], mediates many physiological actions in the
nervous system, such as neuronal signaling and vasodilation [25].
Under pathological conditions, accumulation of this highly reactive
mediator has conversely damaging effects, leading to neurotoxicity
in neurodegenerative diseases and after stroke [6,25]. In IS studies
of NO functions has generated considerable debate as considered to
be either a damaging or protective molecule, depending on the
enzyme/cell source [26,27]. All the NOS isoforms present in the
brain have influence in stroke regulating brain damage. NO
generated primarily by nNOS and iNOS isoforms promotes
neuronal devastation after ischemia. Particularly, uncontrolled up-
regulation of iNOS is responsible for extensive brain damage [11]. In
striking contrast, NO produced by the eNOS isoform in endothelial
cells exert a neuroprotective role [28]. Therefore, the design of
selective pharmacological regulators of the activity of NOS
isozymes is of considerable interest [5,9]. In the field of regulation
of NOS activity, it should be aware that NO is an important mediator
of the brain physiology [9,29], therefore widespread and excessive
down-regulation of NO by non-specific NOS inhibitors, such as
was examined using the
[
14C]-citrulline assay in which the
-[14C]citrulline was measured.
conversion of
L
-[14C]arginine to
L
ONO-1714 potently inhibited iNOS with IC50 value (0.011
accordance with data reported previously [17].
mM) in
2.2.2. Biological in vivo results
Animals were subjected to focal ischemia by pMCAO. After 48 h,
the mice were euthanized, the brain removed, cut in coronal slices
and stained with TTC to recognize and determine infarct volume.
The infarct volume was larger in vehicle-treated mice compared
with chronically ONO-1714 treated subjects (Fig. 2). The size of the
affected brain area was 4.17 mm3 þ 0.2 mm3 in untreated animals
whereas it was faint, under the limit of detection by the TTC
procedure, for the treated group (p < 0.05). The model of pMCAO
substrate analogs of L-arginine, may be detrimental. In fact, inhi-
bition of eNOS worsens injury in experimental stroke, but the use of
the selective nNOS inhibitor 7-nitroindazole significantly decreases

442
A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
Fig. 2. Chronic treatment with ONO-1714 reduces the infarct volume after pMCAO,
frontal branch, in mice. Mice were subjected to 48 h of pMCAO, and volumes of both
the whole brain and the infarcted region were measured from TTC-stained serial
coronal sections. Representative stacks of seven TTC-stained sections are shown for
each group: vehicle treated (A) and ONO-1714 treated (B) mice. No differences
between brain volumes were detected between both groups. However, mice treated
with vehicle (A) reveal a larger unstained area of ischemic tissue in the infarcted
neocortex when compared to their ONO-1714 treated littermates that lack detectable
cortical infarct (B). Data are mean ꢁ SEM, n ¼ 3; (*; p < 0.05).
Fig. 1. A ONO-1714 inhibits mouse iNOS activity in culture medium: Spectrophotom-
eter assay showing the percentage of nitrite release from activated mouse macro-
phages in comparison with non-treated cells (control). Macrophages were incubated,
as described in the material and methods section, in the presence of increasing
concentrations of ONO-1714. Griess assay shows progressive reduction of nitrite-
containing in macrophage samples which correlated well with ONO-1714 concentra-
animal groups. All control animals subjected to pMCAO showed
infarct. In striking contrast, ONO-1714 treated mice consistently
showed no infarct or under the limit of detection using the TTC
procedure, suggesting that ONO-1714 is a neuroprotective molecule
in the context of brain ischemia. It is well known that animals that
underwent focal ischemia induced by pMCAO show significant
changes in iNOS expression [7]. Following this stroke model, we
have recently found no statistically significant changes among
genotypes for nNOS and eNOS isoforms, but a very significant
increase for iNOS when compared with control animals [7]. Now,
we are showing that chronic administration of the iNOS inhibitor
ONO-1714 results in an improvement of the symptoms associated
with focal ischemia, particularly a significant reduction of infarct
volume. Data concerning the effect of ONO-1714 in stroke in this
report are consistent with previous assays that iNOS inhibitors are
candidate treatments for acute IS [5,35].
Acute expression of highly reactive mediators, such as iNOS and
MMP9, participates in brain damage after stroke [6]. Uncontrolled
astrocytic production of NO by iNOS has been found to up-regulate
MMP9 [36], proposed as a marker of brain ischemia [37] and
predictor of poor outcome and mortality in stroke [38]. Also, it has
been proved that chemical or genetic inhibition of MMP9 protects
the brain from ischemic injury [38,39]. Therefore, a mechanism of
action for ONO-1714 in this scenario might be through down-
regulation of MMP9. On the other hand, it has been reported
a neuroprotective effect of ONO-1714 on cytotoxicity induced by N-
tions (0.1, 1, 10, 100 mM). IC50 is calculated at 0.82 mM. Below 0.1 mM of the inhibitor
concentration, nitrite release was unaffected. Data are expressed as percentages of the
control. Values represent mean ꢁ SEM, n ¼ 4. Asterisks show significant differences as
compared to control, p < 0.05 was considered as statistically significant as compared to
control. B ONO-1714 does not affect macrophage cell survival: Spectrophotometer data
obtained with the SRB assay quantitation, showing dose response curves of activated
macrophages treated with different ONO-1714 concentrations: 0.1, 1, 10 and 100 mM.
Experiments were performed at least four times, and the results are expressed as
mean ꢁ standard.
the infarct volume [30]. When assessed by type of inhibitor, total
lesion volume was reduced in permanent models by nNOS and
iNOS inhibitors, but not by nonselective inhibitors [5,17]. Inhibition
of iNOS, for example, has been largely proposed as a neuro-
protective strategy in stroke [31e33]. Therefore, preclinical studies
are concerned to a specific NOS isozyme regulation, means of
increase NO availability to the vasculature or to restore eNOS
activity during IS. Therefore, the discovery and preclinical trial of
new nNOS and iNOS inhibitors exhibiting higher affinity against
one or both isoforms should be promoted. Herein, we report the
preparation of a potent and selective iNOS inhibitor, ONO-1714 [13],
that also exert a potent inhibitory action on nNOS [14,34]. In vitro
results in this study depict ONO-1714 as a potent iNOS inhibitor
with excellent pharmacokinetics which is in good agreement with
previous studies that might potentially be effective as a novel and
potent therapeutic intervention in stroke [13,14].
methyl-
D-aspartate (NMDA) receptor activation, through inhibition
of the nNOS isoform [17]. Following acute ischemic or hypoxic
injury, release of glutamate into the extracellular space triggers the
opening of cation-permeable channels at NMDA receptors
contributing to excytotoxic cell death. By regulating NO levels,
ONO-1714 might exert neuroprotection through these mechanisms,
but further studies are required to confirm this.
We proceed to analyze the in vivo effect of this molecule in
a permanent occlusion model of the frontal branch of the MCA. This
procedure produced a very limited cortical infarct, which made
easy to statistically compare ischemic lesion volumes between

A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
443
3. Experimental protocols
azobisisobutylonitrile (0.04 g, 0.26 mmol). The reaction mixture
was stirred with heating at 80 ꢂC for 4 h under an argon atmosphere.
After cooling in an ice bath, the reaction mixture was diluted with
EtOAc and then washed with 10% aqueous potassium fluoride. The
resulting insoluble substances were removed by filtration. The
organic layer was washed with brine (10 mL), dried over magne-
sium sulfate and the solvents were evaporated under reduced
pressure. The residue was purified by flash column chromatography
on silica gel (hexane/EtOAc 5:1e2:1) to afford (ꢀ)-anti-cis-3 and
(ꢀ)-anti-trans-3 as an inseparable mixture (2:1), (74%). 1H NMR
3.1. Chemistry
Melting points (uncorrected) were determined on a Stuart
Scientific SMP3 apparatus. Infrared (IR) spectra were recorded with
a PerkineElmer 1330 infrared spectrophotometer. 1H and 13C NMR
values were recorded on a Bruker 300-AC instrument. Chemical
shifts (d) are expressed in parts per million relative to internal
tetramethylsilane; coupling constants (J) are in hertz. Mass spectra
were run on a HP 5989A spectrometer. Elemental analyses (C, H, N)
were performed on a PerkineElmer 2400 CHN apparatus at the
Microanalyses Service of the University Complutense of Madrid; all
the values are within ꢁ0.4% of the theoretical compositions. Thin-
layer chromatography (TLC) was run on Merck silica gel 60 F-254
plates. Unless stated otherwise, starting materials used were high-
grade commercial products.
(CDCl3):
d. Data for major isomer assigned as (ꢀ)-anti-cis-3
following assignment of compounds 4: 1.20 (d, 3H, CH3, J ¼ 6.3 Hz),
2.19e2.22 (m, 3H, H4 H6), 2.25e2.30 (m, 1H, H5), 2.75e2.81 (m, 1H,
H7), 3.24 (dd, 1H, H1, J1 ¼ 5.4 Hz, J2 ¼ 7.8 Hz), 3.80 (s, 3H, OCH3), 3.82
(s, 3H, OCH3), 4.00 (d, 1H, CH2, J ¼ 14.6 Hz), 5.20 (d, 1H, CH2,
J ¼ 14.6 Hz), 6.44e6.47 (m, 2H, ArH), 7.22e7.25 (m, 1H, ArH). Data
for minor isomer assigned as (ꢀ)-anti-trans-3 following assignment
of compounds 4:1.20 (d, 3H, CH3, J ¼ 6.3 Hz), 1.74e1.78 (m, 1H, H6),
2.12 (d, 1H, H4, J ¼ 11.1 Hz), 2.28e2.30 (m, 1H, H5), 2.32 (d, 1H, H4’,
J ¼ 11.1 Hz), 2.59e2.61 (m,1H, H7), 2.78e2.81 (m,1H, H1), 3.82 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 4.56 (d, 1H, CH2, J ¼ 14.2 Hz), 4.70 (d, 1H,
CH2, J ¼ 14.2 Hz), 6.44e6.47 (m, 2H, ArH), 7.27e7.28 (m, 1H, ArH).
3.1.1. (1S,5S,6R)-7,7-Dichloro-2-(2,4-dimethoxybenzyl)-5-methyl-
2-azabicyclo[4.1.0]heptan-3-one (ꢀ)-anti-2 and (1S,5S,6R)-7,7-
dichloro-2-(2,4-dimethoxybenzyl)-5-methyl-2-azabicyclo[4.1.0]
heptan-3-one (ꢀ)-syn-2
To a stirred solution of the enamide (ꢀ)-1 (0.48 g, 1.84 mmol) in
CHCl3 (4.42 mL) were added aliquat-336 (0.05 mL) and 50%
aqueous sodium hydroxide (0.97 g) under an argon atmosphere.
The reaction mixture was stirred for 22 h at room temperature.
After completing the reaction, the mixture was treated with satu-
rated aqueous ammonium chloride and extracted with Et2O. The
organic layer was washed with brine, dried over magnesium sulfate
and evaporated under reduced pressure. The residue was purified
by flash column chromatography on silica gel (hexane/EtOAc
4:1e2:1) to afford 0.28 g, (45%) of (ꢀ)-anti-2 as a pale yellow solid
and 0.09 g (15%) of (ꢀ)-syn-2 as a pale yellow oil.
13C NMR (CDCl3)
d: Mixture: 21.4 (CH3), 21.7 (CH3), 22.6, 24.6, 29.4,
31.1, 34.4, 38.8, 39.2, 39.6, 40.0, 40.2, 42.4 (CH2), 42.8 (CH2), 55.3 (4
OCH3), 98.1 (ArCH), 98.2 (ArCH), 103.9 (ArCH), 104.0 (ArCH), 117.1
(ArCipso), 117.2 (ArCipso), 130.9 (ArCH), 131.5 (ArCH), 158.6 (2
ArCeOCH3), 160.2 (2 ArCeOCH3), 170.7 (C ¼ 0), 172.0 (C ¼ 0).
3.1.3. (1S,5S,6R,7S)-7-Chloro-2-(2,4-dimethoxybenzyl)-5-methyl-
2-azabicyclo[4.1.0]-heptan-3-thione (ꢀ)-anti-cis-4 and
(1S,5S,6R,7S)-7-chloro-2-(2,4-dimethoxybenzyl)-5-methyl-2-
azabicyclo[4.1.0]-heptan-3-thione (ꢀ)-anti-trans-4
To a stirred solution of the lactams mixture of (ꢀ)-anti-cis-3 and
(ꢀ)-anti-trans-3 (0.38 g, 1.27 mmol) in benzene (10 mL) was added
Lawesson’s reagent (0.80 mmol). The reaction mixture was stirred
at 80 ꢂC for 2 h. The solvent was evaporated under reduced pres-
sure. The crude residue was separated by flash column chroma-
tography on silica gel (hexane/EtOAc 4:1) to afford 0.20 g (48%) of
(ꢀ)-anti-cis-4 as a white solid and 0.07 g (18%) of (ꢀ)-anti-trans-4 as
a pale yellow oil
(ꢀ)-anti-2: FW ¼ 344.232. Mp ¼ 89e91 ꢂC. IR (KBr) 1665 (C]O)
cmꢀ1. 1H NMR (CDCl3)
d
1.24 (d, 3H, CH3, J ¼ 6.3 Hz),1.74 (dd,1H, H6,
J1 ¼ 9.8 Hz, J2 ¼ 5.4 Hz), 2.11e2.20 (d, 1H, CH2, J1 ¼ 14.2 Hz),
2.07e2.12 (m, 1H, CH), 2.32 (dd, 1H, CH2, J1 ¼ 13.7 Hz, J2 ¼ 2.9 Hz),
3.08 (d, 1H, H1, J ¼ 9.8 Hz), 3.82 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
4.17 (d,1H, CH2, J ¼ 14.2 Hz), 5.14 (d,1H, CH2, J ¼ 13.7 Hz), 6.42e6.45
(m, 2H, ArH), 7.25 (d, 1H, ArH, J ¼ 9.1 Hz). 13C NMR (CDCl3)
d 21.8
(CH3), 27.7 (C5), 35.3 (C4), 39.2 (CH2), 42.5 (C6), 44.3 (C1), 55.3
(OCH3), 55.4 (OCH3), 64, 52 (C7), 98.1 (ArCH), 104.1 (ArCH), 116.6
(ArCipso), 132.2 (ArCH), 158.8 (ArCeOCH3), 160.6 (ArCeOCH3), 170.6
(C]O). MS (ESI) m/z (rel%): 367.13 (4), 366.17 [M þ Na]þ (37), 151
(100), 121.28 (16). Anal. calcd for C16H19Cl2NO3: C, 55.83; H, 5.56; N,
4.07. Found: C, 55.99; H, 5.43, N, 3.96.
(ꢀ)-anti-cis-4: FW ¼ 325.854. Mp ¼ 126e129 ꢂC. IR (KBr) 1605
(C]S) cmꢀ1
.
1H NMR (CDCl3)
d
1.15 (d, 3H, CH3, J ¼ 6.7 Hz),
1.35e1.43 (m, 1H, H6), 2.08e2.13 (m, 1H, CH), 2.53 (t, 1H, CH2,
J1 ¼ 12.6 Hz), 2.84 (m, 1H, H7), 3.12 (dd, 1H, CH2, J1 ¼ 15.2 Hz,
J2 ¼ 3.6 Hz), 3.22 (dd, 1H, H1, J1 ¼ 7.9 Hz, J2 ¼ 5.5 Hz), 3.78 (s, 3H,
OCH3); 3.79 (s, 3H, OCH3), 4.34 (d, 1H, CH2, J ¼ 14.6 Hz), 6.02 (d, 1H,
CH2, J ¼ 14.6 Hz), 6.42e6.44 (m, 2H, ArH), 7.31 (d, 1H, ArH,
(ꢀ)-syn-2: FW ¼ 344.232. IR (KBr) 1668 (C]O) cmꢀ1. 1H NMR
(CDCl3)
d
1.18 (d, 3H, CH3, J ¼ 6.6 Hz), 1.92 (dd, 1H, H6, J1 ¼ 10.4 Hz,
J ¼ 9.1 Hz). 13C NMR (CDCl3)
d 14.0 (CH3), 21.2 (C7), 22.6 (C6), 24.0
J2 ¼ 5.5 Hz), 2.15e2.23 (m, 1H, CH2), 2.36e2.42 (m, 1H, CH),
2.45e2.50 (m, 1H, CH2), 3.38 (d, 1H, H1, J ¼ 10.4 Hz), 3.79 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 4.62 (d, 1H, CH2, J ¼ 14.3 Hz), 4.77 (d, 1H,
CH2, J ¼ 14.3 Hz), 6.40e6.45 (m, 2H, ArH), 7.29 (d,1H, ArH, J ¼ 8.8 Hz).
(C1), 31.5 (C5), 36.4 (C4), 50.1 (CH2), 55.3 (OCH3), 55.4 (OCH3), 98.3
(ArCH), 104.0 (ArCH), 115.4 (ArCipso), 131.1 (ArCH), 158.7
(ArCeOCH3), 160.6 (ArCeOCH3), 203.5 (C]S). Anal. calcd for
C16H20ClNO2S: C, 58.97; H, 6.19; N, 4.30; S, 9.84. Found: C, 58.70; H,
6.02, N, 4.37; S. 9.93.
13C NMR (CDCl3)
d 21.3 (CH3), 22.6 (C5), 36.2 (C4), 40.5 (CH2), 41.6
(C6), 45.1 (C1), 55.2 (OCH3), 55.3 (OCH3), 63.8 (C7), 98.3 (ArCH),104.1
(ArCH), 116.2 (ArCipso), 130.8 (ArCH), 158.4 (ArCeOCH3), 160.0
(ArCeOCH3), 170.1 (C]O). MS (ESI) m/z (rel%): 346.10 (34), 344.21
[M þ H]þ (54),151.10 (100),121.27 (10). Anal. calcd for C16H19Cl2NO3:
C, 55.83; H, 5.56; N, 4.07. Found: C, 54.97; H, 5.65, N, 4.13.
(ꢀ)-anti-trans-4: FW ¼ 325.854. IR (KBr) 1605 (C]S) cmꢀ1. 1H
NMR (CDCl3)
d
1.16 (d, 3H, CH3, J ¼ 6.7 Hz), 1.48e1.51 (m, 1H, H6),
1.71e1.77 (m, 1H, CH), 2.49 (dd, 1H, CH2, J1 ¼ 15.2 Hz, J2 ¼ 10.9 Hz),
2.63e2.65 (m, 1H, H7), 2.84 (dd, 1H, H1, J1 ¼ 9.1 Hz, J2 ¼ 1.8 Hz), 2.98
(dd,1H, CH2, J1 ¼12.2 Hz, J2 ¼ 3.6 Hz), 3.79 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 5.14 (d, 1H, CH2, J ¼ 14.0 Hz), 5.40 (d, 1H, CH2, J ¼ 14.0 Hz),
6.42e6.44 (m, 2H, ArH), 7.33 (d, 1H, ArH, J ¼ 7.9 Hz). 13C NMR
3.1.2. (1S,5S,6R,7S)-7-Chloro-2-(2,4-dimethoxybenzyl)-5-methyl-
2-azabicyclo[4.1.0]-heptan-3-one (ꢀ)-anti-cis-3 and (1S,5S,6R,7S)-
7-chloro-2-(2,4-dimethoxybenzyl)-5-methyl-2-azabicyclo[4.1.0]-
heptan-3-one (ꢀ)-anti-trans-3
(CDCl3)
d 20.8 (CH3), 30.4 (C7), 30.7 (C6), 38.5 (C5), 38.6 (C1), 41.8
(C4), 49.8 (CH2), 55.4 (OCH3), 55.5 (OCH3), 98.3 (ArCH), 104.3
(ArCH), 115.8 (ArCipso), 132.1 (ArCH), 158.8 (ArCeOCH3), 160.9
(ArCeOCH3), 201.2 (C]S). Anal. calcd for C16H20ClNO2S: C, 58.97; H,
6.19; N, 4.30; S, 9.84. Found: C, 58.90; H, 6.23, N, 4.42; S. 9.78.
To a stirred mixture of (ꢀ)-anti-2 (1.00 g, 5.20 mmol) in benzene
(6.60 mL) were added Ph3SnH (2.0 g, 5.7 mmol) and

444
A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
3.1.4. (1S,5S,6R,7R)-7-Chloro-5-methyl-2-azabicyclo[4.1.0]heptan-
3-thione (ꢀ)-anti-cis-5
3.2.2. Cell culture
Resident peritoneal macrophages were recovered from CD-1
mice by instilling and withdrawing 9 mL sterile phosphate-
buffered saline (PBS) (pH 7.4) solution, containing 0.2% heparin,
from the peritoneal cavity. Collected cells were washed, resus-
pended in culture medium, and seeded (1 ꢄ106 cells/well) in tissue
culture plate (Falcon, 24 well), and cultured in Dulbecco’s modified
Eagle medium (DMEM) supplemented with inactivated 10% FBS,
2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin
in a humidified atmosphere with 5% CO2 at 37 ꢂC, for 48 h.
A solution of (ꢀ)-anti-cis-4 (0.12 g, 0.33 mmol) in trifluoroacetic
acid (0.60 mmol) was refluxed for 16 h. The solvent was evaporated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc 4:1) to afford 0.04 g
(70%) of (ꢀ)-anti-cis-5 as a white solid. FW ¼ 175.680. IR (KBr) 1655
(C]S) cmꢀ1
.
1H NMR (CDCl3)
d
1.21 (d, 3H, CH3, J ¼ 6.7 Hz),
1.38e1.44 (m, 1H, H6), 1.95e2.00 (m, 1H, CH), 2.35 (dd, 1H, CH2,
J1 ¼15.6 Hz, J2 ¼ 12.2 Hz), 2.92e2.96 (m, 1H, H7), 2.97e3.01 (m, 1H,
CH2), 2.95 (dd, 1H, H1, J1 ¼ 15.9 Hz, J2 ¼ 3.8 Hz), 8.43 (s(a), 1H). 13
C
NMR (CDCl3)
d
21.2 (CH3), 21.4 (C5), 26.7 (C7), 32.5 (C6), 39.0 (C1),
3.2.3. Nitrite assay. NO production and quantification
47.1 (C4),199.7 (C]S). MS (ESI) m/z (rel%): 312.12 (72), 270.07 (100),
175.98. [M þ H]þ (6), 151.04 (17), 102.19 (15). Anal. calcd for
C7H10ClNS: C, 47.86; H, 5.74; N, 7.97; S, 18.25. Found: C, 47.73; H,
5.62, N, 8.05; S. 18.34.
The inhibitory activity of the test compounds on LPS plus INF-g
induced NO production was evaluated. After 48 h in culture, cell
supernatants were replaced with fresh medium containing LPS
(100 ng/mL) plus IFN-
test material: medium only (negative control) or IFN-
g
(100 ng/mL) in the presence or absence of
and LPS
g
3.1.5. (1S,5S,6R,7R)-7-Chloro-5-methyl-2-azabicyclo[4.1.0]-heptan-
3-imine hydrochloride salt 6
(positive control). After additional 48 h incubation, nitrite released
into the supernatants of mouse macrophages was determined by
A solution of (ꢀ)-anti-cis-5 (0.03 g, 0.15 mmol) in saturated
methanolic ammonia (1.00 mL) was stirred at room temperature
under an argon atmosphere for 46 h. Concentration of the reaction
mixture under reduced pressure gave a residue which was diluted
with acetone (1 mL) with formation of a precipitate which was
removed by filtration. The solid was converted intoits hydrochloride
salt by solving in CH3OH and acidification to pH 3.5 with anhydrous
HCleCH3OH (0.50 mL, prepared from 0.30 mL of AcCl in 8.00 mL of
CH3OH). After concentration under reduced pressure the residue
was disgregated in acetone and filtered to afford the corresponding
hydrochloride salt which was disgregated with acetone to afford
the standard Griess reaction by adding 50
well flat-bottomed plates containing 50
sulfanilamide/0.1%
m
m
L of test solution to 96-
L of Griess reagent [1%
N-(1-naphthyl)ethylenediamine dihydro-
chloride/2.5% H3PO4]. The samples were assayed in quadruplicate.
After 15 min at room temperature, the absorbance of each well was
measured in a FLUOstar OPTIMA-BMG LABTECH microplate reader
at 540 nm and the nitrite concentration was determined from
a standard curve of sodium nitrite. The percentage inhibition was
expressed as: (nitrite level of test samples/nitrite level of vehicle-
treated control) ꢄ 100. All experiments were repeated at least
two times.
0.02 g (45%) of 6 as a white solid. ½a D25
¼ þ62.12 (c 0.03, MeOH), (lit.
ꢃ
½
a 2D5
ꢃ
þ68.10 (c 1.00, MeOH)). FW ¼ 195.089. Mp ¼ 234e235 ꢂC. IR
3.2.4. Determination of cell viability
(KBr) 1690 cmꢀ1. 1H NMR (DMSO-d6)
d
1.18 (d, 3H, CH3, J ¼ 6.7 Hz),
In parallel with the Griess assay, macrophages from each well
were subjected to a cell viability assay using the SRB test. This
method measures the cellular protein content of adherent or
suspension cultures, according to a previous report [40]. The SRB is
a pink aminoxanteno dye with two sulfonic groups. Sulfonic groups
come together, electrostatically with the basic amino acid residues
of the protein in mildly acidic conditions. This technique of staining
allowed us to study the inhibition of cell growth. The SRB bind to
cellular proteins was measured spectrophotometrically at 540 nm,
which provides information on the relative cell growth and
1.43e1.50 (m, 1H, H6); 1.78e1.86 (m, 1H, CH), 2.44 (d, 2H, CH2,
J ¼ 10.9 Hz), 3.05 (dd,1H, H7 J1 ¼8.5 Hz, J2 ¼ 5.5 Hz), 3.65 (dd,1H, H1,
J1 ¼7.3 Hz, J2 ¼ 5.5 Hz), 8.50 (s(a),1H), 9.20 (s(a),1H), 9.70 (s(a),1H).
13C NMR (DMSO-d6)
d 21.2 (CH3), 22.7 (C5), 25.2 (C7), 29.3 (C6), 32.7
(C1), 51.7 (C4), 169.17 (C]NH). MS (ESI) m/z (rel%): 225.21 (13),
161.09 (25), 159.09 [M þ H]þ (100). Anal. calcd for C7H12Cl2N2: C,
43.10; H, 6.20; N, 14.36. Found: C, 43.21; H, 6.36, N, 14.43.
3.2. Biological assays
viability of the cells. Cells were fixed for 1 h at 4 ꢂC by adding 200
mL
DMEM, fetal bovine serum (FBS), penicillin, and streptomycin
were obtained from Gibco BRL (Grand Island, NY, U.S.A.). Lipo-
polysaccharides from Escherichia coli 026:B6 (LPS, Sigma, L8274),
per well of trichloroacetic acid (TCA) to 12.5% (v/v) in double
distilled water. Then, the fixative solution was removed vigorously
and the samples thoroughly washed with tap water. Plates were
then dried with cold air. Once fixed, the cells were stained with 0.4%
interferon-gamma from rat (INF-g), sulforhodamine-B (SRB), Griess
reagent, and 2,3,5-triphenyltetrazolium chloride (TTC) were
purchased form Sigma (St. Louis, MO U.S.A.). Alzet mini-osmotic
pumps (Alzet, model 2004). Isoflurane (IsobarÒVet, Schering-
Plough, Middlesex, UK). Recombinant murine iNOS was
purchased from Cayman Chemical (Cat. No. 60864). Stock solutions
of ONO-1714 were prepared in Dimethyl sulfoxide (DMSO, Appli-
SRB (w/v) in 1% acetic acid (putting 100 mL per well) for 30 min
stirring at room temperature. After this time, the SRB was removed
and the cultures were washed with 1% acetic acid to remove
residual dye. After optimum drying, dye bound to proteins was
solubilized with 10 mM Trizma base (putting 200 mL per well) for
30 min with stirring at room temperature. Then procedure to read
the optical densities was performed by using a plate reader at
540 nm wavelength.
Chem). L
-[14C]arginine (Amersham), Heparin (Chiesi, 962357.9).
Griess’ reagent for nitrite (Fluka, 03553).
3.2.1. Animals
3.2.5. Enzyme assay with recombinant murine iNOS
Hsd:ICR (CD-1) mouse (n ¼ 24) derived from animals from
Charles River Laboratories, Wilmington, Massachusetts, and C57BL/
6JOlaHsd male mice (n ¼ 6) of 12 weeks, were purchased from
The concentration of the inhibitor producing 50% inhibition
(IC50) was obtained by measuring per cent inhibition at least seven
concentrations of inhibitor. The assay media contained 20
-arginine. The IC50 value is the means of three individual experi-
ments. The iNOS enzyme activity was measured by [14C]-citrulline
assay, by monitoring the conversion of [14C]- -arginine to [14C]-
L-
m
M [14C]-
Harlan Laboratories. Animals were kept in
a
temperature-
L
controlled room with a light/dark cycle 12:12, having access to
food and water ad libitum. All procedures comply with the rules of
the European Union Directive (86/609/EEC) and the protocol
approved by the Animal Welfare Committee of the Cajal Institute.
L
citrulline. The details of the procedure have already been described
[41]. The radioactivity was then measured in a liquid scintillation

A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
445
counter (LS 6500, M-P Scintillation Counter, Beckman) and
Acknowledgments
expressed as counts per minute (cpm) per mg of protein per minute
(data not shown).
This study was supported by grants from the Spanish Ministerio
de Educación y Ciencia (MEC) (Project No. CTQ2009-07738/BQU),
the Spanish Ministry for Science and Innovation, (Project No.
SAF2010-15173) and LACER SA. We are very grateful for the excel-
lent technical assistance of Ms. Soledad Martínez Montero.
3.2.6. Alzet osmotic pumps. Permanent focal ischemia model
Chronic administration of the compound was performed
subcutaneously by using Alzet mini-osmotic pumps (model 2004,
200
mL, 0.25
mL/h) placed subcutaneously. The daily pumping rate
was 24
m
g/kg of ONO-1714 dissolved in dimethyl sulfoxide (DMSO).
References
For the ischemic control group, pumps were filled with the vehicle.
The treatment was dispensed for 21 days before and 48 h after
pMCAO. Mice (3 per group) were anesthetized with 3% isoflurane
(in 70% N2O, 30% O2) for induction and with 2% isoflurane for
maintenance. Rectal temperature was maintained at 36.5 ꢂC with
use of a heating pad. The frontal branch of the MCA was exposed
and occluded permanently by suture ligation as previously repor-
ted with modifications [16]. Briefly, an incision perpendicular to the
line connecting the lateral canthus of the left eye and the external
auditory canal was made to expose and retract the temporalis
muscle. A burr hole was drilled, and frontal and parietal branches of
the MCA were exposed by cutting and retracting the dura. The
frontal branch of the MCA was elevated and ligated with a suture
nylon monofilament 8/0. Following ligation, a sharp decrease of
blood flow was evinced by a laser Doppler flowmetry (Järfalla,
Sweden). We selected exclusively for this study animals that
showed post-ligature a drop of blood flow of at least 65% and
exclude animals which have undergone surgery for longer than
15 min. Following surgery, subjects were returned to their cages,
kept at room temperature and allowed free access to food and
water. Physiological parameters: rectal temperature, mean arterial
pressure and blood glucose levels, were not significantly different
between studied groups. Rectal temperature was controlled at
ꢁ37.0 ꢂC throughout experiment by using a temperature-regulated
heating pad. Measurements of hemodynamics parameters, taken
15 min after the onset of pMCAO, were: mean blood pressure
84.4 ꢁ 3.2 and blood glucose level 132.4 ꢁ 2.3 mg/dl. The experi-
ments compared an untreated ischemic control group (n ¼ 3) with
an ischemic group treated with ONO-1714 (n ¼ 3).
[1] R.W. Flynn, R.S. MacWalter, A.S. Doney, The cost of cerebral ischaemia,
Neuropharmacology 55 (2008) 250e256.
[2] A. Kunz, C. Iadecola, Cerebral vascular dysregulation in the ischemic brain,
Handb. Clin. Neurol. 92 (2008) 283e305.
[3] M.D. Ginsberg, Neuroprotection for ischemic stroke: past, present and future,
Neuropharmacology 55 (2008) 363e389.
[4] R.S. Pandya, L. Mao, H. Zhou, S. Zhou, J. Zeng, A.J. Popp, X. Wang, Central
nervous system agents for ischemic stroke: neuroprotection mechanisms,
Cent. Nerv. Syst. Agents Med. Chem. 11 (2011) 81e97.
[5] M. Willmot, C. Gibson, L. Gray, S. Murphy, P. Bath, Nitric oxide synthase
inhibitors in experimental ischemic stroke and their effects on infarct size and
cerebral blood flow: a systematic review, Free Radic. Biol. Med. 39 (2005)
412e425.
[6] Z.G. Del, I. Ginis, J.M. Hallenbeck, C. Iadecola, X. Wang, G.Z. Feuerstein,
Inflammation and stroke: putative role for cytokines, adhesion molecules and
iNOS in brain response to ischemia, Brain Pathol. 10 (2000) 95e112.
[7] O. Hurtado, J. Serrano, M. Sobrado, A.P. Fernandez, I. Lizasoain, R. Martinez-
Murillo, M.A. Moro, A. Martinez, Lack of adrenomedullin, but not complement
factor H, results in larger infarct size and more extensive brain damage in
a focal ischemia model, Neuroscience 171 (2010) 885e892.
[8] P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and
disease, Physiol. Rev. 87 (2007) 315e424.
[9] J.R. Steinert, T. Chernova, I.D. Forsythe, Nitric oxide signaling in brain function,
dysfunction, and dementia, Neuroscientist 16 (2010) 435e452.
[10] A. Aggarwal, V. Gaur, A. Kumar, Nitric oxide mechanism in the protective
effect of naringin against post-stroke depression (PSD) in mice, Life Sci. 86
(2010) 928e935.
[11] R.A. Swanson, W. Ying, T.M. Kauppinen, Astrocyte influences on ischemic
neuronal death, Curr. Mol. Med. 4 (2004) 193e205.
[12] M. Endres, U. Laufs, J.K. Liao, M.A. Moskowitz, Targeting eNOS for stroke
protection, Trends Neurosci. 27 (2004) 283e289.
[13] M. Naka, T. Nanbu, K. Kobayashi, Y. Kamanaka, M. Komeno, R. Yanase,
T. Fukutomi, S. Fujimura, H.G. Seo, N. Fujiwara, S. Ohuchida, K. Suzuki,
K. Kondo, N. Taniguchi, A potent inhibitor of inducible nitric oxide synthase,
ONO-1714, a cyclic amidine derivative, Biochem. Biophys. Res. Commun. 270
(2000) 663e667.
[14] S. Shibuta, S. Varathan, T. Mashimo, The neuroprotective effect of ONO-1714
on NMDA-mediated cytotoxicity in vitro, J. Neurol. Sci. 215 (2003) 31e36.
[15] L. Garcia-Bonilla, A. Rosell, G. Torregrosa, J.B. Salom, E. Alborch, M. Gutierrez,
E. ez-Tejedor, R. Martinez-Murillo, J. Agulla, P. Ramos-Cabrer, J. Castillo,
T. Gasull, J. Montaner, Recommendations guide for experimental animal
models in stroke research, Neurologia 26 (2011) 105e110.
[16] G. Colak, A.J. Filiano, G.V. Johnson, The application of permanent middle
cerebral artery ligation in the mouse, J. Vis. Exp. 53 (Jul 25 2011).
[17] Y. Kawanaka, K. Kobayashi, S. Kusuda, T. Tatsumi, M. Murota, T. Nishiyama,
K. Hisaichi, A. Fujii, K. Hirai, M. Naka, M. Komeno, Y. Odagaki, H. Nakai,
M. Toda, Design and synthesis of orally bioavailable inhibitors of inducible
nitric oxide synthase. Identification of 2-azabicyclo[4.1.0]heptan-3-imines,
Bioorg. Med. Chem. 11 (2003) 1723e1743.
[18] I. Suárez del Villar, A. Gradillas, J. Pérez-Castells, Synthesis of 2-azabicyclo
[4.1.0]heptanes through stereoselective cyclopropanation reactions, Eur. J.
Org. Chem. (2010) 5850e5862.
3.2.7. Determination of infarct size by TTC
Two days after pMCAO, animals were killed by neck dislocation
to assess infarct outcome. Brain was removed and cut into seven
1 mm-thick coronal brain slices (Brain Matrix, WPI, UK) and stained
with TTC (1% TTC in 0.1 M phosphate buffer) [42]. Infarct volumes
were calculated sampling each side of the coronal sections with
a digital camera (Pentax Optio S7), and the images were analyzed
using ImageJ 1.33u software (National Institutes of Health,
Bethesda, MD). The digitized image was displayed on a video
monitor. With the observer masked to the experimental conditions,
the contralateral hemisphere perimeter was overlapped onto the
ipsilateral hemisphere to exclude edema, and infarct borders were
delineated with an operator controlled cursor. The area of infarct,
which was unstained, was determined by counting the pixels
contained within the outlined regions of interest and expressed in
square millimeters. Infarct volumes (in mm3) were integrated from
the infarct areas over the extent of the infarct calculated as an
orthogonal projection. All animals in the control group displayed
infarcts after the occlusion procedure, which included exclusively
the cerebral cortex.
[19] I. Suárez del Villar, A. Gradillas, A. Gómez-Ovalles, R. Martínez-Murillo,
A. Martínez, J. Pérez-Castells, Cyclopropanation reactions for the synthesis of
2-azabicyclo[4.1.0]heptane derivatives with nitric oxide synthase inhibitory
activity, Chem. Lett. 37 (2008) 1222e1223.
[20] Stererochemical assignment for 2.
4.6%
H6
H1
H6
Cl
Cl
Cl
Cl
9.8%
9.9%
+
N
Dmob
O
N
Dmob
O
3.2.8. Statistical analysis
H1
Results are expressed as the means ꢁ S.E.M. t-test was used to
determine the statistical significance of differences between the
means, and p value of <0.05 was considered significant.
anti-2
syn-2

446
A. Pozo-Rodrigálvarez et al. / European Journal of Medicinal Chemistry 54 (2012) 439e446
brain infarction in SOD-1-transgenic mice following transient focal cerebral
[21] Stererochemical assignment for 4.
ischemia, J. Cereb. Blood Flow Metab. 16 (1996) 1153e1157.
[31] S. Parmentier, G.A. Bohme, D. Lerouet, D. Damour, J.M. Stutzmann, I. Margaill,
M. Plotkine, Selective inhibition of inducible nitric oxide synthase prevents
ischaemic brain injury, Br. J. Pharmacol. 127 (1999) 546e552.
4.1%
4.9%
3.6%
7.6%
H5
H6
[32] R. Martinez-Murillo, A.P. Fernández, J. Serrano, J. Rodrigo, E. Salas,
M. Mourelle, A. Martinez, The nitric oxide donor LA 419 decreases brain
damage in a focal ischemia model, Neurosci. Lett. 415 (2007) 149e153.
[33] J. Serrano, J.M. Encinas, A.P. Fernández, J. Rodrigo, A. Martinez, Effects of acute
hypobaric hypoxia on the nitric oxide system of the rat cerebral cortex:
protective role of nitric oxide inhibitors, Neuroscience 142 (2006) 799e808.
[34] F. Sekiguchi, Y. Mita, Y. Kamanaka, N. Kawao, H. Matsuya, C. Taga,
A. Kawabata, The potent inducible nitric oxide synthase inhibitor ONO-1714
inhibits neuronal NOS and exerts antinociception in rats, Neurosci. Lett. 365
(2004) 111e115.
H6
H7
H7
Cl
+
9.4%
7.4%
Cl
N
Dmob
S
N
Dmob
S
H1
H1
(-)-anti-trans-4
(-)-anti-cis-4
[35] V. Danielisova, J. Burda, M. Nemethova, M. Gottlieb, Aminoguanidine
administration ameliorates hippocampal damage after middle cerebral artery
occlusion in rat, Neurochem. Res. 36 (2011) 476e486.
[36] H.H. Wang, H.L. Hsieh, C.M. Yang, Nitric oxide production by endothelin-1
enhances astrocytic migration via the tyrosine nitration of matrix metal-
loproteinase-9, J. Cell Physiol. 226 (2011) 2244e2256.
[37] E. Cuadrado, A. Rosell, A. Penalba, M. Slevin, J. Alvarez-Sabin, A. Ortega-Aznar,
J. Montaner, Vascular MMP-9/TIMP-2 and neuronal MMP-10 up-regulation in
human brain after stroke: a combined laser microdissection and protein array
study, J. Proteome Res. 8 (2009) 3191e3197.
[38] C.A. Graham, R.W. Chan, D.Y. Chan, C.P. Chan, L.K. Wong, T.H. Rainer, Matrix
metalloproteinase 9 mRNA: an early prognostic marker for patients with
acute stroke, Clin. Biochem. (2011).
[22] A.H. Ding, C.F. Nathan, D.J. Stuehr, Release of reactive nitrogen intermediates
and reactive oxygen intermediates from mouse peritoneal macrophages.
Comparison of activating cytokines and evidence for independent production,
J. Immunol. 141 (1988) 2407e2412.
[23] B. Pauwels, A.E. Korst, C.M. de Pooter, G.G. Pattyn, H.A. Lambrechts, M.F. Baay,
F. Lardon, J.B. Vermorken, Comparison of the sulforhodamine B assay and the
clonogenic assay for in vitro chemoradiation studies, Cancer Chemother.
Pharmacol. 51 (2003) 221e226.
[24] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure,
function and inhibition, Biochem. J. 357 (2001) 593e615.
[25] F.X. Guix, I. Uribesalgo, M. Coma, F.J. Munoz, The physiology and patho-
physiology of nitric oxide in the brain, Prog. Neurobiol. 76 (2005) 126e152.
[26] Z. Huang, P.L. Huang, J. Ma, W. Meng, C. Ayata, M.C. Fishman, M.A. Moskowitz,
Enlarged infarcts in endothelial nitric oxide synthase knockout mice are
attenuated by nitro-L-arginine, J. Cereb. Blood Flow Metab. 16 (1996)
981e987.
[27] C. Iadecola, Bright and dark sides of nitric oxide in ischemic brain injury,
Trends Neurosci. 20 (1997) 132e139.
[28] K. Srivastava, P.M. Bath, U. Bayraktutan, Current therapeutic strategies to
mitigate the eNOS dysfunction in ischaemic stroke, Cell Mol. Neurobiol.
(2011).
[39] P. Svedin, H. Hagberg, K. Savman, C. Zhu, C. Mallard, Matrix
metalloproteinase-9 gene knock-out protects the immature brain after cere-
bral hypoxia-ischemia, J. Neurosci. 27 (2007) 1511e1518.
[40] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
[41] M.W. Radomski, P. Vallance, G. Whitley, N. Foxwell, S. Moncada, Platelet
adhesion to human vascular endothelium is modulated by constitutive and
cytokine induced nitric oxide, Cardiovasc. Res. 27 (1993) 1380e1382.
[42] J.B. Bederson, L.H. Pitts, S.M. Germano, M.C. Nishimura, R.L. Davis,
H.M. Bartkowski, Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain
for detection and quantification of experimental cerebral infarction in rats,
Stroke 17 (1986) 1304e1308.
[29] S.R. Vincent, Nitric oxide neurons and neurotransmission, Prog. Neurobiol. 90
(2010) 246e255.
[30] H. Kamii, S. Mikawa, K. Murakami, H. Kinouchi, T. Yoshimoto, L. Reola,
E. Carlson, C.J. Epstein, P.H. Chan, Effects of nitric oxide synthase inhibition on