Novel inhibitors of neuronal nitric oxide synthase with potent antioxidant properties.

Article abstract of DOI:10.1016/S0960-894X(02)00883-1

A series of hybrid compounds possessing an nNOS pharmacophore linked to an antioxidant fragment has been synthesized. Among them, compound 8d, a propofol derivative, displayed the greatest dual potencies against nNOS (IC(50)=0.12 microM) and lipid peroxidation (IC(50)=0.4 microM) accompanied with e/nNOS selectivity (67.5). This shows that nNOS was able to accommodate very bulky groups such as di-tert-butyl or di-iso-propyl phenol in its active site.

Full text of DOI:10.1016/S0960-894X(02)00883-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 209–212
Novel Inhibitors of Neuronal Nitric Oxide Synthase
with Potent Antioxidant Properties
Serge Auvin,a,* Michel Auguet,b Edith Navet,a Jeremiah J. Harnett,a
Isabelle Viossat,b Jocelyne Schulz,b Dennis Bigga and Pierre-E. Chabrierb
aDepartment of Medicinal Chemistry, Beaufour-Ipsen Research Laboratories, Institut Henri Beaufour,
5, Avenue du Canada, 91966 Les Ulis Cedex, France
bDepartment of Biology, Beaufour-Ipsen Research Laboratories, Institut Henri Beaufour,
5, Avenue du Canada, 91966 Les Ulis Cedex, France
Received 28 August 2002; revised 8 October 2002; accepted 10 October 2002
Abstract—A series of hybrid compounds possessing an nNOS pharmacophore linked to an antioxidant fragment has been synthe-
sized. Among them, compound 8d, a propofol derivative, displayed the greatest dual potencies against nNOS (IC50=0.12 mM) and
lipid peroxidation (IC50=0.4 mM) accompanied with e/nNOS selectivity (67.5). This shows that nNOS was able to accommodate
very bulky groups such as di-tert-butyl or di-iso-propyl phenol in its active site.
# 2002 Elsevier Science Ltd. All rights reserved.
A selective or a general loss of neurones is responsible
for many acute or chronic neurological disorders. These
pathophysiological situations, such as cerebral ischemia,
involve an enhanced formation of free radicals in brain
with a basis for further studies towards the development
of a novel strategy for the treatment of stroke that
would consist in the administration of a drug possessing
both activities. Consequently, a series of compounds
was synthesized where the NOS pharmacophore was
covalently linked to the antioxidant. As a prerequisite to
the design of these drugs, they should be devoid of vas-
cular effects. This could be achieved by selectively tar-
geting nNOS without reducing circulating NOꢀ from
eNOS.
ꢀ
-
tissue. Both reactive oxygen species (e.g., O2 ) and reac-
tive nitrogen species (e.g., NOꢀ) participate in the
inflammatory process and contribute to neuronal death.
ꢀ
NOꢀ reacts rapidly with O2 in aqueous media to form
the highly reactive peroxynitrite (ONOO-) with harmful
effects on neuronal cells.
-
NO is synthesised from l-arginine by NO synthase
(NOS). Three isoforms have been identified from three
distinct genes: neuronal NOS (nNOS); inducible NOS
(iNOS) and endothelial NOS (eNOS).
Our first attempt to produce hybrid compounds led to
the discovery of BN 80933.2 This bifunctional agent
provided promising neuroprotection as demonstrated in
a rat model of transient cerebral ischemia.3
Scavenging reactive oxygen species or inhibiting the NO
synthesis has been shown to result in an enhanced neu-
ronal survival after cerebral ischemia. In pioneering
experiments,1 we have shown that the combination of a
NOS inhibitor and an antioxidant (e.g., superoxide sca-
venger) in a model of acute neurodegeneration repre-
sented by focal transient ischemia was not additive but
synergistic to reduce neuronal damage. This provides us
During our investigation towards potent and selective
nNOS inhibitors, phenyl-2-thiophenecarboximidamide4
4a was chosen among NOS pharmacophores which
could be easily incorporated into our structures. It is
now well accepted5 that a selective and/or potent nNOS
inhibition is dependent on the presence of a basic center
in the inhibitor structure. A short structure–activity
study was conducted around 4a in order to determine
the most suitable amino-linker to be used.
These amino-amidine derivatives 4b–e were accessible
through a short, straightforward strategy. As outlined
*Corresponding author. Fax: +33-169-07-3802; e-mail: serge.auvin@
beaufour-ipsen.com
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00883-1

210
S. Auvin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 209–212
in Scheme 1, they were prepared from commercially
available derivatives 1 upon catalytic hydrogenation of
the nitro group that provided the requisite substituted
aniline intermediates 2. The weak nucleophilicity of the
aniline group oriented our chemical strategy towards
reactive amidine precursors such as thioimidate reagents
and in particular S-methyl-2-thiophenethiocarboximide.
HI,6 which allowed us to obtain the desired amidines 3 in
fair to good yields. In the last step, Boc protection was
removed under classical acidic conditions to provide the
hydrochloride salt of the requisite amino-amidine 4b–e.
antioxidant pharmacophore. For the connection
between the NOS pharmacophore and the antioxidant
part of the molecule we used a flexible linker. Optimi-
zation of the size of the molecule was related to the
impact of the bulkiness of the substituted phenols on
the nNOS and lipid peroxidation inhibitory activities.
The synthesis of the dual inhibitors (Scheme 2) was
accomplished in two steps starting from the amino-
amidine derivative 4d and the commercial 5a–c or syn-
thetic phenols 6 (Scheme 3). The peptidic coupling was
achieved using classical carbodiimide/HOBT procedure
to afford 7a–d which were reduced to the required
amines 8a–d using BH3–THF.
The compounds were tested on the two constitutive
isoforms of NO Synthase (nNOS and eNOS) and their
selectivity was calculated. Inhibition was determined by
measuring their effect on the conversion by NOS of
[3H]L-arginine into [3H]L-citrulline.3
The synthesis of the 2,6-di-iso-propylphenol (propofol)8
derivative 6 is illustrated in Scheme 3. After protection
of the hydroxyl group of 9,9 the acetyl derivative 10 was
condensed with (Ph)3P¼CH–CO2Et in refluxing THF.
The cinnamyl intermediate 11 was then successively
hydrogenated and saponified leading to the required
carboxylic acid derivative 6.
As shown in Table 1, different inhibitory potencies on
nNOS were observed as the distance between the amino
group and the arylamidine increased. The different sub-
stitution pattern in 4d and 4e led, however, to similar
activities but with a higher selectivity in favor of 4d.
Hence, 4d was selected as candidate to be linked to the
antioxidant part of the molecule.
Results and Discussion
Of the free radical scavengers described in the literature,
substituted phenols are the best known for their anti-
oxidant properties.7 Among the most potent ones, BHT
(2,6-di-tert-butyl-4-methylphenol) was chosen as the
Table 2 shows the IC50 values for nNOS and eNOS
inhibition. The antioxidant potency of the new com-
pounds was assessed for their ability to inhibit Fe2+
Scheme 1. (i) 10% Pd/C, H2, 1.5 atm, EtOH, rt; (ii) S-methyl-2-thio-
phenethiocarboximide. HI,6 iPrOH, 50–60 ꢁC, 15 h; (iii) HCl in 1,4-
dioxane or EtOAc, rt.
Scheme 2. (i) EDC, HOBT, Et3N, CH2Cl2, rt; (ii) BH3–THF 3 equiv,
THF, reflux, 1.5 h then MeOH and HCl 3 N, reflux, 1 h.
Table 1. nNOS and eNOS inhibition,a results for compounds 4a–e
Compd
R
Isomer
Salt
nNOS Inhibition
IC50, mM
eNOS Inhibition
IC50, mM
Selectivity e/nNOS
4a
4b
4c
4d
4e
H
H2N
H2NCH2
H2NCH2
H2NCH2CH2
1HCl
1HCl
2HCl
2HCl
2HCl
2.2
18
0.2
0.05
0.08
13.9
>100
37.1
6.3
>5.5
195.3
484
4
4
3
4
24.2
25
312.5
aNOS inhibition was determined at least two times according to ref 3.

S. Auvin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 209–212
Table 2. nNOS, eNOS and lipid peroxidation inhibitions for compounds 8a–d
211
Compd
n
R2
Salt
nNOS Inhibition
IC50, mM
eNOS Inhibition
IC50, mM
Selectivity e/nNOS
LPO Inhibition
IC50, mM
AR-R
BHT
8a
8b
8c
—
—
1
2
3
—
—
tBu
tBu
tBu
iPr
2HCl
—
2HCl
—
2HCl
2HCl
0.12
na
1.6
1.4
0.47
0.12
7.2
na
15.7
15.5
4.9
60
—
9.8
11.1
10.4
67.5
na
32.5
4.9
2.4
0.4
0.4
8d
3
8.1
na, not active.
undertaken but we rather focused on the phenol sub-
stitution pattern. In that way, we decided to exchange
the di-tert-butyl group of the phenol for the less bulky
di-iso-propyl, as in propofol.8 Intermediate 6 was read-
ily synthesized as described in Scheme 3 and coupled to
the amino derivative 4d (Scheme 2) to produce 8d. We
could
not
only
enhance
nNOS
inhibition
(IC50=0.12 mM) to a satisfactory level but also achieve
a good selectivity e/nNOS (>67), values which are
similar to those of the reference AR-R17477. A com-
parison between the IC50 of 8c and 8d on nNOS and
eNOS suggested that nNOS was more prone to steric
bulkiness in this region of the active site than eNOS.
The inhibitory potency of 8d in the LPO test was as
good (IC50=0.40 mM) as for 8c, confirming the excellent
antioxidant capacity of this series of compounds. To
complete this study, an iNOS inhibition test was per-
formed on compounds 8a–d showing that all of them
exhibited an IC50 higher than 18 mM.
Scheme 3. (i) CH3COCl, Et3N, CH2Cl2, 69%; (ii) (Ph)3P+CH2CO2Et,
Brꢂ, nBuLi, THF, reflux, 15 h, 71%; (iii) 10% Pd/C, H2, 2 atm, EtOH,
rt, 97%; (iv) LiOH 3 equiv, H2O, THF, 67%.
induced lipid peroxidation in rat brain microsomes.10
AR-R (AR-R174775b) and BHT (2,6-di-tert-butyl-4-
methylphenol) were chosen as reference compounds for
NOS and LPO tests, respectively.
This study demonstrates the synthetic feasibility of dual
nNOS/LPO inhibitors with inhibitory capacities in the
low micromolar range. Unexpectedly nNOS was able to
accommodate very bulky groups such as di-tert-butyl or
di-iso-propyl fragments. Moreover, compound 8d
exhibits potent inhibitory properties versus nNOS and
lipid peroxidation accompanied with acceptable e/
nNOS and i/nNOS selectivity.
At the beginning of our study, we found that compound
8a, could inhibit both NO Synthase and lipid peroxi-
dation. Although the inhibition of nNOS was modest
compared to AR-R17477, 8a demonstrated potent free
radical scavenging activity. These encouraging results
led us to prepare a series of analogues. The one carbon
homologue 8b showed potencies similar to 8a except in
the LPO test where the IC50 was somewhat better.
Increasing the chain length led to the more potent
nNOS inhibitor 8c: IC50=0.47 mM. Although selectivity
between the two constitutive NOS isoforms (10 times)
remained limited, we achieved a dramatic progress in
the LPO test illustrated by the IC50=0.40 mM (80 times
more potent than BHT). Clearly, the di-tert-butyl phe-
nol group linked via a three-methylene chain to the
NOS pharmacophore in 8c greatly enhanced the anti-
oxidant property and improved NOS inhibition by
reaching a more accommodating (lipophilic) region of
the active site of NOS.
Acknowledgements
The authors would like to thank Jean Gregoire Marin
for providing LPO results, Jose Camara and team for
their helpful discussions and advice.
References and Notes
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´
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212
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