N-Substituted acetamidines and 2-methylimidazole derivatives as selective inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2010.09.059

A series of N-substituted acetamidines and 2-methylimidazole derivatives structurally related to W1400 were synthesized and evaluated as Nitric Oxide Synthase (NOS) inhibitors. Analogs with sterically hindering isopropyl and phenyl substituents on the benzylic carbon connecting the aromatic core of W1400 to the acetamidine nitrogen, showed good inhibitory potency for nNOS (IC ₅₀= 0.2 and 0.3 μM) and selectivity over eNOS (500 and 1166) and to a lesser extent over iNOS (50 and 100). A molecular modeling study allowed to shed light on the effects of the structural modifications on the selectivity of the designed inhibitors toward the different NOS isoforms.

Full text of DOI:10.1016/j.bmcl.2010.09.059

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6495–6499
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Substituted acetamidines and 2-methylimidazole derivatives
as selective inhibitors of neuronal nitric oxide synthase
Cristina Maccallini a, , Antonia Patruno a, , Fabio Lannutti , Alessandra Ammazzalorso ,
a,
a
a
a
b
a
a
Barbara De Filippis , Marialuigia Fantacuzzi , Sara Franceschelli , Letizia Giampietro , Simona Masella ,
b
a,
⇑
a,
⇑
Mario Felaco , Nazzareno Re , Rosa Amoroso
a
Dipartimento di Scienze del Farmaco, Università ‘G. d’Annunzio’, via dei Vestini, 66100 Chieti, Italy
Dipartimento di Scienze del Movimento Umano, Università ‘G. d’Annunzio’, 66100 Chieti, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2010
Revised 3 September 2010
Accepted 10 September 2010
Available online 17 September 2010
A series of N-substituted acetamidines and 2-methylimidazole derivatives structurally related to W1400
were synthesized and evaluated as Nitric Oxide Synthase (NOS) inhibitors. Analogs with sterically
hindering isopropyl and phenyl substituents on the benzylic carbon connecting the aromatic core of
W1400 to the acetamidine nitrogen, showed good inhibitory potency for nNOS (IC50 = 0.2 and 0.3 lM)
and selectivity over eNOS (500 and 1166) and to a lesser extent over iNOS (50 and 100). A molecular
modeling study allowed to shed light on the effects of the structural modifications on the selectivity of
the designed inhibitors toward the different NOS isoforms.
Keywords:
Acetamidines
Isoform selectivity
Ó 2010 Elsevier Ltd. All rights reserved.
2
-Methylimidazole derivatives
Nitric oxide synthase (NOS)
NOS inhibitors
1
Nitric oxide (NO) is a short-life molecule derived, in biological
many physiological aspects, including learning and memory, feed-
ing, sleeping and reproductive behavior, as well as in sensory and
systems, from L-arginine in a reaction catalyzed by Nitric Oxide
Synthases (NOS). Three main isoforms have been identified: two
of them are constitutive, the endothelial (eNOS) and the neuronal
nNOS), and one is inducible (iNOS). All three isoforms of NOS
catalyze the oxidation of -arginine to citrulline and NO using
7
motor function; however, excessive production of NO following a
8
pathologic insult can lead to neurotoxicity and contributes to neu-
9
10
(
rodegenerative diseases, including stroke, Parkinson’s disease,
1
1
L
and Alzheimer’s disease. Various mechanisms have been identi-
fied, including the high levels of iNOS expression in activated
glia. Moreover, there is evidence that also NO overproduction
by nNOS plays a key role in these pathologies, and recent studies
report promising results from chronic inhibition of nNOS as an
additional therapeutic strategy for motor nerve repair.14
reducing equivalents from NADPH in the presence of molecular
2
12
oxygen.
1
3
The constitutively expressed isoform eNOS is involved in the
regulation of vascular tone and cell signaling; the other constitu-
3
tive isoform, nNOS, originally identified in neuronal cells, produces
NO that plays important roles in neurotransmitter release and
reuptake, neurodevelopment, synaptic plasticity and regulation
Due to the unique roles played by the three isoforms of NOS in
separate tissues, the selective inhibition of an isoform over the oth-
4
15
of gene expression. The expression of iNOS is largely observed
ers is of paramount importance; in particular, it is essential not to
in macrophages during inflammation and infection related to host
defense; as such, iNOS is a high output enzyme with activity
that can be sustained for days leading to cell death and tissue
damage.
inhibit the eNOS because of its noteworthy role in maintaining
blood pressure. The available X-ray structures show a high degree
of similarity for the active sites of the three NOS isoforms which
makes isoform-selective drug design a challenging task.16 In the last
decade a large amount of studies have been carried out to identify
5
Since NO is an important signaling molecule in cells, its over-
production may result in pathological effects in virtually every
potent and selective inhibitors of iNOS and nNOS as potential treat-
6
17,18
organ system. In particular, in the brain NO is associated with
ment of the above pathological conditions.
Nevertheless, selec-
tive inhibitors have been identified, such as N-(3-(aminomethyl)-
1
9
benzyl)acetamidine (1, W1400), specific for iNOS, and several
N -alkyl and N -nitro-L-arginine derivatives, specific for nNOS.
The key structural features responsible for the selectivity of the
latter inhibitors to nNOS over eNOS and iNOS have been clarified
⇑
Corresponding authors. Tel.: +39 0871 3554686; fax: +39 0871 3554911 (R.A.);
x
x
20
tel.: +39 0871 3554603; fax: +39 0871 3554614 (N.R.).
E-mail addresses: nre@unich.it (N. Re), ramoroso@unich.it (R. Amoroso).
These authors contributed equally to this work.
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.059

6
496
C. Maccallini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6495–6499
by crystallographic and computational studies and rely on subtle
HCl
.
21–23
differences among the active sites.
For instance, the selectivity
.
NH
R
NH HBr
x
of the N -propyl-L-arginine derivatives has been attributed to the
OCH3
different size of the heme ligand binding cavity, which is signifi-
cantly larger for the nNOS rather than for the eNOS and iNOS
N
N
H
H
x
22
and may better accommodate the N -propyl group. On the other
x
rac-9 R = i-Pro
R = Ph
1
2
hand the selectivity of N -nitro-L-arginine containing dipeptide
1
1
inhibitors to nNOS over eNOS recently synthesized by Silverman’s
group directly depends on a single-residue difference in the
R
.
1
8
NH HBr
periphery of the active site, Asp597 in nNOS and Asn368 in eNOS.
N
Recently, we have synthesized and evaluated a series of
O
O
N
N
H
X
N-substituted acetamidines structurally related to 1 (compounds
2
4
2
–7 inFig. 1). Among them, compounds characterized by removal
1
4
R = H,
rac-15 R = CH3, X = CH
R = H, X = N
X = CH
of the 3-aminomethyl group of 1 and addition of a methyl or an
ethyl group on the benzylic carbon connected to the acetamidine
nitrogen, 3 and 4, respectively, showed good inhibitory potencies
toward iNOS and high selectivities over eNOS. A docking study
allowed us to shed light on the main effects of the considered
structural modifications on interaction with the iNOS isoform,
indicating that the interaction of 3-aminomethyl group with the
propionate arms of the heme cofactors of iNOS, like 1, does not
contribute significantly to the binding energy and its removal even
slightly improves inhibitory potency. On the other hand, the
slightly lower selectivity over eNOS indicates a possible role of
the 3-aminomethyl group in determining the high isoform speci-
ficity of 1, as suggested by the comparison of the X-ray structures
with iNOS and eNOS, which points out the different position of the
heme in these two isoforms, leading to different interactions of its
propionate arms with the 3-aminomethyl group.
13
16
Figure 2. Designed NOS inhibitors.
NHOH
MgCl
+
CN
a, b
c
NH2
The interesting results obtained prompted us to increase that
series of molecules, in order to evaluate the effects of the structural
modifications on the inhibitory potency and isoform selectivity of
the new compounds and on their interaction within the heme bind-
ing site. Here we describe the synthesis, the in vitro evaluation, and a
molecular modeling study of new N-substituted acetamidines and
H2N
8
10
d
d
2
(
-methylimidazole derivatives as possible selective nNOS inhibitors
Fig. 2).
Synthesis of compounds rac-9 and 11 was performed according
HBr
.
to Scheme 1. From the reaction between 2-methyl-propanenitrile
and phenylmagnesium chloride, followed by reduction, 2-methyl-
NH
R
1
-phenylpropylamine (8) was obtained, while benzhydrylamine
N
H
(10) was obtained by the reduction of diphenylmethanone oxime.
Amines 8 and 10 were added, under mild conditions, to S-2-
naphthylmethyl thioacetimidate hydrobromide, to give desired
rac-9 R = i-Pro
11 R = Ph
acetamidines rac-9 and 11.25 Condensation of (3-methoxy)benzyl-
amine or (3,4-methylendioxy)benzylamine with ethylacetimidate
Scheme 1. Reagents and conditions: (a) dry THF, N
2 4
, reflux, 2 h; (b) NaBH , dry THF,
2
N , reflux, 5 h; (c) Na, abs EtOH, rt, 3 h; (d) S-2-naphthylmethyl thioacetimidate
hydrobromide, abs EtOH, rt, 2 h.
HBr
.
NH
NH
R
N
H
NH2
N
H
.
NH HCl
O
N
H
2
R = H
1
O
(S)-3 R = Me
(
W1400)
H N
2
rac-4 R = Et
1
2
a
or
or
NH.
HCl
.
O
NH HX
HBr
O
O
.
H N
2
O
N
H
NHR1
NH
N
OEt
N
H
O
N
H
1
5
6
R = CH Pyr, X = Br
2
13
1
7
R = Ts,
X = Cl
Scheme 2. Reagents and conditions: (a) ethylacetimidate hydrochloride, NaH, DMF
dry, 50 °C, 24 h.
Figure 1. W1400 and selective iNOS inhibitors.

C. Maccallini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6495–6499
6497
R
led to a dramatic change in the selectivity of these compounds. In-
deed, rac-9 and 11, containing an isopropyl or a phenyl substituent,
respectively, displayed a good inhibitory potency against nNOS,
and selectivity over iNOS, and especially eNOS. These results show
that the inhibitory potency and the selectivity of the above
compounds are strongly modulated by the steric hindrance of the
substituent on benzylic carbon. While small groups lead to a good
inhibition of iNOS and high selectivity over eNOS and nNOS, as in
2–4, more hindered groups lead to a good nNOS inhibition and
selectivity over eNOS and to a lesser extent over iNOS, as in 9
and 11. We then tried to replace the 3-aminomethyl group on
the phenyl ring of 1 with other groups containing a different het-
eroatom such as a methoxy or a dioxolanic system. The introduc-
tion of m-methoxy group on the phenyl ring of 2, as in 12, led to
a loss of inhibition toward iNOS with only a moderate increase of
inhibition toward nNOS, while that of a dioxolanic system, as in
R
a
NH
N
+
N
N
Cl
X
X
1
4
R = H,
X = CH
rac-15 R = CH3, X = CH
R = H, X = N
16
Scheme 3. Reagents and conditions: (a) NaH, dry DMF, 50 °C, 4–48 h.
hydrochloride provided acetamidines 12 and 13, respectively
Scheme 2). Compounds 14, rac-15 and 16 were obtained by
nucleophilic substitution of chloromethylbenzene, chloroethylben-
zene, or chloromethylpyridine, respectively, with 2-methylimidaz-
2
6
(
2
7
ole (Scheme 3).
All the compounds were biologically evaluated for their capabil-
ity to inhibit iNOS, eNOS and nNOS, using an enzymatic assay; we
used for these isoforms recombinant enzymes from different
sources: murine macrophage iNOS, rat brain nNOS, and bovine
eNOS. The inhibitory activity against NOS isoforms was measured
by following the oxidation of oxyhemoglobin to methemoglobin
by NO and the potency results were expressed as IC50 values,
which are the concentrations that cause 50% loss of activity. The
selectivities for iNOS over eNOS (e/i), and for nNOS over the other
two isoforms (e/n and i/n) were also evaluated. The known inhib-
itor 1 was also subjected to the same test system, for comparison;
we also included molecules 2, (S)-3 and rac-4 (Fig. 1) as they were
not previously tested on nNOS.
The data presented in Table 1 display for acetamidines 2, (S)-3
and rac-4, differing from 1 by the removal of the 3-aminomethyl
group and addition of a methyl or an ethyl to the benzylic carbon
linked to the amidine group, good iNOS inhibition and poor inhibi-
tion of nNOS and eNOS. Our docking studies indicated that the
good inhibitory activity of 3 and 4 to iNOS beneficts from favorable
hydrophobic contacts of the methyl and ethyl groups with the
Pro344 and Val346 residues, belonging to the b-sheet S12 which
flanks the ligand binding cavity around the heme group. Previous
crystallographic studies indicated that the significantly larger size
1
3, led to a complete loss of inhibitory potency toward all isoforms.
A completely different strategy was employed to design com-
pounds 14, rac-15, and 16, where we combined the features of
the above analogs of 1 (without 3-aminomethyl group) with the li-
gands of heme iron of NOS, bearing an imidazolic ring instead of
the acetamidine function. All these 2-methylimidazole derivatives
showed a moderate inhibitory potency against nNOS, with limited
selectivity over the other isoforms.
Docking simulations were first performed to predict the binding
mode in the nNOS, iNOS and eNOS active sites of inhibitors 9 (both
as R and S configurations), 11–13, all the analogs of 1 without the
2
8
3
-aminomethyl group. The effectiveness of the GLIDE program in the
docking of 1 on to its natural iNOS substrate was already shown in
a previous work to give a good agreement between the calculated
pose and the X-ray structure of the 1-iNOS complex, with a root
2
4
mean square deviation (RMSD) of 0.341 Å.
The main docked structures of 9 and 11–13 to the three NOS
isoforms share the hydrogen bond interactions between the com-
mon amidine group and the carboxylate moiety of the Glu371
and Trp366 residues in iNOS or the corresponding Glu592 and
Trp587 in nNOS and Glu363 and Trp358 in eNOS. However, quite
surprisingly, only the docked poses of 12 and 13 show the same
orientation of the benzylic backbone atop the A pyrrole ring of
heme while a completely different orientation was observed for
of this cavity for nNOS than for the iNOS and eNOS isoforms is
responsible for the selectivity for this isoform of N -alkyl-
nine inhibitors with substituents of suitable steric hindrance,
such as the n-propyl group, which fit the nNOS but not the iNOS
and eNOS cavities. We thus decided to replace the methyl or ethyl
groups of 3 and 4 by more sterically hindering substituents, which
could still fit in the cavity of the nNOS but not in those of the iNOS
and eNOS isoforms. In agreement with our hypothesis, the increase
of the steric hindrance of the substituents on benzylic carbon in 2
x
L
-argi-
9
and 11, the compounds with the most sterically hindering iso-
2
2
propyl and phenyl groups and the highest selectivity for nNOS over
iNOS and eNOS. In particular, an analysis of the poses obtained for
these two compounds show that the isopropyl and phenyl groups
apparently do not fit the heme ligand binding cavity as instead ob-
served for the methyl and ethyl groups in our previous docking
studies of 3 and 4. This lack of fit is presumably due to steric
clashes of the isopropyl and phenyl groups with the ‘back wall’
Table 1
Inhibition of NOS by new acetamidines
Compound
IC50
(l
M)a
Selectivity
iNOS
0.2 ± 0.03b
nNOS
eNOS
eNOS/iNOS
eNOS/nNOS
iNOS/nNOS
2
150 ± 23
150 ± 13
90 ± 13
0.2 ± 0.04
0.3 ± 0.03
10 ± 1.5
400 ± 22
5 ± 0.2
350 ± 18b
300 ± 31
1750
666
320
10
11.6
2
2.3
2
0.9
500
1166
10
0.013
0.003
0.003
50
100
5
b
(S)-3
0.45 ± 0.08b
b
80 ± 8b
100 ± 3
350 ± 13
100 ± 9
400 ± 20
90 ± 2
rac-4
rac-9
0.25 ± 0.05
10 ± 1.8
30 ± 3
50 ± 4
800 ± 26
50 ± 4
1
1
1
1
1
2
3
4
0.5
1.8
1
18
2
10
rac-15
100 ± 2
12 ± 2
0.33 ± 0.06
5 ± 0.6
5 ± 0.3
7.3 ± 1.4
80 ± 4
100 ± 3
1100 ± 104
0.8
8.3
3333
16
20
150
20
2.4
0.004
1
1
6
a
The NOS inhibition at 20
Published data, Ref. 24.
lM of arginine. Experiments were independently performed at least three times.
b

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C. Maccallini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6495–6499
Figure 3. Superposition of (a) nNOS and (b) iNOS bound complexes of 1 (turquoise carbons), 9 (orange carbons), and 11 (green carbons).
made up of the b-sheet S12 (Pro344 to Val346 in the iNOS number-
ing) which has been shown in our previous docking studies to give
hydrophobic contacts with the smaller methyl and ethyl substitu-
ents in 3 and 4. As these favorable hydrophobic contacts were
found responsible for the good inhibitory activity of 3 and 4 toward
iNOS, we pursued to seek possible poses of 9 and 11 where the
isopropyl or phenyl groups fit in this cavity. To this purpose we
inhibitory activity (due to the favorable contacts of the isopropyl
and phenyl groups of these ligands with the hydrophobic residues
of the S12 sheet).
In conclusion, in this study we have described a series of
N-substituted acetamidines and 2-methylimidazole derivatives
structurally related to W1400 and screened their activity as selec-
tive NOS inhibitors. Analogs rac-9 and 11 showed good inhibitory
2
9
0
employed a simple method based on the soft-docking approach
to take into account, at least partly, the flexibility of the protein.
potency for nNOS (IC50 = 0.2 and 0.3 lM, respectively) and selectiv-
3
ity over eNOS (500 and 1166, respectively) and to a lesser extent
over iNOS (50 and 100, respectively). A molecular modeling study
allowed to elucidate the effects of the structural modifications on
isoform activity; in particular, the selectivity of 9 and 11 for nNOS
has been attributed to the significantly larger size of the heme
binding cavity for nNOS than for the iNOS and eNOS isoforms,
which better fit the steric hindering isopropyl and phenyl substit-
uents on the benzylic carbon connecting the aromatic core of 1 to
the acetamidine nitrogen.
The soft-docking approach allows to tolerate a significant overlap
between ligand and protein atoms during the docking
procedure—by reducing their Van der Waals radii—thus permitting
the binding of a larger ligand to a pocket structure determined for a
complex with a smaller ligand. The highest ranked poses have been
selected and submitted to an energy minimization to ensure that
any Van der Waals overlaps between protein and ligand atoms
can be resolved. Such an approach has been recently validated giv-
ing very good results in the foreign-docking of several ligands to
moderately flexible proteins.31 We limited our study to the nNOS
and the iNOS which have been shown to possess the largest and
the smallest heme binding cavity, respectively. This approach al-
lowed to find several poses of 9 and 11 to nNOS and iNOS in which
the isopropyl and phenyl groups fit the heme binding cavity and
exploit favorable hydrophobic interactions with the Pro344 and
Val346 residues of the b-sheet S12. The most stable of these poses
are reported in Figure 3 and compared with the lowest poses for 1
calculated with the same procedure. A significantly higher Molec-
ular Mechanics (MM) binding energy has been calculated for 9 and
1
1 with the nNOS enzyme than for the iNOS and eNOS isoenzymes
in agreement with the selectivity of these two compounds toward
nNOS. Moreover, we calculated the reorganization energy required
by the two isoforms to accommodate 9 and 11, and in both cases
the penalty for the iNOS isoform is significantly higher (2–3 kcal -
À1
mol ) than for the nNOS isoform, as expected on the basis of the
smaller heme binding cavity. This higher reorganization energy for
the iNOS isoform is probably due to the smaller size of its heme
binding cavity and in particular to the shift toward the heme group
of the b-sheet S12 in this isoform with respect to nNOS, as shown
by the superposition of the X-ray structures of these two isoforms
(
9
Fig. 4). These results explain both the isoform selectivity shown by
and 11 (due to the highest conformational penalty required by
the iNOS isoform to accommodate these ligands) and their good
Figure 4. Superposition of the X-ray structures of the iNOS and nNOS isoforms
showing the shift of their b-sheet S12.

C. Maccallini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6495–6499
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