Selective Inhibition of iNOS by Benzyl- and Dibenzyl Derivatives of N-(3-Aminobenzyl)acetamidine

Article abstract of DOI:10.1002/cmdc.201100125

Picking your NOS: The effect of amine substitution at the 3-aminomethyl group of known iNOS inhibitor W1400 with mono- or disubstituted benzylic groups was evaluated. The o-nitro-benzyl derivative was identified as a potent inhibitor of iNOS, with good selectivity over eNOS. Docking simulations were performed to predict the binding mode of these derivatives in the iNOS and eNOS active sites.

Full text of DOI:10.1002/cmdc.201100125

DOI: 10.1002/cmdc.201100125
Selective Inhibition of iNOS by Benzyl- and Dibenzyl Derivatives of
N-(3-Aminobenzyl)acetamidine
Marialuigia Fantacuzzi,[a] Cristina Maccallini,[a] Fabio Lannutti,[a] Antonia Patruno,[a] Simona Masella,[a]
Mirko Pesce,[b] Lorenza Speranza,[b] Alessandra Ammazzalorso,[a] Barbara De Filippis,[a] Letizia Giampietro,[a]
Nazzareno Re,[a] and Rosa Amoroso*[a]
Nitric oxide (NO), one of the smallest known bioactive prod-
ucts of mammalian cells, can be produced by almost all cells.[1]
In mammals, NO is synthesized by a family of three NO syn-
thases (NOS): neuronal nNOS, inducible iNOS and endothelial
eNOS, that are products of three genes: NOS1, NOS2, and
NOS3, respectively.[2] The three isoforms generate NO by the
conversion of l-arginine to l-citrulline; they differ in both
structure and function but share about 50% sequence homol-
ogy and are differentially regulated making the catalytic activi-
ty distinct for each isoform. nNOS and eNOS are constitutive
and primarily expressed in neurons and endothelial cells, re-
spectively. These isoforms are calcium/calmodulin dependent
and generate low levels of NO for short periods of time in a
pulsative manner.[3]
nNOS isoforms, which limits their application in vivo. More
recent studies have focused on the design and synthesis of
non-amino acid analogues, such as amino heterocycles, ami-
dines, guanidines, isoquinolinamines, and isothioureas.[11–14]
We previously reported the synthesis, biological evaluation,
and docking studies of a series of N-substituted acetami-
dines,[15] structurally related to the leading scaffold W1400[16]
(1), a potent and selective iNOS inhibitor. Starting from dock-
ing results previously obtained, and with the aim of extending
the study of possible ligand–enzyme interactions, we have
now evaluated the effect of the introduction of several sub-
stituents in ortho, meta or para positions of the leading scaf-
folds 2 and 3, differing from 1 by the amino substitution at
the 3-aminomethyl group with one or two benzylic groups.
iNOS was first identified and characterized in cytokine-acti-
vated murine macrophages.[4] This isoform is not dependent
upon calcium/calmodulin for its enzymatic action and is ex-
pressed in a wide array of cells and tissues, for example, mac-
rophages,[5] hepatocytes,[6] neutrophils, pulmonary epithelium,
colonic epithelium, and vasculature.[7] After induction, iNOS
continuously produces NO until the enzyme is degraded. The
overproduction of NO by iNOS may have detrimental conse-
quences, and this activity seems to be involved in the patho-
physiology of several human diseases, such as asthma, arthritis,
multiple sclerosis, colitis, psoriasis, neurodegenerative diseases,
tumor development, transplant rejection, and septic shock.[8–10]
In recent years, considerable effort has been directed toward
the selective inhibition of iNOS as a strategy for the prevention
of excessive NO production, while maintaining the basal for-
mation of NO from constitutive NOS that is required for
normal physiological function. The first approach to NOS inhib-
itors included analogues of the natural substrate l-arginine
that act competitively at the substrate binding site; however,
these compounds are not selective enough over the eNOS and
Acetamidines 2 and 3 were synthesized as previously de-
scribed,[15] and new compounds 7a–w were obtained accord-
ing to the route shown in Scheme 1. Boc-protected m-xylylen-
diamine was treated with the appropriate ortho-, meta-, or
para-substituted chloromethylbenzene to give N-benzyl deriva-
tives 5a–u. Subsequent removal of the Boc group with tri-
fluoroacetic acid (TFA) and treatment with sodium hydroxide
gave free amines 6a–u. Finally, reaction of 6a–u with S-2-
naphtylmethyl thioacetimidate hydrobromide or ethylacetimi-
date hydrochloride gave 7a–u as hydrobromide or hydrochlo-
ride salts. The syntheses of 5p and 5u also gave large
amounts of N,N-dibenzyl analogues 5v–w, which were isolated
by chromatographic separation; compounds 7v–w were ob-
tained from 5v–w following the same synthetic scheme.
[a] Dr. M. Fantacuzzi,+ Dr. C. Maccallini,+ Dr. F. Lannutti, Dr. A. Patruno,
Dr. S. Masella, Dr. A. Ammazzalorso, Dr. B. De Filippis, Dr. L. Giampietro,
Prof. N. Re, Prof. R. Amoroso
Dipartimento di Scienze del Farmaco
Universitꢀ degli Studi “G. d’Annunzio”
via dei Vestini, 66100 Chieti (Italy)
Fax: (+39)0871-355-4911
E-mail: ramoroso@unich.it
All new compounds are based on the common scaffolds 2
or 3, differing from them in the substitution at the benzyl or
dibenzyl moiety with halogens or small groups, such as
phenyl, methyl, methoxy, thiomethyl, and trifluoromethyl. In
our study, we also considered the possible role of the position
of the substituent (ortho, meta or para) on the aromatic ring.
All new acetamidines were biologically evaluated for their ca-
[b] Dr. M. Pesce, Dr. L. Speranza
Dipartimento di Scienze del Movimento Umano
Universitꢀ degli Studi “G. d’Annunzio”, via dei Vestini
66100 Chieti (Italy)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100125.
ChemMedChem 2011, 6, 1203 – 1206
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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ed an iNOS IC50 value of 40 mm. Furthermore, the
presence of a thiomethyl group in the para position
of the benzyl ring (7o) did not improve the potency
(IC50 =18 mm). The introduction of a methoxy group
in the para position led to a more potent iNOS in-
hibitor (7l: IC50 =5 mm) with improved selectivity
when compared with the parent compound 2 (60-
fold c.f. fivefold). However, when the methoxy group
is in meta and ortho positions (7m–n), the IC50 value
is greater than 20 mm. Conversely, when the sub-
stituent is a phenyl ring in the meta or ortho posi-
tion (7t–u), the iNOS IC50 value is 5 and 1 mm, re-
spectively, while the selectivity (e/i) is 100-fold and
50-fold. Compound 7s, with the phenyl ring in the
para position, exhibited an IC50 value of 30 mm
against iNOS.
The most interesting substituent of the benzyl
ring was certainly the nitro group. The most potent
iNOS inhibitor of this series was compound 7r, con-
taining a nitro group in the ortho position, with an
IC50 value of 0.1 mm against iNOS and 300-fold selec-
tivity for iNOS over eNOS. Conversely, when the
nitro group is in the meta (7q) or para (7p) position,
Scheme 1. Reagents and conditions: a) Et3N, dry CH3CN, N2, 2–4 d, 43–75%; b) CF3COOH,
08C, 1 h, 68–90%; c) NaOH, RT, 5 min, 100%; d) S-2-naphtylmethyltioacetimidate hydro-
bromide or ethylacetimidate hydrochloride, abs EtOH, RT, 7–10 h, 64–97%.
pability to inhibit iNOS, eNOS, and nNOS, in an enzymatic
assay,[17] employing recombinant enzymes from different sour-
ces: murine macrophage iNOS, rat brain nNOS, and bovine
eNOS. The inhibitory activity against NOS isoforms was mea-
sured by following the oxidation of oxyhemoglobin to methe-
moglobin by NO, and the IC50
the IC50 values are 20 and 5 mm, respectively, with no signifi-
cant selectivity for iNOS over other isoforms.
Parent scaffold 3, obtained by dibenzyl substitution of the
primary amino group of 1, showed poor inhibition of iNOS,
with an IC50 value of 100 mm. However, N,N-dibenzyl derivatives
values were calculated from the
Table 1. IC50 and selectivity values for the inhibition of NOS isoforms by compounds 1–3, and 7a–w.
data. The selectivities for iNOS
over eNOS (e/i) and nNOS (n/i),
and for nNOS over eNOS (e/n)
were also evaluated. The known
inhibitor 1 and the scaffolds 2
and 3 were also subjected to the
same test system for compari-
son. The results of the enzymatic
assays are reported in Table 1.
The parent compound 2, with
an unsubstituted benzyl ring,
showed an iNOS IC50 value of
10 mm and selectivity over eNOS
of fivefold. Considering mono-
substitution of the primary
amine group of 1, the introduc-
tion of a chloro (7a–c), trifluoro-
methyl (7g–i) or methyl (7j–k)
group in the ortho, meta or para
position of the benzyl ring did
not improve the IC50 value,
which was greater than 30 mm.
Introduction of a fluoro group in
the para and meta positions
(7d–e) gave compounds with
iNOS IC50 values of 15 mm, while
the ortho derivative (7 f) exhibit-
Compd
R
IC50[a] [mm]
Selectivity
iNOS
eNOS
nNOS
eNOS/iNOS
eNOS/nNOS
nNOS/iNOS
1
2
–
H
4-Cl
3-Cl
2-Cl
4-F
3-F
2-F
4-CF3
3-CF3
2-CF3
4-CH3
3-CH3
4-OCH3
3-OCH3
2-OCH3
4-SCH3
4-NO2
3-NO2
2-NO2
4-Ph
3-Ph
2-Ph
H
0.33Æ0.06
10Æ0.9
100Æ8.9
100Æ7.2
30Æ3.6
15Æ1.2
15Æ1.6
40Æ4.5
40Æ3.8
100Æ9.2
100Æ8.1
30Æ2.9
50Æ4.3
5Æ0.3
1100Æ104
20Æ2.2
7.3Æ1.4
300Æ19.7
150Æ21.8
300Æ6.6
200Æ13.7
20Æ1.7
3333
2
150
0.06
1.33
1
1.5
0.5
1.2
1.33
4
2.75
3.5
0.83
0.6
3.75
0.3
0.5
2.25
0.8
0.4
0.15
0.38
2.5
0.17
8
22
30
1.5
3
6.67
1.33
3.33
0.75
2.5
2
7a
7b
7c
7d
7e
7 f
7g
7h
7i
200Æ13.2
300Æ27.5
300Æ16.2
10Æ1.1
2
3
10
0.67
4
60Æ4.3
50Æ4.3
40Æ3.4
30Æ2.5
1
400Æ24.5
550Æ50.7
350Æ13.2
25Æ2.3
100Æ10.6
200Æ13.9
100Æ7.8
30Æ3.0
10
5.5
3.5
0.83
1.2
60
1.50
1.67
25
16
4
300
1
100
50
4
1
1
2
16
7j
7k
7l
60Æ3.2
100Æ9.2
80Æ7.8
300Æ12.9
30Æ3.2
7m
7n
7o
7p
7q
7r
7s
7t
7u
3
20Æ1.8
60Æ5.6
18Æ1.5
5Æ0.3
100Æ7.6
200Æ12.6
200Æ12.3
100Æ5.5
200Æ19.2
200Æ18.6
80Æ5.2
5
100Æ9.8
450Æ33.2
80Æ7.2
3.33
11
20
20Æ1.7
0.1Æ0.01
30Æ2.5
5Æ0.4
80Æ8.7
10
30Æ2.5
2000
2.67
40
300
0.5
200
13.33
30Æ3.1
500Æ37.7
50Æ2.1
200Æ16.1
300Æ13.2
50Æ4.3
1Æ0.02
100Æ9.2
0.5Æ0.04
15Æ1.3
400Æ35.2
100Æ8.1
400Æ18.3
7v
7w
4-NO2
2-Ph
100Æ11.3
200Æ15.6
200
27
1
2
[a] The NOS inhibition at 10 mm arginine. Values given are the mean ÆSD of experiments performed in tripli-
cate at 7–10 different concentrations.
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ChemMedChem 2011, 6, 1203 – 1206

7v–w showed improved iNOS inhibition compared with lead
compound 3. In particular, derivative 7v, containing two p-
nitro-benzyl rings, was a potent iNOS inhibitor (IC50 =0.5 mm),
with high selectivity (200-fold) for iNOS over eNOS. All tested
compounds were inactive against the nNOS isoform.
mono-nitro-benzyl derivatives 7p–r are predicted to form a
further hydrogen bond between one of the nitro group
oxygen atoms and Asn348 (ortho- and para-nitro-benzyl deriv-
atives 7r and 7q) or Asn115 (meta-nitro-benzyl 7p) in the sub-
strate access channel. The bis-nitro-benzyl derivative 7v is pre-
dicted to form a hydrogen bond between the oxygen atom of
one of the two para-nitro-benzyl groups and Arg382 in the
catalytic site close to Glu371, while the remaining nitro-benzyl
group assumes the same orientation as predicted for com-
pounds 7p–r, pointing in to the substrate access channel with-
out, however, forming a second hydrogen bond with Asn115
or Asn348. The docked pose of 7u does not show any further
hydrogen bond but only an hydrophobic contact with Met114
in the substrate access channel, where the biphenyl moiety re-
sides.
Docking simulations were performed to predict the binding
mode of the two most potent inhibitors 7r and 7v, and the
two most promising nitrobenzyl-containing derivatives 7p and
7q, in the active sites of iNOS and eNOS. Our aim was to shed
light on the reasons for the potent inhibition of iNOS by 7r
and 7v, and for their selectivity for iNOS over eNOS. Further-
more, we aimed to gain insight into the effects of the nitro
group and the influence of its position on the benzyl ring
(ortho, meta, or para) of the leading scaffold 2. We also ad-
dressed the biphenyl derivative 7u to investigate how this
sterically demanding aryl group might be accommodated
within the NOS binding cavity. The effectiveness of the Glide
program in docking compound 1 to iNOS and eNOS isoforms
was shown in previous work, with calculated poses in good
agreement with the X-ray structures of the 1–iNOS (PDB code:
1QW5) and 1–eNOS (PDB code: 1F01) complexes, with a root
mean square deviations (RMSD) of 0.341 ꢁ and 0.242 ꢁ, respec-
tively.[15] Comparison of the docked poses of 7p–r and 7v in
iNOS with that of 1 (Figure 1), provides insight on the main ef-
fects of the presence of one or two nitro-benzyl groups on
their interactions with this NOS isoform.
The docked structures of 7p–r and 7v in the eNOS isoform
predict the same hydrogen-bonding interactions between the
common amidine group and the carboxylate moiety of Glu363
and Trp358, and—for the mono-nitro-benzyl derivatives—be-
tween the 3-aminomethyl group and one propionate arm of
the heme cofactor, observed for the lead compound 1.[18] How-
ever, none of these derivatives are predicted to form hydrogen
bonds between a nitro group and a further amino acid; this
difference could account for the weaker inhibitory potencies of
these compounds against eNOS and thus their selectivity for
iNOS over eNOS. In particular, the ortho- and para-nitro-benzyl
derivatives 7r and 7q are not predicted to form any hydrogen
bonds between the nitro group and Asn340 (corresponding to
Asn348 in iNOS), presumably because the loop to which
Asn340 belongs is shifted towards the heme cofactor in the
eNOS isoform, contributing to the smaller size of its heme
binding cavity.[19] On the other hand, the meta-nitro-benzyl de-
rivative 7p is predicted to be incapable of forming any hydro-
gen bonds with eNOS because the polar Asn115 residue in
iNOS is replaced with the hydrophobic Leu107 in eNOS. Inter-
estingly, replacement of Asn115 (iNOS) by Leu107 (eNOS) and
Leu337 (nNOS) has been proposed as the structural basis for
the isoform selectivity (nNOS and eNOS over iNOS) of dipep-
tide inhibitors, d-Lys-d-ArgNO2-NH2 and d-Phe-d-ArgNO2-
NH2.[20]
The predicted binding geometries of all these compounds,
as that of 7u, share common characteristics: hydrogen-bond-
ing interactions between the amidine group and the carboxyl-
ate moiety of the Glu371 and Trp366 residues; orientation of
the acetamidine-bound benzylic backbone atop of the heme
pyrrole ring; and, for the mono-nitro-benzyl derivatives, inter-
action of the 3-aminomethyl group with one propionate arm
of heme, also observed for the leading scaffold 1.[16] The
Analysis of the calculated docking scores using the EModel
score, which is based on the ChemScore function described by
Eldridge et al.,[21] provides support for the observed higher in-
hibitory potency of 7r and 7v against iNOS and their selectivi-
ty for iNOS over eNOS. The EModel score for the binding of 7r
and 7v to iNOS (100–101) are significantly higher than those
for 7q and 7p (90–94), and indeed, when the values calculated
for the four compounds, together with those for 7u and 1, are
plotted against the experimental pIC50 values, a reasonably
good correlation is obtained with R2 =0.84 (figure S2 in the
Supporting Information). Moreover, a comparative analysis of
the EModel values for the NOS isoforms shows significantly
higher docking scores for the iNOS isoform (90–101) than for
the eNOS isoform (83–88), consistent with the observed selec-
tivity for iNOS over eNOS.
Figure 1. Superimposition of the docked conformation of derivatives 7p
(magenta), 7q (orange), 7r (yellow), and 7v (green) in the active site of the
iNOS isoform.
In conclusion, a series of acetamidines structurally related to
1 was synthesized and evaluated for their biological activity as
ChemMedChem 2011, 6, 1203 – 1206
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selective iNOS inhibitors. Compound 7r, exhibiting an IC50
value of 0.1 mm, is a more potent inhibitor of iNOS than 1, ex-
hibiting good selectivity over eNOS. A docking study predicted
the binding mode of the two most potent inhibitors 7r and
7v in the iNOS active site, suggesting that they form a hydro-
gen bond between their nitro group and an asparagine or ar-
ginine residue.
50.98 (CH2NH), 55.62 (CH2NH), 123.91, 125.53, 126.12, 127.73,
128.62, 128.93, 132.77, 137.54, 141.12, 146.54 (CAr),
164.82 ppm (CNH); IR (KBr): n=1675, 1632 cmÀ1; MS (ESI): m/z
˜
(%): 394.3 (100) [M+H+]; Anal. calcd for C17H21BrN4O2: C 64.41,
H 6.08, N 18.78, O 10.73, found: C 64.21, H 6.09, N 18.83, O
10.76.
See the Supporting Information for experimental details of all
other compounds, biological assays and docking studies.
Experimental Section
General: All chemicals were commercially available and used
without further purification. Flash chromatography was per-
formed on silica gel 60 (Merck) and thin-layer chromatography
(TLC) on F254 silica gel 60 TLC plates. Melting points were deter-
mined on a Buchi apparatus and are uncorrected. Infrared
spectra were recorded on a FT-IR 1600 Perkin–Elmer spectrom-
eter. NMR spectra were run at 300 MHz on a Varian instru-
ment; chemical shifts (d) are reported in ppm. Mass spectra
were obtained on a Thermofinnigan LCQ Advantage spectrom-
eter (ESI). Elemental analyses were carried out using an Euro-
vector Euro EA 3000 model analyzer. The purity of all com-
pounds was ꢀ 98%.
Acknowledgements
This study was supported by grants from the Universita’ degli
Studi G. D’Annunzio di Chieti e Pescara (Italy).
Keywords: acetamidines · inhibitors · inducible nitric oxide
synthases (NOS) · isoform selectivity · nitric oxide
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or ethylacetimidate hydrochloride (0.60 mmol) in EtOH (5 mL)
was added to a solution of 6a–w (0.60 mmol) in EtOH (2 mL)
at RT. After stirring for 7–10 h, the mixture was concentrated.
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with Et2O (3ꢂ15 mL) and EtOAc (2ꢂ15 mL). The aqueous layer
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hydrobromide (7r): hygroscopic yellow solid (180 mg, 75%):
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CH2NH), 4.34 (s, 2H, CH2NH), 4.40 (s, 2H, CH2NH), 7.75 (m, 8H,
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Received: March 4, 2011
Published online on May 12, 2011
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ChemMedChem 2011, 6, 1203 – 1206