1-[(Aryloxy)alkyl]-1H-imidazoles as inhibitors of neuronal nitric oxide synthase

Article abstract of DOI:10.1211/146080899128735225

A series of 1-[(aryloxy)alkyl]-1 H-imidazoles were synthesized from imidazole and various (aryloxy)alkyl bromides and tested for inhibitory activity against the three isoforms of nitric oxide synthase. 1-[2-(4-Bromophenoxy)ethyl]-1 H-imidazole and 1-[2-[4-(trifluoromethyl)phenoxy]ethyl]-1H-imidazole showed inhibitory activity against the neuronal isoform but were less potent against the endothelial isoform. Thus they could be considered interesting for their selectivity. The remaining compounds had only modest activity.

Full text of DOI:10.1211/146080899128735225

Pharm. Pharmacol. Commun. 1999, 5: 491±494
Received May 12, 1999
Accepted June 10, 1999
# 1999 Pharm. Pharmacol. Commun.
1-[(Aryloxy)alkyl]-1H-imidazoles as Inhibitors
of Neuronal Nitric Oxide Synthase
Â
L. SALERNO, V. SORRENTI*, F. GUERRERA, M. C. SARVA, M. A. SIRACUSA, C. DI GIACOMO*
AND A. VANELLA*
Department of Pharmaceutical Sciences and *Institute of Biological Chemistry,
Viale Andrea Doria 6, 95125, University of Catania, Italy
Abstract
A series of 1-[(aryloxy)alkyl]-1 H-imidazoles were synthesized from imidazole and various
(aryloxy)alkyl bromides and tested for inhibitory activity against the three isoforms of
nitric oxide synthase.
1-[2-(4-Bromophenoxy)ethyl]-1H-imidazole and 1-[2-[4-(tri¯uoromethyl)phenoxy]
ethyl]-1H-imidazole showed inhibitory activity against the neuronal isoform but were
less potent against the endothelial isoform. Thus they could be considered interesting for
their selectivity. The remaining compounds had only modest activity.
Nitric oxide (NO) is involved in several biological
functions (Kerwin et al 1995). The production of
NO results from the oxidation of L-arginine to L-
citrulline in a NADPH- and O2-dependent pathway
catalysed by the enzyme nitric oxide synthase
(NOS). Three structurally distinct NOS isoforms
have been identi®ed (Marletta 1993): the con-
stitutive endothelial NOS (eNOS) and neuronal
NOS (nNOS), and the inducible NOS (iNOS). NO
produced from eNOS is responsible for various
biological effects such as the relaxation of vascular
smooth muscle and the inhibition of platelet adhe-
sion and aggregation. NO produced from nNOS is
implicated in pain perception, mediation of long-
term potentiation and memory, and control of cer-
ebral blood ¯ow, and NO synthesized by iNOS is
implicated in the defensive response to infections.
Overproduction of NO by iNOS has been impli-
cated in various pathological processes including
septic shock, tissue damage after in¯ammation and
rheumatoid arthritis. Overproduction of NO from
nNOS has been associated with neurodegeneration
involved in stroke, seizures, schizophrenia and
Alzheimer's, Parkinson's and AIDS dementia. NO
produced from eNOS usually has only physiologi-
cal roles (Moncada et al 1991). Selective NOS
inhibitors are therefore of interest as potential
therapeutical agents (Marletta 1994).
To date, many compounds have been shown to
inhibit NOS, including mono or disubstituted
arginines, guanidines, isothioureas, amidines,
indazoles and imidazoles (Kerwin et al 1995;
Á
Macdonald 1996; Southan & Szabo 1996). The
majority of inhibitors described are non-selective or
iNOS-selective, and only a few compounds, such as
7-nitro-indazole and 1-(2-tri¯uoromethyl-phenyl)-
imidazole, selectively inhibit nNOS (Moore &
Handy 1997).
Imidazole and some of its derivatives are repor-
ted to be inhibitors of various isoforms of NOS
(Wolff et al 1993; Chabin et al 1996) and various
oxido-reductase enzymes (Rogerson et al 1976;
Kato et al 1985). We recently described the
synthesis of a series of N-phenacyl, N-phenethyl-
and N-phenyl-hydroxyethyl-imidazoles and their
inhibitory effects on NOS isoforms (Salerno et al
1999). Some of the compounds tested were con-
sidered selective inhibitors of nNOS because they
showed a signi®cant inhibitory effect against nNOS
compared with eNOS. To explore other molecules
containing the imidazole nucleus as pharmaco-
phore, we describe here the synthesis of a series of
1-[(aryloxy)alkyl]-1H-imidazoles, 3a±m, in which
various phenoxy-ethyl, -propyl or -butyl chains
were linked at the N-1 of the imidazole ring as
potential selective inhibitors of nNOS. Some of
these compounds have already been described in
the literature as thromboxane synthetase inhibitors
(3a, 3d, 3i; Cross et al 1985) or as anticonvulsants
(3c; Robertson et al 1987).
Correspondence: L. Salerno, Dipartimento di Scienze Farma-
ceutiche, Viale Andrea Doria 6, Catania 95125, Italy.
E-Mail: siracusa@mbox.unict.it

492
L. SALERNO ET AL
General procedure for the synthesis of 1-[(aryloxy)-
alkyl]-1H-imidazoles 3a±m
In this study new and previously synthesized
compounds were tested for inhibitory activity
against neuronal rat recombinant NOS (nNOS),
inducible mouse macrophage NOS (iNOS) and
endothelial human platelet NOS (eNOS).
The procedure described by Cross et al (1985) was
adopted with slight modi®cation. Sodium hydride
95% (7Á3 mmol) was added to a solution of imi-
dazole (7Á3 mmol) in dry tetrahydrofuran and the
mixture was stirred at room temperature for
approximately 15 min. A solution of the appro-
priate (aryloxy)alkyl bromides 2a±m (7Á3 mmol)
was then added and the mixture was stirred over-
night. The solvent was evaporated and the residue
was poured into water, alkalynized with 3 M sodium
hydroxide, extracted twice with dichloromethane
and dried. The organic solvent was removed under
vacuum and the residue was puri®ed by ¯ash
chromatography using silica gel and mixtures of
ethyl acetate and methanol.
Materials and Methods
Chemistry
Melting points were determined on a Buchi 510
apparatus and are uncorrected. IR spectra were
recorded on a Perkin-Elmer Model 281 spectro-
meter with KBr disk. 1H NMR spectra were
recorded on a Varian 200 MHz instrument in
CDCl3 solution. Chemical shifts are in ppm (d)
relative to tetramethylsilane as internal standard.
Elemental analyses were performed on a C. Erba
Model 1106 elemental analyser and data for C, H,
N was within 0Á4% of calculated values. Thin layer
chromatography (TLC) was performed on Merck
silica gel 60-F254 precoated aluminium plates.
Flash chromatography using Merck Silica gel 60
(0Á040±0Á063 mm) was used for preparative chroma-
tographic separations. Imidazole and phenols 1a±k
were purchased from Aldrich and (aryloxy)alkyl
bromides 2a±m were prepared according to the
procedure described by Cross et al (1985). Analy-
tical and spectral data were in agreement with data
in the literature. The complete synthetic route for
compounds 3a±m is given in Figure 1.
Proposed structures for all newly synthesized
compounds were con®rmed by elemental analyses
and H NMR. Melting points, analytical and spec-
1
tral data for previously synthesized compounds
were in agreement with data in literature.
1-[2-(4-Bromophenoxy)ethyl]-1H-imidazole (3b).
Yield 65%; mp 78±79ꢀC; 1H NMR (CDCl3): 4Á14±
4Á21 (m, 2H, CH2-O); 4Á29±4Á35 (m, 2H, N-CH2);
6Á70±6Á78 (m, 2H, Ar); 7Á02 (bs, 1H, H5 Im); 7Á07
(bs, 1H, H4 Im); 7Á33±7Á41 (m, 2H, Ar); 7Á59 (bs,
1H, H2 Im). Anal. C11H11BrN2O.
1-[2-[4-(Propyloxy)phenoxy]ethyl]-1H-imidazole
(3f).Yield 40%; mp 45±46ꢀC;1H NMR (CDCl3):
1Á01 (t, 3H, CH3); 1Á72±1Á82 (m, 2H, CH2-CH2-
CH3); 3Á85 (t, 2H, O-CH2); 4Á13±4Á17 (m, 2H,
CH2-O); 4Á18±4Á32 (m, 2H, N-CH2); 6Á75±6Á80 (m,
4H, Ar); 7Á04±7Á07 (m, 2H, H5 and H4 Im); 7Á59 (s,
1H, H2 Im). Anal. C14H18N2O2.
1-[2-[4-(Benzoyl)phenoxy]ethyl]-1H-imidazole (3g).
1
Yield 45%; mp 117±118ꢀC; H NMR (CDCl3):
4Á27±4Á32 (m, 2H, CH2-O); 4Á36±4Á41 (m, 2H, N-
CH2); 6Á91±7Á84 (m, 12H, Ar, H5, H4 and H2 Im).
Anal. C18H16N2O2.
1-[2-(4-Methylphenoxy)ethyl]-1H-imidazole
(3h). Yield 50%; mp 69±70ꢀC; 1H NMR
(CDCl3): 2Á28 (s, 3H, CH3); 4Á15±4Á21 (m, 2H,
CH2-O); 4Á32±4Á34 (m, 2H, N-CH2); 6Á73±6Á78 (m,
2H, Ar); 7Á03±7Á10 (m, 4H, Ar, H5 and H4 Im);
7Á60 (bs, 1H, H2 Im). Anal. C12H14N2O.
1-[2-[4-(Tri¯uoromethyl)phenoxy]ethyl]-1H-
imidazole (3j). Yield 65%; oil; 1H NMR (CDCl3):
4Á21±4Á27 (m, 2H, CH2-O); 4Á33±4Á39 (m, 2H, N-
CH2); 6Á90±6Á95 (m, 2H, Ar); 7Á03±7Á07 (m, 2H,
H5 and H4 Im); 7Á51±7Á57 (m, 2H, Ar); 7Á60 (bs,
1H, H2 Im). Anal. C12H11F3N2O.
Figure 1. Reagents: i. Br(CH2)nBr, K2CO3, 2-butanone,
re¯ux, 48 h; ii. imidazole, NaH, tetrahydrofuran, room
temperature.

NOS INHIBITORS
493
1-[2-[4-(Methoxy)phenoxy]ethyl]-1H-imidazole
(3k). Yield 57%; mp 48±49ꢀC;
sample contained 50 m M L-arginine, 1 mM Mg2‡
(for iNOS only), 170 mM dithiothreitol, 100 mM
NADPH, 12 mM BH4, 1 mM Ca2‡ (for nNOS and
1H NMR (CDCl3): 3Á76 (s, 3H, CH3); 4Á13±4Á19
(m, 2H, CH2-O); 4Á27±4Á33 (m, 2H, N-CH2); 6Á80±
6Á81 (m, 4H, Ar); 7Á03±7Á07 (m, 2H, H5 and H4
Im); 7Á59 (bs, 1H, H2 Im). Anal. C12H14N2O2.
1
eNOS only), 20 units mL calmodulin (for nNOS
and eNOS only), 10 mL DMSO (or the same
volume of DMSO solution of test compounds to a
®nal concentration of 500, 100 and 50 mM), enzym-
1
atic extract (1Á2 units mL for nNOS and iNOS;
cytosol derived from 1Á1 6 1011 platelets for
eNOS) and 5 mM oxyhaemoglobin in 100 mM
HEPES pH 7Á4 in a ®nal volume of 500 mL. The
HEPES buffer was pre-heated before use. NO
formed reacts with oxyhaemoglobin yielding
methaemoglobin which was measured at 401 nm.
Results were the mean (s.d. ꢁ10%) of at least
three assays.
Biology
Enzymatic assay. iNOS isolated from immuno-
stimulated mouse macrophage (RAW 264Á7) cells
and rat recombinant nNOS isolated from a Bacu-
lovirus over-expression system in SF9 cells, were
purchased from Alexis. eNOS was prepared from
washed platelets as described by Radomski et al
(1990). The assay for NOS activity was carried out
by measuring the rate of conversion of oxyhaemo-
globin to methaemoglobin using an Hitachi UV
2000 spectrophotometer. A reference cuvette was
charged with 5 mM oxyhaemoglobin (human A0
ferrous purchased from Sigma) in 100 mM HEPES
(pH 7Á4) in a ®nal volume of 500 mL. A typical
Results and Discussion
The inhibitory effect of 1-substituted imidazoles
3a±m against nNOS, iNOS and eNOS was deter-
mined by monitoring the conversion of oxyhaemo-
globin to methaemoglobin, according to Hevel &
Table 1. Inhibitory activity of compounds 3a±m (500 mM) against neuronal, inducible and endothelial nitric oxide synthase.
Compound
n
R
Inhibition (%)
iNOS
nNOS
eNOS
3aa
3b
2
2
-H
-Br
35Á77
4
25
100
0
(Ki ˆ 125 mM)
(Ki ˆ 4750 mM)
3cb
3da
3ea
3f
2
2
2
2
2
-C6H5
-COOC2H5
-NO2
-OC3H7
-COC6H5
30
15
0
10Á34
17Á2
17Á9
35Á7
64
30Á6
3g
85Á7
0
(Ki ND)
(Ki ˆ 225 mM)
3h
3ia
3j
2
2
2
-CH3
-NH2
-CF3
42Á8
20
22Á69
22Á9
100
4Á6
0
(Ki ˆ ND)
22
(Ki ˆ 45 mM)
56
3k
2
3
4
-OCH3
-H
-H
33
3lc
0
9Á6
3md
Imidazole
45
80
100
90
(Ki ˆ 185 mM)
40
(Ki ˆ 175 mM)
(Ki ˆ 60 mM)
1-Phenyl-imidazole
Nitro-arginine
60
100
(Ki ˆ 430 mM)
100
(Ki ˆ 35 mM)
100
(Ki ˆ 615 mM)
100
(Ki ˆ 0Á5 mM)
(Ki ˆ 7Á6 mM)
(Ki ˆ 0Á5 mM)
b
aAccording to Cross et al (1985). According to Robertson et al (1987) (percentage inhibition is expressed at 50 mM because of
the very low solubility at higher concentrations). cAccording to Dou & Metzger (1976). dAccording to Shimazu et al (1996). Ki was
not calculated for 3g and 3j because they are insoluble at concentrations greater than 500 mM.

494
L. SALERNO ET AL
Cross, P., Dickinson, R., Parry, M., Randall, M. (1985)
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Dixon, M. (1953) The determination of enzyme inhibitor
constants. Biochem. J. 55: 170±171
Marletta (1994). Table 1 shows the percentage
inhibition of NOS activity obtained at a dose of
500 mM. Inhibition constants were calculated for
compounds showing a high degree of inhibition, by
measuring the percentage inhibition of at least three
concentrations of the inhibitor according to Dixon
(1953). Inhibition of eNOS was determined
for only the most active compounds. Imidazole,
1-phenyl-imidazole and nitro-arginine were used as
references in all cases.
From the Ki values (Table 1), it is apparent that
some compounds (3b and 3j), showed a consider-
able degree of selectivity for nNOS. Although less
potent than nitro-arginine, they were more selec-
tive. In addition, they had greater activity against
nNOS than imidazole and 1-phenyl-imidazole,
suggesting that the introduction of an aryloxyalkyl
chain contributes to the inhibition of nNOS.
Structure±activity relationship studies on the
in¯uence on biological activity of the para sub-
stituent on the phenylic ring, suggest the impor-
tance of hydrophobic moieties. Compounds 3b (4-
Br) and 3j (4-CF3) were the most active. This dif-
fers from a previous study on the N-phenacyl ser-
ies, where the most active compound was the
derivative carrying a hydrophilic group (4-NO2)
(Salerno et al 1999).
Dou, H. J. M., Metzger, J. (1976) Catalyse par transfert de
Á
Á Á
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(1985) Thromboxane synthetase inhibitors (TXSI). Design,
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All compounds tested were poor inhibitors of the
inducible NOS.
In conclusion, 3b and 3j are interesting com-
pounds which could be considered for their selec-
tivity for nNOS over eNOS. Further investigation is
necessary to identify the structural features of these
imidazole derivatives and to elucidate their
mechanism of action.
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