N-(imidazolidin-2-ylidene)-1-arylmethanamine oxides: Synthesis, structure and pharmacological evaluation

Article abstract of DOI:10.1002/ardp.201100028

A high yielding three-step procedure was applied for the synthesis of N-(imidazolidin-2-ylidene)-1-arylmethanamine oxides 3 (α-aminonitrones) starting from the easily accessible imidazolidin-2-one O-benzyl oxime 1. The α-aminonitrone-α-iminohydroxyloamine tautomerism of these products was studied theoretically and the structures of the synthesised compounds were confirmed by single crystal X-ray crystallographic analysis. The compounds were evaluated in vitro for their binding affinities to α₁ and α₂ adrenoceptors as well as imidazoline I₁ and I₂ receptors. The highest potencies at the α₂ adrenergic receptors were observed for compounds bearing biphenyl (4h, K _i = 9 nM) and naphthyl (4i, K_i = 92 nM) moieties. Compounds 4h and 4i were further tested in vivo for their cardiovascular and sedative-hypnotic effects in rats. A series of N-(imidazolidin-2-ylidene)-1- arylmethanamine oxides (α-aminonitrones) was prepared and evaluated in vitro for their binding affinities to α-adrenergic and imidazoline receptors. The most potent compounds were tested in vivo for their cardiovascular and sedative-hypnotic effects in rats. The α-aminonitrone- α-iminohydroxylamine tautomerism and the structures of the products were studied using theoretical methods and single crystal X-ray crystallographic analysis Copyright

Full text of DOI:10.1002/ardp.201100028

Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
33
Full Paper
N-(Imidazolidin-2-ylidene)-1-arylmethanamine Oxides:
Synthesis, Structure and Pharmacological Evaluation
1
2
2
3
3
Jarosław Saczewski , Alan Hudson , Shayna Laird , Apolonia Rybczynska , Konrad Boblewski ,
´
Artur Lehmann3, Daqing Ma4, Mervyn Maze4, Helena Watts4, and Maria Gdaniec5
1
´
Department of Chemical Technology of Drugs, Medical University of Gdansk, Gdansk, Poland
2 Department of Pharmacology, University of Alberta, Edmonton, Canada
3 Department of Pathophysiology, Medical University of Gdansk, Gdansk, Poland
4 Department of Surgery and Cancer, Imperial College, London, UK
5 Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland
A high yielding three-step procedure was applied for the synthesis of N-(imidazolidin-2-ylidene)-1-
arylmethanamine oxides 3 (a-aminonitrones) starting from the easily accessible imidazolidin-2-one
O-benzyl oxime 1. The a-aminonitrone–a-iminohydroxyloamine tautomerism of these products was
studied theoretically and the structures of the synthesised compounds were confirmed by single
crystal X-ray crystallographic analysis. The compounds were evaluated in vitro for their binding
affinities to a1 and a2 adrenoceptors as well as imidazoline I1 and I2 receptors. The highest
potencies at the a2 adrenergic receptors were observed for compounds bearing biphenyl
(4h, Ki ¼ 9 nM) and naphthyl (4i, Ki ¼ 92 nM) moieties. Compounds 4h and 4i were further tested
in vivo for their cardiovascular and sedative-hypnotic effects in rats.
Keywords: Alpha-aminonitrones / Alpha-adrenergic receptors / Alpha-iminohydroxylamines / Imidazolines / Imidazoline
receptors
Received: January 26, 2011; Revised: February 24, 2011; Accepted: March 2, 2011
DOI 10.1002/ardp.201100028
Introduction
phenomenon, impotence, obesity and noninsulin-dependent
diabetes [5].
It is well known that imidazoline-containing compounds
such as clonidine, phentolamine and naphazoline constitute
the most vital set of a-adrenergic receptor ligands. The
medical applications of these drugs mediated through acti-
vation of central a2 adrenergic autoreceptors include treat-
ment of hypertension, suppression of opiate withdrawal,
Specific imidazoline receptors have been proposed to be
responsible for various pharmacological effects of imidazo-
line-containing drugs. These receptors constitute a heteroge-
nous group of proteins which are divided into I1, I2 and I3
binding sites [6]. Much work has been focused on the syn-
thesis of selective agents acting at I2 binding sites which
recently have been identified within the brain creatine
kinase enzyme (B-CK). Selective I2 ligands may affect dopa-
mine, 5-HT and noradrenaline release, and therefore can be
used in the treatment of various psychiatric disorders includ-
ing depression and Alzheimer’s disease [7].
anaesthesia,
attention-deficit
hyperactivity
disorder
(ADHD), short statue, glaucoma and diarrhea [1–3]. In
addition, a2 adrenergic agonists have shown organoprotec-
tive effects in various experimental models, and therefore
attracted attention for the treatment of mechanically venti-
lated patients with sepsis [4]. Furthermore, a2 antagonists
have found clinical uses for the treatment of Raynaud’s
The major drawback of these drugs is lack of selectivity at
either a1 and a2 adrenoceptors or imidazoline I1 and I2
receptors [8] which often results in undesired side effects.
For this reason, the search for novel ligands with greater
selectivity is of immense clinical interest [9].
Correspondence: Dr. Jarosław Saczewski, Medical University of Gdansk,
Department of Chemical Technology of Drugs, Al. Gen. J. Hallera 107,
80-416 Gdansk, Poland.
E-mail: js@gumed.edu.pl
Fax: þ48 58 3493257
A typical a2 adrenoceptor ligand consists of an imidazoline
ring attached to an aryl group via a one- or two-atom spacer
(structure A, Figure 1). Previous reports from our laboratories
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34
J. Saczewski et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
have dealt with the discovery of novel imidazoline-containing
agents with a two-atom spacer incorporated into the 1H- or
2H-indazole ring system represented by marsanidine and
character and basicity of the guanidine compounds compared
to classic 2-aryliminoimidazolidines [17, 18].
its analogues which proved to be highly selective at a2 Results and discussion
adrenoceptors [10, 11] and 4-Cl-indazim with selectivity to
imidazoline I2 receptors [12, 13] (Figure 1). Other modifi-
cations that have been investigated include the placement
of the hydroxyl group at the exocyclic nitrogen atom of
2-arylaminoimidazolines leading to structures of type B
(Figure 1) that exhibited in vitro affinity to a2 adrenoceptors
and inhibited human blood platelet aggregation [14], while
in vivo they exhibited moderate hypotensive and tachycardic
effects [15, 16].
Chemistry
Previously, the compounds of type B were obtained by
reacting 2-chloroimidazoline with the corresponding N-aryl-
hydroxylamines [14]; however, analogous reactions of
N-benzylhydroxylamines were unsuccessful due to O-hetero-
alkylation and subsequent retro-ene reactions giving rise to
the formation of aldimine and imidazolidin-2-one [19].
The synthetic approach to hydroxylamine derivatives 3 is
based on our previous observation that O-sulfonyl imidazo-
lidin-2-one oxime undergoes regioselective benzylation at the
exocyclic N2 nitrogen atom [20]. As shown in Scheme 1,
similar reaction of the previously obtained O-benzyl imida-
zolidin-2-one oxime 1 [21] with variously substituted benzyl
and benzhydryl halides produces O-benzyl-N-(4,5-dihydro-1H-
imidazol-2-yl)-N-[alkyl(aryl)]-hydroxylamines 2a–m in high
yields (85–90%). Later compounds were hydrogenated with
use of gaseous hydrogen in the presence of 10% Pd/C to give
the desired debenzylated products 3a–m in excellent yields
(95–99%). Finally, upon treatment of the free bases 3 with a
methanolic solution of hydrochloride or hydrobromide, the
Our present goal was to develop a facile synthetic route to
N-(4,5-dihydro-1H-imidazol-2-yl)-N-(arylmethyl)hydroxylamines
C (Figure 1) to probe the structural requirements for a1 and a2
adrenoceptors as well as imidazoline I1 and I2 receptors.
Chemical modifications included aliphatic and aromatic sub-
stitution at the benzylic carbon atom. The elongation of the
spacer between the aryl and the N-iminoimidazolidine moi-
eties would result in less rigid structures, while the placement
of the hydroxyl group would diminish both the lipophilic
H
N
H
N
Ar
OH
X
N
N
N
Ar
A
B
H
N
H
N
O
Bn
O
R
Bn
c
a, b
N
N
X = NH, CH2, N-CH(R',R'')
Ar = phenyl, naphthyl, heteroaryl
N
H
N
1
2a-m
H
N
H
N
H
N
O
OH
R
OH
R
d
H
N
H
N
O
N
N
N
Y
OH
N
H
N
N
H
R
N
N
R
N
Ar
N
H
C
3a-m
D
4a-m
Ar
R
CH2
e, R =
OCH3
CH2
CH2
CH2
C
D
a, R =
b, R =
i, R =
j, R =
OCH3
α-iminohydroxylamine
α-aminonitrone
CH2
CH2
f
, R =
R = H, alkyl, aryl
Cl
Ar = phenyl, naphthyl
Cl
CH
H3C
CH2
Cl
Cl
H
N
k
, R =
CH2
c, R =
d, R =
g, R =
h, R =
N
H
N
N
N
CH
2
H2C
l, R =
N
N
N
CH2
CH3
CH3
HN
CH
H3C
CH
m, R =
H2C
H3C
marsanidine
4-Cl-indazim
α2-selective agent
I2-selective agent
Scheme 1. Reagents and conditions. (a) RX, DMF, 12 h;
(b) K2CO3; (c) H2, 10% Pd/C; (d) HCl or HBr. X ¼ Br for a–h and
j–m. X ¼ Cl for i. Y ¼ Cl for a–h, j, l and m. Y ¼ Br for i and k.
Figure 1. Imidazoline derivatives with activity at a-adrenergic
and/or imidazoline receptors.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
a-Iminohydroxylamine vs a-Aminonitrone
35
further confirmed by the presence of single resonance
attributable to C4 and C5 imidazolidine carbon atoms in
the 13C-NMR spectrum at 45.1 ppm. Moreover, single crystal
X-ray analysis of the free base 3a obtained by crystallization
from methanol confirmed that in the crystalline phase the
a-aminonitrone form D exists (Figure 2).
To elucidate the possible tautomeric equilibrium we esti-
mated the relative Gibbs free-energies DG (298.15 K) and
dipole moments for 3a tautomers: C (a-iminohydroxylamine)
and D (a-aminonitrone). Calculations were performed using
ab initio Hartree-Fock and DFT methods [23] both in the gas
phase and using SM8 solvation models [24]. From the data
presented in Table 1 one can infer that in polar solvents the
formation of the a-aminonitrone D is energetically favorable.
Nitrone D with higher dipole moment than those found for
hydroxylamine C should also be better stabilized in polar
solvents.
Basicity (pKa value) of the free base 3a was determined by
potentiometric titration. It appeared that the placement of
the hydroxyl group at the nitrogen atom of the guanidine
moiety resulted in the compound with diminished basicity
(pKa ¼ 7.7) compared to classic 2-aryliminoimidazolidines
such as clonidine (pKa ¼ 8.2). Thus, under physiological con-
ditions (pKa ¼ 7.4) compound 3a exists at about 66% in the
free base form D leaving about 34% for the protonated form
of type 4a. The structure of the hydroxylamine hydrochloride
4a resulting from protonation of the oxygen atom of the
nitrone D was confirmed by single crystal X-ray analysis
(Figure 2).
Figure 2. View of the molecular structures of 3a (top) and 4a (bot-
tom) with displacement ellipsoids drawn at the 50% probability level.
water soluble addition salts 4a–m suitable for biological
evaluation were obtained.
Biology
By analogy to the previously investigated N-arylhydroxyl-
amines of type B [22], compounds 3 may exist as a mixture of
two tautomeric forms: a-iminohydroxylamine (C) and a-ami-
nonitrone (D). We therefore felt it necessary to investigate in
detail the structure of both the free base 3a and correspond-
ing salt 4a by means of experimental methods and quantum-
All the obtained compounds of type 4 have been evaluated for
their affinities to a1 and a2 adrenoceptors as well as to
imidazoline I1 and I2 receptors (Table 2). Radioligand binding
assays were performed using P2 membranes prepared from
brains of male Sprague-Dawley rats (for a1, a2 and I2 binding
sites) or crude P2 membranes obtained from kidneys of male
Sprague-Dawley rats (for I2 binding sites). The benzyl deriva-
tives 4a–e exhibited very weak micromolar affinities to
a-adrenergic receptors and imidazoline I1 receptors while
the 4-chloro congener 4b showed a moderate affinity to
1
chemical calculations. Thus, in the H-NMR spectrum of 3a
run in CD3OD a singlet at 3.67 ppm integrated to four
CH2CH2 protons of imidazolidine moiety indicates the pres-
ence of the a-aminonitrone tautomer D. This observation is
Table 1. The relative Gibbs free-energies DG (298.15 K) and dipole moments for 3a tautomers C (a-iminohydroxylamine) and D
(a-aminonitrone) calculated in vacuum and using methanol or water SM8 [24] solvation models.
Method
Vacuum
Methanol
Water
C
D
C
D
C
D
HF/6-31þGꢀ
B3LYP/6-31þGꢀ
DG [kcal/mol]
0
3.54
0
5.4
6.69
0.9
2.5
4.73
3.8
0
9.00
0
1.5
4.69
4.1
0
9.05
0
dipole moment [Debye]
DG [kcal/mol]
dipole moment [Debye]
3.73
5.80
5.14
8.48
5.22
8.55
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36
J. Saczewski et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
Table 2. Experimental binding affinities of salts 4 to a-adrenergic and imidazoline receptors.
Compd 4
R
a1 receptor
Ki (nM)a
a2 receptor
Ki (nM)a
I1 receptor
IC50 (nM)b
I2 receptor
Ki (nM)a
CH2
a
18 100
5440
5150
8260
2210
303
CH2
b
42 600
72.7
Cl
Cl
c
6340
2010
1700
5800
955
2340
5160
CH2
Cl
CH2
d
15 800
CH3
CH3
H3C
CH2
OCH3
e
f
7130
970
19 700
3850
910
105 000
7090
12 000
25 400
794
OCH3
CH2
Ph
CH2
g
h
i
373
77 800
1020
Ph
CH
31
9
46
137
92
1640
41.5
CH2
CH2
j
1040
8680
2090
2660
4880
3220
5990
8500
87 300
144 000
80 600
NDc
2570
38 200
49.4
CH
k
l
H3C
H2CCH2
H3C
H2C
m
517
CH
a
Ki affinity values for a1-adrenoreceptors, a2-adrenoreceptors and I2 imidazoline binding sites were assessed by measuring the ability
of the test compounds to displace [3H]prazosin (rat brain membranes), [3H]RX821002 (rat brain membranes) or [3H]2BFI (rat brain
membranes), respectively.
Molar concentration of the test compounds that displaces 50% of specifically bound [3H]clonidine (rat kidney membranes).
b
c
ND – no displacement
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Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
a-Iminohydroxylamine vs a-Aminonitrone
37
the imidazoline I2 receptor (Ki ¼ 72.7 nM). Poor activity was
also observed for compounds 4f–g incorporating the
diphenyl moiety. The most interesting results, however, were
obtained for lipophilic derivatives 4h–j. Thus, the benzhydryl
congener 4h proved to be a non-selective ligand of a2, a1 and
I2 receptors (Ki values ¼ 9 nM, 31 nM and 46 nM, respect-
ively). When the benzhydryl scaffold was replaced by a 1-
naphthyl structure (4i), the affinity at a1 and a2 adrenocep-
tors was reduced while the activity at the I2 receptor was
retained. In turn, a change from 1-naphthyl (4i) to 2-naphthyl
(4j) structure led to a considerable decrease of activity.
Interestingly enough, elongation of the aliphatic bridge,
i.e. going from N-benzyl derivative 4a to N-phenylethyl deriva-
tive 4l, resulted in an increase of the binding affinity to
imidazoline I2 receptors (Ki ¼ 49 nM vs. 303 nM) which have
been associated with various CNS disorders, such as
Alzheimer’s disease, Huntington’s disease, depression, and
malignancy of human gliomas [25, 26].
to imidazoline I2 receptors. Of special interest are the
selective I2 receptor ligands, i.e. compounds 4b and 4l which,
due to low lipophilicity, are not expected to cross the blood-
brain barrier, and therefore could find applications for the
treatment of vascular hyperplasia [28] or as enhancers of
supraspinal morphine analgesia [29].
The most active compound at a1 and a2 adrenoceptors was
4h which did not show the haemodynamic and sedative
effects. Such pharmacological profile may prove beneficial
for therapeutic applications other than the treatment of
hypertension.
Finally, it is worth noting that the free bases of type 3
bearing nitrone moiety may be regarded as a potential neuro-
protective agent in preventing the death of cells exposed to
enhanced oxidative stress and damage [30]. Further studies
are planned to confirm the above assumptions.
Experimental
The compounds most active at a1 and a2 adrenoceptors, i.e.
4h and 4i, were then evaluated in anaesthetized male Wistar
rats for possible cardiovascular effects. The phentolamine
congener 4h administered intravenously did not affect the
mean arterial blood pressure (MAP) at doses up to 0.1 mg/kg,
however, at that dose it lowered the heart rate (HR) by 30
bpm. As phentolamine blocks a1 adrenergic receptors, lowers
blood pressure and in consequence increases the heart rate
[27], the mechanism underlying the bradycardic action of 4h
should be different and may involve b1 adrenergic receptor
blockage. The naphazoline analogue 4i administered i.v. at
dose 1 mg/kg showed biphasic effect on blood pressure:
initial hypertension (DMAP ¼ 14 mmHg) followed by a pro-
longed hypotension (DMAP ¼ 15 mmHg). Apparently, the
transient vasoconstriction is probably mediated by vascular
a2B adrenoceptors or results from peripheral a1 agonist
activity. Upon crossing the blood-brain barrier, compound
4i elicits a hypotensive effect, which is characteristic of the
centrally acting a2 adrenoceptor agonists.
Melting points were measured on a Boetius 524 apparatus and
are uncorrected. 1H- and 13C-NMR spectra were recorded using a
Varian Unity 200 apparatus at 200 MHz and 50 MHz, respect-
ively, using TMS as an internal standard. Results of C,H,N elemen-
tal analyses were within ꢁ0.4% of theoretical values.
Chemistry
O-Benzyl-N-(arylalkyl)-N-(4,5-dihydro-1H-imidazol-2-yl)-
hydroxylamines 2
General procedure: Imidazolidin-2-one O-benzyl oxime [21]
(0.5 g, 2.61 mmol) and the corresponding arylalkyl bromide or
chloride (2.61 mmol) were dissolved in DMF (2 mL) and stirred
overnight. The resulting suspension was evaporated under
reduced pressure, alkalized with 5% NaOH solution (5 mL) and
extracted with methylene chloride (3 ꢂ 10 mL). The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and evaporated. The oily residue was purified on silica with use
of chromatotron (ethyl acetate/methanol 95:5). Yields 85–90%.
N,O-Dibenzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-
hydroxylamine 2a
Compounds 4h and 4i were further tested for the ability to
activate the non-REM sleep pathway in rats which was deter-
mined by the loss of righting reflex (LORR) test (see
Experimental for details). Both compounds were well toler-
ated at the doses tested (5–500 mg/kg), however, no LORR was
observed for either compound. Thus, we found no evidence
for induction of a sedative-hypnotic state following intraper-
itoneal injection of 4h and 4i.
1
M.p.: 108–110oC; H-NMR (500 MHz, CDCl3) d 3.63 (4H, bs, CH2),
4.51 (2H, s, CH2), 4.65 (2H, s, CH2), 4.70 (1H, bs, NH), 7.16–7.20
(2H, m, CH), 7.31–7.36 (4H, m, CH), 7.39 (2H, t, J ¼ 7.2 Hz, CH),
7.49 (2H, d, J ¼ 7.2 Hz, CH); 13C-NMR (125 MHz, CDCl3) d 46.0,
57.4, 78.4, 128.0, 128.6, 128.8, 128.9, 129.5, 130.1, 135.6, 136.8,
167.4; IR (KBr, cmꢃ1) 3139, 3064, 2948, 2848, 1592, 1582, 1497,
1454, 1277, 1089, 1002, 990, 840, 749, 698.
O-Benzyl-N-(4-chlorobenzyl)-N-(4,5-dihydro-1H-imidazol-
2-yl)hydroxylamine 2b
M.p.: 76–78oC; 1H-NMR (200 MHz, CDCl3) d 3.65 (4H, bs, CH2), 4.56
(2H, s, CH2), 4.59 (2H, s, CH2), 4.70 (1H, bs, NH), 7.15–7.45 (9H, m,
CH); 13C-NMR (50 MHz, CDCl3) d 46.0, 56.8, 78.6, 128.9, 129.1,
Conclusion
129.2, 129.7, 131.6, 134.0, 135.6, 135.8, 167.3; IR (KBr, cmꢃ1
)
All the synthesized congeners of the series 4a–m exhibited
very weak binding affinities to imidazoline I1 receptors.
Compounds 4b, 4h, 4i and 4l demonstrated good affinity
3184, 3031, 2933, 2870, 1603, 1498, 1283, 1088, 800, 752, 734,
698.
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38
J. Saczewski et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
O-Benzyl-N-(2,6-dichlorobenzyl)-N-(4,5-dihydro-1H-
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
imidazol-2-yl)hydroxylamine 2c
(naphthalen-1-ylmethyl)hydroxylamine 2i
M.p.: 140–1448C; 1H-NMR (500 MHz, CDCl3) d 3.40–3.49 (2H, m,
CH2), 3.85–3.94 (2H, m, CH2), 4.33 (2H, s, CH2), 4.59 (1H, bs, NH),
5.01 (2H, s, CH2), 7.13–7.17 (2H, m, CH), 7.26 (1H, t, J ¼ 7.8 Hz,
CH), 7.30–7.34 (3H, m, CH), 7.40 (2H, d, J ¼ 7.8 Hz, CH); 13C-NMR
(125 MHz, CDCl3) d 45.0, 52.6, 79.1, 128.7, 128.8, 128.9, 129.5,
130.0, 132.3, 135.2, 137.8, 167.3; IR (KBr, cmꢃ1) 3155, 2981, 2922,
2856, 1604, 1562, 1491, 1459, 1437, 1279, 1091, 1003, 991, 780,
754, 692.
Obtained as an oil, 1H-NMR (500 MHz, CDCl3) d 3.71 (4H, bs, CH2),
4.13 (2H, s, CH2), 5.16 (2H, s, CH2), 6.94–6.98 (2H, m, CH),
7.22–7.30 (3H, m, CH), 7.49–7.64 (4H, m, CH), 7.88–7.93
(2H, m, CH), 8.32–8.35 (1H, m, CH).
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
(naphthalen-2-ylmethyl)hydroxylamine 2j
Obtained as an oil; 1H-NMR (200 MHz, CDCl3) d 3.65 (4H, s, CH2),
4.51 (2H, s, CH2), 4.60 (1H, bs, NH), 4.82 (2H, s, CH2), 7.09–7.18
(2H, m, CH), 7.22–7.32 (3H, m, CH), 7.43–7.55 (2H, m, CH),
7.61–7.68 (1H, m, CH), 7.80–7.91 (4H, m, CH); 13C-NMR
(50 MHz, CDCl3) d 45.0, 57.9, 78.7, 126.4, 126.6, 128.1, 128.2,
128.4, 128.5, 129.0, 129.1, 129.2, 129.7, 133.4, 133.8, 134.6, 135.8,
167.5.
O-Benzyl-N-(4-tert-butylbenzyl)-N-(4,5-dihydro-1H-
imidazol-2-yl)hydroxylamine 2d
M.p.: 96–988C; 1H-NMR (500 MHz, CDCl3) d 1.36 (9H, s, CH3), 3.64
(4H, bs, CH2), 4.54 (2H, s, CH2), 4.60 (1H, bs, NH), 4.63 (2H, s, CH2),
7.15–7.17 (2H, m, CH), 7.33–7.35 (4H, m, CH), 7.41–7.43 (3H, m,
CH); 13C-NMR (50 MHz, CDCl3) d 31.9, 35.0, 45.0, 57.2, 78.7, 125.7,
129.0, 129.2, 129.8, 130.1, 133.8, 135.8, 151.1, 167.4; IR (KBr,
cmꢃ1) 3172, 2962, 2865, 1595, 1492, 1457, 1280, 1007, 986,
830, 757, 749, 698.
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
(1-phenylethyl)hydroxylamine 2k
M.p.: 72–738C; 1H-NMR (200 MHz, CDCl3) d 1.62 (3H, d, J ¼ 6.8 Hz,
CH3), 3.61 (4H, s, CH2), 4.35 (1H, bs, NH), 4.50–4.60 (2H, m, CH2),
5.34 (1H, q, J ¼ 6.8 Hz, CH), 7.15–7.22 (2H, m, CH), 7.29–7.43
(6H, m, CH), 7.52–7.60 (2H, m, CH); 13C-NMR (50 MHz, CDCl3)
d 15.2, 45.0, 61.3, 79.5, 128.1, 128.6, 129.0, 129.1 (two signals),
129.6, 135.9, 140.8, 167.0; IR (KBr, cmꢃ1) 3156, 3030, 2934, 2880,
1591, 1497, 1453, 1371, 1279, 989, 752, 699.
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
(3,5-dimethoxybenzyl)hydroxylamine 2e
M.p.: 66–688C; 1H-NMR (200 MHz, CDCl3) d 3.63 (4H, bs, CH2), 3.79
(6H, s, OCH3), 4.15 (1H, bs, NH), 4.58 (2H, s, CH2), 4.59 (2H, s, CH2),
6.43 (1H, t, J ¼ 2.3 Hz, CH), 6.64 (2H, d, J ¼ 2.3 Hz, CH), 7.19–7.35
(5H, m, CH); 13C-NMR (50 MHz, CDCl3) d 44.5, 55.8, 57.9, 78.7,
100.5, 108.0, 129.0, 129.1, 129.7, 135.9, 139.4, 161.2, 167.5; IR
(KBr, cmꢃ1) 3157, 2946, 2846, 1597, 1463, 1345, 1283, 1203, 1146,
1052, 824, 752, 700.
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
phenethylhydroxylamine 2l
M.p.: 76–798C; 1H-NMR (500 MHz, CDCl3) d 3.02 (2H, t, J ¼ 7.5 Hz,
CH2), 3.59 (4H, bs, CH2), 3.78 (2H, t, J ¼ 7.5 Hz), 4.84 (2H, s, OCH2),
4.86 (1H, bs, NH), 7.15–7.45 (10H, m, CH); 13C-NMR (50 MHz,
CDCl3) d 32.4, 42.8, 48.0, 77.5, 126.2, 128.4, 128.7, 128.8,
129.0, 129.1, 135.6, 139.2, 166.1; IR (KBr, cmꢃ1) 3234, 3024,
2930, 2867, 1645, 1607, 1494, 1458, 1374, 1275, 1215, 1063,
1005, 740, 697.
O-Benzyl-N-(biphenyl-2-ylmethyl)-N-(4,5-dihydro-1H-
imidazol-2-yl)hydroxylamine 2f
M.p.: 165–1668C; 1H-NMR (200 MHz, DMSO-d6) d 3.41 (4H, bs,
CH2), 4.48 (2H, s, CH2), 4.50 (2H, s, CH2), 6.00 (1H, bs, NH),
7.03–7.07 (2H, m, CH), 7.25–7.40 (11H, m, CH), 7.59–7.61
(1H, m, CH); 13C-NMR (50 MHz, DMSO-d6) d 45.2, 53.8, 77.1,
127.3, 127.4, 127.5, 128.3 (two signals), 128.4, 129.3, 129.5,
O-Benzyl-N-(4,5-dihydro-1H-imidazol-2-yl)-N-
130.0, 130.1, 134.4, 135.7, 140.8, 142.1, 166.4; IR (KBr, cmꢃ1
3167, 2956, 2854, 1590, 1492, 1456, 1278, 1004, 991, 758, 699.
)
(2-phenylpropyl)hydroxylamine 2m
Obtained as an oil; 1H-NMR (200 MHz, CDCl3) d 1.34 (3H, d,
J ¼ 7.3 Hz, CH3), 3.29 (1H, q, J ¼ 7.3 Hz, CH), 3.52 (4H, s, CH2),
3.75 (2H, d, J ¼ 7.3 Hz, CH2), 4.78 (1H, bs, NH), 4.62–4.78 (2H, m,
OCH2), 7.18–7.40 (10H, m, CH); 13C-NMR (50 MHz, CDCl3) d 20.5,
38.3, 49.5, 60.6, 77.8, 126.8, 128.0, 128.9, 129.1 (two signals),
129.5, 136.1, 145.4, 167.5.
O-Benzyl-N-(biphenyl-3-ylmethyl)-N-(4,5-dihydro-1H-
imidazol-2-yl)hydroxylamine 2g
Obtained as an oil; 1H-NMR (200 MHz, CDCl3) d 3.64 (4H, bs, CH2),
4.45 (1H, bs, NH), 4.56 (2H, s, CH2), 4.71 (2H, s, CH2), 7.14–7.72
(14H, m, CH); 13C-NMR (50 MHz, CDCl3) d 45.0, 57.7, 78.7, 127.0,
127.7, 127.8, 129.1, 129.2 (two signals), 129.3 (two signals), 129.8
(two signals), 135.8, 137.5, 141.4, 141.7, 167.5.
N-(Imidazolidin-2-ylidene)-1-arylalkylamine oxides 3
General procedure: The corresponding hydroxylamine 2 (0.5 g)
was dissolved in methanol (5 mL). The substrate was hydrogen-
ated in the presence of 10% Pd/C (0.05 g) by passing gaseous
hydrogen through the solution (1 h) or with use of pressurized
reactor (0.5 h). The progress of the reaction was monitored by
TLC. The filtration and evaporation of the reaction mixture
furnished a solid residue which upon rinsing with diethyl ether
gave pure product 3. Yields 95–99%. The free bases 3 thus
obtained were converted into corresponding addition salts 4
upon treatment with methanolic solutions of gaseous hydrochlo-
ride or hydrobromide.
N-Benzhydryl-O-benzyl-N-(4,5-dihydro-1H-imidazol-2-
yl)hydroxylamine 2h
M.p.: 118–1208C; 1H-NMR (500 MHz, CDCl3) d 3.28–3.37 (2H, m,
CH2), 3.72–3.81 (2H, m, CH2), 4.30 (2H, bs, CH2), 4.68 (1H, bs, NH),
6.49 (1H, s, CH2), 7.10–7.12 (2H, m, CH), 7.29–7.36 (9H, m, CH),
7.44–7.46 (4H, m, CH); 13C-NMR (125 MHz, CDCl3) d 45.3, 54.4,
69.2, 79.1, 127.5, 128.4, 128.7, 128.8, 129.4, 129.8, 135.5, 139.7,
166.3; IR (KBr, cmꢃ1) 3159, 3062, 3027, 2940, 2870, 1605, 1494,
1454, 1285, 1001, 750, 723, 699.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
a-Iminohydroxylamine vs a-Aminonitrone
39
N-(Imidazolidin-2-ylidene)-1-phenylmethanamine oxide 3a Hydrochloride of 1-(4-tert-butylphenyl)-N-(imidazolidin-2-
1
M.p.: 192–1978C; H-NMR (200 MHz, CD3OD) d 3.67 (4H, s, CH2),
4.63 (2H, s, CH2), 4.94 (2H, s, NH), 7.33–7.37 (5H, m, CH); 13C-NMR
(50 MHz, CD3OD) d 45.1, 57.6, 129.1, 129.3, 129.9, 137.1, 158.8; IR
(KBr, cmꢃ1) 3028, 2858, 2656, 1694, 1519, 1494, 1480, 1290, 1264,
1111, 741, 702.
ylidene)methanamine oxide 4d
M.p.: 178–1808C; 1H-NMR (200 MHz, DMSO-d6) d 1.27 (9H, s, CH3),
3.67 (4H, s, CH2), 4.82 (2H, s, CH2), 7.29 (2H, d, J ¼ 8.3 Hz, CH),
7.39 (2H, d, J ¼ 8.3 Hz, CH); 8.87 (bs, 2H, NH), 11.14 (s, 1H, OH);
13C-NMR (50 MHz, DMSO-d6) d 31.4, 34.6, 43.4, 55.6, 125.5, 128.5,
132.1, 150.7, 160.6. Anal. calcd. for C14H22ClN3O: C, 59.25;
H, 7.81; N, 14.81; found: C, 59.01; H, 8.06; N, 14.69.
Hydrochloride of N-(imidazolidin-2-ylidene)-1-
phenylmethanamine oxide 4a
M.p.: 129–1318C; 1H-NMR (500 MHz, DMSO-d6) d 3.69 (4H, s, CH2),
4.87 (2H, s, CH2), 4.94 (2H, s, NH), 7.33–7.39 (5H, m, CH), 8.98 (2H,
bs, NH), 11.17 (1H, s, OH); 13C-NMR (125 MHz, DMSO-d6) d 43.9,
56.3, 128.7, 129.1, 129.2, 135.5, 161.1. Anal. calcd. for
C10H14ClN3O: C, 52.75; H, 6.20; N, 18.45; found: C, 52.44; H,
6.51; N, 18.31.
1-(3,5-Dimethoxyphenyl)-N-(imidazolidin-2-ylidene)-
methanamine oxide 3e
M.p.: 172–1758C.
Hydrochloride of 1-(3,5-dimethoxyphenyl)-N-(imidazolidin-
2-ylidene)methanamine oxide 4e
M.p.: 179–1818C; 1H-NMR (200 MHz, DMSO-d6) d 3.68 (4H, s, CH2),
3.74 (6H, s, OCH3), 4.78 (2H, s, CH2), 6.45–6.47 (1H, m, CH),
6.54–6.55 (2H, m, CH), 8.96 (2H, bs, NH), 11.18 (1H, s, OH);
13C-NMR (50 MHz, DMSO-d6) d 43.4, 55.5, 56.0, 99.7, 106.6,
137.2, 160.8 (two signals); IR (KBr, cmꢃ1) 3122, 2844, 1647,
1629, 1609, 1597, 1474, 1429, 1351, 1208, 1164, 1069, 833.
Anal. calcd. for C12H18ClN3O3: C, 50.09; H, 6.31; N, 14.60; found:
C, 49.98; H, 6.68; N, 14.39.
1-(4-Chlorophenyl)-N-(imidazolidin-2-ylidene)-
methanamine oxide 3b
1
M.p.: 159–1638C; H-NMR (500 MHz, CD3OD) d 3.70 (4H, s, CH2),
4.62 (2H, s, CH2), 4.97 (2H, s, NH), 7.34–7.40 (4H, m, CH); 13C-NMR
(50 MHz, CD3OD) d 45.1, 56.9, 129.9, 131.1, 134.9, 135.9, 159.3; IR
(KBr, cmꢃ1) 3058, 2886, 1686, 1522, 1489, 1292, 1113, 1017, 801.
Hydrochloride of 1-(4-chlorophenyl)-N-(imidazolidin-2-
ylidene)methanamine oxide 4b
1-(Biphenyl-2-yl)-N-(imidazolidin-2-ylidene)methanamine
oxide 3f
Obtained as an oil.
M.p.: 155–1608C; 1H-NMR (200 MHz, DMSO-d6) d 3.67 (4H, s, CH2),
4.87 (2H, s, CH2), 7.36–7.48 (4H, m, CH), 9.01 (2H, bs, NH), 11.18
(1H, bs, OH); 13C-NMR (50 MHz, DMSO-d6) d 43.5, 55.2, 128.7,
130.7, 132.9, 134.0, 160.7; IR (KBr, cmꢃ1) 3198, 3123, 3023,
2833, 1661, 1626, 1570, 1492, 1290, 1115, 1093, 1014, 802.
Anal. calcd. for C10H13Cl2N3O: C, 45.82; H, 5.00; N, 16.03; found:
C, 45.76; H, 5.12; N, 15.94.
Hydrochloride of 1-(biphenyl-2-yl)-N-(imidazolidin-2-
ylidene)methanamine oxide 4f
M.p.: 165–1708C; 1H-NMR (500 MHz, DMSO-d6) d 3.65 (4H, b, CH2),
4.84 (2H, s, CH2), 7.27–7.49 (9H, m, CH), 8.81 (2H, bs, NH), 11.01
(1H, s, OH); 13C-NMR (50 MHz, DMSO-d6) d 43.4, 54.1, 127.6, 127.8,
128.0, 128.6, 129.3, 130.4, 131.9, 140.0, 141.6, 160.7; IR (KBr,
cmꢃ1) 3120, 2857, 1638, 1573, 1480, 1294, 1106, 1010, 744,
702. Anal. calcd. for C16H18ClN3O: C, 63.26; H, 5.97; N, 13.83;
found: C, 63.41; H, 6.05; N, 13.58.
1-(2,6-Dichlorophenyl)-N-(imidazolidin-2-ylidene)-
methanamine oxide 3c
1
M.p.: 176–1828C; H-NMR (200 MHz, CD3OD) d 3.79 (4H, s, CH2),
4.92 (2H, s, CH2), 4.95 (2H, s, NH), 7.27–7.45 (3H, m, CH); 13C-NMR
(50 MHz, CD3OD) d 44.9, 51.5, 129.7, 131.4, 131.7, 138.2, 159.1; IR
(KBr, cmꢃ1) 3420, 2945, 2866, 1685, 1582, 1563, 1495, 1436, 1289,
1208, 1006, 835, 782, 769.
1-(Biphenyl-3-yl)-N-(imidazolidin-2-ylidene)methanamine
oxide 3g
Obtained as an oil.
Hydrochloride of 1-(2,6-dichlorophenyl)-N-(imidazolidin-2-
ylidene)methanamine oxide 4c
M.p.: 248–2528C; 1H-NMR (200 MHz, DMSO-d6) d 3.72 (4H, s, CH2),
5.06 (2H, s, CH2), 7.40–7.56 (5H, m, CH), 8.99 (2H, bs, NH), 10.86
(1H, s, OH); 13C-NMR (50 MHz, DMSO-d6) d 43.5, 51.2, 129.1, 129.3,
131.5, 136.6, 160.8; IR (KBr, cmꢃ1) 3115, 3031, 2852, 1660, 1564,
1435, 1295, 1114, 1088, 935, 777, 770. Anal. calcd. for
C10H12Cl3N3O: C, 40.50; H, 4.08; N, 14.17; found: C, 40.45;
H, 4.14; N, 14.02.
Hydrochloride of 1-(biphenyl-3-yl)-N-(imidazolidin-2-
ylidene)methanamine oxide 4g
M.p.: 161–1638C; 1H-NMR (200 MHz, DMSO-d6) d 3.69 (4H, s, CH2),
4.93 (2H, s, CH2), 7.36–7.54 (5H, m, CH), 7.60–7.72 (4H, m, CH),
9.01 (2H, bs, NH), 11.21 (1H, s, OH); 13C-NMR (50 MHz, DMSO-d6)
d 43.2, 55.7, 126.3, 126.7, 126.8, 127.4, 127.6, 129.0, 129.1, 135.4,
139.9, 140.3, 160.5; IR (KBr, cmꢃ1) 3113, 3029, 2857, 1636, 1569,
1480, 1454, 1292, 1109, 759, 725, 704. Anal. calcd. for
C16H18ClN3O: C, 63.26; H, 5.97; N, 13.83; found: C, 63.46; H,
6.13; N, 13.86.
1-(4-tert-Butylphenyl)-N-(imidazolidin-2-ylidene)-
methanamine oxide 3d
M.p.: 188–1938C; 1H-NMR (500 MHz, DMSO-d6) d 1.28 (9H, s, CH3),
3.42 (4H, s, CH2), 4.44 (2H, s, CH2), 7.25 (2H, d, J ¼ 8.3 Hz, CH),
7.33 (2H, d, J ¼ 8.3 Hz, CH); 13C-NMR (50 MHz, DMSO-d6) d 31.2,
N-(Imidazolidin-2-ylidene)-1,1-diphenylmethanamine
oxide 3h
34.1, 48.4, 56.5, 124.7, 128.3, 134.8, 149.2, 166.7; IR (KBr, cmꢃ1
)
3054, 2962, 2866, 1694, 1516, 1477, 1289, 1267, 1008, 831, 680,
548.
M.p.: 162–1648C; IR (KBr, cmꢃ1) 3413, 3041, 2931, 2884, 1663,
1514, 1445, 1289, 1143, 1095, 866, 741, 728, 703.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

40
J. Saczewski et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
Hydrochloride of N-(imidazolidin-2-ylidene)-1,1-
N-(Imidazolidin-2-ylidene)-2-phenylethanamine oxide 3l
M.p.: 148–1558C; 1H NMR (200 MHz, DMSO-d6) d 2.84 (2H, t,
J ¼ 7.5 Hz, CH2), 3.37 (4H, s, CH2), 3.48 (2H, t, J ¼ 7.5 Hz, CH2),
7.15–7.32 (5H, m, CH), 7.60 (2H, bs, NH); IR (KBr, cmꢃ1) 3022,
2930, 2870, 1682, 1519, 1498, 1284, 1201, 1108, 755, 707,
670.
diphenylmethanamine oxide 4h
M.p.: 198–2058C; 1H-NMR (200 MHz, DMSO-d6) d 3.69 (4H, s, CH2),
6.82 (1H, s, CH), 7.33–7.39 (10H, m, CH), 9.30 (2H, bs, NH), 10.80
(1H, s, OH); 13C-NMR (50 MHz, DMSO-d6) d 43.4, 67.1, 128.2, 128.7,
128.9, 137.9, 161.8; IR (KBr, cmꢃ1) 3062, 1624, 1582, 1568, 1496,
1454, 1292, 1106, 1030, 748, 729, 700. Anal. calcd. for
C16H18ClN3O: C, 63.26; H, 5.97; N, 13.83; found: C, 63.03; H,
5.64; N, 13.66.
Hydrochloride of N-(imidazolidin-2-ylidene)-2-
phenylethanamine oxide 4l
M.p.: 152–1578C; 1H-NMR (200 MHz, DMSO-d6) d 2.93 (2H, t,
J ¼ 7.5 Hz, CH2), 3.56 (4H, s, CH2), 3.83 (2H, t, J ¼ 7.5 Hz, CH2),
7.21–7.32 (5H, m, CH), 8.73 (2H, bs, NH), 11.19 (1H, s, OH);
13C-NMR (50 MHz, DMSO-d6) d 32.1, 43.3, 53.6, 126.7, 128.6,
129.3, 138.0, 160.5; IR (KBr, cmꢃ1) 3088, 3036, 2899, 2844,
1673, 1561, 1453, 1296, 1206, 1112, 1078, 943, 756, 706, 646.
Anal. calcd. for C11H16ClN3O: C, 54.66; H, 6.67; N, 17.38; found:
C, 54.50; H, 6.98; N, 17.13.
N-(Imidazolidin-2-ylidene)-1-(naphthalen-1-yl)-
methanamine oxide 3i
Obtained as an oil.
Hydrobromide of N-(imidazolidin-2-ylidene)-1-
(naphthalen-1-yl)methanamine oxide 4i
M.p.: 222–2278C; 1H-NMR (500 MHz, DMSO-d6) d 3.65 (4H, s, CH2),
3.84 (2H, bs, NH), 4.90 (2H, s, CH2), 7.49–7.56 (2H, m, CH),
7.58–7.66 (2H, m, CH), 7.92–7.96 (1H, m, CH), 7.98–8.06
(2H, m, CH), 8.77 (1H, bs, OH); 13C-NMR (50 MHz, DMSO-d6)
d 42.9, 44.0, 123.6, 125.2, 125.7, 126.4, 126.8, 128.6, 128.9,
130.8, 132.5, 133.6, 159.8; IR (KBr, cmꢃ1) 3169, 3068, 1682,
1602, 1380, 1066, 807, 780. Anal. calcd. for C14H16BrN3O: C,
52.19; H, 5.01; N, 13.04; found: C, 52.02; H, 5.24; N, 13.09.
N-(Imidazolidin-2-ylidene)-2-phenylpropan-1-amine
oxide 3m
M.p.: 132–1378C; 1H-NMR (200 MHz, CDCl3) d 1.34 (3H, d,
J ¼ 7.5 Hz, CH3), 2.00 (2H, bs, NH), 3.30–3.55 (5H, m, CH2, CH),
3.96 (2H, d, J ¼ 7.5 Hz, CH2), 7.15–7.45 (5H, m, CH); 13C-NMR
(50 MHz, CDCl3) d 18.2, 36.8, 43.3, 59.3, 126.7, 127.8, 128.3, 142.6,
160.2; IR (KBr, cmꢃ1) 3158, 3062, 2964, 2899, 1680, 1553, 1452,
1378, 1292, 1086, 1015, 773, 707.
N-(Imidazolidin-2-ylidene)-1-(naphthalen-2-yl)-
methanamine oxide 3j
Hydrochloride of N-(imidazolidin-2-ylidene)-2-
M.p.: 170–1748C; 1H-NMR (200 MHz, DMSO-d6) d 3.43 (4H, s, CH2),
4.64 (2H, s, CH2), 7.42–7.56 (3H, m, CH), 7.80–7.93 (4H, m, CH),
8.00 (2H, bs, NH); IR (KBr, cmꢃ1) 3042, 2943, 2896, 1682, 1525,
1296, 1113, 819, 752.
phenylpropan-1-amine oxide 4m
M.p.: 62–658C; 1H-NMR (500 MHz, DMSO-d6) d 1.20 (3H, d,
J ¼ 6.8 Hz, CH3), 3.21–3.25 (1H, m, CH), 3.54 (4H, m, CH2),
3.62–3.66 (1H, m, CH), 3.80–3.84 (1H, m, CH), 7.20–7.34
(5H, m, CH) 8.65 (2H, bs, NH), 11.04 (1H, s, OH); 13C-NMR
(50 MHz, DMSO-d6) d 18.8, 36.6, 43.3, 58.3, 126.9, 127.8, 128.6,
143.4, 160.5. Anal. calcd. for C12H18ClN3O: C, 56.36; H, 7.09;
N, 16.43; found: C, 56.12; H, 7.27; N, 16.30.
Hydrochloride of N-(imidazolidin-2-ylidene)-1-
(naphthalen-2-yl)methanamine oxide 4j
M.p.: 2068C dec.; 1H-NMR (200 MHz, DMSO-d6) d 3.70 (4H, s, CH2),
5.04 (2H, s, CH2), 7.50–7.55 (3H, m, CH), 7.91–7.95 (4H, m, CH),
9.03 (2H, bs, NH), 11.22 (1H, s, OH); 13C-NMR (50 MHz, DMSO-d6)
d 43.5, 56.1, 126.5 (two signals), 126.7, 127.6, 127.9, 128.0, 128.4,
132.6, 132.8, 133.0, 160.7; IR (KBr, cmꢃ1) 3146, 3035, 1680, 1561,
1300, 1117, 811, 756, 716, 602. Anal. calcd. for C14H16ClN3O: C,
60.54; H, 5.81; N, 15.13; found: C, 60.48; H, 5.90; N, 15.02.
Radioligand binding assays
I1 binding site assay
Kidneys were obtained post mortem from male Sprague-Dawley
rats (250–280 g) and crude P2 membranes were prepared accord-
ing to methods of Lione et al. [31]. [3H]Clonidine (3 nM, Perkin
Elmer) was bound in the presence of 10 mM rauwolscine to
preclude binding to a2 adrenoceptors. The specific component
was defined by 10 mM rilmenidine; under these conditions, the
site labelled represents a model of the central I1 binding site [32].
Membrane aliquots (400 mL, 0.2–0.5 mg protein) were incubated
with 11 concentrations of the test compounds over the range
0.01 mM to 100 mM. Incubations were carried out in 50 mM Tris-
HCl buffer (pH 7.4) at room temperature for 45 min. Bound
radioligand and free radioactivity were separated by rapid filtra-
tion through pre-soaked (0.5% polyethylamine) glass-fibre filters
(Whatman GFB). Trapped radioligand was determined by liquid
scintillation counting and the data were analysed with
GraphPad Prism version 3.02 for Windows (GraphPad
Software, San Diego, CA, USA) to yield IC50 values (the concen-
tration of tested ligand that displaces 50% of specifically bound
[3H]clonidine).
N-(Imidazolidin-2-ylidene)-1-phenylethanamine oxide 3k
Obtained as an oil; 1H-NMR (200 MHz, CDCl3) d 1.55 (3H, d,
J ¼ 6.8 Hz, CH3), 3.38 (4H, s, CH2), 4.92 (2H, bs, NH), 5.01 (1H,
q, J ¼ 6.8 Hz, CH), 7.20–7.46 (5H, m, CH); 13C-NMR (50 MHz,
CDCl3) d 17.7, 45.7, 60.2, 127.4, 127.9, 128.8, 141.3, 160.0.
Hydrobromide of N-(imidazolidin-2-ylidene)-1-
phenylethanamine oxide 4k
M.p.: 147–1548C; IR (KBr, cmꢃ1) 3114, 2882, 2853, 1634, 1559,
1452, 1297, 1130, 1069, 987, 703; 1H-NMR (500 MHz, DMSO-d6) d
1.55 (3H, d, J ¼ 6.8 Hz, CH3), 3.69 (4H, s, CH2), 5.24 (1H, q,
J ¼ 6.8 Hz, CH), 7.32–7.43 (5H, m, CH); 13C-NMR (125 MHz,
DMSO-d6) d 17.9, 43.8, 59.9, 127.9, 128.7, 129.2, 139.6, 161.3.
Anal. calcd. for C11H16BrN3O: C, 46.17; H, 5.64; N, 14.68; found:
C, 45.87; H, 5.99; N, 14.41.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
a-Iminohydroxylamine vs a-Aminonitrone
41
test compound or vehicle administration. When the effect was
significant, post hoc comparisons were performed using Fisher’s
test. A value of p <0.05 was considered statistically significant.
This allowed direct comparison of responses to treatment
between groups during the entire experiment or for selected
time-points.
a1, a2 and I2 binding site assays
Brains were obtained post mortem from male Sprague-Dawley rats
(250–280 g) and crude P2 membranes were prepared according
to methods of Lione et al. [31]. Membrane aliquots (400 mL, 0.2–
0.3 mg protein) were incubated with 11 concentrations of the
tested compounds over the range 0.01 nM to 100 mM in the
presence of the selective I2 binding site ligand [3H]2BFI [33]
(1 nM), the a1 adrenoceptor antagonist [3H]prazosin (1nM) or
the a2 adrenoceptor antagonist [3H]RX821002 [34] (1 nM) in a
final volume of 500 mL. Non-specific binding was determined
using 10 mM BU224 [35] for defining I2 binding, 10 mM phenyl-
ephrine for a1-adrenoceptors and 10 mM rauwolscine to define
a2 adrenoceptor binding. Each incubation was performed in
triplicate at room temperature and was allowed to reach equi-
librium (45 min). Bound and free radioactivity were separated by
rapid filtration through pre-soaked (0.5% polyethyleneimine)
glass-fibre filters (Whatman GF/B). Filters were then washed twice
with 5 mL of ice-cold buffer and membrane-bound radioactivity
remaining on the filters was determined by liquid scintillation
counting. Data were analysed by iterative non-linear regression
curve fitting procedures with GraphPad Prism. Each experiment
was analysed individually and equilibrium dissociation con-
stants (Ki) were determined by the method of Cheng and
Prusoff [36]. The resulting values are given as means of 3 or 4
separate experiments.
Loss of righting reflex (LORR) test
The ability of compounds to activate the non-REM sleep pathway
in animals is determined by induction of the loss of righting
reflex (LORR). If an animal is placed on its back, it will normally
turn over immediately, returning to an upright position (the
righting reflex). However, with increasing loss of consciousness
(such as during sedation) this ability is lost and the animal will
remain in a supine position. The novel compounds 4h and 4i
were solubilised in sterile saline for intra-peritoneal (i.p.) injec-
tion at the following doses, (i) 5 mg/kg; (ii) 50 mg/kg and (iii)
500 mg/kg. The dose range was based upon previous studies of
a2 agonist sedative-hypnotic efficacy in rats [38]. Adult male
Sprague-Dawley rats weighing 250–280 g received a single i.p.
injection and were observed for LORR (n ¼ 3 per dose). If,
after 5 min, no LORR was observed, the dose was repeated,
up to a maximum dosage of 1000 mg/kg. A selection of
brains were retained following perfusion-fixation with 4%
paraformaldehyde.
X-ray crystal structure analysis
In vivo studies: Mean arterial blood pressure (MAP)
and heart rate (HR) in rats
The diffraction data for single crystals of compounds 3a and 4a
were collected with an Oxford Diffraction SuperNova diffracto-
meter using Cu Ka radiation. The intensity data were collected
and processed using Oxford Diffraction CrysAlis Software [39].
The structures were solved by direct methods with the program
SHELXS-97 [40] and refined by full-matrix least-squares method
on F2 with SHELXL-97 [40].
Male Wistar rats, weighing 200–250 g, were purchased from the
Animal House of the Medical University of Gdansk, Poland. All
experiments were approved by the Local Ethical Committee on
Animal Experiments. The animals were fed a commercial rodent
chow (Labofeed–B, Poland). Tap water was available ad libitum.
Rats were anaesthetized by i.p. injection of thiopental (Sandoz,
Austria) at the dose 70 mg/kg body weight and maintained under
anaesthesia by thiopental supplementation (30 mg/kg/min)
during the experiment. The animals were placed on a heated
table, and body temperature was maintained between 36 and
378C. Tracheostomy was performed. Catheters were inserted into
the carotid artery for blood pressure and heart rate monitoring,
into a jugular vein for infusions, and into the bladder for free
diuresis. After all surgical procedures, a 40 min recovery period
was allowed to establish steady state. The rats were infused with
isotonic saline (Fresenius Kabi, Poland) supplemented with thio-
pental at the rate of 1.2 mL/h. After 40 min of saline infusion, the
tested compound was administered as a 100 mL bolus through
the venous catheter at a dose of 0.1 mg/kg. Arterial blood pres-
sure and heart rate were monitored directly and sampled con-
tinuously at 100 Hz, as described previously [37], using Biopac
Systems, Inc., Model MP 100 (Goleta, CA, USA). The results of
measurements were elaborated with the help of the
ACQKnowledge (Goleta, CA, USA) measurement system and were
selected, scaled and filtered to remove accidental signal disturb-
ances. The recorded time domain transient data are presented as
graphs with the help of Excel (Microsoft, USA).
Crystal data for 3a C10H13N3O, orthorhombic, space group Pbca,
3
˚
˚
a ¼ 9.4805(1), b ¼ 10.1116(1), c ¼ 20.1214(1) A, V ¼ 1928.90(3) A ,
Z ¼ 8, T ¼ 130 K, dx ¼ 1.317 g cmꢃ3, m(Cu Ka) ¼ 0.718 mmꢃ1
,
25743 data were collected up to umax ¼ 75.558 for a crystal with
dimensions 0.69 ꢄ 0.08 ꢄ 0.08 mm3 (Rint ¼ 0.0253, Rs ¼ 0.0083).
Final R indices for 1887 reflections with I > 2s(I) and 136 refined
parameters are: R1 ¼ 0.0317, wR2 ¼ 0.0823 (R1 ¼ 0.0330,
wR2 ¼ 0.0833 for all 1987 data).
Crystal data for 4a C10H14ClN3O, triclinic, space group P-1,
˚
a ¼ 8.3310(4), b ¼ 8.7350(5), c ¼ 9.6385(5) A, a ¼ 63.059(6),
3
˚
b ¼ 83.530(4), g ¼ 67.902(5)8, V ¼ 577.93(7) A , Z ¼ 2, T ¼
293 K, dx ¼ 1.308 g cmꢃ3, m(Cu Ka) ¼ 2.759 mmꢃ1, 8341 data
were collected up to umax ¼ 75.168 for a crystal with dimensions
0.49 ꢄ 0.27 ꢄ 0.22 mm3 (Rint ¼ 0.0151, Rs ¼ 0.0084). Final
R
indices for 2217 reflections with I > 2s(I) and 148 refined
parameters are: R1 ¼ 0.0416, wR2 ¼ 0.01017 (R1 ¼ 0.0422,
wR2 ¼ 0.1022 for all 2267 data).
Crystallographic data for compounds 3a and 4a have
been deposited with the Cambridge Crystallographic Data
Centre, with the deposition numbers CCDC 785379 and
785378, respectively [41].
ANOVA was performed for DMAP and DHR, calculated as the
difference in MAP and in HR from baseline measurements (‘‘time
0’’) for each group, as described previously [37]. This allowed for
direct comparison of responses to treatment between groups.
Data were analysed by ANOVA with repeated measurements,
using Statistica StatSoft software (StatSoft, Inc., Tulsa, USA), after
This work was supported in part by National Science Engineering Research
Council, Canada (A. H., S. L.) and by the proof concept grant from
Innovation Centre, Imperial College London, UK (D. M., M. M., H. W.).
The authors have declared no conflict of interest.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

42
J. Saczewski et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 33–42
[22] M. Gdaniec, F. Saczewski, T. Debowski, J. Chem. Crystallogr.
1995, 25, 813–821.
References
[23] Spartan ’08 for Windows, Wavefunction, Inc., 18401 Von
Karman Ave., suite 370, Irvine, CA 92612, USA;
www.wavefun.com.
[1] B. Szabo, Pharmacol. Ther. 2002, 93, 1–35.
[2] a) L. E. Nelson, B. A. Jun Lu, T. Guo, C. B. Saper, N. P. Franks,
M. Maze, Anesthesiology 2003, 98, 428–436; b) D. Ma, N.
Rajakumaraswamy, M. Maze, British Medical Bulletin 2005,
71, 77–92.
[24] A. V. Marenich, R. M. Olson, C. P. Kelly, C. J. Cramer, D. G.
Truhlar, J. Chem. Theory Comput. 2007, 3, 2011–2033.
[25] K. Kawamura, M. Naganawa, F. Konno, J. Yui, H. Wakizaka,
T. Yamasaki, K. Yanamoto, A. Hatori, M. Takei, Y. Yoshida, K.
Sakaguchi, T. Fukumura, Y. Kimura, M.-R. Zhang, Nuclear
Medicine and Biology 2010, 37, 625–635.
[3] M. P. Giovannoni, C. Ghelardini, C. Vergelli, V. Dal Piaz, Med.
Res. Rev. 2009, 29, 339–368.
[4] a) B. Weber, M. Steinfath, J. Scholz, B. Bein, Drug News
Perspect. 2007, 20, 149–154; b) T. V. S. P. Murthy, R. Singh,
J. Anaesth. Clin. Pharmacol. 2009, 25, 267–272.
[26] L. F. Callado, J. I. Martin-Gomez, J. Ruiz, J. M. Garibi, J. J.
Meana, J. Neurol. Neurosurg. Psychiatry 2004, 75, 785–787.
[5] a) M. Berlan, J. L. Montastruc, M. Lafontan, Trends Pharmacol.
Sci. 1992, 13, 277–282; b) R. Aantaa, Bailliere’s Clinical
Anaesthesiology 2000, 14, 285–292.
[27] M. A. Stern, H. K. Gohlke, H. S. Loeb, R. P. Croke, R. M.
Gunnar, Circulation 1978, 58, 157–163.
[28] S. Regunathan, D. J. Reis, Hypertension 1997, 30, 295–300.
[6] G. A. Head, D. N. Mayorov, Cardiovasc. Hematol. Agents Med.
Chem. 2006, 17–32.
[29] P. Sanchez-Blazquez, M. A. Boronat, G. Olmos, J. A. Garcia-
Sevilla, J. Garzon, Br. J. Pharmacol. 2000, 130, 146–152.
[7] A. Kimura, R. J. Tyacke, J. J. Robinson, S. M. Husbands,
M. C. W. Minchin, D. J. Nutt, A. L. Hudson, Brain Res.
2009, 1279, 21–28.
[30] W. Porcal, P. Hernandez, M. Gonzalez, A. Ferreira, C.
Olea-Azar, H. Cerecetto, A. Castro, J. Med. Chem. 2008, 51,
6150–6159.
[8] J. E. Piletz, H. Zhu, D. N. Chikkala, J. Pharmacol. Exp. Ther. 1996,
279, 694–702.
[31] L. A. Lione, D. J. Nutt, A. L. Hudson, Eur. J. Pharmacol. 1998,
353, 123–135.
[9] Q.-M. Zhu, J. D. Lesnick, J. R. Jasper, S. J. MacLennan, M. P.
Dillon, R. M. Eglen, D. R. Blue, Jr, Br. J. Pharmacol. 1999, 126,
1522.
[32] P. Ernsberger, M. E. Graves, L. M. Graff, N. Zakieh, P. Nguyen,
L. A. Collins, K. L. Westbrooks, Ann. N. Y. Acad. Sci. 1995, 763,
22–43.
[10] F. Saczewski, A. Kornicka, A. Rybczynska, A. L. Hudson, S. S.
Miao, M. Gdaniec, K. Boblewski, A. Lehmann, J. Med. Chem.
2008, 51, 3599–3608.
[33] A. L. Hudson, C. B. Chapleo, J. W. Lewis, S. Husbands, K.
Grivas, N. J. Mallard, D. J. Nutt, Neurochem. Int. 1997, 30,
47–54.
[11] F. Saczewski, P. Tabin, R. J. Tyacke, A. Maconie, J. Saczewski,
A. Kornicka, D. J. Nutt, A. L. Hudson, Bioorg. Med. Chem. 2006,
14, 6679–6685.
[34] D. Langin, M. Lafontan, M. R. Stillings, H. Paris, Eur. J.
Pharmacol. 1989, 167, 95–104.
[12] F. Saczewski, J. Saczewski, M. Gdaniec, Chem Pharm. Bull.
2001, 49, 1203–1206.
[35] D. J. Nutt, N. French, S. Handley, A. L. Hudson, S. Husbands,
H. Jackson, S. Jordan, M. Lalies, J. Lewis, L. Lione, N. Mallard,
J. Pratt, Ann. N. Y. Acad. Sci. 1995, 763, 125–139.
[13] F. Saczewski, A. L. Hudson, R. J. Tyacke, D. J. Nutt, J. Man, P.
Tabin, J. Saczewski, Eur. J. Pharm. Sci. 2003, 20, 201–208.
[36] Y. C. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22,
3099–3108.
[14] F. Sa˛czewski, T. Debowski, M. Gdaniec, J. Petrusewicz, M.
Turowski, B. Damasiewicz, Eur. J. Pharm. Sci. 1996, 4, 85–93.
´
[37] A. Rybczynska, K. Boblewski, A. Lehmann, C. Orlewska, H.
Foks, K. Drewnowska, A. Hoppe, Am. J. Hypertens. 2005, 18,
[15] F. Sa˛czewski, E. Kobierska, T. Debowski, H. Trzeciak, E.
Krzystanek, M. Krzystanek, M. Gdaniec, E. Nowak,
Nadcisnienie Tetnicze (nt.viamedica.pl) 2000, 4, 173–180.
364–371.
[38] N. D. Franken, H. van Oostrom, P. J. Stienen, A. Doornenbal,
L. J. Hellebrekers, Vet. Anaesth. Analg. 2008, 35, 424–431.
[16] E. Cohnen, A. G. Patent Beiersdorf, DE 2457979; Chem.
Abstr. 1976, 85, 1927 25.
[39] CrysAlis Pro software, ver. 1.171.33, Oxford Diffraction Ltd,
Abingdon, Oxfordshire, UK, 2008.
[17] E. L. Conway, B. Jarrott, Journal of Pharmacokinetics and
Biopharmaceutics 1982, 10, 187–200.
[40] G. M. Sheldrick, A short history of SHELX, Acta Cryst. 2008,
A64, 112–122.
[18] P. B. M. W. M. Timmermans, A. Brands, P. A. van Zwieten,
Arch. Pharmacol. 1977, 300, 217–226.
[41] Crystallographic data for the structures of 3a and 4a have
been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication number CCDC 7853
78. Copies of the data can be obtained, free of charge,
on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK [fax: þ44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
[19] F. Sa˛czewski, E. Kobierska, T. Debowski, S. Charakchiewa-
Minol, M. Mokrosz, M. Gdaniec, E. Nowak, Arch. Pharm. 2000,
333, 425–430.
[20] J. Sa˛czewski, M. Gdaniec, Pol. J. Chem. 2008, 82, 2107–2113.
[21] J. Sa˛czewski, Z. Brzozowski, M. Gdaniec, Tetrahedron 2005, 61,
5303–5309.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com