3-[(Imidazolidin-2-yl)imino]indazole ligands with selectivity for the α2-adrenoceptor compared to the imidazoline I1 receptor

Article abstract of DOI:10.1016/j.bmc.2010.11.020

A series of 3-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles has been synthesized as positional analogues of marsanidine, a highly selective α₂-adrenoceptor ligand. Parent compound 4a and its 4-chloro (4c) and 4-methyl (4d) derivatives display α₂-adrenoceptor affinity at nanomolar concentrations (K_i = 39.4, 15.9 and 22.6 nM, respectively) and relatively high α₂/I₁ selectivity ratios of 82, 115 and 690, respectively. Evidence was obtained that these compounds act as partial agonists at α_2A-adrenoceptors. Compound 4d with intrinsic activity comparable with that of marsanidine, but lower than that of clonidine, elicited pronounced cardiovascular effects in anesthetized rats at doses as low as 0.01 mg/kg iv.

Full text of DOI:10.1016/j.bmc.2010.11.020

Bioorganic & Medicinal Chemistry 19 (2011) 321–329
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-[(Imidazolidin-2-yl)imino]indazole ligands with selectivity
for the a2-adrenoceptor compared to the imidazoline I1 receptor
Franciszek Sa˛czewski a, , Anita Kornicka a, , Alan L. Hudson b, Shayna Laird b, Mika Scheinin c,
⇑
⇑
c
d
d
d
e
´
Jonne M. Laurila , Apolonia Rybczynska , Konrad Boblewski , Artur Lehmann , Maria Gdaniec
a Department of Chemical Technology of Drugs, Medical University of Gdan´sk, 80-416 Gdan´sk, Poland
b Department of Pharmacology, University of Alberta, Edmonton, Canada T6G 2R3
c Department of Pharmacology, Drug Development and Therapeutics, University of Turku, and Turku University Hospital, FI-20014 Turku, Finland
d
´
´
Department of Patophysiology, Medical University of Gdansk, 80-416 Gdansk, Poland
Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland
e
´
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2010
Revised 4 November 2010
Accepted 8 November 2010
Available online 11 November 2010
A
series of 3-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles has been synthesized as positional
analogues of marsanidine, a highly selective 2-adrenoceptor ligand. Parent compound 4a and its
4-chloro (4c) and 4-methyl (4d) derivatives display 2-adrenoceptor affinity at nanomolar concentra-
tions (Ki = 39.4, 15.9 and 22.6 nM, respectively) and relatively high 2/I1 selectivity ratios of 82, 115
and 690, respectively. Evidence was obtained that these compounds act as partial agonists at 2A-adreno-
a
a
a
a
ceptors. Compound 4d with intrinsic activity comparable with that of marsanidine, but lower than that of
clonidine, elicited pronounced cardiovascular effects in anesthetized rats at doses as low as 0.01 mg/kg iv
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Imidazolines
Indazoles
Alpha2-adrenenoceptor agonists
Hypotensive agents
Synthesis
X-ray structure
1. Introduction
However, the above hypothesis appears to be uncertain, espe-
cially in view of the recent evidence12,13 derived from experiments
Imidazole and imidazoline-containing agents acting at
2-adrenoceptors exhibit several important effects in vivo and
using D79N a2-adrenoceptor transgenic mice (functional a2-adre-
noceptor ‘knock-out’ mice) that demonstrated that hypotensive
a
could find potential applications as hypotensive, analgesic, seda-
tive, anxiolytic, hemodynamic-stabilizing and organ-protective
agents.1–7 Other effects of these agents, mediated by central and
peripheral autoreceptors located on noradrenergic nerve endings
and by postsynaptic receptors on target cells, include stimulation
of growth hormone secretion and decreased output of endocrine
and exocrine secretory glands, such as decreased insulin secretion
and decreased salivation.8,9
It has been hypothesised that compounds that selectively
activate a2-adrenoceptors, and not imidazoline I1 receptors, would
be likely to have decreased hypotensive and bradycardic effects
compared with some non-selective agents that are now in clinical
use,10,11 such as clonidine and dexmedetomidine that may be
compounds such as dexmedetomidine, brimonidine, clonidine,
rilmenidine and moxonidine act as
a2A-adrenoceptor agonists
in vivo to lower blood pressure. No evidence of I1-imidazoline recep-
tor-mediated hypotensive effects of these compounds was obtained.
These observations did not support the imidazoline mode of action
of these agents.14
Our previous investigations on
a2-adrenoceptor ligands led to
the discovery of some highly selective 1-[(4,5-dihydroimidazoli-
din-2-yl)imino]indazoles15 (Fig. 1, structure A, marsanidine). Based
on analysis of the relative binding affinities of these compounds,
we reasoned that one of the structural features which may contrib-
ute to a2-adrenoceptor activity is the presence of an @C–H group in
the indazole moiety which might participate in hydrogen bonding
or stacking interactions with an aromatic ring side chain of the
receptor protein (Fig. 1). Such a hypothesis was strongly supported
by the observation that introduction of a methyl group at position
considered as mixed a2/I1 agonists.
3 of the indazole ring was detrimental for a2-adrenoceptor affinity
(Fig. 1, structure B).
⇑
Corresponding authors. Tel.: +48 58 3493250; fax: +48 58 3493257 (F.S.);
tel.: +48 58 3493256; fax: +48 58 3493257 (A.K.).
E-mail addresses: saczew@gumed.edu.pl (F. Sa˛czewski), a.kornicka@gumed.
edu.pl (A. Kornicka).
It was not obvious, however, what effect translocation of the imi-
noimidazolidine moiety might have with regard to 2-adrenoceptor
a
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.020

322
F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
Compound 3a crystallizes as an imidazolidin-2-imine tautomer.
H
CH3
7
H
4
R
R
1
N
The imidazolidine ring is in a flattened envelope conformation
with the C13 atom forming a flap; the highest absolute value of
the endocyclic torsion angle in the five-member ring is 17.5°. The
indazole fragment of the molecule is virtually planar with the
amino N1 atom slightly pyramidized. The indazole and imidazoli-
din-2-imine fragments are mutually twisted around the formally
single C3–N10 bond by À18.7° and the conformation of 3a is stabi-
lized by an intramolecular N15–HÁ Á ÁN2 hydrogen bond. The
conformation of 3a in solid state differs strongly from that of 4-
chloromarsanidine,15 where indazole and imidazolidin-2-imine
fragments where nearly perpendicular and no intramolecular
hydrogen bond was formed. In the crystals of 3a, intermolecular
N–HÁ Á ÁN hydrogen bonds join the individual molecules into a
two-dimensional polymeric structure and the amino N1–H group
of the indazole ring plays an essential role in the recognition pro-
cess. It acts as a hydrogen-bond donor to the imine N10 atom of the
neighbouring molecule and also as a weak acceptor of the hydro-
gen bond from the N12–H group from the imidazolidine unit. In
4-chloromarsanidine the @C–H group, replacing the N1–H group
C
3
C
6
5
2
N
N
2
N
5
H
N
H
N
6
N
N
3
H
N
N
1
4
N
7
N
HN
HN
HN
A, R = H, marsanidine
B, 3-Me-marsanidine
C
α
α
2, Ki = 14.05 nM
I1, Ki = 54500 nM
2, Ki = 71070 nM
I1, Ki = >100000 nM
Figure 1. Structure of the selective
a2-adrenoceptor ligand A (marsanidine), its
inactive and analogue B and positional isomer C showing common structural
features: a C–H group and weakly acidic N–H group.
affinity, and therefore, we were interested in analogues of type C (
Fig. 1) wherein the iminoimidazolidine moiety had been shifted to
the 3-position of the indazole ring in order to obtain a new series
of marsanidine analogues incorporating @N–H functionality at posi-
tion 1. In the present work, the importance of the substitution pat-
tern of the indazole ring for
a2-adrenoceptor affinity and
of 3a, is involved in an intermolecular C–HÁ Á Á
p interaction to the
selectivity has been explored. The pharmacological effects of such li-
gand modifications incorporated into the newly prepared positional
analogues of type C, including functional characterization at recom-
benzene ring.
The molecular properties of marsanidine A (the most selective
2/I1 agent) and its positional analogue 3a were also investigated
a
binant
a2A-adrenoceptors and hemodynamic assessment in anes-
by comparison of their 3D electrostatic potential maps.20 As shown
in Figure 3, electrostatic potential maps of both free base A and free
base 3a show superimposable negative wells positioned around
the aromatic indazole ring and nitrogen atoms of the 2-iminoimi-
dazolidine moiety. Therefore, a similar mode of interactions with
thetized rats, were also investigated.
2. Results and discussion
2.1. Chemistry
a
2-adrenoceptors may be assumed by marsanidine-like ligands
The known 3-aminoindazoles 2a–c16 and 2f–i,17 as well as the
novel derivatives 2d–e and 2j–k were prepared according to
previously described procedures by reacting the corresponding
2-fluorobenzonitriles with hydrazine18 As shown in Scheme 1,
3-aminoindazoles 2a–k reacted smoothly with N-tert-buthoxycar-
bonyl-4,5-dihydro-1H-imidazole19 to give, after deprotection, the
desired free bases 3a–k in good yields. For pharmacological tests,
water-soluble hydrochloride salts 4a–k were prepared upon treat-
ment of the free bases with methanolic HCl solution.
and their 3-(2-iminoimidazolidine) analogues 3. However, in the
case of 3a the negative potential is more pronounced, especially
in close proximity to the indazole N2 nitrogen atom and the exocy-
clic nitrogen atom of the 2-iminoimidazolidine moiety. This obser-
vation is further confirmed by the more basic character of 3a21
compared to marsanidine (pKa = 8.02 vs 6.32). Consequently, a dif-
ference of 1.7 log units when comparing the basicity of 3a to
marsanidine should influence the pharmacokinetic profiles of the
newly prepared compounds. At physiological pH, marsanidine
with a pKa of 6.32 exists preferably in neutral free base form
(92%) which, in turn, facilitates its ability to cross the blood–brain
barrier (BBB). Instead, the compound 3a with pKa = 8.02 bears
resemblance to the well known aryliminoimidazolidines such as
clonidine, and is mostly ionized at physiological pH (only 19% in
non-ionized form at pH 7.4).
Structures of the free bases 3a–k and the hydrochlorides 4a–k
were confirmed by elemental analysis, IR and NMR spectroscopy,
as well as X-ray structure analysis of the compound 3a (Fig. 2).
R
1a
1b
1g: R = 5-CH3
1h: R = 5-OCH3
F
: R = H
: R = 2-Br
1i
: R = 4-Cl
1c: R = 2-Cl
CN
1j
1d
1e
: R = 4-CH3
: R = 6-CH3
: R = 6-OCH3
2.2. Radioligand binding assays
1a-k
1k: R = 3-Cl
1f: R = 5-Cl
a
Radioligand binding experiments were conducted using crude
P2 rat brain membranes for both
I2 receptors and crude P2 rat kidney membranes for
a
2-adrenoceptors and imidazoline
1-adrenocep-
a
H
N
H
N
H
N
R
R
R
b, c
d
Cl
N
N
N
H
N
H
N
NH2
N
N
2a-k
3a-k
4a-k
H
N
H
N
H
2a, 3a, 4a
2b, 3b, 4b
2c, 3c, 4c
2g, 3g, 4g
: R = H
: R = 4-Br
: R = 4-Cl
: R = 5-CH3
2h, 3h, 4h: R = 5-OCH3
2i, 3i, 4i
: R = 6-Cl
2j, 3j, 4j: R = 6-CH3
2k, 3k, 4k
2d, 3d, 4d: R = 4-CH3
2e, 3e, 4e
: R = 4-OCH3
2f, 3f, 4f: R = 5-Cl
: R = 7-Cl
Scheme 1. Reagents and conditions: (a) NH2NH2Á Á ÁH2O, n-BuOH, 5 h, reflux;
42–79%; (b) N-Boc-2-methylthio-4,5-dihydro-1H-imidazole, acetic acid, 60–62 °C
(oil bath), 16 h; (c) 15% Na2CO3/H2O, crystallization from suitable solvent, 22–59%;
(d) HCl/MeOH, 20–22 °C, 30 min, crystallization from suitable solvent, 41–53%.
Figure 2. Molecular structure of 3a with atom labelling scheme and displacement
ellipsoids shown at the 50% probability level. The intramolecular N–HÁ Á ÁN hydrogen
bond is shown with a dashed line.

F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
323
to be less detrimental, especially at
a
2-adrenoceptors. It should be
pointed out that the most active compounds at
a2-adrenoceptors,
4c and 4d, also displayed good affinities to
a1-adrenoceptors
(Ki = 69.3 and 46.9 nM, respectively), while the 7-Cl congener 4k
exhibited relatively high affinity for the imidazoline I2 receptor
(Ki = 30.8 nM).
In Table 1 the selected results obtained for analogous 1-[(imidaz-
olidin-2-yl)imino]indazoles of type A are included (values given in
parentheses). From comparison of the relevant data it can be con-
cluded that translocation of the 2-iminoimidazolidine moiety from
position 1 to position 3 of the indazole ring yields compounds with
high or moderate binding affinity to
a2-adrenoceptors and relatively
high I1/ 2 selectivity ratios, depending on the substitution pattern at
a
the indazole ring.
2.3. Functional [35S]GTP
cS binding assays
Figure 3. Comparison of the electrostatic potential maps of marsanidine A (left) and
its positional analogue 3a (right). Electrostatic potential map isocontoured at
À10 kcal/mol (top) and electrostatic potential mapped onto the surface of electron
density (bottom).
Ligands 4a, 4c, 4d and 4k that displayed affinities towards rat
brain 2-adrenoceptors within the nanomolar range were sub-
jected to [35S]GTP
a
c
S binding experiments to determine their
intrinsic activity and agonist potency.23–25 For SAR purposes,
marsanidine and its 4-Cl, 7-Me and 7-Cl derivatives representing
an indazole series of type A were also included.
tors and I1 receptors. Equilibrium dissociation constants (Ki) were
determined by the method of Cheng and Prusoff22 and the result-
ing values are given as the mean sem for three or four separate
experiments.
All of the investigated compounds were found to stimulate
binding of [35S]GTP
cS to CHO cell membranes expressing recombi-
As shown in Table 1, the unsubstituted compound 4a showed
nant human
a2A-adrenoceptors and showed typical agonist dose–
good affinity for
ate I1/a2 selectivity ratio of 82. The 4-Cl and 4-CH3-substituted
indazoles 4c and 4d displayed enhanced 2-adrenoceptor affinities
(Ki = 15.9 and 20.0 nM, respectively) and higher selectivity with re-
spect to I1 receptors (I1/ 2 selectivity ratios of 115 and 690, respec-
tively). Worth noting is a noticeable difference in binding between
4b (4-Br) and 4c (4-Cl) at the 2-receptor (6420 vs 15.9 nM). This
a2-adrenoceptors with Ki = 39.4 nM and a moder-
response plots. Table 2 presents their agonist potencies expressed
as EC50 values and their relative efficacy compared to the natural
full agonist noradrenaline.
a
When compared to the full agonist noradrenaline, the reference
drugs dexmedetomidine and clonidine as well as the two series of
indazole derivatives were identified as partial agonists. Agonist
potencies of both series were in the submicromolar range with the
newly prepared 4d being the most potent agonist (EC50 = 14 nM).
On the other hand, in both series, the highest intrinsic activity was
displayed by congeners containing a Cl-substituent at a similar
position relative to the iminoimidazolidine moiety, that is, by
4-Cl-marsanidine and 7-chloro-3-[(4,5-dihydroimidazol-2-yl) imino]
indazole 4k. Moreover, their intrinsic activities were found to be
on the same level as those of clonidine and dexmedetomidine
(efficacy estimates relative to noradrenaline were 62%, 66%, 53%
and 60%, respectively).
a
a
result, however, is difficult to rationalize based on both the elec-
tronic properties (similar electronegativity and identical Hammet
constant
r), and steric factors, since the compound 4e bearing
4-OCH3 group is much more active (Ki = 196 nM). Perhaps, the
hydrophobic pocket in this region appears to be size-limited and
bromine atom is least favourable at the C4 position.
On the other hand, 5-substituted analogues 4f–h showed affini-
ties about 6–285 times lower at both a2 and I1 receptors, while sub-
stitution at the indazole C-6 and C-7 positions (entries 4i–k) proved
Table 1
Binding affinities of 3-[(imidazolidin-2-yl)imino]indazole hydrochlorides 4a–k at
a
-adrenorceptors and imidazoline receptors
Compound
Binding affinitiesa
I1 IC50 (nM)c
a
2-selectivityd
No.
R
a1 Ki (nM)b
a2 Ki (nM)b
I2 Ki (nM)b
4a (13a)
4b
4c
4d (13k)
4e
H (H)
4-Br
NTe (NT)
984 142
69.3 19.8 (1010)
46.5 1.6 (NT)
196 11.8
39.4 7.7 (14)
6420 829
15.9 2.3 (30.3)
22.6 6.5 (53.5)
748 33.7
3219.0 271 (54,500)
7560 2550
1840.0 580 (46,800)
32,500 2100 (387)
25,600
133.6 19.6 (16,900)
5380 916
45.2 14.2 (15,000)
132.7 8.3 (2520)
167.0 18.9
82 (3879)
1
115 (1544)
690 (7.2)
34
4-Cl (7-Cl)f
4-CH3 (7-CH3)
4-OCH3
4f
5-Cl
121.0 8.8
10600.0 3700
11400.0 (49)
1460 310 ((96)
502 110 (114)
>100,000
21,200 (NT)
>100,000 (NT)
2010 430 (16,980)
93000.0
10,200 (2520)
9910.0 360 (33,640)
410 73.0 (20,620)
>9.4
2
>68
4
4g (13i)
4h (13j)
4i (13h)
5-CH3 (6-CH3)
5-OCH3 (6-OCH3)
6-Cl (5-Cl)
29,000 (NT)
244 58.0 (NT)
386 27.0 (NT)
148
4j (13g)
4k (13e)
6-CH3 (5-CH3)
7-Cl (4-Cl)
508 166 (NT)
684 48.3 (NT)
176 37.4 (20,310)
133 15.3 (61)
16,600 4770 (7159)
4010 599 (31,460)
325 198
30.8 2.9 (11,080)
94.3 (459)
30 (510)
a
Values shown are mean sem from 3 to 4 experiments, otherwise values represent the mean of two separate assays. In parentheses the corresponding values for
1-substituted indazoles of type A are shown (see Ref. 15).
b
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.
c
Molar concentration of the test compounds that displaces 50% of specifically bound [3H]clonidine (rat kidney membranes).
d
a
2-Selectivity was determined from the ratio of the equilibrium constants: I1 IC50
NT: not tested.
The previously not described analogue 7-chloro-1-[(imidazolidin-2-yl)imino]indazole hydrochloride was prepared and tested according to the procedures presented in
/a2 Ki.
e
f
Ref. 15; mp: 223–224 °C; 1H NMR (DMSO-d6, 200 MHz): d = 12.55 (br s, 1H), 9.09 (s, 2H), 8.37 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.29 (t, 1H), 3.75 (s, 4H).

324
F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
Table 2
subtype as compared to 4k and the previously studied marsanidine
analogues.15 Further receptor binding and functional studies on the
cloned 2-adrenoceptor subtypes will be necessary to examine this
Characterization of [35S]GTP
cS binding to CHO cell membranes expressing recombi-
nant human
a2A-adrenoceptors: estimates of agonist potency (EC50) and intrinsic
a
activity relative to the natural full agonist noradrenaline
preference in detail.
Ligand
EC50 (nM)
Intrinsic activity (% of noradrenaline)
100
Noradrenaline
Dexmedetomidine
Clonidine
Marsanidine
4-Cl-Marsanidine
7-Me-Marsanidine
7-Cl-Marsanidinea
4a
110 14
1.5 0.19
28 4.3
160 66
180 38
37 9.0
15 1.9
82 16
3. Conclusion
60
53
18
62
32
48
55
13
40
66
2
3
2
6
5
3
6
1
5
3
We have here described a facile synthesis of 3-[(4,5-dihydroim-
idazolidin-2-yl)imino]indazoles, which constitute a new series of
positional analogues of the previously reported highly a2-adreno-
ceptor selective 1-[(4,5-dihydroimidazolidin-2-yl)imino]indazoles
(marsanidine-like ligands). Compounds 4a and 4c–d showed nano-
4c
4d
4k
240 140
14 2.1
73 10
molar affinities to
a2-adrenoceptors in in vitro receptor binding
experiments and relatively high I1/a2 selectivity ratios ranging
from 82 to 690. In terms of pharmacological activity, compounds
Values shown are mean sem from 3 to 16 independent experiments.
7Cl-Marsanidine has been prepared according to general procedure described
in Ref. 15.
a
4a, 4c, 4d and 4k are partial
a2-adrenoceptor agonists as deter-
mined in functional [35S]GTP
c
S binding experiments in vitro.
When systemically administered to anaesthetized rats, the com-
pound 4d elicited pronounced hemodynamic effects and may
prove useful in the treatment of hypertension. On the other hand,
the weak partial agonist 4c was shown to possess very weak hypo-
tensive properties. Such a pharmacological profile may prove ben-
eficial for therapeutic applications other than the treatment of
hypertension.
2.4. In vivo cardiovascular effects
Compounds 4c, 4d and 4k, with relatively high affinity for
a2-adrenoceptors (Table 1) and clonidine-like partial agonist activity
at a2A-adrenoceptors (Table 2) were evaluated in anaesthetized rats
for possible cardiovascular effects.26 As shown in Table 3, neither 4c
nor 4k exerted significant effects on mean arterial blood pressure
after intravenous infusion at doses up to 0.1 mg/kg. Much higher car-
diovascular activity was observed for the 4-methylated congener 4d
(Figs. 4 and 5). The maximal hypotensive and negative chronotropic
4. Experimental
4.1. Chemistry
effect of 0.1 mg/kg 4d administration reached
and
ture–activity relationships were previously found for the 1-substi-
tuted indazole series A, wherein the greatest hypotensive activity
was noted for the congener bearing a methyl group at the peri posi-
tion, that is, 7-Me-marsanidine.15
Weaker in vivo hemodynamic effects of the systemically admin-
istered compounds 4c and 4k compared to 4d (Fig. 4) could not be
explained by their inability to cross the BBB, as the calculated lipo-
philicity of the former compounds is comparable to that of 4d (log P
(Ghose–Crippen) = 2.07 vs 2.0). Since 4d exhibited the highest I1/a2
selectivity ratio of the tested compounds, 550 (Table 1), and proved
D
MAP = À53.5 mmHg
Melting points were measured on a Büchi 535 apparatus and are
not corrected. IR spectra were taken in KBr pellets on a Perkin–Elmer
FTIR 1600 spectrometer. NMR spectra were recorded on a Varian
Gemini 200 or a Varian Unity 500 apparatus. 1H and 13C chemical
shifts were measured relative to the residual solvent signal at
2.50 ppm and 39.5 ppm (DMSO-d6). Mass spectra were recorded
on a FinniganMAT 95 mass spectrometer at 70 eV. The results of ele-
mental C, H, N analyses of all newly prepared compounds were with-
in 0.4% of the theoretical values. The following compounds were
obtained according to previously described procedures: 3-aminoin-
dazoles (2a–c),16 (2f–i)17 by reacting the commercially available
2-fluorobenzonitrile 1a–c, f–i with 98% hydrazine hydrate.18 The
same procedure was applied to the synthesis of the previously not
described 3-aminoindazole derivatives 2d–e and 2j–k. N-tert-Buth-
oxycarbonyl-4,5-dihydro-1H-imidazole were obtained by reacting
2-methylthio-2-imidazoline hydroiodide with di-tert-butyl
dicarbonate.19
D
HR = À132 bpm, respectively(Tables 3 and 4). Similar struc-
to be
a potent agonist of the a2A-adrenoceptor subtype
(EC50 = 14 nM, Table 2), we may conclude that the hypotensive
effect of this compound is best explained by activation of
a2A-adrenoceptors.
As to the blood pressure effects elicited by the active compounds
4d and 4k (Fig. 4), a classical biphasic response characteristic for
activation of 2-adrenoceptors was observed for compound 4k,
while in the case of the more potent 4d, a hypertensive phase was
a
4.2. General procedure for the preparation of 3-aminoindazoles
(2d–e, j–k)
much less pronounced. The initial hypertensive phase is probably
at least in part mediated by vascular
a
2B-adrenoceptors (but
The title compounds were obtained according to the procedure
described by Vasudevan et al.18 A solution of the commercially
available 2-fluorobenzonitrile (1d–e, j–k) (22.0 mmol) and 98%
hydrazine hydrate (90.0 mmol) in n-butanol (15 ml) was stirred
a
1-adrenoceptors may also contribute), whereas the long-lasting
hypotension is mediated by central
a
2A-adrenoceptors.9 Thus,
compound 4d may in functional terms prefer the a2A-adrenoceptor
Table 3
The maximal hypotensive effect of synthesized compounds at 0.01, 0.1 and 1.0 mg/kg in anesthetized rats
Compound
0.01 mg/kg
MAP (mmHg)
p
0.1 mg/kg
MAP (mmHg)
p
1.0 mg/kg
MAP (mmHg)
D
(n)
D
(n)
D
(n)
4c
4d
4k
À10.8 4.2
À17.1 1.9
À12.2 4.5
3
4
3
NS
<0.001
NS
À10.8 2.9
À53.5 2.6
À21.7 3.7
4
4
5
NS
<0.001
NS
À23.6 7.0
À22.0 2.6
À37.2 6.7
4
4
5
Values are mean sem; n, number of animals; p, significance; NS, not statistically significant;
DHR, the difference of heart rate (HR) between the moment of maximal
hypotensive effect and the baseline (time 0 of experiment).

F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
325
Figure 4. Effect of 0.1 mg/kg of 4c, 4d and 4k on
D
MAP (calculated as the difference of MAP from baseline) in rats. Each point represents the mean value of
D
MAP for four or
five experiments. Comparisons were made using ANOVA with repeated measures and Fisher test. Significances (&) p <0.001 were found for comparisons of 4d versus 4c, 4d
versus 4k and 4d versus control groups. Significance (#) p <0.01 was found for comparison of 4k versus control group.
Figure 5. Effect of 0.1 mg/kg of 4c, 4d and 4k on
DHR (calculated as the difference of HR from baseline) in rats. Each point represents the mean value of DHR for four or five
experiments. Comparisons were made using ANOVA with repeated measures and Fisher test. Significances (&) p <0.001 were found for comparisons of 4d and 4k versus
control group and of 4k versus 4c group.
Table 4
The maximal negative chronotropic effect of synthesized compounds at 0.01, 0.1 and 1.0 mg/kg in anesthetized rats
Compound
0.01 mg/kg
HR (bpm)
À37 20
À43
À35 11
p
0.1 mg/kg
HR (bpm)
p
1.0 mg/kg
HR (bpm)
D
(n)
D
(n)
D
(n)
4c
4d
4k
3
4
3
NS
<0.001
<0.01
À40 15
À132 10
À92 22
4
4
5
NS
NS
NS
À68 25
À155 13
À117 23
4
4
5
8
Values are mean sem; n, number of animals; p, significance; NS, not statistically significant;
DHR, the difference of heart rate (HR) between the moment of maximal
hypotensive effect and the baseline (time 0 of experiment).

326
F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
under reflux for 7 h. After cooling to room temperature, the result-
ing mixture was concentrated under reduced pressure to a volume
of 5 ml, and then water was added. The solid product that was pre-
cipitated was filtered, washed with water, dried and purified by
crystallization from methanol. In this manner, the following com-
pounds were obtained.
4.3.2. 4-Bromo-3-[(imidazolidin-2-yl)imino]indazole (3b)
Yield: 0.20 g (22%). White solid; mp: 200–203 °C (MeOH). IR
(KBr): 3335, 3195, 3155 (NH), 2940, 2885 (CH), 1630, 1605
(C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz) d: 3.44 (s, 4H, CH2),
.
6.89 (br s, 1H, NH), 7.07–7.14 (m, 2H, aromat.), 7.22 (br s, 1H,
NH), 7.26–7.31 (m, 1H, aromat.), 12.09 (br s, 1H, NH). 13C NMR
(DMSO-d6, 50 MHz) d: 41.8 (two overlapping signals), 109.1,
114.6, 115.7, 122.3, 127.1, 142.1, 151.8, 159.1. Anal. Calcd for
4.2.1. 3-Amino-4-methylindazole (2d)
C10H10BrN5 (280.12): C, 42.88; H, 3.60; N, 25.00. Found: C, 42.62;
Yield: 2.1 g (65%). Beige solid; mp: 153–156 °C. IR (KBr): 3454,
H, 3.43; N, 25.32.
3335, 3183 (NH2, NH), 2964, 2922 (CH), 1609 (d NH2, C@N) cmÀ1
.
1H NMR (DMSO-d6, 500 MHz): d 2.59 (s, 3H, CH3), 4.92 (s, 2H,
NH2), 6.60 (d, J = 6.8 Hz, 1H, aromat.), 7.04–7.07 (m, 2H, aromat.),
11.41 (s, 1H, NH). Anal. Calcd for C8H9N3 (147.18): C, 65.29; H,
6.16; N 28.25. Found: C, 65.32; H, 5.98; N, 28.01.
4.3.3. 4-Chloro-3-[(imidazolidin-2-yl)imino]indazole (3c)
ZYield: 0.32 g (42%). White solid; mp: 227–229 °C (EtOH). IR
(KBr): 3400, 3350, 3120 (NH), 3085, 2880 (CH), 1610 (C@N) cmÀ1
.
Widmo 1H NMR (DMSO-d6, 200 MHz) d: 3.45 (s, 4H, CH2), 6.87–
6.91 (m, 1H, aromat.), 7.00 (br s, 2H, NH), 7.12–7.25 (m, 2H, aromat.),
12.09 (br s, 1H, NH). Anal. Calcd for C10H10ClN5 (235.67): C, 50.96; H,
4.28; N, 29.72. Found: C, 50.63; H, 3.96; N, 29.58.
4.2.2. 3-Amino-4-methoxyindazole (2e)
Yield: 1.6 g (44%). Beige solid; mp: 163–166 °C. IR (KBr): 3467,
3437, 3184 (NH2, NH), 3006, 2955 (CH), 1625 (d NH2), 1600
(C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz): d 3.84 (s, 3H, OCH3),
.
4.3.4. 3-[(Imidazolidin-2-yl)imino]-4-methylindazole (3d)
Yield: 0.41 g (59%). White solid; mp: 199–203 °C (acetonitrile). IR
(KBr): 3390, 3345 (NH), 3090, 2945, 2880 (CH), 1610 (C@N) cmÀ1. 1H
NMR (DMSO-d6, 500 MHz) d: 2.71 (s, 3H, CH3), 3.54 (br s, 4H, CH2),
6.27 (br s, 1H, NH), 6.65–6.67 (m, 1H, aromat.), 7.06–7.10 (m, 2H,
aromat.), 7.62 (br s, 1H, NH), 11.69 (br s, 1H, NH). 13C NMR
(DMSO-d6, 125 MHz) d: 19.2, 41.9, 43.9, 107.4, 117.6, 119.7, 126.8,
133.8, 141.8, 153.8, 159.8. Anal. Calcd for C11H13N5 (215.25): C,
61.38; H, 6.08; N, 32.53. Found: C, 61.02; H, 5.73; N, 32.21.
4.97 (s, 2H, NH2), 6.30 (d, J = 7.7 Hz, 1H, aromat.), 6.76
(d, J = 8.1 Hz, 1H, aromat.), 7.09 (t, 1H, aromat.), 11.39 (s, 1H,
NH). Anal. Calcd for C8H9N3O (163.18): C, 58.88; H, 5.56; N,
25.75. Found: C, 58.71; H, 5.23; N, 25.42.
4.2.3. 3-Amino-6-methylindazole (2j)
Yield: 2.5 g (79%). Beige solid; mp: 199–201 °C. IR (KBr): 3444,
3309, 3194 (NH2, NH), 2916, 2858 (CH), 1625 (d NH2, C@N) cmÀ1
.
1H NMR (DMSO-d6, 200 MHz): d 2.35 (s, 3H, CH3), 5.23 (s, 2H,
NH2), 6.71 (d, J = 8.1 Hz, 1H, aromat.), 6.99 (s, 1H, aromat.), 7.53 (d,
J = 8.1 Hz, 1H, aromat.), 11.17 (s, 1H, NH). Anal. Calcd for C8H9N3
(147.18): C, 65.29; H, 6.16; N, 28.55. Found: C, 65.01; H, 5.98; N,
28.32.
4.3.5. 3-[(Imidazolidin-2-yl)imino]-4-methoxyindazole (3e)
Yield: 0.23 g (31%). Beige solid; mp: 266–268 °C (DMF). IR
(KBr): 3390, 3340, 3160 (NH), 2960, 2880 (CH), 1635, 1615
(C@N) cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 3.41 (br s, 4H,
.
CH2), 3.80 (s, 3H, OCH3), 6.33 (d, J = 7.8 Hz, 1H, aromat.), 6.56 (br
s, 1H, NH), 6.83 (d, J = 8.3 Hz, 1H, aromat.), 7.12 (t, 1H, aromat.),
7.59 (br s, 1H, NH), 11.78 (br s, 1H, NH). 13C NMR (DMSO-d6,
125 MHz) d: 41.9, 44.1, 55.6, 99.1, 102.8, 108.9, 127.9, 143.8,
152.5, 155.8, 160.1. Anal. Calcd for C11H13N5O (231.25): C, 57.13;
H, 5.67; N, 30.28. Found: C, 56.87: H, 5.41; N, 29.95.
4.2.4. 3-Amino-7-chloroindazole (2k)
Yield: 1.92 g (52%). White solid; mp: 187–188 °C. IR (KBr):
3402, 3169, 3148 (NH2, NH), 2941, 2854 (CH), 1626 (d NH2, C@N)
cmÀ1 1H NMR (DMSO-d6, 200 MHz): d 5.51 (s, 2H, NH2), 6.90 (t,
.
1H, aromat.), 7.30 (d, J = 7.3 Hz, 1H, aromat.), 7.67 (d, J = 8.0 Hz,
1H, aromat.), 11.84 (s, 1H, NH). Anal. Calcd for C7H6ClN3
(167.60): C, 50.17; H, 3.61; N, 25.07. Found: C, 50.01; H, 3.36; N,
24.89.
4.3.6. 5-Chloro-3-[(imidazolidin-2-yl)imino]indazole (3f)
Yield: 0.22 g (29%). White solid; mp: 272–274 °C (DMF/MeOH).
IR (KBr): 3330, 3140 (NH), 2880 (CH), 1620 (C@N) cmÀ1 1H NMR
.
4.3. General procedure for the preparation of 3-[(imidazolidin-
2-ylo)imino]indazoles (3a–k)
(DMSO-d6, 200 MHz) d: 3.47 (br s, 4H, CH2), 6.75 (br s, 1H, NH),
7.23 (d, J = 8.8 Hz, 1H, aromat.), 7.32 (d, J = 8.8 Hz, 1H, aromat.),
7.42 (br s, 1H, NH), 7.52 (s, 1H, aromat.), 11.95 (br s, 1H, NH). Anal.
Calcd for C10H10ClN5 (235.67): C, 50.96; H, 4.28; N, 29.72. Found: C,
50.62; H, 3.97; N, 29.67.
A
suspension of the appropriate 3-aminoindazole (2a–k)
(3.2 mmol) and N-tert-butoxycarbonyl-2-methylthio-4,5-dihydro-
1H-imidazole (0.83 g, 3.84 mmol) in acetic acid (2 ml) was stirred
at 60–62 °C (oil bath) for 16 h and then the solvent was evaporated
under reduced pressure. The viscous residue was treated with
water (7 ml) and to the resulting solution or mixture was added
dropwise 15% aqueous Na2CO3 solution to pH 9.5–10. The precipi-
tate thus obtained was filtered, washed with water, dried and puri-
fied by crystallization from suitable solvent. In this manner, the
following compounds were obtained.
4.3.7. 3-[(Imidazolidin-2-yl)imino]-5-methylindazole (3g)
Yield: 0.27 g (39%). White solid; mp: 236–239 °C (EtOH). IR
(KBr): 3395, 3300 (NH), 3080, 2880 (CH), 1615 (C@N) cmÀ1 1H
.
NMR (DMSO-d6, 200 MHz) d: 2.35 (s, 3H, CH3), 3.43 (br s, 4H,
CH2), 6.60 (br s, 1H, NH), 7.07 (d, J = 8.2 Hz, 1H, aromat.), 7.18 (d,
J = 8.2 Hz, 1H, aromat.), 7.36 (s, 1H, aromat.), 7.57 (br s, 1H, NH),
11.60 (br s, 1H, NH). 13C NMR (DMSO-d6, 50 MHz) d: 21.2, 42.0
(two overlapping signals), 109.4, 119.1, 119.8, 126.8, 128.2,
139.8, 152.0, 159.7. Anal. Calcd for C11H13N5 (215.25): C, 61.38;
H, 6.08; N, 32.53. Found: C, 61.11; H, 5.86; N, 32.50.
4.3.1. 3-[(Imidazolidin-2-yl)imino]indazole (3a)
Yield: 0.35 g (54%). White solid; mp: 218–221 °C. IR (KBr):
3390, 3335 (NH), 2890, 2745 (CH), 1615 (C@N) cmÀ1 1H NMR
.
(DMSO-d6, 200 MHz): d 3.46 (br s, 4H, CH2), 6.65 (br s, 1H, NH),
6.93 (t, 1H, aromat.), 7.20–7.52 (m, 2H, aromat.), 7.41 (br s, 1H,
NH), 7.59 (d, J = 7.8 Hz, 1H, aromat.), 11.75 (br s, 1H, NH). MS (EI)
m/z = 201.1 (M+, 100), 144.1 (17), 118.1 (12). Anal. Calcd for
4.3.8. 3-[(Imidazolidin-2-ylo)imino]-5-methoxyindazole (3h)
Yield: 0.16 g (22%). Beige solid; mp: 219–223 °C (i-PrOH). IR
(KBr): 3395, 3360 (NH), 3050, 2945, 2880 (CH), 1625 (C@N) cmÀ1
.
1H NMR (DMSO-d6, 500 MHz) d: 3.57 (br s, 4H, CH2), 3.75 (s, 3H,
OCH3), 6.51 (br s, 1H, NH), 6.93–6.97 (m, 2H, aromat.), 7.21 (d,
J = 5.4 Hz, 1H, aromat.), 7.56 (br s, 1H, NH), 11.64 (br s, 1H, NH).
C10H11N5 (201.23): C, 59.69; H, 5.51; N 34.80. Found: C, 59.32; H,
5.21; N, 34.92.

F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
327
Anal. Calcd for C11H13N5O (231.25): C, 57.13; H, 5.67; N, 30.28.
Found: C, 56.89; H, 5.32; N, 30.11.
4.4.3. 4-Chloro-3-[(imidazolidin-2-yl)imino]indazole
hydrochloride (4c)
Yield: 0.12 g (43%). White solid; mp: 265–267 °C (EtOH/Et2O).
IR (KBr): 3325, 3160, 2990, 2890 (NH, NHÈ, CH), 1650, 1625
4.3.9. 6-Chloro-3-[(imidazolidin-2-yl)imino]indazole (3i)
Yield: 0.32 g (42%). White solid; mp: 280–282 °C (DMF). IR (KBr):
3435, 3375 (NH), 2955, 2890 (CH), 1625, 1610 (C@N) cmÀ1. 1H NMR
(DMSO-d6, 500 MHz) d: 3.48 (br s, 4H, CH2), 6.73 (br s, 1H, NH), 6.93
(d, J = 8.5 Hz, 1H, aromat.), 7.34 (s, 1H, aromat.), 7.50 (br s, 1H, NH),
7.57 (d, J = 8.5 Hz, 1H, aromat.), 11.91 (br s, 1H, NH). Anal. Calcd for
(C@N) cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 3.70 (s, 4H, CH2),
.
7.25 (d, J = 7.3 Hz, 1H, aromat.), 7.41 (t, 1H, aromat.), 7.57 (d,
J = 8.3 Hz, 1H, aromat.), 8.53 (s, 2H, NH), 10.66 (s, 1H, NH), 13.67
(s, 1H, NHÈ). Anal. Calcd for C10H11Cl2N5 (272.14): C, 44.13; H,
4.07; N, 25.73. Found: C, 43.92; H, 3.89; N, 25.48.
C10H10ClN5 (235.67): C, 50.96; H, 4.28; N, 29.72. Found: C, 50.67; H,
3.99; N, 29.89.
4.4.4. 3-[(Imidazolidin-2-yl)imino]-4-methylindazole
hydrochloride (4d)
Yield: 0.12 g (50%). White solid; mp: 209–211 °C (EtOH/Et2O).
4.3.10. 3-[(Imidazolidin-2-yl)imino]-6-methylindazole (3j)
IR (KBr): 3340, 3215, 2965, 2900 (NH, NHÈ, CH), 1655, 1630
Yield: 0.31 g (45%). White solid; mp: 265–267 °C (DMF). IR
(C@N) cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 2.61 (s, 3H, CH3),
.
(KBr): 3385, 3345 (NH), 3080, 2970 (CH), 1610 (C@N) cmÀ1 1H
.
3.70 (s, 4H, CH2), 6.91 (d, J = 6.8 Hz, 1H, aromat.), 7.27–7.30 (m,
1H, aromat.), 7.37 (d, J = 8.8 Hz, 1H, aromat.), 8.48 (s, 2H, NH),
10.86 (s, 1H, NH), 13.23 (s, 1H, NHÈ). Anal. Calcd for C11H14ClN5
(251.71): C, 52.49; H, 5.61; N, 27.82. Found: C, 52.19; H, 5.38; N,
27.56.
NMR (DMSO-d6, 200 MHz) d: 2.37 (s, 3H, CH3), 3.45 (br s, 4H,
CH2), 6.62 (br s, 1H, NH), 6.75 (d, J = 8.1 Hz, 1H, aromat.), 7.05 (s,
1H, aromat.), 7.45 (d, J = 8.1 Hz, 1H, aromat.), 7.56 (br s, 1H, NH),
11.56 (br s, 1H, NH). 13C NMR (DMSO-d6, 50 MHz) d: 21.5, 42.5
(two overlapping signals), 108.8, 116.8, 120.0, 120.2, 135.6,
141.5, 152.1, 159.6. Anal. Calcd for C11H13N5 (215.25): C, 61.38;
H, 6.08; N, 32.53. Found: C, 61.12; H, 5.76; N, 32.60.
4.4.5. 3-[(Imidazolidin-2-yl)imino]-4-methoxyindazole
hydrochloride (4e)
4.3.11. 7-Chloro-3-[(imidazolidin-2-yl)imino]indazole (3k)
Yield: 0.14 g (52%). White solid; mp: 229–230 °C (EtOH/Et2O).
IR (KBr): 3350, 3230, 3020, 2925 (NH, NHÈ, CH), 1655, 1620
Yield: 0.32 g (42%). White solid; mp: 223–225 °C (EtOH). IR
(KBr): 3350, 3245 (NH), 3075, 2880 (CH), 1635, 1615 (C@N) cmÀ1
.
(C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz) d: 3.74 (s, 4H, CH2),
.
1H NMR (DMSO-d6, 500 MHz) d: 3.50 (br s, 4H, CH2), 6.84 (br s,
1H, NH), 6.93–6.96 (m, 1H, aromat.), 7.34 (d, J = 7.3 Hz, 1H, aro-
mat.), 7.46 (br s, 1H, NH), 7.57 (d, J = 7.8 Hz, 1H, aromat.), 12.25
(br s, 1H, NH). 13C NMR (DMSO-d6, 125 MHz) d: 42.2 (two overlap-
ping signals), 114.8, 119.7, 120.1, 121.5, 126.2, 138.8, 153.8, 160.6.
Anal. Calcd for C10H10ClN5 (235.67): C; 50.96; H, 4.28; N, 29.72.
Found: C, 50.72; H, 3.97; N, 29.98.
3.93 (s, 3H, OCH3), 6.55 (d, J = 7.7 Hz, 1H, aromat.), 7.03 (d,
J = 8.4 Hz, 1H, aromat.), 7.32 (t, 1H, aromat.), 8.76 (s, 2H, NH),
10.45 (s, 1H, NH), 13.02 (s, 1H, NHÈ). Anal. Calcd for C11H14ClN5O
(267.71): C, 49.35; H, 5.27; N, 26.16. Found: C, 49.02; H, 4.98; N,
26.08.
4.4.6. 5-Chloro-3-[(imidazolidin-2-yl)imino]indazole
hydrochloride (4f)
4.4. General procedure for the preparation of 3-[(imidazolidin-
2-yl)imino]indazole hydrochlorides (4a–k)
Yield: 0.12 g (43%). White solid; mp: 253–256 °C (i-PrOH). IR
(KBr): 3390, 3320, 3190, 2980, 2885 (NH, NHÈ, CH), 1660 (C@N)
cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 3.75 (s, 4H, CH2), 7.44 (d,
.
To
a
suspension of the appropriate 3-[(imidazolidin-
J = 8.5 Hz, 1H, aromat.), 7.56 (d, J = 8.5 Hz, 1H, aromat.), 8.14 (s,
1H, aromat.), 8.46 (s, 2H, NH), 12.34 (br s, 1H, NH), 13.22 (s, 1H,
NHÈ). Anal. Calcd for C10H11Cl2N5 (272.14): C, 44.13; H, 4.07; N,
25.73. Found: C, 43.92; H, 3.82; N, 25.78.
2-yl)imino]indazole (3a–k) (1 mmol) in anhydrous methanol
(5 ml) was added dropwise HCl/MeOH solution (5.98 g/100 ml,
0.74 ml, 1.2 mmol). The cooling bath was removed, and the result-
ing solution or mixture was stirred at 20–22 °C for 30 min. Then
the solvent was evaporated under reduced pressure to dryness.
The crude product thus obtained was purified by crystallization
from suitable solvent. In this manner the following compounds
were obtained.
4.4.7. 3-[(Imidazolidin-2-yl)imino]-5-methylindazole
hydrochloride (4g)
Yield: 0.10 g (42%). White solid; mp: 233–236 °C (EtOH/Et2O).
IR (KBr): 3300, 3215, 2920 (NH, NHÈ, CH), 1670, 1625 (C@N) cm
À1
1H NMR (DMSO-d6, 200 MHz) d: 2.40 (s, 3H, CH3), 3.74 (s, 4H,
4.4.1. 3-[(Imidazolidin-2-yl)imino]indazole hydrochloride (4a)
.
Yield: 0.12 g (50%). White solid; mp: 196–199 °C (EtOH/Et2O).
CH2), 7.26 (dd, J = 8.5 Hz, J = 1.3 Hz, 1H, aromat.), 7.41 (d,
J = 8.5 Hz, 1H, aromat.), 7.72 (s, 1H, aromat.), 8.42 (s, 2H, NH),
12.04 (br s, 1H, NH), 12.86 (s, 1H, NHÈ). Anal. Calcd for C11H14ClN5
(251.71): C, 52.49; H, 5.61; N, 27.82. Found: C, 52.27; H, 5.32; N,
27.71.
IR (KBr): 3250, 2975, 2865 (NH, NHÈ, CH), 1660, 1615 (C@N) cm
À1
.
1H NMR (DMSO-d6, 200 MHz) d: 3.80 (s, 4H, CH2), 7.21 (t, 1H,
aromat.), 7.44–7.59 (m, 2H, aromat.), 8.07 (d, J = 8.4 Hz, 1H, aro-
mat.), 8.50 (s, 2H, NH), 12.37 (s, 1H, NH), 13.07 (s, 1H, NHÈ). Anal.
Calcd for C10H12ClN5 (237.69): C, 50.53; H, 5.09; N, 29.46. Found: C,
50.23; H, 4.78; N, 29.21.
4.4.8. 3-[(Imidazolidin-2-ylo)imino]-5-methoxyindazole
hydrochloride (4h)
4.4.2. 4-Bromo-3-[(imidazolidin-2-yl)imino]indazole
hydrochloride (4b)
Yield: 0.13 g (48%). White solid; mp: 210–213 °C (EtOH/Et2O).
IR (KBr): 3390, 3310, 3190, 2910, 2830 (NH, NHÈ, CH), 1670,
Yield: 0.13 g (41%). White solid; mp: 256–260 °C (EtOH/Et2O).
1635 (C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz) d 3.73 (s, 4H,
.
IR (KBr): 3305, 3075, 3030, 2985, 2890 (NH, NHÈ, CH), 1660,
1625 (C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz) d: 3.67 (s, 4H,
.
CH2), 3.78 (s, 3H, OCH3), 7.08 (dd, J = 9.1 Hz, J = 2.3 Hz, 1H,
aromat.), 7.42 (d, J = 9.1 Hz, 1H, aromat.), 7.46 (d, J = 2.3 Hz, 1H,
aromat.), 8.40 (s, 2H, NH), 12.14 (s, 1H, NH), 12.87 (s, 1H, NHÈ).
Anal. Calcd for C11H14ClN5O (267.71): C, 49.35; H, 5.27; N, 26.16.
Found: C, 49.11; H, 5.08; N, 26.32.
CH2), 7.28–7.43 (m, 2H, aromat.), 7.61 (d, J = 8.0 Hz, 1H, aromat.),
8.49 (s, 2H, NH), 10.62 (br s, 1H, NH), 13.75 (s, 1H, NHÈ). Anal.
Calcd for C10H11BrClN5 (316.58): C, 37.94; H, 3.50; N, 22.12. Found:
C, 37.77; H, 3.34; N, 21.97.

328
F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
4.4.9. 6-Chloro-3-[(imidazolidin-2-yl)imino]indazole
hydrochloride (4i)
was allowed to reach equilibrium (45 min). Bound and free radio-
activity were separated by rapid filtration through pre-soaked
(0.5% polyethyleneimine) glass-fibre filters (Whatman GF/B). Fil-
ters were then washed twice with 5 ml of ice-cold buffer and
membrane-bound radioactivity remaining on the filters was deter-
mined by liquid scintillation counting. Data were analysed by iter-
ative non-linear regression curve fitting procedures with GraphPad
Prism. Each experiment was analysed individually and equilibrium
dissociation constants (Ki) were determined by the method of
Cheng and Prusoff.22 The resulting values are given as means of
three or four separate experiments.
Yield: 0.15 g (53%). White solid; mp: 309–311 °C (EtOH/Et2O).
IR (KBr): 3220, 3135, 2860, 2850 (NH, NHÈ, CH), 1655, 1615
(C@N) cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 3.74 (s, 4H, CH2),
.
7.20 (d, J = 8.8 Hz, 1H, aromat.), 7.59 (s, 1H, aromat.), 8.05 (d,
J = 8.8 Hz, 1H, aromat.), 8.46 (s, 2H, NH), 12.39 (s, 1H, NH), 13.17
(s, 1H, NHÈ). Anal. Calcd for C10H11Cl2N5 (272.14): C, 44.13; H,
4.07; N, 25.73. Found: C, 42.93; H, 3.87; N, 25.57.
4.4.10. 3-[(Imidazolidin-2-yl)imino]-6-methylindazole
hydrochloride (4j)
Yield: 0.12 g (50%). White solid; mp: 223–228 °C (EtOH/Et2O).
4.6. Estimation of agonist potency and efficacy
IR (KBr): 3305, 3155, 2925, 2850 (NH, NHÈ, CH), 1665, 1620
(C@N) cmÀ1 1H NMR (DMSO-d6, 200 MHz) d: 2.43 (s, 3H, CH3),
.
4.6.1. Cell culture and transfections
3.74 (s, 4H, CH2), 6.99 (d, J = 8.4 Hz, 1H, aromat.), 7.27 (s, 1H, aro-
mat.), 7.85 (d, J = 8.4 Hz, 1H, aromat.), 8.42 (s, 2H, NH), 12.16 (s, 1H,
NH), 12.80 (s, 1H, NHÈ). Anal. Calcd for C11H14ClN5 (251.71): C,
52.49; H, 5.61; N, 27.82. Found: C, 52.12; H, 5.42; N, 27.61.
Adherent Chinese hamster ovary (CHO) cells (K1 strain)
(American Type Culture Collection, Manassas, VA, USA), transfected
to express the human
-minimum essential medium supplemented with 2 mM
glutamine, 20 mM NaHCO3, 5% heat-inactivated foetal calf serum,
penicillin (50 units mlÀ1) and streptomycin (50 g mlÀ1). The pc
a
2A-adrenoceptor subtype,23 were cultured in
a
4.4.11. 7-Chloro-3-[(imidazolidin-2-yl)imino]indazole
hydrochloride (4k)
l
DNA3.1+-based expression constructs were originally transfected
into CHO cells using the Lipofectamine™ 2000 reagent kit (Invitro-
gen Life Technologies, Inc., Rockville, MD, USA). Stable transfections
Yield: 0.12 g (43%). White solid; mp: 277–280 °C (EtOH/Et2O).
IR (KBr): 3280, 3130, 2980, 2895, 2860 (NH, NHÈ, CH), 1665,
1630 (C@N) cmÀ1 1H NMR (DMSO-d6, 500 MHz) d: 3.76 (s, 4H,
.
were selected using 800
(Calbiochem, San Diego, CA, USA). After selection, transfected cell
cultures were examined for their ability to bind the 2-adrenoceptor
antagonist radioligand [3H]RX821002. The cells were subsequently
maintained in 200
g mlÀ1 G418. Confluent cells were harvested
l
g mlÀ1 of the neomycin analogue G418
CH2), 7.17–7.20 (m, 1H, aromat.), 7.56 (d, J = 7.3 Hz, 1H, aromat.),
8.01 (d, J = 7.8 Hz, 1H, aromat.), 8.45 (s, 2H, NH), 12.34 (br s, 1H,
NH), 13.48 (s, 1H, NHÈ). Anal. Calcd for C10H11Cl2N5 (272.14): C,
44.13; H, 4.07; N, 25.73. Found: C, 44.01; H, 3.92; N, 25.63.
a
l
into chilled phosphate-buffered saline, pelleted and frozen at
À70 °C.
4.5. Radioligand binding assays
4.6.2. Membrane preparation
4.5.1. I1-Binding site assay
All procedures were performed on ice. The harvested recombi-
nant CHO cell pellets were thawed and suspended in hypotonic lysis
buffer (10 mM Tris–HCl, 0.1 mM EDTA, 0.32 mM sucrose, pH 7.4)
and homogenised using an Ultra-Turrax homogeniser (3 Â 10 s at
800 rpm). The homogenate was centrifuged at 23,000g for 30 min,
and the pellet was re-homogenised and again centrifuged at
23,000g for 30 min. The membrane pellet was suspended in hypo-
tonic lysis buffer and stored at À70 °C until used. Protein concentra-
tions were determined with the method of Bradford24 using bovine
serum albumin as reference.
Kidneys were obtained post mortem from male Sprague–Dawley
rats (250–280 g) and crude P2 membranes prepared according to
methods of Lione et al.27 [3H]clonidine (3 nM, Perkin–Elmer) was
bound in the presence of 10
l
M rauwolscine to preclude binding
2-adrenoceptors. The specific component was defined by
lM rilmenidine; under these conditions, the site labelled repre-
to
a
10
sents a model of the central I1 binding site.28 Membrane aliquots
(400
l
l, 0.2–0.5 mg protein) were incubated with 11 concentra-
M–100 mM.
tions of the test compounds over the range 0.01
l
Incubations were carried out in 50 mM Tris–HCl buffer (pH 7.4)
at room temperature for 45 min. Bound radioligand and free radio-
activity were separated by rapid filtration through pre-soaked
(0.5% polyethyleneimine) glass-fibre filters (Whatman GFB).
Trapped radioligand was determined by liquid scintillation count-
ing and the data were analysed with GraphPad Prism version 3.02
for Windows (GraphPad Software, San Diego, CA, USA) to yield IC50
values (the concentration of tested ligand that displaces 50% of
specifically bound [3H]clonidine).
4.6.3. [35S]GTP
cS Binding assay
Agonist-induced stimulation of [35S]GTP
cS binding was mea-
sured essentially as described previously.25 Briefly, membranes
were thawed and diluted with binding buffer (50 mM Tris, 1 mM
EDTA, 5 mM MgCl2, 150 mM NaCl, 10 lM GDP, 1 mM DTT, 30 lM
ascorbic acid, pH 7.4). Incubations were performed on 96-well
Millipore MultiScreen MAFB glass-fibre filter plates (Millipore
Corp., Bedford, MA, USA). Samples containing 5
protein were preincubated with agonists for 30 min at 37 °C prior
to addition of 0.1 nM [35S]GTP
S. Reactions were terminated after
lg of membrane
4.5.2. a1-, a2- and I2-Binding site assays
c
Brains were obtained post mortem from male Sprague–Dawley
60 min incubation at 37 °C by vacuum filtration using a Millipore
MultiScreen Vacuum Manifold. The filter plates were washed three
times with cold wash buffer (20 mM Tris, 1 mM EDTA, 5 mM
rats (250–280 g) and crude P2 membranes prepared according to
methods of Lione et al.27 Membrane aliquots (400
l
l, 0.2–0.3 mg
protein) were incubated with 11 concentrations of the tested com-
pounds over the range 0.01 nM–100 M in the presence of the
selective I2 binding site ligand [3H]2BFI28 (1 nM), the
1-adreno-
ceptor antagonist [3H]prazosin (1 nM) or the
2-adrenoceptor
antagonist [3H]RX82100229 (1 nM) in a final volume of 500
l.
Non-specific binding was determined using 10
M BU22430 for
defining I2 binding, 10 M phenylephrine for 1-adrenoceptors
and 10 M rauwolscine to define 2-adrenoceptor binding. Each
incubation was performed in triplicate at room temperature and
MgCl2, pH 7.4). Filters were dried, and 50 ll SuperMix scintillation
l
cocktail was added into each well. The incorporated radioactivity
was measured using a MicroBeta scintillation counter (Perkin–El-
mer Wallac, Turku, Finland). All experiments were performed in
duplicate and repeated at least three times. Analysis of the results
a
a
l
l
with GraphPadPrism yielded estimates of agonist potency (EC50
)
l
a
and efficacy (intrinsic activity in comparison to the natural full
agonist noradrenaline).
l
a

F. Sa˛czewski et al. / Bioorg. Med. Chem. 19 (2011) 321–329
329
4.6.4. In vivo studies mean arterial blood pressure (MAP) and
Acknowledgements
heart rate (HR) in rats
Male Wistar rats, weighing 200–250 g, were purchased from the
This research was supported in part by the Polish Ministry of
Science and Higher Education, Poland (Grant N40500532/0458).
The authors (A.R., K.B., A.L.) gratefully acknowledge the technical
assistance of Ewa Maciejewska.
´
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 ip injection of thiopental (Sandoz,
Austria) at the dose 70 mg/kg body weight and maintained under
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.020. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
anaesthesia by thiopental supplementation (30 lg/kg/min) during
the experiment. The animals were placed on a heated table, and
body temperature was maintained between 36 and 37 °C. Trache-
ostomy 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 thiopental at the rate
of 1.2 ml/h. After 40 min of saline infusion, the tested compound
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4.7. X-ray structure analysis
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149 refined parameters are: R1 = 0.0335, wR2 = 0.0878 (R1 = 0.0455,
wR2 = 0.0983 for all 2258 data). Atom labelling is shown in Figure 1.
Tables of atomic coordinates, bond lengths, and bond angles
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