Fluorinated analogues of marsanidine, a highly α2-AR/imidazoline I1 binding site-selective hypotensive agent. Synthesis and biological activities

Article abstract of DOI:10.1016/j.ejmech.2014.09.083

The aim of these studies was to establish the influence of fluorination of the indazole ring on the pharmacological properties of two selective α₂-adrenoceptor (α₂-AR) agonists: 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine,

Full text of DOI:10.1016/j.ejmech.2014.09.083

Accepted Manuscript
Fluorinated analogues of marsanidine, a highly α -AR / imidazoline I binding site -
2
1
Selective hypotensive agent. Synthesis and biological activities
Aleksandra Wasilewska, Franciszek Sączewski, Alan L. Hudson, Mehnaz Ferdousi,
Mika Scheinin, Jonne M. Laurila, Apolonia Rybczyńska, Konrad Boblewski, Artur
Lehmann
PII:
S0223-5234(14)00910-6
10.1016/j.ejmech.2014.09.083
EJMECH 7390
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2014
Revised Date: 25 September 2014
Accepted Date: 26 September 2014
Please cite this article as: A. Wasilewska, F. Sączewski, A.L. Hudson, M. Ferdousi, M. Scheinin, J.M.
Laurila, A. Rybczyńska, K. Boblewski, A. Lehmann, Fluorinated analogues of marsanidine, a highly
α -AR / imidazoline I binding site - Selective hypotensive agent. Synthesis and biological activities,
2
1
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.083.
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ACCEPTED MANUSCRIPT
Fluorinated analogues of marsanidine, a highly α2-AR / imidazoline I1 binding
site - selective hypotensive agent. Synthesis and biological activities
Aleksandra Wasilewskaa*, Franciszek Sączewskia, Alan L. Hudsonb, Mehnaz Ferdousib, Mika Scheininc,
Jonne M. Laurilac, Apolonia Rybczyńskad, Konrad Boblewskid, Artur Lehmannd
a Department of Chemical Technology of Drugs, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416
Gdańsk, Poland
b Department of Pharmacology, 947 Medical Sciences Building, University of Alberta, Edmonton, Canada T6G
2H7
c Department of Pharmacology, Drug Development and Therapeutics, University of Turku and Turku University
Hospital, Fl-20014 Turku, Finland
d Department of Pathophysiology, Medical University of Gdańsk, Dębinki 7, Building 27, 80-211 Gdańsk, Poland
*Corresponding author. Tel.: +48 58 349 19 57; fax: +48 58 349 16 54.
E-mail address: alwas@gumed.edu.pl
Abstract
The aim of these studies was to establish the influence of fluorination of the indazole ring on
the pharmacological properties of two selective α2-adrenoceptor (α2-AR) agonists: 1-[(imidazolidin-2-
yl)imino]-1H-indazole (marsanidine, A) and its methylene analogue 1-[(4,5-dihydro-1H-imidazol-2-
yl)methyl]-1H-indazole (B). Introduction of fluorine into the indazole ring of A and B reduced both
binding affinity and α2-AR/I1 imidazoline binding site selectivity. The most α2-AR-selective ligands
were 6-fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c) and 7-fluoro-1-[(imidazolidin-2-yl)imino]-
1H-indazole (6d). The in vivo cardiovascular properties of fluorinated derivatives of A and B revealed
that in both cases the C-7 fluorination leads to compounds with the highest hypotensive and
bradycardic activities. The α2-AR partial agonist 6c was prepared as a potential lead compound for
development of a radiotracer for PET imaging of brain α2-ARs.
Keywords: marsanidine, indazole, α2-adrenoceptor, Selectfluor
1. Introduction
α2-Adrenoceptors (α2-ARs) represent a subfamily of G-protein coupled receptors mediating
many of the central and peripheral actions of the endogenous catecholamines norepinephrine and
epinephrine. In humans and other mammals, they are present as three subtypes, α2A-, α2B- and α2C-
ARs [1], with α2A responsible for most of the hitherto recognized physiological functions of α2-ARs,
which include regulation of sympathetic nervous system activity, arterial blood pressure, insulin
secretion, gastrointestinal motility, body temperature and seizure threshold [2–6]. α2-ARs are also
involved in different aspects of central nervous system functioning. They regulate noradrenergic
neurotransmission via two mechanisms: somato-dendritic autoreceptors inhibit the firing of
noradrenergic neurons in the locus coeruleus and in other noradrenergic brain nuclei, and activation
of presynaptic α2-autoreceptors inhibits norepinephrine release from nerve endings as part of a
negative feedback loop [2,7]. The pharmacological effects of activation of central α2-heteroreceptors

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include sedation, anxiolysis, analgesia and anaesthetic-sparing activity [8]. α2-ARs also contribute to
the modulation of a number of mental functions, among others mood, cognition and behaviour
[2,5,9–14]. α2-AR functions may be disturbed in many neurological and psychiatric disorders such as
Alzheimer’s disease [15–19], depression [20,21], long-term stress responses [22–24] and anxiety
disorders [25,26], where they also represent potential therapeutic drug targets. It is thus considered
important to try to develop approaches to investigate brain α2-ARs in vivo, in order to enable
investigation and diagnostics of and drug development for these conditions.
There are numerous reports on the alteration of pharmacological properties of biologically
active compounds on fluorination that could be ascribed to changes in pKa [27–29], lipophilicity [30],
conformation [31,32], chemical/metabolic stability [33–39] or propensity to participate in
intermolecular interactions [40–43]. According to the results obtained by Kirk et al. [44], fluorine
substitution of the aromatic ring of adrenergic agonists may have marked and site-of-fluorination-
dependent influences on their receptor affinity and selectivity. Therefore, it seemed reasonable to
expand our previous SAR studies of 1-[(imidazolidin-2-yl)imino]-1H-indazoles A (Fig. 1) [45–47] and 1-
[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazoles B (Fig. 1) [48], α2-AR ligands, by including
fluorine-substituted derivatives 6a–e and 8a–c (Fig. 1).
Positron emission tomography (PET) is a molecular imaging technique that offers the
potential to reveal distribution and concentration of protein targets (receptors, transporters,
enzymes) in living organisms by the use of specific ligands [49,50] labelled with short-lived positron
emitting radionuclides, most commonly 11C or 18F [51,52]. Imaging of brain receptors with PET is
dependent on the availability of selective high-affinity ligands that are amenable for labelling with a
positron emitter and demonstrate good permeability of the blood-brain barrier. For example, the
possibility of imaging of α2-ARs with PET has been extensively investigated in recent years and
several α2-AR ligands have been labelled with positron emitters and evaluated in animals and
humans [53–62]. Promising results were obtained for [11C]yohimbine [61] and [11C]MPTQ ([N-methyl-
11C]
c]quinolone) [62].
In view of the above information our next goal was to develop a fluorination method suitable
5-methyl-3-[4-(3-phenylallyl)-piperazin-1-ylmethyl]-3,3a,4,5-tetrahydroisoxazolo[4,3-
for 18F labelling of compounds A or B.
Fig. 1. Structures of marsanidine and its analogues.
2. Results and Discussion
2.1 Chemistry
The fluorinated derivatives of marsanidine (6a–e) were obtained according to the procedure
previously described [45] and depicted in Scheme 1. Condensation of 2-fluorobenzaldehydes 1a–e

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with hydrazine furnished the respective fluoro-1H-indazoles 2a–e [63]. The concurrent 1- and 2-
amination of the latter was accomplished by the use of hydroxylamine-O-sulfonic acid (HOSA). The
isolated 1-amino-1H-indazoles (3a–e) were then reacted with N,N′-bis-Boc-imidazolidine-2-thione
[64] in the presence of HgCl2 to obtain bis-Boc-protected compounds 5a–e. Deprotection of the latter
with TFA/CH2Cl2 afforded the target fluoro-substituted marsanidine analogues 6a–e.
Scheme 1. Synthesis of fluorinated derivatives of marsanidine. Reagents and conditions: a) NH2NH2∙H2O, Δ, 12–
48h, 20–40 % yield for 2a–d or NH2NH2∙H2O/THF, 0 ºC then 70 °C, 48 h, 13 % yield for 2e; b) HOSA (2.6 molar
equiv.), 6 % NaOH/H2O, EtOH, 55 °C, 20 min., then room temperature 1.5 h, 20–40 % yield for 3a–e; c) N,N’-bis-
Boc-imidazolidine-2-thione (1.5 molar equiv.), HgCl2 (1.5 molar equiv.), Et3N (3.5 molar equiv.), anhydrous DMF,
0 °C, 20 min., room temperature 5–7 days, 20–70 % yield for 5a–e; d) 50 % TFA/CH2Cl2, room temperature, 2 h;
e) 10 % NaOH/H2O, 60–88 % yield for 6a–e; f) 2.6 M HCl/Et2O.
The synthesis of monofluoro-substituted derivatives of B was accomplished according to
Scheme 2 [48]. The indazole sodium salts, generated upon treatment of the corresponding indazoles
with sodium hydride in THF, were reacted with the preformed 2-chloromethylimidazoline [65]. From
the resulting mixture of 1- and 2-alkylated indazoles compounds 8a–c were isolated in 25 to 40 %
yield.

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Scheme 2. Synthesis of fluorinated derivatives 8a–c of 1-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-indazole.
Reagents and conditions: a) NaH, THF; b) 2-chloromethylimidazoline, 25–40 % yield for 8a–c; c) 2.5 M HCl/Et2O.
For biological studies, compounds 6a–e and 8a–c were converted into hydrochloride salts
7a–e and 10a–c, respectively, with the use of ethereal solution of hydrochloride. Structures of all
newly prepared compounds were confirmed by C, H, N elemental analyses as well as IR and NMR
spectroscopic data.
2.2 Pharmacological evaluation
2.2.1. Radioligand binding assays
In vitro radioligand binding assays involved the investigation of the affinity and selectivity of
the newly prepared compounds 6a–e and 8a–c for α1- and α2-ARs as well as imidazoline I1 and I2
binding sites. A summary of all these properties together with the data obtained for previously
reported marsanidine [45,46], B and 8d [48] are displayed in Table 1. All of the monofluoro-
substituted marsanidine derivatives 6a–d and analogues 8a–d were bound with high to moderate
affinity to α2-ARs. In both series of compounds, fluorination at C-4 proved to be detrimental for α2-AR
binding, as 8a (Ki = 187.3 nM) and 6a (Ki = 416 nM) displayed 10- to 30-fold higher Ki values than B (Ki
= 18.5 nM) and marsanidine (Ki = 14.0 nM). On the contrary, substitution at C-6 or C-7 yielded
compounds with the highest activity at α2-AR in both series. Given the above results, the inactivity of
the perfluorinated 6e at α2-AR was rather surprising.
Apart from being high to moderate affinity α2-AR ligands, compounds 8a–c displayed high
affinity for I2 imidazoline binding sites with Ki values ranging from 8.193 to 21.87 nM. This property
may be ascribed to fluorine substitution of the indazole moiety since B is inactive at I2 binding sites
(Ki = 42 200 nM).
In comparison to the parent marsanidine, compounds 6a–d displayed definitely lower I1/α2
selectivity, with Ki ratios ranging from 1.4 to 789; 6b had nearly similar affinity for I1 binding sites and
α2-ARs. Worth noting is also their moderate (6b–d) or low (6a) selectivity for α2- versus α1-ARs. The
highest affinity for α2-ARs, close to that of marsanidine, was observed for compounds 6c and 6d (Ki =
26.2 and 31.0 nM). These two compounds also displayed the highest overall selectivity within the 6a–
d and 8a–d series. Their α1/α2, I1/α2 and I2/α2 selectivity ratios all exceeded 35 (α1/α2 for 6c) and
ranged up to 11 263 (I2/α2 for 6d).
Table 1.
Binding affinity data for marsanidine, B, 6a–e and 8a–d

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Compd
α1 Ki (nM)a,c
ndf
α2 Ki (nM)a,c
14.05 ± 2.7
416.0 ± 16.2
64.33 ± 4.39
26.20 ± 6.30
30.97 ± 1.93
3717 ± 220.2
18.5 ± 5.9
187.3 ± 40.26
32.17 ± 9.60
29.73 ± 3.04
12.6 (n=1)g
I1 IC50 (nM)b,c
I2 Ki (nM)a,c
marsanidined
54550 ± 16730
14776 ± 7893
91.65 ± 16.54
20675 ± 20513
7740 ± 5522
5827 ± 3402
19400 (n=1)g
1765 ± 1200
2419 ± 2057
33456 ± 24023
16400 (n=1)g
16900 ± 5900
6177 ± 4962
5703 ± 2656
21967 ± 3735
348833 ± 306639
22733 ± 817.2
42200 (n=1)g
21.87 ± 4.703
10.38 ± 5.44
8.193 ± 3.43
156 (n=1)g
6a
6b
6c
6d
6e
Be
8a
8b
8c
8de
3807 ± 1342
2320 ± 293
918.3 ± 52.6
1625 ± 475.2
19793 ± 18003
272 ± 42
534.7 ± 118.4
476.3 ± 267.5
518.3 ± 159.9
107 (n=1)g
a
Ki affinity values for α1-adrenoceptors, α2-adrenoceptors, and I2 imidazoline binding sites were assessed by
measuring the ability of the tested compounds to compete with [3H]prazosin, [3H]RX821002 or [3H]2BFI binding
b
to rat brain membranes. Molar concentration of the tested compounds that displaces 50% of specifically
bound [3H]clonidine in rat kidney membranes in the presence of rauwolscine (I1 imidazoline binding sites).
c
d
Values given are means ± s.e.m. from 3–4 independent experiments. The results have been published earlier
e
f
in Sączewski F. et al. [45]. The results have been published earlier in Sączewski J. et al. [48]. nd: not
determined. g n: number of experiments.
The most α2-AR-selective ligands 6c and 6d were further evaluated for their affinity for the
different human α2-AR subtypes (Table 2). In this analysis, 6c and 6d displayed selectivity towards
α2A- versus α2C-ARs and were slightly more active at α2A- than at α2B-ARs. Since α2A-ARs are the most
abundant in the brain [3,66–71], while the density of α2B-ARs in the brain is too small to be detected
with PET [62], one can expect that compounds 6c and 6d could be suitable ligands for selective α2A-
AR imaging.
Table 2.
Binding affinities of 6c and 6d obtained with [3H]RS-79948-197 to human α2-AR subtypes expressed in CHO cell
membranes. Results expressed as Ki (nM) and their 95 % confidence intervals of three independent
experiments.
Human
Compd
α2A-AR
α2B-AR
α2C-AR
marsanidine
6c
6d
52 (39–70)
33 (27–41)
65 (54–77)
79 (63–99)
72 (54–92)**
158 (130–190)**
640 (520–780)***
600 (510–710)***
1200 (960–1400)**
Statistical significance of differences from the α2A-AR is shown by symbols: **P<0.01; ***P<0.001 tested with
unpaired t-tests.
2.2.2. In vivo cardiovascular effects
The potential pharmacological effects of centrally acting α2-AR agonists include hypotension
and bradycardia [2–6]. To determine the influence of fluorination of the indazole ring on the
cardiovascular properties of compounds A and B the high-affinity α2-AR ligands 6b–d, 8b and 8c were
subjected to in vivo studies in anesthetized rats after intravenous infusion of the tested compounds
using previously described procedures [72]. Changes in mean arterial blood pressure (MAP) and
heart rate (HR) were measured and compared to baseline values. The results are expressed as ΔMAP
and ΔHR and are presented in Table 3.

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The previously reported [45,46,48] SAR analysis concerning marsanidine and its methylene
analogue B indicated that substitution at the C-7 atom of the indazole nucleus with chlorine or
methyl group resulted in compounds with high cardiovascular activity, exceeding that of the
congeners. The same rule applied to C-7 fluorination as compounds 6d and 8d [48] displayed the
largest hemodynamic effects in the respective 6b–6d and 8b–8d series. It is worth noting that
hypotensive effects of 6d administered at a dose of 0.1 mg/kg were larger than those of 7-chloro-
(ΔMAP = –49.6 mmHg, unpublished data) and 7-methylmarsanidine (ΔMAP = –43.5 mmHg [45]). On
the other hand, 8d (ΔMAP = – 29.5 mmHg) reduced arterial blood pressure to a lesser extent than its
7-chloro- (ΔMAP = –47.7 mmHg [48]) and 7-methyl- (ΔMAP = –49.7 mmHg, unpublished data)
analogues.
Comparison of 6c with 6d or 8c with 8d reveals marked differences in cardiovascular
properties that are in contradiction with the similar receptor affinity profiles. Another rule that can
be seen in the 6b–6d and 8b–8d series of compounds is that C-6-substituted derivatives are more
active than C-5-substituted ones. Indeed, 6b was associated with relatively small MAP responses
even though it showed moderately high affinity for α2-ARs and I1 imidazoline binding sites both of
which are involved in central blood pressure regulation [73,74]. On the other hand, 8b had no effects
on MAP and HR, which is exceptional among the compounds studied.
Table 3.
Effects of compounds 6b–d and 8b–d at 0.1 mg/kg i.v. on mean arterial blood pressure (MAP) and heart rate
(HR) in anesthetized rats
.
a,d
b,d
c,d
+ΔMAPmax
-ΔMAPmax
-ΔHRmax
ne
f
f
f
compd
6b
(tmax
)
(tmax
)
(tmax)
14.6 ± 5.9 (2) *%
24.8 ± 4.6 (3) *§
21.1 ± 2.2 (4) *§
0.8 ± 1.0 (2)
–8.6 ± 2.3 (11) $!@
–36.0 ± 2.3 (35) *!
–59.4 ± 5.7 (33) *$@
–7.2 ± 1.6 (40) $!@
–12.7 ± 1.8 (36) #$!@
–29.5 ± 4.0 (34) *$!
–2.7 ± 1.2 $!@
–61 ± 7 (5) *§
4
4
4
4
5
5
5
6c
–118 ± 15 (2) *§^&
–128 ± 9 (10) *§^&
–12 ± 3 (38) ^&
–52 ± 9 (5) *§
6d
8b
8c
8dg
controlh
a
23.6 ± 3.6 (4) *§
17.7 ± 7.1 (1) *§
0.4 ± 0.7
–88 ± 21 (21) *§^
–9 ± 2 ^&
b
The maximal hypertensive effect [mmHg] of a compound observed during 60 minutes after injection. The
maximal hypotensive effect [mmHg] of a compound observed during 60 minutes after injection. c The maximal
bradycardic effect [bpm] of a compound observed during 60 minutes after injection. d Values given are means ±
e
s.e.m from n independent experiments. Number of experiments. f tmax: time (in minutes) after the injection
g
h
when the maximal effect was observed. The results have been published earlier in Sączewski J. et al. [48].
Saline vehicle injection. (*) p <0.001, (#) p <0.05 vs control. (&) p <0.001 vs 6b. ($) p<0.001 vs 6c. (!) p <0.001 vs
6d. (§) p <0.001, (%) p <0.05 vs 8b. (^) p <0.001 vs 8c. (@) p <0.001 vs 8d.

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Fig. 2. Effects of 0.1 mg/kg of 6c, 6d and 8d on MAP (∆MAP calculated as the difference of MAP between
sequential measurements and time 0 of the experiment) in anesthetized rats. The lines represent the mean
values of ∆MAP for four or five animals. Comparisons were made using ANOVA with repeated measures and
Fisher and Duncan tests. Significant differences (*) p <0.001 were found for comparisons 6c versus control, 6d
versus control, 8d versus control, 6c versus 6d and 6d versus 8d.
Fig. 3. Effects of 0.1 mg/kg of 6c, 6d and 8d on HR (∆HR calculated as the difference of HR between sequential
measurements and time 0 of the experiment) in anesthetized rats. The lines represent mean values of ∆HR for
four or five animals. Comparisons were made using ANOVA with repeated measures and Fisher and Duncan

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tests. Significant differences (*) p <0.001 were found for comparisons 6c versus control, 6d versus control, 8d
versus control, 6c versus 6d and 6d versus 8d.
2.2.3. Functional [35S]GTPγS binding assays
To explain the marked difference in the hemodynamic effects of compounds 6c and 6d that
disagreed with their similar receptor affinity profiles, [35S]GTPγS binding experiments were
conducted to compare their agonist potency and intrinsic activity at recombinant human α2A-ARs
[75]. The intrinsic activity was measured relative to the full agonist norepinephrine. The results
presented in Table 4 indicate that, similarly to marsanidine, compounds 6c and 6d are only relatively
weak partial agonists of human α2A-ARs. It is however of note that 6d is markedly more efficacious
than 6c which may at least in part account for its greater cardiovascular activity.
Table 4
Characterization of [35S]GTPγS binding to CHO cell membranes expressing recombinant human α2A-ARs:
estimates of agonist potency (EC50) and intrinsic activity relative to the natural full agonist norepinephrine.
Values shown are means ± s.e.m. from 3 independent experiments for compounds 6c, 6d and marsanidine and
19 independent experiments for norepinephrine.
Compound
pEC50
Intrinsic activity
(% of norepinephrine)
100
Norepinephrine 6.89 ± 0.06
Marsanidinea
6c
6d
7.03 ± 0.29
7.41 ± 0.23
6.70 ± 0.33
14 ± 2
5.2 ± 2.9
18 ± 4
a The results have been published earlier in Sączewski F. et al. [46].
2.3. Procedure for late stage fluorination
A candidate for PET radiotracer development should be amenable to labelling with a positron
emitter. Given the short half-lives of β+-emitting radionuclides [76] their introduction into the target
molecules should take place as late as possible in the course of the synthesis.
Fluorination of aromatic rings can be accomplished with aromatic nucleophilic substitution
(SNAR) [77–79]. This approach, though in many cases successful, requires aromatic moieties that carry
good leaving groups and are suitably activated by the presence of electron-withdrawing substituents.
The electron-rich indazole ring seems a poor substrate for SNAR. The scarce reports [80–84] on SNAR
within the benzene portion of an indazole moiety concern molecules that have one or two nitro
substituents functioning as activating or both activating and leaving groups. We therefore turned our
attention to electrophilic fluorination methods and decided to explore the utility of site-selective
fluorodestannylation reported by Ritter et al. [85] that uses Selectfluor as a fluorinating agent. The
method seemed attractive since it yields the products in 20 minutes under mild conditions and is
applicable to a wide scope of aromatic and heteroaromatic substrates. The potential usefulness of
this method for 18F-labelling was pioneered by Teare et al. [86] who developed the procedure for
[18F]Selectfluor radiosynthesis (using high specific activity [18F]F2) and demonstrated its usefulness for
fluorodemetalation of variously substituted phenylstannanes [86,87]. Fluorination of 2-methyl-1-
trimethylsilyloxytetral-1-ene with [18F]Selectfluor yielded 2-[18F]fluoro-2-methyl tetralone exhibiting
specific activity of 20 GBq/μmol [86]. Based on the above, we have designed and tested the
effectiveness of a potential procedure (Scheme 3) for the late stage fluorination of marsanidine
leading to 6c. In the first step we obtained 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-6-

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bromo-1H-indazole (5f) as a substrate for aryl stannane 11 synthesis according to published
procedures [85]. The latter was treated with Selectfluor in the presence of silver triflate using
conditions described by Ritter et al. [85]. The reaction, conducted for 20 minutes, yielded the desired
5c together with 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (12) - a
hydrodestannylation product - that could not be separated from 5c by preparative thin layer
chromatography. Formation of 12-like side-products has been reported by Ritter et al. [85]. Then,
however, deprotection of 5c contaminated with 12 gave a mixture of marsanidine and 6c that was
separable by flash column chromatography. The desired compound 6c was obtained from 11 with
16 % overall yield.
Scheme 3. The late stage fluorination leading to 6c. Reagents and conditions: a) HOSA (2.6 molar equiv.), 6%
NaOH/H2O, EtOH, 55 °C, 20 min., then room temperature, 2 h, 46 % yield; b) N,N’-bis-Boc-imidazolidine-2-
thione (1.5 molar equiv.), HgCl2 (1.5 molar equiv.), Et3N (3.5 molar equiv.), anhydrous DMF, 0 °C, 20 min., room
temperature 5 days, 56 % yield; c) Sn2Bu6, Pd[P(C6H5)3]4, toluene, 100 °C, 24 h, 70 % yield; d) AgOTf (2 molar
equiv.), Selectfluor (1.2 molar equiv.), acetone, room temperature, 20 min., 42 % yield; e) neat TFA, room
temperature, 40 min.; f) 10 % NaOH/H2O, 53 % yield.
Fig. 4. The structure of 12.
3. Conclusions
This study aimed to determine the influence of fluorination of the indazole nucleus on the
pharmacological properties of A and B. Accordingly, the fluorinated compounds 6a–e and 8a–c were
synthesized and evaluated for their affinity and selectivity towards α1- and α2-ARs as well as
imidazoline I1 and I2 binding sites. In the case of compounds 6a–d, decreased α2-AR affinity as well as
α2/I1 selectivity were observed.The results obtained for 8a–d indicated that on fluorination the α2-

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AR-selectivity of B was lost due to markedly enhanced affinity for I2 binding sites. From all of the
compounds studied, 6c and 6d displayed the highest selectivity towards α2-ARs. The in vivo
cardiovascular effects of the high-affinity α2-ARs ligands 6b–d, 8b and 8c confirmed our previous
observation that C-7 substitution of the indazole ring leads to congeners with relatively high
hypotensive and bradycardic activity.
In the synthetic part of our studies, we have established the utility of silver-mediated
fluorination of aryl stannanes [85] for the two-step preparation of 6c from 11. These results revealed
that the reaction conditions and the chemical properties of Selectfluor [88,89] were tolerated by the
iminoimidazolidine moiety. The present study thus widens the scope of the relatively new
fluorination procedure developed by Ritter et al. [85].
4. Experimental section
4.1. Chemistry
Melting points were measured on a Boetius apparatus and are uncorrected. IR spectra were
taken on a Perkin-Elmer FT-IR 1600 spectrometer. NMR spectra were recorded on a Varian Gemini
1
200 or a Varian Unity 500 apparatus. H and 13C NMR chemical shifts were measured relative to
residual solvent signal at 7.26 ppm and 77.0 ppm (CDCl3) or 2.50 ppm and 39.5 ppm (DMSO-d6).
Preparative thin layer chromatography was performed on silica gel 60 PF254 containing gypsum
(Merck) with aid of Chromatotron® using the reported solvent systems. 5-Fluoro- and 6-bromo-1H-
indazole were purchased from Sigma-Aldrich and used as obtained. N,N’-bis(tert-
butoxycarbonyl)imidazolidine-2-thion [64], 2-(chloromethyl)-4,5-dihydro-1H-imidazole [65], 4-fluoro-
1H-indazole [63] and 4,5,6,7-tetrafluoro-1H-indazole [90] were obtained according to published
methods.
4.1.1. General procedure for the synthesis of fluoro-1H-indazoles 2c, 2d
The title compounds were obtained by modifying a method described by Lukin et al. [63]. To
the properly substituted 2-fluorobenzaldehyde (5 g, 35 mmol) was added hydrazine hydrate (12.9 g,
258 mmol, 12.5 ml). After stirring for 12–48 hours (TLC control) at reflux water (20 ml) was added
and the resulting mixture was cooled to 5 °C. The precipitate was collected by vacuum filtration. The
filtrate was extracted with dichloromethane and the residue after evaporation was combined with
the precipitate.
4.1.1.1. 6-Fluoro-1H-indazole (2c). The combined solids were extracted with diethyl ether. The
ethereal extracts were evaporated to dryness and the resulting yellow solid was purified by flash
column chromatography (silica gel) eluting with dichloromethane and then chloroform. Yield: 1 g (20
%); mp 131–132 °C (lit. [91] 126 ºC); IR (KBr, cm-1) 3195, 3162, 3071, 3016, 2945, 2863, 1632, 1358,
1145, 947, 849; 1H NMR (200 MHz, CDCl3) δ 6.97 (dt, J1=8.8 Hz, J2=2.2 Hz, 1H), 7.15–7.20 (m, 1H), 7.72
(dd, J1=8.8 Hz, J2=5.2 Hz, 1H), 8.11 (1H, s), 10.98 (1H, s); 13C NMR (50 MHz, CDCl3) δ 95.8 (d, J=26 Hz),
111.8 (d, J=26 Hz), 120.2, 122.5 (d, J=11 Hz), 134.8, 140.5 (d, J=11 Hz); 162.7 (d, J=243.9 Hz, CF); Anal.
calcd. for C7H5FN2 (136.13): C, 61.76; H, 3.70; N, 20.58. Found: C, 61.94; H, 3.63; N, 20.79.
4.1.1.2. 7-Fluoro-1H-indazole (2d). The combined solids were purified by flash column
chromatography (silica gel) eluting with petroleum ether/acetone/ethyl acetate (100:4:4). Yield: 1.95
g (41 %); mp 124–125 °C (lit. [91] 120 ºC); IR (KBr, cm-1) 3193, 3145, 3018, 2960, 2854, 1589, 1522,

ACCEPTED MANUSCRIPT
1
1357, 1241, 1059, 727; H NMR (200 MHz, CDCl3) δ 7.06–7.16 (m, 2H), 7.55–7.59 (m, 1H), 8.25 (d,
J=3.3 Hz, 1H), 11.97 (br s); 13C NMR (50 MHz, CDCl3) δ 110.8 (d, J=15.7 Hz), 116.6 (d, J=4.4 Hz), 121.4
(d, J=5.2 Hz), 126.8, 130.0 (d, J=15.2 Hz), 134.9, 148.3 (d, J=248.0 Hz, CF). Anal. calcd. for C7H5FN2
(136.13): C, 61.76; H, 3.70; N, 20.58. Found: C, 61.44; H, 3.93; N, 20.47.
4.1.2. General procedure for the preparation of 1-aminofluoro-1H-indazoles 3a–e and 1-amino-6-
bromo-1H-indazole 3f
The title compounds were obtained from the appropriate indazoles (10 mmol) using
hydroxylamine-O-sulfonic acid (2.94 g, 26 mmol) in aqueous NaOH solution (2.2 g, 55 mmol in 34 ml
of H2O) and EtOH (9.6 ml) according to the procedure described by Adger et al. [92] To the aqueous-
alcoholic solution of NaOH indazole was added and the resulting mixture was heated to 55 °C. HOSA
was added in portions to keep the temperature at 55–57 °C. The reaction mixture was left to cool
down to room temperature and then kept at this temperature for 1.5 h. The precipitate (1-amino-1H-
indazole alone or its mixture with 2-amino-2H-indazole) was collected by vacuum filtration and if
necessary subjected to flash column chromatography (silica gel, 1-amino-1H-indazole was eluted
first) or recrystallized. The filtrate was extracted with dichloromethane and the residue after
evaporation (the mixture of 1- and 2-aminoindazole and unreacted indazole) was separated by flash
column chromatography (silica gel, 1-amino-1H-indazole was eluted first).
4.1.2.1. 1-Amino-4-fluoro-1H-indazole (3a). Eluted with CH2Cl2/petroleum ether (1:1). Yield 0.61 g (40
1
%); IR (KBr, cm-1) 3326, 3210, 3093, 3057, 1654, 1577, 1514, 1385, 1225, 1000, 774, 683; H NMR
(200 MHz, CDCl3) δ 5.26 (s, 2H), 6.71–6.81 (m, 1H), 7.24–7.37 (m, 2H), 7.92 (s, 1H); 13C NMR (50 MHz,
CDCl3) δ 105.2 (d, J=18.4 Hz), 106.0 (d, J=4.1 Hz), 112.6 (d, J=20 Hz), 127.3, 127.7 (d, J=7.6 Hz), 142.2
(d, J=8.5 Hz), 156.0 (d, J=252.4 Hz, CF). Anal. calcd. for C7H6FN3 (151.14): C, 55.63; H, 4.00; N, 27.80.
Found: C, 55.44; H, 3.92; N, 27.67.
4.1.2.2. 1-Amino-5-fluoro-1H-idazole (3b). Eluted with CH2Cl2/petroleum ether (2:1→5:1). Yield 0.68 g
1
(45 %); IR (KBr, cm-1) 3325, 3205, 1516, 1381, 1223, 856, 800, 575; H NMR (200 MHz, CDCl3) δ 5.36
(s, 2H), 7.17 (td, J1=9.0 Hz, J2=2.5 Hz, 1H), 7.24–7.30 (m, 1H), 7.53 (dd, J1=9.0 Hz, J2=4.2 Hz, 1H), 7.81
(s, 1H); 13C NMR (50 MHz, CDCl3) δ 104.7 (d, J=24 Hz), 111.2 (d, J=9.6 Hz), 116.7 (d, J=26.7 Hz), 122.2
(d, J=10.5 Hz), 130.4 (d, J=5.7 Hz), 136.5, 158.2 (d, J=237 Hz, CF). Anal. calcd. for C7H6FN3 (151.14): C,
55.63; H, 4.00; N, 27.80. Found: C, 55.67; H, 4.12; N, 27.74.
4.1.2.3. 1-Amino-6-fluoro-1H-indazole (3c). Eluted with chloroform. Yield 0.42 (30 %); mp 146–147 °C;
1
IR (KBr, cm–1) 3322, 3208, 1655, 1474, 1215, 848, 620; H NMR (200 MHz, CDCl3) δ 5.25 (s, 2H), 6.90
(td, J1=9.0 Hz, J2=2.0 Hz, 1H), 7.21 (dd, J1=9.0 Hz, J2=2.2 Hz, 1H), 7.58 (dd, J1=9.0 Hz, J2=5 Hz, 1H), 7.82
(s, 1H); 13C NMR (50 MHz, CDCl3) δ 95.6 (d, J=27 Hz), 111.2 (d, J=26 Hz), 119.3, 122.3 (d, J=10.9 Hz),
131.2, 140 (d, J=10 Hz), 162.7 (d, J=244.2 Hz, CF). Anal. calcd. for C7H6FN3 (151.14): C, 55.63; H, 4.00;
N, 27.80. Found: C, 55.85; H, 3.83; N, 27.91.
4.1.2.4. 1-Amino-7-fluoro-1H-indazole (3d). Eluted with methylene chloride/petroleum ether
(1:2→2:1). Yield 0.36 g (25 %); mp 84–86 °C; IR (KBr, cm–1) 3326, 3210, 1581, 1318, 1223, 1180, 842,
731; 1H NMR (200 MHz, CDCl3) δ 5.47 (s, 2H), 6.98–7.09 (m, 2H), 7.37–7.43 (m, 1H), 7.84 (d, J=1.9 Hz,
1H); 13C NMR (50 MHz, CDCl3) δ 112.2 (d, J=16.7 Hz), 112.8, 117.1 (d, J=4.5 Hz), 121.8 (d, J=5.5 Hz),
127.0 (d, J=10 Hz), 131.5, 148.7 (d, J=249.5, CF). Anal. calcd. for C7H6FN3 (151.14): C, 55.63; H, 4.00; N,
27.80. Found: C, 55.91; H, 4.13; N, 27.98.

ACCEPTED MANUSCRIPT
4.1.2.5. 1-Amino-4,5,6,7-tetrafluoro-1H-indazole (3e). Eluted with petroleum ether/ethyl acetate
(10:1→5:1). Yield 0.48 g (22 %); mp 135–136 °C; IR (KBr, cm–1) 3316, 3206, 1559, 1496, 1355, 1160,
1
965; H NMR (200 MHz, CDCl3) δ 6.82 (s, 2H), 8.24 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 109.5 (dd,
J1=21.5, J2=3.0 Hz), 127.4 (d, J=5.9 Hz), 129.9–130.3 (m), 131.6 (t, J=15.5 Hz), 134.9–135.3 (m),
136.07–136.91 (m), 139.8–140.2 (m), 141.2–141.8 (m). Anal. calcd. for C7H3F4N3 (205.11): C, 40.99; H,
1.47; N, 20.49. Found: C, 40.86; H, 1.62; N, 20.55.
4.1.2.6. 1-Amino-6-bromo-1H-indazole (3f). Obtained by crystallization of the crude product from
methanol. Yield 0.99 g (46 %); mp 179–180 °C; IR (KBr, cm–1): 3321, 1647, 1610, 1357, 1034, 905, 794,
615; 1H NMR (200 MHz, DMSO-d6) δ 6.48 (s, 2H), 7.23 (dd, J1=8.3 Hz, J2=1.7 Hz, 1H), 7.68 (d, J=8.3 Hz,
1H), 7.72 (s, 1H), 7.94 (s, 1H); 13C NMR (50 MHz, DMSO-d6) δ 112.2, 119.5, 120.7, 122.5, 123.4, 129.6,
139.6; Anal. calcd. for C7H6BrN3 (212,05): C, 39.65; H, 2.85; N, 19.82. Found: C, 39.42; H, 2.99; N,
19.71.
4.1.3. General procedure for the synthesis of 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-
1H-indazoles 5a–f
The title compounds were obtained according to the method described by F. Sączewski et al.
[45]. To a stirred solution of the appropriate 1-amino-1H-indazole (0.5 g, 3.3 mmol), N,N’-bis(tert-
butoxycarbonyl)imidazolidine-2-thion (1.5 g, 4.97 mmol) and triethylamine (1.16 g, 1.6 ml, 11.5
mmol) in DMF (5.5 ml) was added HgCl2 (1.32 g, 4.97 mmol) at 0 °C. The reaction mixture was stirred
for 20 min. at 0 °C and then for 5 days at room temperature. The resulting dark-grey/black reaction
mixture was diluted with ethyl acetate (30 ml) and filtered through a celite pad. The filtrates were
washed with brine (3×) and water (1×), dried over MgSO4 and concentrated under vacuum. The
viscous residue was separated through preparative thin layer chromatography. The unreacted N,N’-
bis(tert-butoxycarbonyl)imidazolidine-2-thion was eluted first followed by the product.
4.1.3.1. 4-Fluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5a). Eluted with
petroleum ether/ethyl acetate (9:1→4:1). Yield 0.74 g (53 %); mp 118–119 °C; IR (KBr, cm-1) 3008,
1
2976, 2933, 2892, 1724, 1711, 1653, 1629, 1368, 1314, 1147, 995, 781; H NMR (200 MHz, CDCl3) δ
1.05 (s, 9H), 1.58 (s, 9H), 3.81–4.02 (m, 4H), 6.74 (dd, J1=7.7 Hz, J2=9.2 Hz, 1H), 7.20–7.30 (m, 1H),
7.42 (dd, J1=9.2 Hz, J2=1.3 Hz, 1H), 7.91 (d, J=1 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 27.5, 28.4, 43.5,
44.1, 83.2, 83.4, 105.2 (d, J=18.4 Hz), 106.9 (d, J=4 Hz), 112.8 (d, J=24 Hz), 126.1, 127.5 (d, J=7.5 Hz),
140.1 (d, J=10 Hz), 143.9, 150.1, 150.3, 155.6 (d, J=251.4). Anal. calcd. for C20H26FN5O4 (419.45): C,
57.27; H, 6.25; N, 16.70. Found: C, 57.17; H, 6.54; N, 16.63.
4.1.3.2. 5-Fluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5b). Eluted with
petroleum ether followed by petroleum ether/ethyl acetate (9:1→7:1). Yield 0.5 g (36 %); mp 157–
1
159 °C; IR (KBr, cm-1) 2979, 2901, 1728, 1693, 1655, 1324, 1155, 1051, 848, 581; H NMR (200 MHz,
DMSO-d6) δ 0.96 (s, 9H), 1.53 (s, 9H), 3.84 (br s, 4H), 7.29–7.33 (m, 1H), 7.46–7.56 (m, 2H), 7.94–7.96
(m, 1H); 13C NMR (50 MHz, DMSO-d6) δ 26.8, 27.7, 43.1, 43.8, 81.9, 82.0, 104.9 (d, J=24 Hz), 111.6 (d,
J=10 Hz), 116.0 (d, J=28 Hz), 121.6 (d, J=11 Hz), 129.2 (d, J=5.5 Hz), 133.6, 144.0, 149.3, 149.5, 157.5
(d, J=240 Hz). Anal. calcd. for C20H26FN5O4 (419.45): C, 57.27; H, 6.25; N, 16.70. Found: C, 57.04; H,
7.34; N, 16.58.
4.1.3.3. 6-Fluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5c). Eluted with
petroleum ether/ethyl acetate (8:1→4:1). Yield 1 g (70 %); mp 139–141 °C; IR (KBr, cm-1) 3088, 2986,

ACCEPTED MANUSCRIPT
1
2896, 1728, 1702, 1658, 1625, 1331, 1161, 1029, 952; H NMR (200 MHz, DMSO-d6) δ 0.96 (s, 9H),
1.52 (s, 9H), 3.83 (br s, 4H), 6.97–7.14 (m, 2H), 7.75–7.83 (m, 1H), 8.00 (d, J=1.2 Hz, 1H); 13C NMR (50
MHz, DMSO-d6) δ 26.9, 27.8, 43.2, 43.8, 81.9, 82.0, 94.9 (d, J=26.1 Hz), 110.4 (d, J=26.1 Hz), 118.8,
122.8 (d, J=10.9 Hz), 129.8, 136.5 (d, J=13.5 Hz), 144.1, 149.3, 149.5, 161.4 (d, J=242.4 Hz) Anal. calcd.
for C20H26FN5O4 (419.45): C, 57.27; H, 6.25; N, 16.70. Found: C, 57.61; H, 6.43; N, 16.77.
4.1.3.4. 7-Fluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5d). Eluted with
petroleum ether/ethyl acetate (10:1→4:1). Yield 0.6 g (42 %); mp 118–119 °C; IR (KBr, cm-1) 2984,
1
1716, 1648, 1309, 1151, 1066, 729; H NMR (200 MHz, CDCl3) δ 1.01 (s, 9H), 1.56 (s, 9H), 3.80–4.00
(m, 4H), 6.94–7.04 (m, 2H), 7.36–7.41 (m, 1H), 7.85 (d, J=2.1 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 27.4,
28.3, 43.4, 44.1, 83.3, 83.5, 111.5 (d, J=17 Hz), 116.3 (d, J=5 Hz), 121.4 (d, J=6 Hz), 126.6 (d, J=4 Hz),
130.0, 144.6, 149.2 (d, J=252 Hz), 150.18, 150.2. Anal. calcd. for C20H26FN5O4 (419.45): C, 57.27; H,
6.25; N, 16.70. Found: C, 57.39; H, 6.03; N, 16.95.
4.1.3.5. 4,5,6,7-Tetrafluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5e).
Eluted with petroleum ether/ethyl acetate (10:1→1:1). Yield: 0.047 g from 0.1 g of the substrate (20
%); mp 156–158 °C, IR (KBr, cm-1) 2981, 2925, 1753, 1625, 1552, 1493, 1300, 1152, 946; 1H NMR (200
MHz, CDCl3) δ 1.52 (s, 18H), 3.73 (s, 4H), 7.96 (d, J=1.7 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 28.2, 39.8,
43.5, 44.03, 83.5, 149.1 (C=N), 150.6 (C=O) The other signals could not be detected due to their high
multiplicity and thus low intensity. Anal. calcd. for C20H23F4N5O4 (473.42): C, 50.74; H, 4.90; N, 14.79.
Found: C, 50.36; H, 5.11; N, 14.66.
4.1.3.6. 6-Bromo-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-1H-indazole (5f). Eluted with
petroleum ether/ethyl acetate (20:1→5:1) and then crystallized from methanol. Yield 0.625 g (56 %);
1
mp 133–135 °C; IR (KBr, cm-1) 3108, 2983, 1740, 1709, 1302, 1154, 984, 763; H NMR (200 MHz,
DMSO-d6) δ 0.99 (s, 9H), 1.53 (s, 9H), 3.84 (br s, 4H), 7.27 (dd, J1=4.8 Hz, J2=1.7 Hz, 1H), 7.61 (br s,
1H), 7.72 (d, J=8.3 Hz, 1H), 8.01 (s, 1H); 13C NMR (50 MHz, DMSO-d6) δ 26.9, 27.8, 43.2, 43.8, 81.9,
82.0, 112.2, 119.7, 120.5, 122.7, 123.8, 129.7, 136.9, 144.4, 149.2, 149.4. Anal. calcd. for
C20H26BrN5O4 (480.36): C, 50.01; H, 5.46; N, 14.58. Found: C, 50.20; H, 5.40; N, 14.81.
4.1.4. General procedure for the synthesis of fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazoles 6a–e
and their hydrochloride salts 7a–e.
A mixture of the appropriate fluoro-1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-
1H-indazole (1.36 g, 3.24 mmol.) and 50% trifluoroacetic acid in dichloromethane (13 ml) was stirred
for 2 h at room temperature. The mixture was evaporated to dryness and the resulting oily residue
was treated with water (5 ml) and then made alkaline to pH≈11 with 10 % aqueous NaOH. The
precipitated solid was collected by vacuum filtration, dried and crystallized from acetonitrile.
The free bases were then converted into their hydrochloride salts by adding 1.5 molar equiv.
of the ethereal solution of hydrochloride (2.6 M) to the solution of the appropriate fluoro-1-
[(imidazolidin-2-yl)imino]-1H-indazole in dichloromethane.
4.1.4.1. 4-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6a). Yield: 0.55 g (77 %); mp 204–205 °C; IR
(KBr,cm-1) 3227, 3173, 3008, 2875, 2830, 1636, 1622, 1513, 1403, 1288, 999, 778; 1H NMR (CDCl3, 200
MHz) δ 3.61 (br s, 4H), 5.40 (br s, 1H), 6.40 (br s, 1H), 3.72 (ddd, J1=10.2 Hz, J2=7.1 Hz, J3=1 Hz, , 1H),
7.17–7.34 (m, 2H), 7.92 (s, 1H); 13C NMR (CDCl3, 50 MHz) δ 42.4, 43.7, 104.8 (d, J=18.4 Hz), 106.7 (d,
J=4.1 Hz), 124.9, 126.8 (d, J=7.7 Hz), 139.3, 155.9 (d, J=251.7 Hz), 162.2. Anal. calcd. for C10H10FN5

ACCEPTED MANUSCRIPT
(219.22): C, 54.79; H, 4.60; N, 31.95; Found: C, 54.61; H, 4.63; N, 32.12. Hydrochloride 7a. Mp 217–
219 °C; IR (KBr, cm-1) 3102, 2903, 2613, 1650, 1514, 1289, 995, 775. Anal. calcd. for C10H11ClFN5
(255.68): C, 46.98; H, 4.34; N, 27.39. Found: C, 46.90; H, 4.16; N, 27.36.
4.1.4.2. 5-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6b). Yield: 0.45 g (63 %); IR (KBr, cm-1)
3344, 3239, 3170, 2967, 2896, 1632, 1500, 1288, 1129, 796; 1H NMR (CDCl3, 200 MHz) δ 3.59 (s, 4H),
5.50 (br s, 1H), 6.35 (br s, 1H), 7.09 (dt, J1=8.8 Hz, J2=2.2 Hz, 1H), 7.26 (dd, J1=8.8 Hz, J2=2.2 Hz, 1H),
7.46 (dd, J1=9.2 Hz, J2=4.4 Hz, 1H), 7.77 (s, 1H); 13C NMR (CDCl3, 50 MHz) δ 42.6 (br s), 104.3 (d, J=24
Hz), 111.6 (d, J=9.6 Hz), 116.0 (d, J=27.7 Hz), 128.3 (d, J=6 Hz), 133.8, 158.5 (d, J=238 Hz), 162.1. Anal.
calcd. for C10H10FN5 (219.22): C, 54.79; H, 4.60; N, 31.95; Found: C, 54.86; H, 4.23; N, 31.86.
Hydrochloride 7b. Mp 215–217 °C; IR (KBr, cm-1) 3118, 2933, 1655, 1626, 1513, 1215. Anal. calcd. for
C10H11ClFN5 (255.68): C, 46.98; H, 4.34; N, 27.39. Found: C, 47.12; H, 4.49; N, 27.22.
4.1.4.3. 6-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6c). Yield: 0.52 g (73 %); mp 182–184 °C; IR
(KBr, cm-1) 3248, 3162, 2999, 2886, 2808, 1635, 1618, 1473, 1289, 1203, 952, 839. 1H NMR (DMSO-d6,
200 MHz) δ 3.38 (br s), 6.48 (br s, 1H), 6.68 (br s, 1H), 6.89–7.02 (m, 2H), 7.73 (dd, J1= 5.1 Hz, J2= 8.3
Hz, 1H), 7.91 (s, 1H). 13C NMR (DMSO-d6, 50 MHz) δ 42.6, 43.7, 95.7 (d, J=26.3 Hz), 110.5 (d, J=26.5
Hz), 119.6, 123.4 (d, J=11.1 Hz), 129.2, 136.7, 161.8 (d, J=240.8 Hz), 165.1. Anal. calcd. for C10H10FN5
(219.22): C, 54.79; H, 4.60; N, 31.95; Found: C, 54.91; H, 4.66; N, 31.73. Hydrochloride 7c. Mp 202–
204 °C; IR (KBr, cm-1) 3194, 2926, 1648, 1624, 1477, 1215, 950. Anal. calcd. for C10H11ClFN5 (255.68):
C, 46.98; H, 4.34; N, 27.39. Found: C, 46.99; H, 4.16; N, 27.51.
4.1.4.4. 7-Fluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6d). Yield 0.49 g (70 %); mp 253–255 °C; IR
(KBr, cm-1) 3243, 3167, 3060, 3012, 2878, 2842, 1632, 1519, 1289, 1214, 1069, 729; H1 NMR (DMSO-
d6, 200 MHz) δ 3.31 (br s, 4H), 6.38 (br s, 1H), 6.72 (br s, 1H), 6.95–7.09 (m, 2H), 7.51 (d, J=7.8 Hz,
1H), 7.95 (d, J=2.0 Hz, 1H); 13C NMR (DMSO-d6, 50 MHz) δ 41.3, 42.2, 109.4 (d, J=16.8 Hz), 116.2 (d,
J=4.5 Hz), 119.5 (d, J=5.4 Hz), 125.5 (d, J=4.2 Hz), 128.2, 147.5 (d, J=249 Hz), 164.8. Anal. calcd. for
C10H10FN5 (219.22): C, 54.79; H, 4.60; N, 31.95; Found: C, 55.10; H, 4.28; N, 32.16. Hydrochloride 7d.
Mp 228–230 °C; IR (KBr, cm-1) 3297, 3099, 2888, 1655, 1628, 1214, 1070, 859, 734. Anal. calcd. for
C10H11ClFN5 (255.68): C, 46.98; H, 4.34; N, 27.39. Found: C, 46.73; H, 4.50; N, 27.14.
4.1.4.5. 4,5,6,7-Tetrafluoro-1-[(imidazolidin-2-yl)imino]-1H-indazole (6e). Obtained from 0.047 g (0.1
mmol) of 5e and 0.4 ml of 50% TFA in CH2Cl2. Yield 0.024 g (88 %); mp 210–212 °C; IR (KBr, cm-1)
3332, 3254, 3184, 1634, 1547, 1489, 1345, 955; 1H NMR (200 MHz, CDCl3) δ 3.64 (br s, 4H), 5.02 (br s,
1H), 6.18 (br s, 1H), 7.95 (d, J=1.5 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 42.2 (br s), 43.6 (br s), 162.2 (d,
J=6.5 Hz), 137.3, 162.4. The other signals could not be detected due to their high multiplicity and
thus low intensity. Anal. calcd. for C10H7F4N5 (273.19): C, 43.96; H, 2.58; N, 25.64. Found: C, 44.13; H,
2.37; N, 25.79. Hydrochloride 7e. Mp 216–218 °C; IR (KBr, cm-1) 3075, 3003, 2876, 1648, 1533, 1488,
1349, 944. Anal. calcd. for C10H8ClF4N5 (309.65): C, 38.79; H, 2.60; N, 22.62. Found: C, 38.88; H, 2.64;
N, 2.37.
4.1.5. The general procedure for the synthesis of fluoro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-
indazoles 8a–c and their hydrochloride salts 10a–c.
The title compounds were obtained according to a method described by J. Sączewski et al.
[48]. To the stirred solution of properly substituted fluoroindazole (0.5 g, 3.7 mmol) in anhydrous THF
(5 ml) sodium hydride (0.22 g, 5.5 mmol, 60 % oil dispersion) was added in one portion. After 15 min.

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freshly prepared 2-(chloromethyl)-4,5-dihydro-1H-imidazole was added and the reaction mixture was
stirred at room temperature for 12 hours. After this time the reaction was quenched with water (10
ml). The layers were separated and the aqueous one was extracted with dichloromethane (3×10 ml).
The combined organic layers were dried (Na2SO4) and evaporated under vacuum. The oily residue
thus obtained was purified by preparative thin layer chromatography eluting first with ethyl acetate
and then with ethyl acetate/methanol/triethylamine 50:5:3. The N1-alkylated products were eluted
first, while the N2-alkylated ones had considerably lower Rf and were not isolated in pure form.
Compounds 8a–8c were then converted into their hydrochloride salts 10a–10c by adding 1.5
molar equiv. of the ethereal solution of hydrochloride (2.6 M) to the solution of the appropriate
fluoro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole in dichloromethane.
4.1.5.1. 4-Fluoro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (8a). Yield (0.2 g, 25%); mp
1
96–97 °C, IR (KBr, cm-1) 3258, 2938, 1612, 1493, 1238, 1188, 1000, 779; H NMR (200 MHz, CDCl3) δ
3.60 (s, 4H), 4.48 (br s, 1H), 5.21 (s, 2H), 6.80–6.86 (m, 1H), 7.30–7.36 (m, 2H), 8.11 (s, 1H); 13C NMR
(50 MHz, CDCl3) δ 47.9, 50.1, 105.5, 105.6 (d, J=20.8 Hz), 113.9 (d, J=4.7 Hz), 128.2 (d, J=7.8 Hz), 155.9
(d, J=253.1 Hz), 162.7. Anal. calcd. for C11H11FN4 (218.23): C, 60.54; H, 5.08; N, 25.67. Found: C, 60.66;
H, 5.15; N, 25.31. Hydrochloride 10a. Mp 197–199 °C; IR (KBr, cm-1) 3411, 3339, 3074, 2960, 1634,
1242, 779. Anal. calcd. for C11H12ClFN4 (254.69): C, 51.87; H, 4.75; N, 22.00. Found: C, 51.93; H, 4.63;
N, 22.26.
4.1.5.2. 5-Fluoro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (8b). Yield (0.2 g, 25 %); mp
1
103–105 °C; IR (KBr, cm-1) 3164, 3129, 2946, 2874, 1618, 1508, 1216, 847, 799; H NMR (200 MHz,
CDCl3) δ 3.59 (s, 4H), 5.14 (br s, 1H), 5.20 (s, 2H), 7.13–7.23 (m, 1H), 7.31–7.37 (m, 1H), 7.44–7.50 (m,
1H), 7.98 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 47.8, 49.9, 105.2 (d, J=23.6 Hz), 110.9 (d, J=9.7 Hz), 116.8
(d, J=27.8), 124.3 (d, J=10.2), 134.1 (d, J=50.6 Hz), 137.0, 158.3 (d, J=239), 163.0. Anal. calcd. for
C11H11FN4 (218.23): C, 60.54; H, 5.08; N, 25.67. Found: C, 60.58; H, 4.90; N, 25.80. Hydrochloride 10b.
Mp 257–260 °C; IR (KBr, cm-1) 3059, 2949, 2918, 1623, 1508, 1290, 1210, 851; Anal. calcd. for
C11H12ClFN4 (254.69): C, 51.87; H, 4.75; N, 22.00. Found: C, 52.02; H, 4.58; N, 21.79.
4.1.5.3. 6-Fluoro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (8c). Yield (0.4 g, 41 %); mp
1
114–116 °C; IR (KBr, cm-1) 3093, 2931, 2862, 1620, 1505, 1477, 1247, 955; H NMR (200 MHz, CDCl3)
δ 3.59 (s, 4H), 4.65 (br s, 1H), 5.14 (s, 2H), 6.95 (td, J1=9.3, J2=2.4 Hz, 1H), 7.16 (d, J=9.3 Hz, 1H), 7.67
(dd, J1=8.8 Hz, J2=5.4, 1H), 8.01 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 47.7, 50.0, 95.3 (d, J=26.7), 111.4
(d, J=26.1 Hz), 121.1, 134.6, 140.3, 162.6 (d, J=246.4), 162.8. Anal. calcd. for C11H11FN4 (218.23): C,
60.54; H, 5.08; N, 25.67. Found: C, 60.71; H, 5.31; N, 25.49. Hydrochloride 10c. Mp 270–272 °C; IR
(KBr, cm-1) 3053, 2946, 1620, 1249, 847. Anal. calcd. for C11H12ClFN4 (254.69): C, 51.87; H, 4.75; N,
22.00. Found: C, 51.52; H, 4.84; N, 21.72.
4.1.6. The procedure for fluorination leading from 5f to 6c
4.1.6.1. 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-6-(tributylstannyl)indazole (11)
The title compound was prepared analogously to a method described by Ritter et al. [85]. To
5f (480 mg, 1.00 mmol, 1.00 molar equiv.) and tetrakis(triphenylphosphine)palladium (116 mg, 0.1
mmol, 10 mol%) dissolved in toluene (10 ml) was added bis(tri-n-butyltin) (1.01 ml, 2.00 mmol, 2.00
molar equiv.) under argon. After stirring for 24 h at 100 °C under argon atmosphere, the reaction
mixture was cooled to room temperature and concentrated under vacuum. The residue was purified

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by flash column chromatography (silica gel) eluting with petroleum ether/acetone/ethyl acetate
(100:5:5) to afford 480 mg of the title compound as a colourless oil (yield 70 %). IR (oil fim, cm–1)
1
2957, 2928, 2871, 1719, 1651, 1313, 1153, 759; H NMR (500 MHz, CDCl3) δ 0.88 (t, J=7.2 Hz, 9H),
0.98 (s, 9H), 1.04–1.12 (m, 6H), 1.23–1.38 (m, 6H), 1.41–1.67 (m, 6H), 1.60 (s, 9H), 3.85 (m, 4H), 7.19
(d, J=7.8 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 7.72 (s, 1H), 7.83 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 9.7,
13.7, 27.2, 27.4, 28.3, 29.1, 43.1, 43.7, 82.8, 83.0, 118.2, 119.3, 122.0, 128.0, 129.5, 137.5, 140.2,
142.5, 150.5 (two overlapping signals). Anal. calcd. for C32H53N5O4Sn (690.50): C, 55.66; H, 7.74; N,
10.14. Found: C, 55.48; H, 7.91; N, 9.85.
4.1.6.2. 6-Fluoro-1-[(imidazolidin-2-yl)imino]indazole (6c) from 11
The multistep synthesis was accomplished using the procedures desribed by Ritter et al. [85]
and F. Sączewski et al. [45]. To 1-{[1,3-di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-6-
(tributylstannyl)indazole (11) (0.4 g, 0.58 mmol, 1.00 molar equiv.) in acetone (12 ml) at room
temperature under argon atmosphere was added silver triflate (0.30 g, 1.16 mmol, 2.00 molar equiv.)
and 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(trifluoroborate) (0.25 g, 0.696
mmol, 1.20 molar equiv.). The reaction mixture was stirred for 20 min. at room temperature, filtered
through a celite pad and evaporated to dryness. The residue was suspended in dichloromethane and
washed with water. The organic phase was dried (MgSO4), concentrated under vacuum and
subjected to preparative thin layer chromatography eluting with petroleum ether/ethyl acetate
1
(6:1→1:1) to afford a 2:1 (according to H NMR) mixture (110 mg) of 5c and 1-{[1,3-di(tert-
butoxycarbonyl)imidazolidin-2-yl]imino}indazole (12) as a white solid. The mixture (110 mg) was
added to trifluoroacetic acid (0.9 ml), stirred for 40 min. at room temperature and then evaporated
to dryness. The residue was treated with water (1 ml) and then made alkaline to pH≈11 with 10 %
aqueous NaOH. The precipitated solid (40 mg) was extracted to methylene chloride (3×10 ml). The
organic phase was dried (MgSO4), concentrated under vacuum and subjected to flash column
chromatography
(Silica
gel
60,
less
than
0.063
mm)
eluting
with
ethyl
acetate/methanol/triethylamine (50:1:1). 6-fluoro-1-[(imidazolidin-2-yl)imino]indazole (6c) was
eluted first (22 mg, yield 16 %) followed by 1-[(imidazolidin-2-yl)imino]indazole (6) (8 mg, yield 5 %,
mp 175–177 ºC, lit. 172–174 ºC [45]). For the spectroscopic data of 6c see 4.1.4.3.
4.2 Pharmacological methods
4.2.1. Radioligand binding assays
4.2.1.1. I1-Imidazoline site binding assay
Kidneys were obtained post mortem from male Sprague-Dawley rats (250–280 g) and crude
P2 membranes prepared according to Lione et al. [93]. Binding of [3H]clonidine (3 nM, Perkin–Elmer)
was investigated in the presence of 10 μM rauwolscine to preclude binding to α2-ARs. The specific
component was defined by 10 μM rilmenidine; under these conditions, the site labelled represents a
model of the central I1 binding site [94]. Membrane aliquots (400 μl, 0.2–0.5 mg protein) were
incubated with 11 concentrations of the test compounds over the range 0.1 nM–100 μM. 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 filtration through pre-soaked (0.5 %
polyethyleneimine) glass-fibre filters (Whatman GFB). Trapped radioligand was determined by liquid
scintillation counting and the data were analysed with GraphPad Prism version 4.03 for Windows

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(GraphPad Software, San Diego, CA, USA) to yield IC50 values (the concentration of tested ligand that
displaces 50 % of specifically bound [3H]clonidine).
4.2.1.2. α1-AR, α2-AR and I2-imidazoline site binding assays
Brains were obtained post mortem from male Sprague-Dawley rats (250–280 g) and crude P2
membranes were prepared [93]. Membrane aliquots (400 μl, 0.2–0.3 mg protein) were incubated
with 11 concentrations of the tested compounds over the range 0.1 nM–100 μM in the presence of
the selective I2 binding site radioligand [3H]-2BFI (2-(benzofuranyl)-2-imidazoline) [95] (1 nM), α1-AR
antagonist radioligand [3H]prazosin (1 nM) or α2-AR antagonist radioligand [3H]RX821002 (2-(2,3-
dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole) (1 nM) in a final volume of 500
μl. Non-specific binding was determined using 10 μM BU224 (2-(4,5-dihydroimidazol-2-yl)quinoline)
[96] for I2 binding, 10 μM phenylephrine for α1-ARs and 10 μM rauwolscine to define α2-AR binding.
Incubations were performed in triplicate at room temperature and were allowed to reach
equilibrium (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. The data were analyzed by iterative non-linear regression
analysis with GraphPad Prism version 4.03 for Windows (GraphPad Software, San Diego, CA, USA).
Each experiment was analyzed individually and equilibrium dissociation constants (Ki) were
determined by the method of Cheng and Prusoff [97]. The resulting values are given as means of
three or four separate experiments except where compounds demonstrated low affinity and were
tested in a singular experiment as indicated.
4.2.1.3. α2A-, α2B- and α2C-adrenoceptor binding assays
Adherent Chinese hamster ovary (CHO) cells (K1 strain) (American Type Culture Collection,
Manassas, VA, USA), stably expressing cDNAs encoding each of the human α2-AR subtypes were
produced and treated as described by Pohjanoksa et al. [98]. Receptor density and the affinity of the
subtype-nonselective radioligand [3H]RS-79948-197 were determined for each human α2-AR subtype
with saturation binding assays, as described previously by Fallarero et al. [99]. The Kd and Bmax values
were as follows: α2A, 0.20 ± 0.04 nM; α2B, 0.33 ± 0.04 nM and α2C, 0.18 ± 0.02 nM; and α2A, 4.2 ± 0.3
pmol/mg protein; α2B, 9.6 ± 2.0 pmol/mg protein and α2C, 8.6 ± 1.1 pmol/mg protein. Competition
binding assays were implemented using a MultiScreen Vacuum Manifold system (Millipore
Corporation, Bedford, MA, USA) with Millipore MultiScreen MSFBN 96-well glass fibre filtration
plates. The experiments were performed in a total assay volume of 180 µl (in 50 mM potassium
phosphate
buffer,
pH
7.4)
using
0.2
nM
[3H]RS-79948-197
((8aR,12aS,13aS)-
5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-
g][1,6]naphthyridine; GE Healthcare, London, U.K.), 8 serial dilutions of the competitor ligands and
crude cell membrane preparations containing 6-10 µg of protein per sample. Non-specific binding
was determined in parallel wells in the presence of 100 µM oxymetazoline. Bound radioactivity was
measured with a Wallac 1450 MicroBeta scintillation counter (PerkinElmer Wallac, Turku, Finland).
All experiments were performed in duplicate and repeated at least three times. The apparent affinity
(apparent Ki) of each ligand was determined using nonlinear regression analysis (GraphPad Prism),
assuming one-site binding. For conversion of IC50 estimates to Ki values, the Cheng-Prusoff equation
was applied [97].

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4.2.2. In vivo cardiovascular effects
Male Wistar rats, weighing 200–290 g, were purchased from the Animal House of the
Medical University of Gdańsk, Poland. All in vivo experiments were approved by the Local Ethical
Committee on Animal Experiments. The animals were fed commercial rodent chow (Labofeed-B,
Poland). Tap water was available ad libitum. Rats were anaesthetized by i.p. injection of thiopental
(Sandoz, Austria) at 70 mg/kg body weight and anaesthesia was maintained by thiopental
supplementation (30 μg/kg/min). The animals were placed on a heated table, and body temperature
was maintained between 36 and 37 °C. Tracheotomy was performed. Catheters were inserted into
the carotid artery for monitoring of MAP and HR, 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 a stable baseline. The rats were infused with isotonic saline (Fresenius Kabi, Poland)
supplemented with thiopental at a rate of 1.2 ml/h. After 40 min of saline infusion, the tested
compound was administered as a 100 μl bolus through the venous catheter at doses of 0.1 mg/kg.
The time of administration of a compound was recorded as “time 0”. MAP and HR were monitored
directly and sampled continuously at 100 Hz as described previously [72], using Biopac Systems, Inc.,
Model MP 100 (Goleta, CA, USA). The recorded results were elaborated with the help of the
ACQKnowledge (Goleta, CA, USA) analysis system and were selected, scaled and filtered to remove
accidental signal disturbances. The recorded time domain transient data are presented as graphs
with the help of Excel (Microsoft, USA).
ANOVA was performed for ΔMAP and ΔHR, calculated as the difference in MAP and in HR
from baseline measurements (“time 0”) for each group, as described previously [72]. This allowed for
direct comparison of responses to treatment between groups. Data were analyzed by ANOVA for
repeated measurements, using Statistica StatSoft software (StatSoft, Inc., Tulsa, OK, USA), after test
compound or vehicle administration. When a treatment effect was significant, post hoc comparisons
were performed using Fisher’s test. A value of p <0.05 was considered statistically significant.
4.2.3. In vitro estimation of agonist potency and efficacy
4.2.3.1. Cell culture
CHO cells stably expressing human α2A-ARs were cultured as described by Pohjanoksa et al.
[98]. Before the functional [35S]GTPγS binding assays, the cultured cells were tested for their capacity
to bind the α2-AR antagonist radioligand [3H]RS-79948-197. Confluent cells were harvested into
chilled phosphate-buffered saline, pelleted and frozen at –70 °C.
4.2.3.2. Membrane preparation
All procedures were performed on ice. 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 × 10 s at 8000 rpm). The homogenate was centrifuged at 180 g
for 15 min to remove cell nuclei, unbroken cells and aggregates. The supernatants were pooled and
centrifuged at 50,200 g for 30 min. The pellet was washed with TE buffer (10 mM Tris, 0.1 mM EDTA)
and re-centrifugated as above. The membranes were then suspended in TE buffer, aliquoted and
stored at –70 °C until used. Protein concentrations were determined with the method of Bradford
[100] using bovine serum albumin as reference.
4.2.3.4. [35S]GTPγS binding assay

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Agonist-induced stimulation of [35S]GTPγS binding was measured essentially as described
previously [75]. Briefly, membranes were thawed and diluted with binding buffer (25 mM Tris, 1 mM
EDTA, 5 mM MgCl2, 20 mM NaCl, 1 μM GDP, 1 mM DTT, 30 μM ascorbic acid, pH 7.4). Incubations
were performed on 96-well Millipore MultiScreen MSFBN glass-fibre filter plates. Samples containing
5 μg of membrane protein were incubated with 7 or 8 serial dilutions of the test compounds and 0.1
nM [35S]GTPγS. Reactions were terminated after 30 min incubation at RT by rapid vacuum filtration
using a Millipore MultiScreen Vacuum Manifold. The filter plates were washed three times with ice-
cold wash buffer (20 mM Tris, 1 mM EDTA, 5 mM MgCl2, pH 7.4). Filters were dried and 50 μl
SuperMix scintillation cocktail was added into each well. The incorporated radioactivity was
measured using a Wallac 1450 Betaplate scintillation counter. All experiments were performed in
duplicate and repeated at least three times. Analysis of the results with GraphPad Prism software
yielded estimates of agonist potency (EC50) and efficacy (intrinsic activity in comparison to the natural
full agonist norepinephrine).
Acknowledgements
This research project was supported financially by the National Science Centre, Poland, on the basis
of the decision number DEC-2011/01/B/NZ4/00520 (A. Wasilewska). Additional grant support was
received from Turku University Hospital (to M. Scheinin). M. Ferdousi was supported with discovery
grant from Natural Sciences and Engineering Research Council of Canada 371863-2009.
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Table 1.
Binding affinity data for marsanidine, B, 6a–e and 8a–d
Compd
α1 Ki (nM)a,c
ndf
α2 Ki (nM)a,c
14.05 ± 2.7
416.0 ± 16.2
64.33 ± 4.39
26.20 ± 6.30
30.97 ± 1.93
3717 ± 220.2
18.5 ± 5.9
187.3 ± 40.26
32.17 ± 9.60
29.73 ± 3.04
12.6 (n=1)g
I1 IC50 (nM)b,c
54550 ± 16730
14776 ± 7893
91.65 ± 16.54
20675 ± 20513
7740 ± 5522
5827 ± 3402
19400 (n=1)g
1765 ± 1200
2419 ± 2057
33456 ± 24023
16400 (n=1)g
I2 Ki (nM)a,c
marsanidined
16900 ± 5900
6177 ± 4962
5703 ± 2656
21967 ± 3735
348833 ± 306639
22733 ± 817.2
42200 (n=1)g
21.87 ± 4.703
10.38 ± 5.44
8.193 ± 3.43
156 (n=1)g
6a
6b
6c
6d
6e
Be
8a
8b
8c
8de
3807 ± 1342
2320 ± 293
918.3 ± 52.6
1625 ± 475.2
19793 ± 18003
272 ± 42
534.7 ± 118.4
476.3 ± 267.5
518.3 ± 159.9
107 (n=1)g
a Ki affinity values for α1-adrenoceptors, α2-adrenoceptors, and I2 imidazoline binding sites were assessed by
measuring the ability of the tested compounds to compete with [3H]prazosin, [3H]RX821002 or [3H]2BFI binding
to rat brain membranes. b Molar concentration of the tested compounds that displaces 50% of specifically
bound [3H]clonidine in rat kidney membranes in the presence of rauwolscine (I1 imidazoline binding sites). c
Values given are means ± s.e.m. from 3–4 independent experiments. d The results have been published earlier
in Sączewski F. et al. [45]. e The results have been published earlier in Sączewski J. et al. [48]. f nd: not
determined. g n: number of experiments.

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Table 2.
Binding affinities of 6c and 6d obtained with [3H]RS-79948-197 to human α2-AR subtypes expressed in CHO cell
membranes. Results expressed as Ki (nM) and their 95 % confidence intervals of three independent
experiments.
Human
Compd
α2A-AR
α2B-AR
α2C-AR
marsanidine
6c
6d
52 (39–70)
33 (27–41)
65 (54–77)
79 (63–99)
72 (54–92)**
158 (130–190)**
640 (520–780)***
600 (510–710)***
1200 (960–1400)**
Statistical significance of differences from the α2A-AR is shown by symbols: **P<0.01; ***P<0.001 tested with
unpaired t-tests.

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Table 3.
Effects of compounds 6b–d and 8b–d at 0.1 mg/kg i.v. on mean arterial blood pressure (MAP) and heart rate
(HR) in anesthetized rats
.
a,d
b,d
c,d
+ΔMAPmax
-ΔMAPmax
-ΔHRmax
ne
f
f
f
compd
6b
(tmax
)
(tmax
)
(tmax)
14.6 ± 5.9 (2) *%
24.8 ± 4.6 (3) *§
21.1 ± 2.2 (4) *§
0.8 ± 1.0 (2)
–8.6 ± 2.3 (11) $!@
–36.0 ± 2.3 (35) *!
–59.4 ± 5.7 (33) *$@
–7.2 ± 1.6 (40) $!@
–12.7 ± 1.8 (36) #$!@
–29.5 ± 4.0 (34) *$!
–2.7 ± 1.2 $!@
–61 ± 7 (5) *§
4
4
4
4
5
5
5
6c
–118 ± 15 (2) *§^&
–128 ± 9 (10) *§^&
–12 ± 3 (38) ^&
–52 ± 9 (5) *§
6d
8b
8c
8dg
controlh
23.6 ± 3.6 (4) *§
17.7 ± 7.1 (1) *§
0.4 ± 0.7
–88 ± 21 (21) *§^
–9 ± 2 ^&
a The maximal hypertensive effect [mmHg] of a compound observed during 60 minutes after injection. b The
maximal hypotensive effect [mmHg] of a compound observed during 60 minutes after injection. c The maximal
bradycardic effect [bpm] of a compound observed during 60 minutes after injection. d Values given are means ±
s.e.m from n independent experiments. e Number of experiments. f tmax: time (in minutes) after the injection
when the maximal effect was observed. g The results have been published earlier in Sączewski J. et al. [48]. h
Saline vehicle injection. (*) p <0.001, (#) p <0.05 vs control. (&) p <0.001 vs 6b. ($) p<0.001 vs 6c. (!) p <0.001 vs
6d. (§) p <0.001, (%) p <0.05 vs 8b. (^) p <0.001 vs 8c. (@) p <0.001 vs 8d.