Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines

Article abstract of DOI:10.1016/j.bmc.2011.11.025

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α₁- and α₂- adrenoceptors and imidazoline I₁ and I₂ receptors. The most active 2-[(indazol-1-yl)methyl]imidazolin

Full text of DOI:10.1016/j.bmc.2011.11.025

Bioorganic & Medicinal Chemistry 20 (2012) 108–116
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activities of 2-[(heteroaryl)methyl]imidazolines
Jaroslaw Saczewski a, , Alan Hudson b, Mika Scheinin c, Apolonia Rybczynska d, Daqing Ma e,
⇑
Franciszek Saczewski a, Shayna Laird b, Jonne M. Laurila c, Konrad Boblewski d,
Artur Lehmann d, Jianteng Gu e, Helena Watts e
a
´
´
Department of Chemical Technology of Drugs, Medical University of Gdansk, Al. Gen. J. Hallera 107, Gdansk 80-416, Poland
b Department of Pharmacology, 947 Medical Sciences Building, 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 Pathophysiology, Medical University of Gdansk, Poland
e Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Imperial College London, Chelsea and Westminster Hospital, London, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2011
Revised 12 November 2011
Accepted 14 November 2011
Available online 25 November 2011
A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at a1- and
a
2-adrenoceptors and imidazoline I1 and I2 receptors. The most active 2-[(indazol-1-yl)methyl]imidaz-
olines showed high or moderate affinities for 1- and 2-adrenoceptors. However, their intrinsic activ-
ities at 2A-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent
1-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats.
Ó 2011 Elsevier Ltd. All rights reserved.
a
a
a
a
Keywords:
Imidazolines
Indazoles
Alpha1-adrenenoceptor
Alpha2-adrenenoceptor
Hypotensive agents
1. Introduction
7-methyl-marsanidine9 and its positional analogues E,10 com-
pounds with potent hypotensive and diuretic activities.
Imidazoline-containing compounds constitute a valuable class
of agents acting at
-adrenergic and/or imidazoline receptors.1–4
For example, 2-(anilino)imidazolines such as clonidine and its ana-
logues (structure A, Fig. 1) are non-selective 2-adrenoceptor/I1
In this report we describe the synthesis and biological activities
of previously unexplored 2-(anilinomethyl)-imidazoline analogues
of type F with a nitrogen atom incorporated into a heterocyclic ring
system such as indazole, benzimidazole or benzotriazole. These
a
a
receptor agonists widely used in clinical practice for the treatment
of hypertension and in intensive care units as sedative, anxiolytic
and analgesic agents, while other analogues of clonidine with a
methylene bridge connecting the imidazoline ring with an aromatic
moiety, represented by, for example, tolazoline (structure B), are
newly prepared variants of 2-(anilinomethyl)imidazolines
(X = N) with partially restricted conformational freedom were
tested for their in vitro binding affinities at 1- and 2-adrenocep-
tors and imidazoline I1 and I2 receptors, their intrinsic activity at
2A-adrenoceptors as well as their in vivo effects on circulatory
C
a
a
a
a
1-adrenoceptor antagonists which behave as vasodilators. On
the other hand, cirazoline with a methylenoxy spacer (structure
C, X = O) is known to behave as an 1-adrenoceptor agonist and
and CNS functions in the rat.
a
2. Results and discussion
an a2 antagonist with vasoconstricting properties, and a series
of 2-(anilinomethyl)imidazolines of type C (X = NH) has been
2.1. Chemistry
described as potent a1-adrenoreceptor agonists useful for the treat-
ment of benign prostatic hyperplasia.5–8
As shown in Schemes 1 and 2, the reaction procedure leading to
target compounds 3, 5, and
Recently, we have disclosed a highly selective imidazoline-
7 consisted in the reaction of
based partial
a2-adrenoceptor agonist marsanidine (1-[(imidazoli-
corresponding azole sodium salts, generated by the treatment of
indazoles 1a–i, benzimidazoles 4a–h or benzotriazoles 6a–c with
sodium hydride in anhydrous THF, with 2-chloromethylimidazo-
line 2 at ambient temperature. Thus, indazoles 1a–i gave mixtures
of 1- and 2-substituted alkylation products, from which indazol-
1-yl compounds 3a–i were separated in pure form in 26–34% yield
din-2-yl)imino]indazole, compound D, Fig. 1), from which emerged
⇑
Corresponding author. Tel.: +48 58 349 3252; fax: +48 58 349 3257.
E-mail address: js@gumed.edu.pl (J. Saczewski).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.025

J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
109
R
R
N
N
X
R
N
H
N
N
H
N
H
HN
A
B
C
X = O, R - cyclopropyl (cirazoline
)
R = H (tolazoline)
R = 2,6-Cl (clonidine
)
α
α
1 agonist, 2 antagonist
X = NH, R = COOMe, heteroaryl
α
α
1 antagonist
2, I1 agonist
(
2-(anilinomethyl)imidazolines)
α
1 agonists
R
H
C
H
N
Y
X
N
N
N
N
N
N
N
HN
HN
R
R
HN
HN
HN
D
E
F
R = H (marsanidine)
R = CH3, Cl
α2
R = alkyl, halogen, alkoxyl
X = N or CH
R = 7-CH3
(
7Me-marsanidine)
agonists
Y = N or CH
α
2 agonists
Figure 1. Structures of
a-adrenergic ligands A–C and their analogues D–F with partially restricted conformational freedom.
H
R2
R5
R1
N
Cl
N
N
R2
R5
R1
N
R3
R4
R3
R4
2
N
THF, NaH
rt, 6h
N
N
H
HN
1a-i
3a i
-
H
N
Cl
R2
R2
R3
R4
N
N
R3
R4
N
N
R1
2
R1
THF, NaH
rt, 6h
N
H
N
R5
R5
HN
4a h
-
5a-h
1
2
1a 3a
,
, R = R = R3 = R4 = R5 = H
4a, 5a, R1 = R2 = R3 = R4 = R5 = H
4b 5b
1b, 3b, R1 = Me, R2 = R3 = R4 = R5 = H
,
, R1 = Me, R2 = R3 = R4 = R5 = H
1
2
3
4
1c 3c
,
, R = H, R = Me, R = R = R5 = H 4c, 5c, R1 = Et, R2 = R3 = R4 = R5 = H
, R1 = H, R2 = Cl, R3 = R4 = R5 = H
1e, 3e, R1 = H, R2 = OMe, R3 = R4 = R5 = H
4d, 5d, R1 = Ph, R2 = R3 = R4 = R5 = H
1d 3d
,
1
4e 5e
,
, R = Bz, R2 = R3 = R4 = R5 = H
, R1 = R2 = H, R3 = Me, R4 = R5 = H
1
2
,
, R = H, R = Me, R3 = R4 = R5 = H
1f 3f
,
4f 5f
1g, 3g, R1 = R2 = R3 = R4 = H, R5 = Me
,
, R1 = R2 = H, R3 = R4 = Me, R5 = H
4h, 5h, R1 = Et, R2 = H, R3 = R4 = Me, R5 = H
4g 5g
1h 3h
,
, R1 = R2 = R3 = R4 = H, R5 = Cl
, R1 = R2 = R3 = R4 = H, R5 = F
1i 3i
,
Scheme 1. Preparation of indazol-1-yl derivatives 3a–i and benzimidazol-1-yl derivatives 5a–h.
by preparative thin layer chromatography on silica gel using
chromatotron. In a similar manner, benzimidazol-1-yl derivatives
5a–h were obtained in 42–83% yield. On the other hand, from
the reaction of sodium salts of benzotriazoles 6a–c with 2-chlo-
romethylimidazoline 2, both the 1-substituted products 7a–b and
2-substituted products 8a–b could be separated in 34–35% and
10–12% yield, respectively. For biological testing, the products 3,
5, 7 and 8 were further converted into the corresponding
water-soluble hydrochlorides with use of methanolic hydrochloric
acid solution or by passing gaseous hydrogen chloride through
dichloromethane solutions of the free base.
Structures of all newly prepared compounds were confirmed by
C, H, N elemental analyses as well as IR and NMR spectroscopic
data (see Section 4).

110
J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
R1
The most interesting results, however, were found within a
series of indazole derivatives 3a–i which were characterised by
moderate or high affinity for -adrenoceptors, depending on the
substitution pattern. The unsubstituted compound 3a showed high
affinity for 2-adrenoceptors (Ki = 18.5 nM) and moderate affinity
for 1-adrenoceptors (Ki = 272 nM), while the 3-, 4- and 5-substi-
tuted analogues 3b–g showed affinities about 7–33 times lower
at 2-adrenoceptors (Ki values in the range of 130–607 nM) and
comparable or lower affinities at 1-adrenoceptors (Ki in the range
of 95–1670 nM). It should be pointed out that the highest affinity
at both the 1- and 2-adrenoceptors was exhibited by compound
3h with a chlorine atom placed at position 7 of the indazole ring
(Ki = 14 and 6.4 nM, respectively). Compound 3h also displayed
submicromolar affinities to imidazoline I1 and I2 receptors (Ki
values = 856 and 554 nM, respectively).
Summing up, from an analysis of the results of in vitro binding
assays presented in Table 1 it can be concluded that indazole-con-
taining compounds 3 constitute an interesting class of nonselective
a1a2-adrenoceptor ligands of which the most active was the 7-Cl
derivative 3h that bears resemblance to the previously described
agents of type C, D and E (Fig. 1).
R2
R3
N
N
a
N
a
N
a
HN
7a, R1 = R2 = R3 = H
H
N
Cl
a
R1
a
, R1 = H, R2 = R3 = Me
7b
R2
R3
N
2
N
a
a
N
R1
THF, NaH
rt, 6h
N
H
R2
R3
N
N
6a, R1 = R2 = R3 = H
N
N
6b, R1 = Me, R2 = R3 = H
6c, R1 = H, R2 = R3 = Me
HN
8a, R1 = Me, R2 = R3 = H
8b, R1 = H, R2 = R3 = Me
Scheme 2. Preparation of benzotriazol-1-yl derivatives 7a,b and benzotriazol-2-yl
derivatives 8a,b.
2.3. Functional [35S]GTP
cS binding assays
Compounds 3a (R = H) and 3h (R = 7-Cl) with the highest bind-
ing affinity at 2-adrenoceptors and the compound 3g (R = 7-Me)
which displayed higher 2-adrenoceptor binding affinity compared
to its affinity at
1-adrenoceptors were subjected to [35S]GTP
binding experiments to determine their intrinsic activity and ago-
nist potency at recombinant human
2A-adrenoceptors.11,12 For
SAR purposes, noradrenaline, clonidine as well as marsanidine
and its 7-Me and 7-Cl derivatives representing nitrogen-bridged
analogues of type E were also included as controls.
2.2. Radioligand binding assays
a
a
The in vitro assay involved the investigation of the affinities of
2-[(heteroaryl)methyl]-imidazolines 3a–i, 5a–h, 7a–b and 8a–b
for a1- and a2-adrenoceptors as well as imidazoline I1 and I2 recep-
a
cS
a
tors. All ligands were used in form of water soluble hydrochloride
salts and the results of the receptor binding assays are presented in
Table 1.
In general, all of the tested compounds exhibited very low
affinity for imidazoline I1 receptors (Ki values in the range of
646–96400 nM). Similar results were obtained for imidazoline I2
receptors with the only the exception of the 5-methylindazole
derivative 3f, which exhibited high affinity (Ki = 22 nM).
As shown in Figure 2 and Table 2, in contradistinction to the
natural full agonist noradrenaline as well as the partial agonists
clonidine and the marsanidine derivatives, the newly prepared
compounds of type 3 only weakly stimulated the binding of
[
35S]GTP
man
c
S to CHO cell membranes expressing recombinant hu-
a
2A-adrenoceptors. Their intrinsic activities estimated relative
Table 1
to noradrenaline were found to be very low (6.3, 6.3 and 7.4% for
compounds 3a, 3g and 3h, respectively). Moreover, their agonist
potencies expressed as EC50 values were in the micromolar range.
Binding affinity data for compounds 3a–i, 5a–h, 7a–b, and 8a–b
Compound a1 Ki (nM)a
a2 Ki (nM)a
I1 IC50 (nM)b
I2 Ki (nM)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
8a
8b
272 42
95 27
409 39
187 77
1670 393
363 89
101 28
14.1 3.9
107 (n = 1)
929 48
1290 599
5500 610
1110 440
30900 (n = 1)
1500 (n = 1)
1500 740
18.5 5.9
435 99
197 28
130 27
305 59
268 59
607 97
6.4 2.3
12.6 (n = 1)
3250 479
1020 163
5000 900
7440 680
11200 (n = 1) 21000 (n = 1)
273 (n = 1)
2260 490
19400 (n = 1)
21300 (n = 1)
12900 (n = 1)
5150 688
32900 (n = 1)
45400 (n = 1)
96400 (n = 1)
856 239
16400 (n = 1)
43900 (n = 1)
9390 1059
44200 (n = 1)
28100 (n = 1)
42200 (n = 1)
359 88
116 44
2850 492
382 72
22 7.4
6840 879
554 288
156 (n = 1)
6100 410
NDc
2.4. In vivo cardiovascular effects
Based on the results of the in vitro experiments described above,
the most interesting compound 3h (R = 7-Cl) was selected for
400
300
200
100
0
34100 (n = 1)
NDc
NDc
77700 (n = 1)
74000 (n = 1)
93300 (n = 1)
56800 (n = 1)
16800 (n = 1)
48800 (n = 1)
646 (n = 1)
24400 (n = 1)
28000 (n = 1)
81 12
810 269
26300 (n = 1)
78 55
12900 5300 7410 620
1340 547
8040 (n = 1)
1540 (n = 1)
6070 (n = 1)
5110 1189
1560 (n = 1)
499 (n = 1)
1320 (n = 1)
-12
-10
-8
-6
-4
-2
233 (n = 1)
log conc. (M)
a
Ki affinity values for
a1-adrenoceptors, a2-adrenoceptors and I2 imidazoline
Figure 2. Graphical presentation of
[
35S]GTP
membranes expressing recombinant human
noradrenaline (d) and the partial agonist clonidine (ꢀ) dose-dependently stimu-
lated [35S]GTP
cS
binding results with CHO cell
binding sites were assessed by measuring the ability of the test compounds to
compete with [3H]prazosin, [3H]RX821002 or [3H]2BFI binding to rat brain
membranes.
a
2A-adrenoceptors: the full agonist
b
Molar concentration of the test compounds that displaces 50% of specifically
c
S binding but compounds 3a (h), 3g (N) and 3h (5) were almost
bound [3H]clonidine in rat kidney membranes in the presence of rauwolscine.
inactive in this assay. Values shown are means s.e.m. from
3 independent
c
ND—no displacement up to 1,00,000 nM.
experiments.

J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
111
Table 2
negligible intrinsic activity at
a
2A-adrenoceptors we put forward
Characterization of [35S]GTP
cS binding to CHO cell membranes expressing recombi-
a hypothesis that the possible mechanism of the observed hypo-
tensive effect might involve blockade of peripheral 1-adrenocep-
tors and/or 2-adrenoceptors that are widely expressed in vascular
beds. Testing of this hypothesis was performed by examining the
interaction of 3h with the selective 1-adrenoceptor agonist
nant human
a2A-adrenoceptors: estimates of agonist potency (EC50) and intrinsic
a
activity relative to the natural full agonist noradrenaline. Values shown are
means s.e.m. from 3 independent experiments for compounds 3a, 3g and 3h and
3–19 independent experiments for the other compounds
a
a
Ligand
EC50 (nM)
Intrinsic activity
methoxamine that has vasoconstrictor and positive inotropic prop-
(% of Noradrenaline)
erties. As shown in Figure 3, the intravenous infusion of methoxa-
Noradrenaline
Clonidine
Marsanidinea
7-Me-marsanidinea
7-Cl-marsanidinea
3a
118 14
100
mine at 10
attenuated the hypotensive action of the compound. A further in-
crease of the methoxamine infusion rate to 20 g/kg/min almost
lg/kg/min, 10 min prior to 3h administration, markedly
30
4
51
18
32
48
6.3 1.6
6.3 0.6
7.4 1.0
2
2
5
3
163 66
37
15
9
2
l
completely abolished the hypotensive effect of 3h.
4500 4300
17,400 15,500
16,600 5800
On the other hand, as shown in Figure 4, co-administration of
methoxamine with 3h only slightly attenuated the decrease in
heart rate (this difference did not reach statistical significance in
3g
3h
These results have been published previously in Sa˛czewski F. et al. 2011.10
a
comparison to the 3h group). Since activation of a1-adrenoceptors
in the myocardium by methoxamine causes a positive chrono-
tropic response14,15 and this effect was not fully inhibited by 3h,
evaluation of possible cardiovascular effects in anaesthetized rats.
We reasonedthatthis compound, characterizedby very weak intrin-
the newly prepared imidazoline compound may produce
a
sic activity at
a2A-adrenoceptors, could still prove to be vasoactive
negative chronotropic effect on the heart independent of
by being a mixed
a
1-adrenoceptor/ 2-adrenoceptor antagonist. Its
a
a
-adrenoceptors. A possible mechanism may, for example, involve
b-adrenoceptors16 or sodium channel blockade.17
in vivo hemodynamic effects were evaluated in anaesthetized rats
by monitoring changes in blood pressure and heart rate after
intravenous infusion of 3h using procedures described previously.13
The post-infusion data were compared to baseline values and are
2.5. Loss of righting reflex (LORR) test
presented as
found that administration of 3h at a dose of 0.1 mg/kg induced
highly significant decreases in blood pressure
MAP = ꢀ47/
4.3 mmHg) and heart rate (
HR = ꢀ131.8 11/5 bpm).
Clonidine-like centrally acting antihypertensive agents, with
their main pharmacological actions mediated by 2A-adrenoceptor
activation, exert biphasic hemodynamic effects after intravenous
administration: an initial short-lasting increase in arterial blood
pressure resulting from a peripheral vasoconstrictor response is
followed by a prolonged centrally mediated hypotensive effect.
Upon administration of 3h, however, an immediate fall of blood
pressure was observed with no initial pressor response. In view
of the in vitro finding that the imidazoline derivative 3h has
DMAP and DHR in Figures 3 and 4, respectively. We
a
2-Adrenoceptor agonists such as clonidine and dexmedetomi-
dine have been used in anaesthesiology to induce a sedative-
hypnotic state somewhat similar to physiological non-rapid eye
movement (non-REM) sleep.18 Therefore, the compound 3h with
(
D
7
D
high affinity and poor efficacy at
a2-adrenoceptors was investi-
a
gated to determine whether or not it shows capacity to activate
the non-REM sleep pathway in rats. This anesthetic state was
evaluated by induction of loss of the righting reflex (LORR) over a
wide range of doses. The results presented in Table 3 indicate that
at low doses of 10–500
at the highest administered doses of 1000 and 2000
lg/kg, the righting reflex was present, and
lg/kg
compound 3h produced marked CNS actions with rapid onset.
Within 5–8 min post-injection rats displayed loss of spontaneous
Figure 3. Effect of 0.1 mg/kg of 3h in the absence and presence of 10 (ME10) and 20 (ME20)
l
g/kg/min of methoxamine (ME) on
D
MAP (mean arterial blood pressure)
MAP from five to seven
calculated as the difference of MAP between sequential measurements and time 0 of the experiment. Each point represents a mean value of
D
experiments. Comparisons were made using ANOVA for repeated measurements and Fisher’s test. Significant differences (⁄) p <0.001 were found for comparisons 3h versus
control, 3h versus ME20+3h and 3h versus ME10+3h; (&) p <0.01 was found for comparison of ME10+3h versus the control group. Significance (#) p <0.05 was found for
ME20+3h versus the control group between 3 and 5 min of the experiment (shaded part).

112
J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
Figure 4. Effect of 0.1 mg/kg of 3h in the absence and presence of 10 (ME10) and 20 (ME20)
between sequential measurements and time 0 of the experiment. Each point represents a mean value of
ANOVA for repeated measurements and Fisher’s test. Significant differences (⁄) p <0.001 were found for comparisons 3h, ME20+3h and ME10+3h versus control.
lg/kg/min of methoxamine on
DHR (heart rate) calculated as the difference of HR
D
HR from five to seven experiments. Comparisons were made using
Table 3
have previously been reported to produce increases, decreases or
even biphasic effects on body temperature.25 Therefore, given the
fairly rapid onset and short-lasting action (<30 min) of the effects
of 3h noted in the present study, further testing using a higher ini-
tial dose (>500 mg/kg) and temperature monitoring will be neces-
sary for a more comprehensive assessment. Changes in the release
of dopamine, 5-HT and other neurotransmitters leading to the
hyperthermic effect of 3h cannot be excluded.25
LORR dose–response results for compound 3h at 30 min after i.p. injection
Animal
Weight (g)
Dose (
lg/kg)
LORR
1
2
3
4
5
260
253
258
260
251
10
No
No
No
Yes
Yes
100
500
1000
2000
3. Conclusion
locomotor activity and reduced motor co-ordination, suggestive of
locomotor impairment without a true hypnotic action as the right-
ing reflex was slowed but not completely lost. After 30 min, the
rats remained in a calm, subdued state, remaining still with no
exploratory behaviour and showing little resistance to handling.
However, reflex responses such as paw grasping and the righting
reflex had returned to normal.
The above results regarding reduced locomotor activity after
systemic administration of 3h require further investigation but
are briefly commented here. They agree with previously described
In this paper we have described the synthesis and binding
affinities of four series of novel analogues of 2-(anilinomethyl)
imidazolines with partially restricted conformational freedom.
Indazol-1-yl derivatives of type 3 exhibited high or moderate
affinity to both
pounds showed negligible intrinsic activity at
a
1- and
a
2-adrenoceptors. However, these com-
2A-adrenoceptors.
a
Hypotensive responses to the most active derivative 7-chloro-1-
((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-indazole (3h) were
progressively reduced by increasing doses of the a1-adrenoceptor
agonist methoxamine, which suggests that this effect may result
from peripheral vascular effects rather than central sympathetic
depression, and the newly prepared imidazoline compound 3h
findings that also
locomotor behaviour of rats and mice.19–21 Moreover, it was found
that both pharmacologic and genetic inhibition of 1-adrenoceptor
activation inhibited dopamine-mediated locomotor hyperactiv-
ity.22,23 Hence,
1-antagonists were considered as a new approach
a1-adrenoceptors participate in regulation of
a
may be regarded as a prototype of a new class of
antagonists.
a1-adrenoceptor
a
to treat dopamine-induced dyskinesia. However, since decreased
locomotor activity was observed only in animals treated with rel-
atively high doses of 3h (1–2 mg/kg), we cannot exclude that this
depressant effect may result from a profound decrease in blood
pressure. Such observations have been made previously upon
treatment of experimental animals with various hypotensive
agents.24
Interestingly, the onset of CNS effects was accompanied by
changes in body temperature, which suggests activity in the hypo-
thalamus and possibly in the same brain nuclei that are involved in
regulation of non-REM sleep. In contrast to clonidine and dex-
medetomidine, the effect on temperature regulation appeared to
be hyper- rather than hypothermic. Although sedation is usually
accompanied by a decrease in body temperature, hypnotic agents
4. Experimental
4.1. Chemistry
Melting points were measured on a Buchi 535 apparatus and
are uncorrected. IR spectra were taken in KBr pellets on a Per-
kin–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 7.26 ppm (CDCl3) or 2.50 ppm and 39.5 ppm (DMSO-d6).
Chromatography was performed on silica gel 60 PF254 containing
gypsum (Merck) using the reported solvent systems. Indazoles
3c, 3f, 3g and 3d, 3e, 3h, 3i were prepared according to the

J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
113
Rüchardt and Hassmann procedure26 or using o-fluorobenzaldehy-
des,27 respectively. The benzimidazoles 4a–h and benzotriazoles
6a–c were obtained from the corresponding o-phenylenediamines.
2-(Chloromethyl)-4,5-dihydro-1H-imidazole hydrochloride was
synthesized from 2-chloroacetonitrile and ethylenediamine.28
127.7, 132.1, 133.7, 140.4, 163.8; mmax 3180, 2952, 2881, 2794,
1633, 1506, 1439, 1295, 1237, 1154, 978, 847, 784, 771 cmꢀ1
;
MS (ESI) m/z: 215 [M+H]+. Hydrochloride of 3c; mp = 234–
238 °C; Anal. Calcd (C12H15ClN4): C, 57.48; H, 6.03; N, 22.35.
Found: C, 57.17; H, 6.22; N, 22.29.
4.2. General procedure for the preparation of compounds3, 5, 7,
and 8
4.2.4. 4-Chloro-1-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-
indazole (3d)
A white solid (0.24 g, 31%), mp = 103–109 °C; 1H NMR (CDCl3): d
3.61 (s, 4H, CH2), 4.92 (br s, 1H, NH), 5.24 (s, 2H, CH2), 7.18 (d,
J = 8.0 Hz, 1H, CH), 7.35 (dd, J = 8.0 Hz, J = 8.0 Hz, 1H, CH), 7.45 (d,
J = 8.0 Hz, 1H, CH), 8.14 (s, 1H, CH); 13C NMR (CDCl3): d 47.8,
49.9, 108.2, 121.2, 123.7, 127.0, 128.0, 133.4, 141.1, 163.1; mmax
3212, 2960, 2940, 2866, 1613, 1496, 1355, 1281, 1163, 982,
Sodium hydride (0.53 g, 6.6 mmol, 60% oil dispresion) was
added to the stirred solution of the corresponding azole (3.3 mmol)
in anhydrous THF (2 mL) at room temperature. After 15 min,
freshly prepared 2-(chloromethyl)-4,5-dihydro-1H-imidazole
2
(0.47 g, 4.0 mmol) was added and the reaction mixture was stirred
at ambient temperature for 6 h. The N-alkylation products 3, 5, 7
and 8 were isolated upon quenching the reaction mixture with
water (5 mL) followed by extraction with dichloromethane
(3 ꢁ 5 mL). The combined organic layers were dried over anhy-
drous sodium sulfate and evaporated under reduced pressure.
The oily residue thus obtained was purified on silica with use of
chromatotron (Et3N/MeOH/AcOEt 1:5:100). All products were very
polar with Rf values close to 0.05.
The N-alkylation reaction of indazoles (1) provided the desired
N1 substituted products (3) along with the N2 substituted side
product. The latter compounds demonstrated considerably lower
Rf than 3 and were not isolated in pure form. The alkylation reac-
tions of benzotriazoles 6a and 6c allowed the isolation of N1
substituted products 7a–b (higher Rf) and N2 isomer 8b (lower
Rf). However, in the case of 4-methyl-benzotriazole 6b only the
N2 substituted isomer 8a was isolated as a pure product.
The products 3, 5, 7 and 8 were then converted into water-sol-
uble hydrochloride salts suitable for biological tests with use of
methanolic hydrochloric acid solution or by passing gaseous
hydrogen chloride through dichloromethane solution of the corre-
sponding free base.
;
776 cmꢀ1 MS (ESI) m/z: 235 [M+H]+. Hydrochloride of 3d;
mp = 225–231 °C; Anal. Calcd (C11H12Cl2N4): C, 48.74; H, 4.46; N,
20.66. Found: C, 48.58; H, 4.71; N, 20.61.
4.2.5. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-4-methoxy-
1H-indazole (3e)
A white solid (0.20 g, 26%), mp = 94–97 °C; 1H NMR (CDCl3): d
3.50 (br s, 1H, NH), 3.57 (s, 4H, CH2), 3.96 (s, 3H, OCH3), 5.24 (s,
2H, CH2), 6.49 (d, J = 7.7 Hz, 1H, CH), 7.07 (d, J = 7.7 Hz, 1H, CH),
7.32 (t, J = 7.7 Hz, 1H, CH), 8.10 (s, 1H, CH); 13C NMR (CDCl3): d
48.0, 50.36, 55.9, 100.5, 102.4, 116.6, 128.9, 132.8, 142.2, 154.3,
163.6; mmax 3475, 3113, 2940, 2871, 1625, 1601, 1513, 1392,
1268, 1055, 986, 772 cmꢀ1; MS (ESI) m/z: 231 [M+H]+. Dihydro-
chloride of 3e; mp = 96–99 °C; Anal. Calcd (C12H16Cl2N4O): C,
47.54; H, 5.32; N, 18.48. Found: C, 47.80; H, 5.41; N, 18.74.
4.2.6. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5-methyl-1H-
indazole (3f)
A white solid (0.19 g, 27%), mp = 135–140 °C; 1H NMR (CDCl3): d
2.45 (s, 3H, CH3), 3.57 (s, 4H, CH2), 5.02 (br s, 1H, NH), 5.18 (s, 2H,
CH2), 7.24 (d, J = 8.4 Hz, 1H, CH), 7.40 (d, J = 8.4 Hz, 1H, CH), 7.49 (s,
1H, CH), 7.94 (s, 1H, CH); 13C NMR (CDCl3): d 21.7, 47.8, 50.2, 109.4,
120.6, 125.0, 129.6, 131.2, 134.2, 139.1, 163.9; mmax 3106, 3073,
2928, 2868, 2795, 1615, 1509, 1293, 1240, 1175, 1142, 985, 825,
790, 578 cmꢀ1; MS (ESI) m/z: 215 [M+H]+. Dihydrochloride of 3f;
mp = 100–103 °C; Anal. Calcd (C12H16Cl2N4): C, 50.19; H, 5.62; N,
19.51. Found: C, 50.26; H, 5.91; N, 19.62.
4.2.1. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-1H-indazole
(3a)
A white solid (0.20 g, 30%), mp = 132–135 °C; 1H NMR (CDCl3): d
3.59 (s, 4H, CH2), 4.57 (br s, 1H, NH), 5.21 (s, 2H, CH2), 7.15–7.23
(m, 1H, CH), 7.38–7.54 (m, 2H, CH), 7.72–7.77 (m, 1H, CH), 8.05
(s, 1H, CH); 13C NMR (CDCl3): d 47.9, 50.4, 109.7, 121.6, 121.7,
124.6, 127.5, 134.8, 140.4, 163.6; mmax 3082, 2926, 2856, 2797,
1617, 1508, 1413, 1239, 1173, 985, 907, 831, 752, 741 cmꢀ1; MS
(ESI) m/z: 201 [M+H]+. Hydrochloride of 3a; mp = 196 °C dec.; Anal.
Calcd (C11H13ClN4): C, 55.82; H, 5.54; N, 23.67. Found: C, 55.65; H,
5.63; N, 23.39.
4.2.7. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-7-methyl-1H-
indazole (3g)
A white solid (0.22 g, 31%), mp = 143–147 °C; 1H NMR (CDCl3): d
2.77 (s, 3H, CH3), 3.57 (s, 4H, CH2), 4.47 (br s, 1H, NH), 5.40 (s, 2H,
CH2), 7.02–7.15 (m, 2H, CH), 7.57 (d, 1H, CH), 8.01 (s, 1H, CH); 13C
NMR (CDCl3): d 19.9, 50.2, 50.4, 119.5, 121.1, 122.0, 125.4, 129.4,
135.2, 139.9, 164.7; mmax 3094, 2933, 2870, 2786, 1623, 1502,
1426, 1408, 1292, 1238, 986, 833, 778, 749 cmꢀ1; MS (ESI) m/z:
215 [M+H]+. Hydrochloride of 3g; mp = 248–255 °C; Anal. Calcd
(C12H15ClN4): C, 57.48; H, 6.03; N, 22.35. Found: C, 57.39; H,
6.24; N, 22.33.
4.2.2. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-3-methyl-1H-
indazole (3b)
A white solid (0.23 g, 33%), mp = 141–144 °C; 1H NMR (CDCl3): d
2.58 (s, 3H, CH3), 3.62 (s, 4H, CH2), 4.75 (br s, 1H, NH), 5.20 (s, 2H,
CH2), 7.14–7.21 (m, 1H, CH), 7.37–7.49 (m, 2H, CH), 7.64–7.69 (m,
1H, CH); 13C NMR (CDCl3): d 12.4, 47.5, 50.3, 109.6, 120.9, 124.1,
127.5, 141.3, 143.5, 164.0; mmax 3146, 2926, 2859, 1620, 1499,
1274, 1176, 919, 750 cmꢀ1; MS (ESI) m/z: 215 [M+H]+. Dihydro-
chloride of 3b; mp = 210 °C dec.; Anal. Calcd (C12H16Cl2N4): C,
50.19; H, 5.62; N, 19.51. Found: C, 50.33; H, 5.81; N, 19.76.
4.2.8. 7-Chloro-1-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-
indazole (3h)
A white solid (0.26 g, 34%), mp = 159–163 °C; 1H NMR (CDCl3): d
3.59 (s, 4H, CH2), 4.90 (br s, 1H, NH), 5.60 (s, 2H, CH2), 7.09 (t,
J = 7.6 Hz, 1H, CH), 7.38 (d, 7.6 Hz, 1H, CH), 7.64 (d, 7.6 Hz, 1H,
CH), 8.07 (s, 1H, CH); 13C NMR (CDCl3): d 44.1, 44.7, 111.1, 115.1,
117.1, 121.9, 123.2, 129.9, 131.2, 159.0; mmax 3156, 3104, 2932,
4.2.3. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-4-methyl-1H-
indazole (3c)
A white solid (0.22 g, 31%), mp = 133–135 °C; 1H NMR (CDCl3): d
2.60 (s, 3H, CH3), 3.57 (s, 4H, CH2), 4.94 (br s, 1H, NH), 5.20 (s, 2H,
CH2), 6.93–6.97 (m, 1H, CH), 7.30–7.32 (m, 2H, CH), 8.05 (s, 1H,
CH); 13C NMR (CDCl3): d 19.0, 47.9, 50.2, 107.1, 121.7, 125.1,
2872, 2799, 1620, 1503, 1236, 1133, 963, 839, 773, 733 cmꢀ1
;
MS (ESI) m/z: 235 [M+H]+. Hydrochloride of 3h; mp = 242–
250 °C; Anal. Calcd (C11H11ClN4): C, 56.30; H, 4.72; N, 23.87.
Found: C, 56.02; H, 4.99; N, 23.61.

114
J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
4.2.9. 7-Fluoro-1-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-
indazole (3i)
CH2), 7.20–7.36 (m, 7H, CH), 7.38–7.40 (m, 1H, CH), 7.79–7.81 (m,
1H, CH); 13C NMR (CDCl3): d 34.8, 43.1, 50.3, 109.9, 120.2, 123.1,
123.6, 127.7, 129.2, 129.4, 135.9, 136.4, 143.0, 153.7, 162.5; mmax
3219, 3031, 2939, 2869, 1618, 1506, 1466, 1411, 1234, 1165,
765, 746, 711 cmꢀ1; MS (ESI) m/z: 291 [M+H]+. Dihydrochloride
of 5e; mp = 158–163 °C; Anal. Calcd (C18H20Cl2N4): C, 59.51; H,
5.55; N, 15.42. Found: C, 59.71; H, 5.68; N, 15.79.
A white solid (0.39 g, 54%), mp = 171–173 °C; 1H NMR (CDCl3): d
3.57 (s, 4H, CH2), 5.01 (br s, 1H, NH), 5.33 (s, 2H, CH2), 7.00–7.09
(m, 2H, CH), 7.43–7.52 (m, 1H, CH), 8.03 (d, 1H, CH); 13C NMR
(CDCl3): d 49.7 (d, J = 4.8 Hz), 50.1, 112.3 (d, J = 16.9 Hz), 117.4 (d,
J = 4.5 Hz), 122.2 (d, J = 5.5 Hz), 128.5 (d, J = 3.9 Hz), 129.9 (d,
J = 12.9 Hz), 135.5, 140.9 (d, J = 247.2 Hz), 164.0; mmax 3143, 3102,
2937, 2866, 2794, 1621, 1583, 1501, 1434, 1412, 1316, 1248,
1236, 1174, 866, 836, 783, 733 cmꢀ1; MS (ESI) m/z: 219 [M+H]+.
Hydrochloride of 3i; mp = 244–248 °C; Anal. Calcd (C11H11FN4):
C, 60.54; H, 5.08; N, 25.67. Found: C, 60.46; H, 5.21; N, 25.51.
4.2.15. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-4-methyl-1H-
benzimidazole (5f)
A white solid (0.30 g, 42%), mp = 152–159 °C; 1H NMR (CDCl3): d
2.58 (s, 3H, CH3), 3.62 (s, 4H, CH2), 5.01 (s, 2H, CH2), 5.10 (br s, 1H,
NH), 7.06–7.10 (m, 1H, CH), 7.18–7.30 (m, 2H, CH), 7.91 (s, 1H, CH);
13C NMR (CDCl3): d 16.8, 43.9, 50.1, 107.4, 123.3, 123.8, 130.6,
133.6, 142.5, 143.1, 162.7; mmax 3075, 2937, 2867, 1620, 1498,
1360, 1243, 986, 762 cmꢀ1; MS (ESI) m/z: 215 [M+H]+. Dihydro-
chloride of 5f; mp = 235–240 °C; Anal. Calcd (C12H16Cl2N4): C,
50.19; H, 5.62; N, 19.51. Found: C, 50.11; H, 5.82; N, 19.66.
4.2.10. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-1H-benzimid-
azole (5a)
A white solid (0.50 g, 76%), mp = 197–201 °C; 1H NMR (CDCl3): d
3.60 (br s, 1H, NH), 3.63 (s, 4H, CH2), 4.99 (s, 2H, CH2), 7.31–7.37
(m, 2H, CH), 7.47–7.49 (m, 1H, CH), 7.80–7.81 (m, 1H, CH), 7.88–
7.89 (m, 1H, CH); 13C NMR (CDCl3): d 44.0, 50.4, 109.9, 120.8,
123.0, 123.9, 134.0, 143.3, 144.0, 162.3; mmax 3066, 2930, 2870,
2803, 1616, 1516, 1498, 1459, 1423, 1367, 1247, 1206, 1193,
1175, 988, 766, 753, 740 cmꢀ1; MS (ESI) m/z: 201 [M+H]+. Dihydro-
chloride of 5a; mp = 285–288 °C; Anal. Calcd (C11H14Cl2N4): C,
48.37; H, 5.17; N, 20.51. Found: C, 48.21; H, 5.34; N, 20.37.
4.2.16. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5,6-dimethyl-
1H-benzimidazole (5g)
A white solid (0.63 g, 83%), mp = 180–185 °C; 1H NMR (CDCl3): d
2.36 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.62 (s, 4H, CH2), 4.28 (br s, 1H,
NH), 4.82 (s, 2H, CH2), 7.21 (s, 1H, CH), 7.50 (s, 1H, CH), 7.72 (s, 1H,
CH); 13C NMR (CDCl3): d 20.7, 21.0, 44.2, 50.5, 110.2, 120.9, 132.2,
132.7, 133.4, 142.7, 163.0; mmax 3082, 2930, 2864, 1617, 1498,
1239, 1222, 986, 981 cmꢀ1; MS (ESI) m/z: 229 [M+H]+. Dihydro-
chloride of 5g; mp = 219–223 °C; Anal. Calcd (C13H18Cl2N4): C,
51.84; H, 6.02; N, 18.60. Found: C, 51.73; H, 6.13; N, 18.56.
4.2.11. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-2-methyl-1H-
benzimidazole (5b)
A white solid (0.57 g, 81%), mp = 151–156 °C; 1H NMR (CDCl3): d
2.59 (s, 3H, CH3), 3.60 (br s, 1H, NH), 3.63 (s, 4H, CH2), 4.92 (s, 2H,
CH2), 7.23–7.38 (m, 3H, CH), 7.66–7.70 (m, 1H, CH); 13C NMR
(CDCl3): d 14.3, 43.0, 50.5, 109.3, 119.9, 123.0, 123.2, 135.5,
143.1, 152.3, 162.8; mmax 3091, 2928, 2870, 1613, 1515, 1467,
1450, 1407, 1289, 1244, 1160, 1037, 768, 752, 741 cmꢀ1; MS
(ESI) m/z: 215 [M+H]+. Dihydrochloride of 5b; mp = 173–178 °C;
Anal. Calcd (C12H16Cl2N4): C, 50.19; H, 5.62; N, 19.51. Found: C,
50.03; H, 5.88; N, 19.45.
4.2.17. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-2-ethyl-5,6-
dimethyl-1H-benzimidazole (5h)
A white solid (0.68 g, 80%), mp = 185–188 °C; 1H NMR (CDCl3): d
1.42 (t, J = 7.8 Hz, 3H, CH3), 2.37 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.86
(q, J = 7.8 Hz, 2H, CH2), 3.60 (br s, 4H, CH2), 4.35 (br s, 1H, NH), 4.88
(s, 2H, CH2), 7.12 (s, 1H, CH), 7.49 (s, 1H, CH); 13C NMR (CDCl3): d
11.9, 20.5, 20.8, 21.0, 42.5, 109.3, 119.9, 131.4, 132.0, 133.8, 141.4,
155.6, 163.0; mmax 3082, 2930, 2864, 1617, 1498, 1239, 1222, 986,
981 cmꢀ1; MS MS (ESI) m/z: 257 [M+H]+. Dihydrochloride of 5h;
mp = 252–260 °C; Anal. Calcd (C15H22Cl2N4): C, 54.72; H, 6.73; N,
17.02. Found: C, 54.48; H, 6.95; N, 17.01.
4.2.12. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-2-ethyl-1H-
benzimidazole (5c)
A white solid (0.59 g, 78%), mp = 162–168 °C; 1H NMR (CDCl3): d
1.33 (t, J = 7.5 Hz, 3H, CH3), 2.78 (q, J = 7.5 Hz, 2H, CH2), 3.59 (s, 4H,
CH2), 4.46 (br s, 1H, NH), 4.89 (s, 2H, CH2), 7.21–7.36 (m, 3H, CH),
7.66–7.70 (m, 1H, CH); 13C NMR (CDCl3): d 11.9, 21.2, 42.6, 50.4,
109.4, 119.9, 122.9, 123.2, 135.6, 143.0, 156.7, 163.0; mmax 3084,
2975, 2935, 2868, 1613, 1518, 1465, 1415, 1243, 1206, 985,
743 cmꢀ1; MS (ESI) m/z: 229 [M+H]+. Dihydrochloride of 5c;
mp = 254–259 °C; Anal. Calcd (C13H18Cl2N4): C, 51.84; H, 6.02; N,
18.60. Found: C, 51.70; H, 6.32; N, 18.51.
4.2.18. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-1H-benzo-
triazole (7a)
A white solid (0.23 g, 35%), mp = 180–183 °C; 1H NMR (CDCl3): d
3.62 (s, 4H, CH2), 4.86 (br s, 1H, NH), 5.48 (s, 2H, CH2), 7.35–7.44
(m, 1H, CH), 7.49–7.57 (m, 1H, CH), 7.65–7.69 (m, 1H, CH), 8.02–
8.07 (m, 1H, CH); 13C NMR (CDCl3): d 46.8, 50.24, 110.2, 120.5,
124.9, 128.6, 133.6, 146.5, 161.8; mmax 3067, 2936, 2863, 2797,
4.2.13. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-2-phenyl-1H-
benzimidazole (5d)
1618, 1509, 1456, 1415, 1240, 1161, 1101, 983, 786, 745 cmꢀ1
;
MS (ESI) m/z: 202 [M+H]+. Hydrochloride of 7a; mp = 255–
258 °C; Anal. Calcd (C10H12ClN5): C, 50.53; H, 5.09; N, 29.46. Found:
C, 50.47; H, 5.23; N, 29.13.
A white solid (0.63 g, 69%), mp = 151–155 °C; 1H NMR (DMSO-
d6): d 3.45 (s, 4H, CH2), 4.84 (s, 2H, CH2), 6.69 (s, 1H, CH), 7.22–
7.33 (m, 2H, CH), 7.45–7.60 (m, 4H, CH), 7.64–7.75 (m, 1H, CH),
7.85–7.91 (s, 2H, CH); 13C NMR (DMSO-d6): d 40.1, 43.0, 111.1,
119.3, 122.3, 122.7, 128.9, 129.5, 130.0, 130.4, 136.6, 142.8,
153.5, 163.4; mmax 3067, 2929, 2859, 1607, 1463, 1443, 1392,
1237, 974, 745, 692 cmꢀ1; MS (ESI) m/z: 277 [M+H]+. Dihydrochlo-
ride of 5d; mp = 171–175 °C; Anal. Calcd (C17H18Cl2N4): C, 58.46;
H, 5.19; N, 16.04. Found: C, 58.52; H, 5.33; N, 15.82.
4.2.19. 1-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5,6-dimethyl-
1H-benzotriazole (7b)
A white solid (0.26 g, 34%), mp = 154–157 °C; 1H NMR (CDCl3): d
2.37 (s, 3H, CH3), 2.39 (s, 3H, CH3), 3.59 (s, 4H, CH2), 4.63 (br s, 1H,
NH), 7.36 (s, 1H, CH), 7.72 (s, 1H, CH); 13C NMR (CDCl3): d 20.4,
20.9, 46.4, 50.0, 108.9, 118.9, 132.1, 134.2, 138.5, 145.2, 161.4; mmax
3260, 2952, 2871, 1613, 1499, 1454, 1284, 1226, 1104, 977, 850
4.2.14. 2-Benzyl-1-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-
benzimidazole (5e)
cmꢀ1
;
MS (ESI) m/z: 230 [M+H]+. Dihydrochloride of 7b;
A white solid (0.70 g, 73%), mp = 147–152 °C; 1H NMR (CDCl3): d
3.42 (s, 4H, CH2), 3.61 (br s, 1H, NH), 4.33 (s, 2H, CH2), 4.85 (s, 2H,
mp = 249–251 °C; Anal. Calcd (C12H17Cl2N5): C, 47.69; H, 5.67; N,
23.17. Found: C, 47.55; H, 5.72; N, 23.16.

J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
115
4.2.20. 2-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-4-methyl-2H-
benzotriazole (8a)
Each experiment was analysed individually and equilibrium dissoci-
ation constants (Ki) were determined by the method of Cheng and
Prusoff.33 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.
A white solid (0.07 g, 10%), mp = 118–120 °C; 1H NMR (CDCl3): d
2.67 (s, 3H, CH3), 3.67 (s, 4H, CH2), 4.75 (br s, 1H, NH), 5.58 (s, 2H,
CH2), 7.14–7.18 (m, 1H, CH), 7.28–7.36 (s, 1H, CH), 7.68–7.72 (m,
1H, CH); 13C NMR (CDCl3): d 17.8, 50.4, 54.2, 117.6, 128.3, 128.5,
130.2, 141.4, 144.1, 161.9; mmax 3160, 2945, 2869, 1620, 1503,
1276, 1239, 981 cmꢀ1; MS (ESI) m/z: 216 [M+H]+. Dihydrochloride
of 8a; mp = 205–210 °C; Anal. Calcd (C11H15Cl2N5): C, 45.85; H,
5.25; N, 24.30. Found: C, 45.94; H, 5.41; N, 24.51.
4.4. Estimation of agonist potency and efficacy
4.4.1. Cell culture and transfections
Adherent Chinese hamster ovary (CHO) cells (K1 strain) (Amer-
ican Type Culture Collection, Manassas, VA, USA), stably expressing
4.2.21. 2-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5,6-dimethyl-
2H-benzotriazole (8b)
cDNAs encoding the human a2A-adrenoceptor subtype were pro-
duced previously by Pohjanoksa et al. using the expression vector
pMAMneo (Clontech, Palo Alto, CA, USA) that contains a neomycin
analogue G418 (GeneticinÒ, Calbiochem, San Diego, CA, USA) resis-
A white solid (0.17 g, 22%), mp = 167–170 °C; 1H NMR (CDCl3): d
2.40 (s, 6H, CH3), 3.66 (s, 4H, CH2), 4.20 (br s, 1H, NH), 5.57 (s, 2H,
CH2), 7.59 (s, 2H, CH); 13C NMR (CDCl3): d 21.4, 50.5, 54.8, 117.0,
137.9, 144.7, 161.7; mmax 3201, 2949, 2866, 1631, 1553, 1504,
1365, 1273, 1236, 1170, 997, 852 cmꢀ1; MS (ESI) m/z: 230
[M+H]+. Dihydrochloride of 8a; mp = 231–235 °C; Anal. Calcd
(C12H17Cl2N5): C, 47.69; H, 5.67; N, 23.17. Found: C, 47.76; H,
5.83; N, 23.14.
tance gene.35 Cells were cultured in
a-minimum essential medium
(GIBCO™, Invitrogen, Carlsbad, CA, USA) supplemented with
26 mM NaHCO3, 5% heat-inactivated foetal bovine serum, penicil-
lin (50 units/mL), streptomycin (50
Before the functional [35S]GTP
S binding assays, the cultured cells
were tested for their capacity to bind the 2-adrenoceptor
lg/mL) and 200 lg/mL G418.
c
a
antagonist radioligand [3H]RX821002 (2-(2,3-dihydro-2-meth-
oxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imidazole). Thereafter,
confluent cells were harvested into chilled phosphate-buffered
saline, pelleted and frozen at ꢀ70 °C.
4.3. Radioligand binding assays
4.3.1. I1-Binding site assay
Kidneys were obtained post mortem from male Sprague–Dawley
rats (250–280 g) and crude P2 membranes were prepared accord-
ing to the methods of Lione et al.29 Binding of [3H]clonidine
4.4.2. 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 800 rpm). The
homogenate was centrifuged at 23,000 g for 30 min, and the pellet
was re-homogenised and again centrifuged at 23,000 g for 30 min.
The membrane pellet was suspended in hypotonic lysis buffer and
stored at ꢀ70 °C until used. Protein concentrations were deter-
mined with the method of Bradford12 using bovine serum albumin
as reference.
(3 nM, Perkin–Elmer) was investigated in the presence of 10
rauwolscine to preclude radioligand binding to 2-adrenoceptors.
The specific component was defined by 10 M rilmenidine; under
these conditions, the site labelled represents a model of the central
I1 binding site.30 Membrane aliquots (400
L, 0.2–0.5 mg protein)
were incubated with 11 concentrations of the test compounds over
the range 0.01–100 M. Incubations were carried out in 50 mM
lM
a
l
l
l
Tris–HCl buffer (pH 7.4) at room temperature for 45 min. Bound
radioligand and free radioactivity were separated by rapid filtra-
tion through pre-soaked (0.5% polyethyleneimine) glass-fibre fil-
ters (Whatman GFB). Trapped radioligand was determined by
liquid scintillation counting and the data were analysed with
GraphPad Prism version 3.02 for Windows (GraphPad Software,
San Diego, CA, USA) to yield IC50 values (the concentration of tested
ligand that displaces 50% of specifically bound [3H]clonidine).
4.4.3. [35S]GTP
cS binding assay
Agonist-induced stimulation of [35S]GTP
cS binding was mea-
sured essentially as described previously.11 Briefly, membranes
were thawed and diluted with binding buffer (25 mM Tris, 1 mM
EDTA, 5 mM MgCl2, 20 mM NaCl, 1 lM GDP, 1 mM DTT, 30 lM
ascorbic acid, pH 7.4). Incubations were performed on 96-well Mil-
4.3.2. a1-, a2- and I2-Binding assays
lipore MultiScreen MSFBN glass-fibre filter plates (Millipore Corp.,
Brains were obtained post mortem from male Sprague–Dawley
Bedford, MA, USA). Samples containing 5
were incubated with 8–7 serial dilutions of the test compounds
and 0.1 nM [35S]GTP
S. Reactions were terminated after 30 min
incubation at RT by rapid vacuum filtration using a Millipore Mul-
tiScreen Vacuum Manifold. The filter plates were washed three
times with ice-cold wash buffer (20 mM Tris, 1 mM EDTA, 5 mM
lg of membrane protein
rats (250–280 g) and crude P2 membranes were prepared.29 Mem-
brane aliquots (400
11 concentrations of the tested compounds over the range 0.01
nM–100 M in the presence of the selective I2 binding site radioli-
lL, 0.2–0.3 mg protein) were incubated with
c
l
gand [3H]2BFI (2-(2-benzofuranyl)-2-imidazoline)30 (1 nM), the
a
a
1-adrenoceptor antagonist radioligand [3H]prazosin (1 nM) or the
2-adrenoceptor antagonist radioligand [3H]RX821002 (2-(2,3-
MgCl2, pH 7.4). Filters were dried and 50 ll SuperMix scintillation
cocktail was added into each well. The incorporated radioactivity
was measured using a Wallac 1450 Betaplate scintillation counter
(PerkinElmer Wallac, Turku, Finland). All experiments were per-
formed in duplicate and repeated at least three times. Analysis of
the results with GraphPad Prism software yielded estimates of ago-
nist potency (EC50) and efficacy (intrinsic activity in comparison to
the natural full agonist noradrenaline).
dihydro-2-methoxy-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-imid-
azole)31 (1 nM) in a final volume of 500
L. Non-specific binding was
determined using 10 BU224 (2-(4,5-dihydroimidazol-2-
yl)quinoline)32 for I2 binding, 10
M phenylephrine for 1-adreno-
ceptors and 10 M rauwolscine to define 2-adrenoceptor binding.
l
lM
l
a
l
a
Incubations were performed in triplicate at room temperature and
were 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). Filters
were then washed twice with 5 mL of ice-cold buffer and mem-
brane-bound radioactivity remaining on the filters was determined
by liquid scintillation counting. The data were analysed by iterative
non-linearregression curve fittingprocedures with GraphPad Prism.
4.5. In vivo studies: mean arterial blood pressure (MAP) and
heart rate (HR) in rats
Male Wistar rats, weighing 200–290 g, were purchased from the
´
Animal House of the Medical University of Gdansk, Poland. All
in vivo experiments were approved by the Local Ethical Committee

116
J. Saczewski et al. / Bioorg. Med. Chem. 20 (2012) 108–116
on Animal Experiments. The animals were fed a commercial rodent
chow (Labofeed–B, Poland). Tap water was available ad libitum.
Rats were anaesthetized by i.p. injection of thiopental (Sandoz,
Austria) at a dose 70 mg/kg body weight and maintained under
375637), by a Grant from the Jusélius Foundation, Finland, to
M.S., and by a Grant from Natural Sciences and Engineering Re-
search Council of Canada to S.L.
anaesthesia by thiopental supplementation (30 lg/kg/min) during
Supplementary data
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 a rate
of 1.2 mL/h. After 40 min of saline infusion, the tested compound
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.025. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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Acknowledgments
This research was supported in part by the Polish Ministry of
Science and Higher Education, Poland (Grant No.
N N405