1-[(imidazolidin-2-yl)imino]indazole. Highly α2/I 1 selective agonist: Synthesis, X-ray structure, and biological activity

Article abstract of DOI:10.1021/jm800112s

Novel benzazole derivatives bearing a (imidazolidin-2-yl)imino moiety at position 1 or 2 were synthesized by reacting 1-amino- or 2-aminobenzazoles with N,N′-bis(tert-butoxycarbonyl)imidazolidine-2-thione in the presence of HgCl₂. Structures of

Full text of DOI:10.1021/jm800112s

J. Med. Chem. 2008, 51, 3599–3608
3599
1-[(Imidazolidin-2-yl)imino]indazole. Highly r2/I1 Selective Agonist: Synthesis, X-ray Structure,
and Biological Activity
Franciszek Sa¸czewski,*,† Anita Kornicka,*,† Apolonia Rybczyn´ska,‡ Alan L. Hudson,§ Shu Sean Miao,§ Maria Gdaniec,#
Konrad Boblewski,‡ and Artur Lehmann‡
Department of Chemical Technology of Drugs and Department of Pathophysiology, Medical UniVersity of Gdan´sk, 80-416 Gdan´sk, Poland,
Department of Pharmacology, UniVersity of Alberta, Edmonton, T6G 2R3, Canada, and Faculty of Chemistry, A. Mickiewicz UniVersity,
60-780 Poznan´, Poland
ReceiVed February 3, 2008
Novel benzazole derivatives bearing a (imidazolidin-2-yl)imino moiety at position 1 or 2 were synthesized
by reacting 1-amino- or 2-aminobenzazoles with N,N′-bis(tert-butoxycarbonyl)imidazolidine-2-thione in the
presence of HgCl2. Structures of 1-[(imidazolidin-2-yl)imino]indazole (marsanidine, 13a) and free base of
the 4-Cl derivative 12e were confirmed by X-ray single crystal structure analysis. Compound 13a was found
to be the selective R2-adrenoceptor ligand with R2-adrenoceptor/imidazoline I1 receptor selectivity ratio of
3879, while 1-[(imidazolidin-2-yl)imino]-7-methylindazole (13k) proved to be a mixed R2-adrenoceptor/
imidazoline I1 receptor agonist with R2/I1 selectivity ratio of 7.2. Compound 13k when administered
intravenously to male Wistar rats induced a dose-dependent decrease in mean arterial blood pressure (ED50
) 0.6 µg/kg) and heart rate, which was attenuated following pretreatment with R2A-adrenoceptor antagonist
RX821002. Compound 13a may find a variety of medical uses ascribed to R2-adrenoceptor agonists, and its
7-methyl derivative 13k is a good candidate for development as a centrally acting antihypertensive drug.
Introduction
ceptors or imidazoline receptors within the central nervous
system, and a potentiating interaction exists between these two
receptor classes.12 Whether the mechanism of this interaction
involves molecular receptor-receptor interference on the ventral
medullary cardiovascular neurons or an interaction between
receptors located on different neuronal structures but with effects
converging to inhibit the vasomotor tone remains to be
demonstrated. To achieve this goal, however, new compounds
with imidazoline structure and a very high selectivity for R2-
adrenoceptors vis-a`-vis imidazoline I1 receptors will be required.
Currently, a growing interest in the field of developing highly
selective R2-adrenoceptor agonists has been stimulated by their
potential applications as analgesic, sedative, anxiolytic, hemo-
dynamic-stabilizing, and organ-protective agents.13,14 Prominent
examples of such compounds include imidazole-containing
dexmedetomidine (1)15,16 and spirooxazoline derivative S18616
(2).17 Mivazerol (3) is an example of the imidazole derivative
designed for the prevention of myocardial infarction in perio-
perative patients,18,19 while spiroimidazoline S19014 (4) has
been assessed as antimigraine agent.20–22 Other effects of
clonidine-like agents on central noradrenergic transmitter and
modulator functions that are not related to hypertension include
the stimulation of human growth hormone and the decrease of
the output of nearly all secretory glands, such as lacrimal,
salivary, gastric secretory, and sweat glands.23
Recently, in connection with our previous studies on novel
imidazoline compounds24–26 we have turned our attention to
the [(imidazolidin-2-yl)imino]indazoles. As shown in Figure 1,
the previously described 4-substituted indazole derivative 5
(indanidine) exhibited selectivity for R1-adrenoceptors27 while
its 6-substituted analogues of type 6 were claimed in patent
literature to be agonists of R2-adrenoceptors,28 although no data
regarding R2/I1 selectivity ratio were given.
A priori, it was not obvious what effect translocation of the
iminoimidazolidine moiety might have with regard to R-adreno-
ceptor agonism. As such, we were interested in analogues of
type 7 (Figure 1) wherein the iminoimidazolidine moiety had
R2-Adrenoceptors are widely expressed in many tissue types
and mediate a multitude of functions in both peripheral organs
and within the central nervous system, and the R2-adrenoceptor
agonists represent a unique class of compounds because of a
wide range of medicinal uses reported in the literature.1–5 For
example, activation of central R2-adrenoceptors in the brain stem
has been utilized clinically for many years in the treatment of
hypertension.2,3 However, this original explanation has later been
challenged by a concurrent “imidazoline hypothesis” that
assumes the existence of the imidazoline receptors and attributes
the sympathoinhibition to activation of I1 imidazoline receptors
in the medulla oblongata.6–10
Nevertheless, because of some degree of similarity between
the two receptor classes and the fact that most R2-adrenoceptor
agonists, including prototypical clonidine, also have comparable
affinity for I1 imidazoline receptors, it is still difficult to
dissociate imidazoline receptors from R2-adrenoceptors both
pharmacologically and functionally. Moreover, R2-adrenoceptors
and imidazoline receptors usually colocalize in the central
nervous system, which raises a question as to whether a link
between these two types might exist. In this context, a
comprehensive review by Szabo should be mentioned,11 in
which a critical analysis of the above hypotheses led the author
to a conclusion that the sympathoinhibitory effects of clonidine-
like drugs are best explained by activation of R2-adrenoceptors.
On the other hand, Bousquet pointed out that it is possible to
produce a hypotensive effect by activating either R2-adreno-
* To whom correspondence should be addressed. For F.S.: phone, (48-
58)-3493250; fax, (48-58)-3493257; e-mail, saczew@amg.gda.pl. For A.K.:
phone, (48-58)-3493250; fax, (48-58)-3493257; e-mail, a.kornicka@
amg.gda.pl.
†
Department of Chemical Technology of Drugs, Medical University of
Gdan´sk.
‡
Department of Pathophysiology, Medical University of Gdan´sk.
University of Alberta.
A. Mickiewicz University.
§
#
10.1021/jm800112s CCC: $40.75
2008 American Chemical Society
Published on Web 06/03/2008

3600 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Sa¸czewski et al.
Figure 1. Ligands of medicinal interest selective for R1- and R2-adrenoceptors.
Scheme 1. Synthesis of the Aminoindazolesa
reagents. However, treatment of 9 or 10 with 2-chloroimidazo-
line30,31 at mild as well as at elevated temperature and prolonged
reaction time led to the extensive decomposition of starting
material. Because of a poor nucleophilic character of N-
aminoazoles,32 similar difficulty in guanylation was observed
with 2-methylthioimidazoline. Therefore, the alternative route
for preparation of compounds 12a-k and 15a-f was considered
by employing Kim’s guanidine synthesis.33 As shown in Scheme
2, aminoindazoles 9 and 1034 were treated with the preformed
N,N′-bis-Boc-imidazolidine-2-thione35 in the presence of HgCl2
to give bis-Boc-protected guanidines 11a-k and 14a-f in
30-59% and 45-61% yields, respectively. This method fol-
lowed by deprotection with TFA resulted in the desired
1-[(imidazolidin-2-yl)imino]indazoles 12a-k and 2-[(imidazo-
lidin-2-yl)imino]indazoles 15a-f, which upon treatment with
methanolic solution of HCl were converted into the correspond-
ing hydrochlorides 13a-k and 16a-f.
a Reagents and conditions: (a) hydroxylamine-O-sulfonic acid (HOSA)
(2.6 molar equiv), 6% NaOH/H2O, EtOH, 20 min, 55 °C, 1 h, room temp,
then 1 h, 10 °C; (b) CH2Cl2, chromatography on silica gel, 21-60% for
9a-k, 8-30% for 10a-k.
For SAR studies involving variation of the azolyl moiety,
the previously described 1-aminobenzimidazoles 17a,b36,37 (X
) CH) and 1-aminobenzotriazole 17c38 (X ) N) were prepared
by amination of corresponding azoles and, as shown in Scheme
3, subjected to the reaction with N,N′-bis(tert-butoxycarbonyl)-
imidazolidine-2-thione to give Boc-protected 1-(iminoimidazo-
lidine)azoles 18a-c. The free bases 19a-c were obtained upon
cleavage of the protecting group and were in turn used to prepare
the desired hydrochloride salts 20a-c.
been shifted to the 1- or 2-position in order to obtain a new
series of clonidine-like compounds with decreased basicity of
the guanidine grouping and, hopefully, with a new pharmaco-
logical profile. In this work, the importance of the properly
substituted indazole ring for R2-adrenoceptor affinity and
selectivity has been explored. The in vivo biological effects of
these newly prepared positional analogues of 5 and 6 have also
been investigated.
Structures of the final free bases and corresponding hydro-
chloride salts were confirmed by elemental analyses as well as
IR and NMR spectroscopic data (see Experimental Section).
Results and Discussion
Synthesis. Target compounds of type 7 may be considered
as cyclic N,N′,N′′-trisubstituted guanidines. Most methods for
preparation of guanidine derivatives are based on the reaction
of the corresponding amine with electrophilic precursors of the
guanidine moiety.29 Therefore, as depicted in Scheme 1, the
synthesis of our target molecules commenced with the N-
amination of corresponding indazole 8a-k to afford mixtures
of 1-amino- and 2-aminoindazoles 9a-k and 10a-k, which
could be separated by column chromatography.
Crystal Structure Analysis and Molecular Modeling. The
molecular structures of the free bases 12 and hydrochlorides
13 were further confirmed by X-ray crystal structure analysis
of 12e and 13a, which are shown in Figures 2 and 3,
respectively.
The molecule of 12e (Figure 2) consists of two fragments, a
planar indazole ring and a roughly planar imidazolidin-2-imine
group, which form dihedral angle of 80.75(5)°. The torsion
angles along the C-N and N-N bonds connecting these two
fragments are 0.0(3)° and 107.3(2)°. The last value indicates
that there is practically no conjugation between the p orbitals
Then an effort was made to introduce the imidazoline group
under variety of conditions and with a number of guanylating

1-[(Imidazolidin-2-yl)imino]indazole
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3601
Scheme 2. Synthesis of the 1- and 2-[(Imidazolidin-2-yl)imino]indazolesa
a Reagents and conditions: (a) 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, then room temp, 5 days or room temp, 3 days then 6 h, 85 °C for 11k; (b) EtOAc, chromatography on silica gel, then crystallization from
suitable solvent, 30-59% for 11a-k, 45-61% for 14a-f; (c) 50% TFA/CH2Cl2, 2 h, room temp; (d) 10% NaOH/H2O, 5 °C, 50-76% for 12a-k, 51-72%
for 15a-f; (e) HCl/MeOH, 5 °C, then room temp, 30 min, 56-75% for 13a-k, 59-77% for 16a-f.
Scheme 3. Synthesis of the 1-[(Imidazolidin-2-yl)imino]azolesa
Figure 2. X-Ray structure of compound 12e showing atomic labels
and displacement ellipsoids at the 50% probability level. Bond lengths
(Å): C3-N2 1.325(2), N2-N1 1.3594(18), N1-N10 1.4019(17),
N10-C11 1.307(2), C11-N12 1.333(2), C11-N15 1.3480(19). Torsion
angles (deg): N12-C11-N10-N1 0.0(3), C11-N10-N1-N2
107.3(2).
length of 1.402(2) Å agrees well with the standard single bond
length between the sp2-hybridized N atoms (1.401 Å).39 The
indazole N1 atom is slightly pyramidal, being displaced 0.093(2)
Å from the plane defined by its three bonding atoms. The twist
a Reagents and conditions: (a) N,N′-bis-Boc-imidazolidine-2-thione (1.5
around the exocyclic N-N bond, which renders the molecule
molar equiv), HgCl2 (1.5 molar equiv), Et3N (3.5 molar equiv), anhydrous
of 12e chiral in the solid state, determines also distances of the
imidazoline N atoms to the centroid of the indazole benzene
ring (4.41 and 5.64 Å). In compound 13a (Figure 3), which is
a close analogue of 12e, the exocyclic N atom is protonated
and the two N-N bonds become of equal length. Nonetheless,
the degree of pyramidalization of the indazole N1 atom remains
practically unaltered, with N1 displaced 0.115(2) Å from the
DMF, 0 °C, 20 min, then room temp, 5 days; (b) EtOAc, chromatography
on silica gel, then crystallization from suitable solvent, 40-62%; (c) 50%
TFA/CH2Cl2, 2 h, room temp; (d) 10% NaOH/H20, 5 °C, 65-80%; (e)
HCl/MeOH, 5 °C, then room temp, 30 min, 59-77%.
of the sp2 hybridized indazole N1 atom and the exocyclic N
atom of the imidazoline ring. Indeed, the exocyclic N-N bond

3602 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Sa¸czewski et al.
Biological Activity. In Vitro Radioligand Binding Study. The
in vitro assays involved the investigation of the affinity and
selectivity of the newly prepared imidazoline ligands for the
R2-adrenoceptor, imidazoline I1, and imidazoline I2 binding sites,
closely related receptor types. All compounds were used in form
of hydrochloride salts. A summary of all these biological
properties is displayed in Table 1.
In general, all of the tested compounds showed very low or
no affinity for imidazoline I2 receptors. Initial evaluation of the
two isomeric indazole compounds 13a and 16a established that
only 1-substituted isomer 13a possessed significant activity at
R2-adrenoceptors (Ki ) 14.0 nM) and a very high R2/I1
selectivity ratio of 3879 (Table 1). Compound 16c, which can
be regarded as a partially constrained analogue of the well-
known antihypertensive drug guanabenz, showed very weak
affinity for R2-adrenoceptors (Ki ) 3136 nM) and extremely
low activity at imidazoline I1 receptors (Ki ) 47 840 nM). The
poor activity of the 1-substituted benzimidazoles 20a,b and
1-substituted benzotriazole analogue 20c was also surprising
because the active indazole 13a possessed the same substitution
pattern.
Further SAR studies suggested that the R2-adrenoceptor
affinity of the 1-substituted indazole derivatives 13 was strongly
influenced by the nature and position of substituent, and
therefore, our work focused on the modification of the substitu-
tion pattern. First, placement of either methyl or phenyl group
at position 3 was not tolerated, as evidenced by considerable
decrease in affinity for R2-adrenoceptors ranging from 1838-
to 5058-fold for compounds 13c and 13b, respectively. Trans-
location of the methyl group from position 3 to 4, 5, or 6 led to
active compounds 13d, 13g, and 13i (Ki of 37.1, 44.2, and 49.1
nM, respectively). However, corresponding 4-Cl and 5-Cl-
substituted analogues 13e and 13h were 2- to 3-fold less active
(Ki of 61.6 and 114.2 nM, respectively) and compound 13f
bearing electron-donating OMe group at position 4 proved to
be inactive at R2-adrenoceptors (Ki ) 69.63 µM).
Interestingly, within a homologous series of Me-substituted
derivatives, the 7-Me analogue 13k retained activity at R2-
adrenoceptors (Ki ) 53.5 nM), whereas its affinity for imida-
zoline I1 receptor increased by about 2 orders of magnitude (Ki
) 387 nM, R2/I1 selectivity ratio ) 7.2). The graphical
representations (Figures 6 and 7) illustrate the differences in
selective profiles of these compounds.
Summing up, the above results suggest that binding of the
indazole derivatives 13 may occur at multiple classes of receptor
sites, depending on the substitution pattern. Compound 13a
shows highest affinity to rat brain R2-adrenoceptors and a very
high R2/I1 selectivity ratio. On the other hand, 7-Me-analogue
13k is characterized by good affinity to R2-adrenoceptors and
moderate affinity to imidazoline I1 receptors, and therefore, it
bears resemblance to clonidine-like “hybrid drugs”.
Figure 3. X-Ray structure of compound 13a showing atomic labels
and displacement ellipsoids at the 50% probability level. Bond lengths
(Å): C3-N2 1.3159(19), N2-N1 1.3739(16), N1-N10 1.3758(16),
N10-C11 1.3365(18), C11-N12 1.3184(17), C11-N15 1.3241(18).
Torsion angles (deg): N12-C11-N10-N1 -3.5(2), C11-N10-N1-N2
-72.51(17).
plane of the three bonding atoms. A strong twist of the
imidazolidin-2-iminium fragment relative to the indazole ring
[dihedral angle of 65.18(4)°] also makes this molecule chiral;
however, an opposite direction of the twist compared with 12e
increases the distances of the imidazoline N atoms from the
centroid of the indazole benzene ring (5.07 and 5.95 Å).
The series of 1-[(imidazolidin-2-yl)imino]indazoles 12 share
structural similarities with the antihypertensive clonidine-like
drugs,23 which in solution exists in the imino form. We have
examined tautomers of 12a by quantum chemical calculations
using density functional (B3LYP/6.31G**) calculations.40 As
shown in Figure 4, the imino-imidazolidine tautomer A was
calculated to be substantially lower in energy than the amino-
imidazoline tautomer B (∆E ) 11.05 kcal/mol). Moreover, on
the basis of their calculated dipole moments, tautomer A (3.34
D) would be predicted to predominate over B (1.99 D) in polar
solvents.
As shown by conformational analysis and crystallographic
data, 12 can adopt a variety of conformations that involve a
relatively small energy expense. Thus, puckered conformation
required for interaction with R2-adrenoceptors23 can be achieved
at an energy expense of about 7 kcal/mol by rotation around
N1-N10 single bond. In order to compare the global energy
minimum planar geometry of 12a with those of clonidine, the
electrostatic potential was generated. Comparison between 3D
electrostatic potential maps40 of 12a and clonidine (Figure 5)
shows that superimposable negative wells appear around the
exocyclic nitrogen atoms. However, separated electrostatic
regions are positioned around the phenyl moiety of indazole
ring of 12a.
Previously established model for central antihypertensive
agents also demonstrated the importance of decreased basicity
of the guanidine moiety by introduction of electronegative
substituents into the aromatic ring.23 We expected that unlike
other arylimino-imidazolidines, which are ionized at physi-
ological pH (pKa of clonidine of 8.2; 14% of un-ionized form
at pH 7.4), the corresponding 1-[(imidazolidin)imino]indazoles
13 would have much lower basicity (pKa < 8) because of the
electron withdrawing effect of the azaaromatic ring. Indeed, at
physiological pH it is very likely that agonists 13a and 13k
with pKa values of 6.32 and 6.53, respectively (Table 1), exist
primarily as the neutral free bases (92% and 88%, respectively).
This would likely facilitate their ability to cross the blood-brain
barrier.
In Vivo Cardiovascular Activities. The cardiovascular
properties of 13a and its selected derivatives were evaluated
after intravenous infusion in anaesthetized rats using procedure
previously described.41 Changes in mean arterial blood pressure
and heart rate were measured directly. These measurements were
compared to the baseline values and are presented as ∆MAP
and ∆HR in Table 2.
In general, most of the tested compounds showed a biphasic
effect on blood pressure: transient hypertension followed by
prolonged hypotension effect. However, no clear correlation was
found between in vivo potencies and affinities of the compounds
studied to R2-adrenoceptors. For example, cardiovascular evalu-
ation of 13a showed that this compound exerts significant effect

1-[(Imidazolidin-2-yl)imino]indazole
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3603
Figure 4. Molecular diagram of 1-[(imidazolidin-2-yl)imino]indazole (12a): N ) natural charges; M ) charges derived from Mulliken population
analysis; E ) charges derived from electrostatic potential.
stimulation of R2A-adrenoceptors.46 As shown in Figure 10,
imidazoline-containing R2A-antagonist RX821002 at 5 µg/kg,
administered iv 5 min prior to 13k (administered at dose 10
µg/kg iv), attenuated hypotensive action of 13k, and at dose 10
µg/kg it was even more effective at reversing 13k-induced
hypotensive effect.
The data presented above support the hypothesis according
to which an interaction between imidazoline I1 receptors and
R2-adrenoceptors might explain the mechanism of the hypoten-
sive effects of clonidine-like “hybrid drugs” binding to both
R2-adrenoceptors and imidazoline I1 receptors.47,48
In summary, we have shown that methyl substituent at
position 7 has a significant role in determining overall cardio-
Figure 5. Comparison of the electrostatic potential maps of marsani-
dine free base (12a) (right) and clonidine free base (left) isocontoured
vascular profile of the investigated indazole derivatives 13.
Compound 13k with affinities for both R2-adrenoceptors and
imidazoline I1 receptors confers the most potent hypotensive
and negative chronotropic properties. However, whether the
long-lasting hypotension of these compounds is likely to be
attributable to their central sympathetic depression rather than
peripheral vascular effects11 remains to be established.
at -20 kcal/mol.
on blood pressure at the dose of 0.1 mg/kg (∆MAP ) -30.9
mmHg) and heart rate (∆HR ) -49.0 bpm). Similar or slightly
lower hypotensive activity was observed for 4- and 6-methylated
derivatives 13d and 13i (∆MAP ) -29.0 and -23.4 mmHg,
respectively). On the other hand, 5-Me congener 13g, which
was equally active at R2-adrenoceptors, exhibited a very weak
cardiovascular activity (∆MAP ) -4.9 mmHg). The most
striking discrepancy was observed for the 7-Me derivative 13k,
which exhibited similar affinity for R2-adrenoceptors and much
higher hypotensive effect when administered intravenously at
the same dose (∆MAP ) -43.5 mmHg, ED50 ) 0.6 µg/kg,
iv).
As shown in Figure 8, changes of ∆MAP induced by 13a,
13k, and standard drug moxonidine were highly significant in
comparison to control group and reached p < 0.01, p < 0.001,
and p < 0.001, respectively. Similarly, 13k and moxonidine
injections induced immediate decrease in ∆HR, reaching high
significance: p < 0.001 and p < 0.01 in comparison to control
and 13a groups, respectively (Figure 9). Nevertheless, 13a
administration resulted in weaker but also significant (p < 0.05)
decrease of ∆HR in comparison to control animals.
Conclusions
From the above-discussed results one can draw the following
conclusions: (i) placement of iminoimidazolidine moiety at the
N1 nitrogen atom of an azaaromatic ring, such as indazole,
benzimidazole, and benzotriazole, decreases basicity of formal
guanidine grouping, which is a prerequisite for hypotensive
properties of clonidine-like drugs; (ii) 1-[(imidazolidin-2-
yl)imino]indazoles (13) exhibit structure-dependent affinities for
R2-adrenoceptors and R2/I1 selectivity ratio. The most active at
R2-adrenoceptors unsubstituted indazole derivative 13a (Ki )
14 nM) shows a very high R2/I1 selectivity ratio of 3879 vs
dexmedetomidine’s ratio of 32 (i.e., over 100-fold difference)
and therefore may serve as a pharmacological tool and find a
variety of medical uses, especially as a more cardiostable R2
agonist for organoprotection and anesthesia; (iii) 7-Me analogue
It was previously found42,43 that the selective R2-adrenoceptor
antagonists could reverse the central hypotensive effects of
highly selective R2-adrenoceptor agonists or prevent hypotension
elicited by clonidine-like drugs binding to R2-adrenoceptors and
to imidazoline I1 receptors. Therefore, we have examined the
interaction of 13k with RX821002,a a selective R2A-adrenoceptor
antagonist.44,45 This compound was chosen because of its
imidazoline structure and previous finding that hypotensive
effects of clonidine-like drugs may be mediated through
13k, the most potent hypotensive agent in this series (ED50
)
0.6 µg/kg), is characterized by good affinity for R2-adrenoceptors
(Ki ) 53 nM) and moderate affinity for imidazoline I1 receptors
(Ki ) 387 nM). Compound 13k may therefore serve as a lead
structure for further development of novel antihypertensive
drugs.
To explain the differences observed between in vitro binding
affinities and in vivo circulatory activities of 13, further studies
on their pharmacokinetics are needed. Binding affinities of 13a
for human R1-adrenoceptor as well as R2-adrenoceptor subtypes
a Abbreviations: Tris, 2-amino-2-(hydroxymethyl)propane-1,3-diol;
RX821002, 2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; 2BFI, 2-(2-
benzofuranyl)-2-imidazoline; BU224, 2-(4,5-dihydroimidazol-2-yl)quinoline.

3604 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Sa¸czewski et al.
Table 1. Binding Affinities, R2/I1 Selectivity, and pKa Data for Compounds 13a-k, 16a-f, and 20a-c
c
compd
R2 Ki (nM)a
I1 IC50 (nM)b
I2 Ki (nM)a
R2/I1 selectivity ratio
pKa
13a
13b
13c
13d
13e
13f
13g
13h
13i
14.05 ( 2.7
54500.0 ( 16730
Nd
16900.0 ( 5900
32610.0 ( 8130
684.0 ( 74.3
3879.0
6.32
71070.0 ( 5080
25830.0 ( 4508
37.1 ( 9.8
76510.0 ( 29880
38530.0 ( 7930
31460.0 ( 2348
13500.0 ( 2880
20310.0 ( 7840
16980.0 ( 3670
Nd
2.96
1038.54
510.71
0.19
459.50
148.69
10370.0 ( 2860
11080.0 ( 3930
5984.0 ( (n ) 1)
7159.0 ( 1277
20620.0 ( 1393
19170.0 ( 1730
33640.0 ( 6330
2520.0 ( 593
77826.7 (n ) 2)
35110.0 ( 14190
13140.0 ( 2990
11440.0 ( 2830
33200.0 (n ) 1)
42880.0 ( 11830
>10000
6.20
5.77
6.03
6.30
5.88
61.6 ( 22.6
69630.0 ( 15570
44.2 ( 8.3
114.2 ( 39.4
49.1 ( 7.8
96.2 ( 28.4
53.5 ( 18.4
78343.3 ( 21851
11850.0 ( 19320
3136.0 ( 793
45620.0 ( 18670
74090.0 ( 25069
50680.0 ( 21188
6016.3 ( 895
10072.7 ( 13930
>10000
13j
Nd
6.21
6.53
13k
16a
16b
16c
16d
16e
16f
20a
20b
20c
387.0 ( 97.3
Nd
Nd
47840.0 ( 8210
13420.0 ( 5567
24670.0 (n ) 1)
19730.0 (n ) 1)
>100000
7.23
15.26
0.29
0.33
0.39
>16.62
Nd
84633.4
>10000
>10000
<8.46
a Ki affinity values for R2-adrenoreceptors and I2 imidazoline binding sites were assessed by measuring the ability of test compounds to displace [3H]RX821002
and rauwolscine (rat brain membranes) or [3H]2BFI and BU224 (rat brain membranes), respectively. b Molar concentration of test compounds that displaces
50% of specifically bound [3H]clonidine. Nd: not determined. c pKa values were determined at 25 °C by potentiometric titration with TiNet 2.5 software.
Indazole (8a),49 3-methylindazole (8b),34 3-phenylindazole
(8c),34 4-methylindazole (8d),49 4-chloroindazole (8e),49 4-meth-
oxyindazole (8f),50 5-methylindazole (8g),49 5-chloroindazole
(8h),49 6-methylindazole (8i),49 6-methoxyindazole (8j),50 7-me-
thylindazole (8k),49 1-aminoindazole (9a),34 1-amino-3-methylin-
dazole (9b),34 1-amino-3-phenylindazole (9c),34 2-aminoindazole
(10a),34 2-amino-3-methylindazole (10b),34 2-amino-3-phenylin-
dazole (10c),34 N,N′-bis(tert-butoxycarbonyl)imidazolidine-2-
thione,35 1-aminobenzimidazole (17a),36 1-amino-5,6-dimethyl-
benzimidazole (17b),37 and 1-aminobenzotriazole (17c)38 were
obtained as previously reported.
General Procedure for the Preparation of 1-[(Imidazolidin-
2-yl)imino]indazoles 12a-k and Their Hydrochlorides 13a-
k. Step 1. To a stirred solution of the appropriate 1-aminoindazole
Figure 6. Competition binding curves of marsanidine (13a) on R2-
9a-k (2.5 mmol), N,N′-bis(tert-butoxycarbonyl)imidazolidine-2-
thione35 (1.12 g, 3.7 mmol), and Et3N (0.88 g, 1.21 mL, 8.7 mmol)
adrenoceptors, imidazoline I1, and imidazoline I2 receptors.
in anhydrous DMF (4 mL) was added HgCl2 (1.0 g, 3.7 mmol) at
0 °C. The reaction mixture was stirred for an additional 20 min at
0 °C and then at room temperature for 5 days or for 3 days followed
by heating at 85 °C for 6 h (in the case of 9k). The resulting dark-
gray reaction mixture was diluted with EtOAc (40 mL), filtered
off, and washed with EtOAc (3 × 10 mL). The filtrates were washed
successively with brine (3 × 20 mL) and water (3 × 20 mL), dried
over MgSO4, and finally concentrated under vacuum. The viscous
residue (a mixture of unreacted N,N′-bis-Boc-imidazolidine-2-thione
and Boc-protected 2-iminoimidazolidines 11a-k) thus obtained was
separated by flash column chromatography on silica gel. N,N′-Bis-
Boc-imidazolidine-2-thione was eluted first and isolated in 17-25%
yields.
In this manner the following compounds were obtained.
Figure 7. Competition binding curves of 7-Me-marsanidine (13k) on
1-{[1,3-Di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}indazole (11a).
11a was obtained from 1-aminoindazole (9a,34 0.33 g) and EtOAc/
CHCl3 (0.1:10) as eluent: yield 0.58 g (59%); mp 128-130 °C
(n-heptane); 1H NMR (500 MHz, CDCl3) δ 1.03 (s, 9H, CH3), 1.6
(s, 9H. CH3) 3.9 (t, 2H, CH2), 4.03 (t, 2H, CH2), 7.16 (m, 1H),
7,38 (m, 1H) 7.66 (m, 2H) 7.9 (s, 1H). Anal. (C20H27N5O4 (401.46))
C, H, N.
R2-adrenoceptors, imidazoline I1, and imidazoline I2 receptors.
R
2A, R2B, and R2C are also planned, and the results of these
studies will be described elsewhere.
Experimental Section (See Also Supporting Information)
Chemistry. Melting points were measured on a Bu¨chi 535
apparatus and are not corrected. IR spectra were taken in KBr pellets
on a Perkin-Elmer FTIR 1600 spectrometer. NMR spectra were
recorded on a Varian Gemini 200 or 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 and 39.5 ppm (DMSO-
d6). Chromatography was performed on silica gel 60 (230-400
mesh ASTM, Fluka) using the reported solvent systems. Analysis
results of C, H, N were within (0.4% of the theoretical values.
1-{[1,3-Di(tert-butoxycarbonyl)imidazolidin-2-yl]imino}-7-meth-
ylindazole (11k). 11k was obtained from 1-aminoindazole 9k (0.37
g) and EtOAc/CHCl3 (0.1:3) as eluent: yield 0.31 g (30%); mp
1
160-162 °C (n-heptane); H NMR (500 MHz, CDCl3) δ 1.02 (s,
9H, CH3), 1.57 (s, 9H. CH3), 2.84 (s, 3H, CH3), 3.9 (t, 2H, CH2),
4.06 (t, 2H, CH2), 7.04 (m, 1H), 7,14 (m, 1H), 7.49 (m, 1H), 7.89
(s, 1H). Anal. (C21H29N5O4 (415.49)) C, H, N.
Step 2. A solution of the appropriate Boc-protected 2-iminoimi-
dazolidine 11a-k (2.0 mmol) from step 1 in 50% trifluoroacetic

1-[(Imidazolidin-2-yl)imino]indazole
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3605
Table 2. Effects of Compounds 13a,c-k at 0.1 mg/kg iv on Mean Arterial Blood Pressure (MAP) and Heart Rate (HR) in Anesthetized Rats
time after application of tested compounds (min),
∆MAP (mmHg)a and ∆HR (bpm)a,b
compd
10
30
60
90
nc
13a
13c
13d
13e
13f
13g
13h
13i
-6.9 ( 4.4 (-48.5 ( 23.5)
-4.1 ( 0.9 (-17.7 ( 3.5)
-17.7 ( 4.2 (-104.2 ( 10.5)
-10.9 ( 7.4 (-60.8 ( 18.0)
-3.7 ( 1.8 (-18.6 ( 5.4)
-4.1 ( 1.7 (-104.2 ( 10.5)
-0.2 ( 0.7 (0.6 ( 1.2)
-13.2 ( 7.2 (-71.8 ( 14.9)
-16.5 ( 5.7 (-33.0 ( 13.9)
-14.8 ( 6.9 (-110.0 ( 16.7)
-1.2 ( 0.8 (-4.9 ( 1.7)
-30.9 ( 4.7 (-49.0 ( 26.5)
-7.6 ( 1.9 (-10.1 ( 4.9)
-29.0 ( 4.3 (-88.3 ( 11.4)
-14.0 ( 4.1 (-32.2 ( 15.5)
-7.6 ( 1.7 (-9.7 ( 3.9)
-3.6 ( 2.3 (-3.2 ( 4.5)
-1.4 ( 1.4 (7.2 ( 3.4)
-23.4 ( 0.9 (-43.5 ( 14.8)
-17.1 ( 5.4 (-23.6 ( 12.1)
-40.6 ( 4.2 (-115.0 ( 12.6)
-1.3 ( 2.0 (-8.6 ( 2.7)
-29.0 ( 9.3 (-31.4 ( 11.1)
-10.0 ( 2.4 (-11.1 ( 8.1)
-14.1 ( 3.2 (-22.7 ( 17.2)
-12.3 ( 2.7 (-24.3 ( 11.4)
-11.0 ( 2.5 (-12.5 ( 6.4)
-4.6 ( 3.0 (-1.0 ( 9.5)
-2.8 ( 1.4 (12.8 ( 7.8)
-20.8 ( 2.6 (-26.4 ( 11.0)
-11.6 ( 4.6 (-4.1 ( 16.0)
-43.5 ( 4.6 (-122.9 ( 12.6)
-3.5 ( 1.8 (-6.2 ( 6.3)
-22.1 ( 6.7 (-9.7 ( 8.6)
-11.4 ( 3.0 (-12.7 ( 10.2)
-10.1 ( 1.6 (-2.1 ( 12.1)
-10.9 ( 3.8 (-19.0 ( 7.9)
-12.9 ( 2.0 (-11.6 ( 8.5)
-4.9 ( 3.6 (-3.0 ( 10.1)
-1.1 ( 1.8 (28.6 ( 4.9)
-11.0 ( 3.6 (-22.2 ( 6.0)
-5.3 ( 5.7 (14.3 ( 15.3)
-43.2 ( 4.1 (-119.9 ( 13.5)
-9.1 ( 2.9 (-19.8 ( 11.9)
4
5
4
5
5
5
4
4
4
7
5
13j
13k
control
a Values are the mean ( SE. b Values in parentheses. c Number of experiments.
Figure 10. Effect of 10 µg/kg of 13k in the absence and presence of
5 (RX5) and 10 (RX10) µg/kg of RX 821002 on ∆MAP (calculated as
the difference of MAP between sequential measurement and time 0 of
experiment) in rats. Each point represents mean value of ∆MAP for
four to seven experiments. Comparisons were made using ANOVA
with repeated measures and Fisher and Duncan tests. Significance (&)
p < 0.001 was found for comparisons of the following: 13k versus
control, 13k + RX5 (5 µg/kg of RX 821002), and 13k + RX10 (10
µg/kg of RX 821002); control versus 13k + RX5 (5 µg/kg of RX
821002). Significance (#) p < 0.01 was found for comparison of 13k
+ RX10 (10 µg/kg of RX 821002) and control.
Figure 8. Effect of 0.1 mg/kg of 13a, 13k, and moxonidine on ∆MAP
(calculated as the difference of MAP between sequential measurement
and time 0 of experiment) in rats. Each point represents the mean value
of ∆MAP for four to seven experiments. Comparisons were made using
ANOVA with repeated measures and Fisher and Duncan tests.
Significances (&) p < 0.001, (&) p < 0.001, and (#) p < 0.01 were
found for comparisons of 13k, moxonidine, and 13a versus control
group, respectively.
The precipitate thus obtained was filtered off and purified by
crystallization from suitable solvent.
In this manner the following compounds were obtained.
1-[(Imidazolidin-2-yl)imino]indazole (12a). 12a was obtained
from 11a (0.80 g): yield 0.24 g (60%); mp 172-174 °C (2-
1
propanol); H NMR (500 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.69
(d, J ) 7.8 Hz, 1H), 7.34 (d, J ) 8.3 Hz, 1H), 7.27 (t, 1H), 7.05
(t, 1H), 6.65 (s, 1H), 6.47 (s, 1H), 3.61-3.41 (m, 4H). Anal.
(C10H11N5 (201.23)) C, H, N.
1-[(Imidazolidin-2-yl)imino]-7-methylindazole (12k). 12k was
obtained from 11k (0.83 g): yield 0.30 g (70%); mp 201-204 °C
(MeCN); 1H NMR (500 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.47 (d,
J ) 7.8 Hz, 1H), 6.96-6.88 (m, 2H), 6.62 (s, 1H), 6.20 (s, 1H),
3.40-3.36 (m, 4H), 2.61 (s, 3H). Anal. (C11H13N5 (215.25)) C, H,
Figure 9. Effect of 0.1 mg/kg of 13a, 13k, and moxonidine on ∆HR
(calculated as the difference of HR between sequential measurement
N.
and time 0 of experiment) in rats. Each point represents mean value of
Step 3. To an ice-cold suspension of the appropriate 2-iminoimi-
dazolidine 12a-k (1.0 mmol) from step 2 in anhydrous methanol
(7 mL) was added dropwise HCl/MeOH solution (d ) 5.67 g/100
mL, 0.78 mL, 1.2 mmol). The cooling bath was removed, and the
resulting solution was stirred at room temperature for 30 min. Then
the solvent was evaporated under reduced pressure to dryness. The
crude product thus obtained was purified by crystallization from
suitable solvent.
In this manner the following compounds were obtained.
1-[(Imidazolidin-2-yl)imino]indazole Hydrochloride (13a). 13a
was obtained from 2-iminoimidazolidine 12a (0.20 g): yield 0.18 g
(75%); mp 195-196 °C (EtOH/Et2O); 1H NMR (200 MHz, DMSO-
∆HR for four to seven experiments. Comparisons were made using
ANOVA with repeated measures and Fisher and Duncan tests.
Significances (&) p < 0.001, ( &) p < 0.001, and (/) p < 0.05 were
found for comparisons of 13k, moxonidine, and 13a versus control
group, respectively. Significance (#) p < 0.01 was found for compari-
sons of 13a versus 13k and moxonidine groups.
acid in CH2Cl2 (8 mL) was stirred at room temperature for 2 h,
and then the solvent and excess of trifluoroacetic acid were
evaporated under reduced pressure. The viscous residue was treated
with water (7 mL), and the resulting mixture or solution was made
alkaline (pH 10-10.5) with 10% aqueous NaOH solution at 5 °C.

3606 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Sa¸czewski et al.
d6) δ 12.59 (bs, 1H), 8.97 (s, 2H), 8.25 (s, 1H), 7.86 (d, J ) 8.0
Hz, 1H), 7.54 (d, J ) 4.0 Hz, 2H), 7.33-7.25 (m, 1H), 3.72 (s,
4H). Anal. (C10H12ClN5 (237.69)) C, H, N.
1-[(Imidazolidin-2-yl)imino]-7-methylindazole Hydrochloride
(13k). 13k was obtained from 2-iminoimidazolidine 12k (0.21 g):
yield 0.15 g (63%); mp 189-190 °C (EtOH/Et2O); 1H NMR (200
MHz, DMSO-d6) δ 12.78 (bs, 1H), 8.98 (s, 2H), 8.21 (s, 1H), 7.67
(d, J ) 7.7 Hz, 1H), 7.29-7.12 (m, 2H), 3.74 (s, 4H), 2.55 (s,
3H). Anal. (C11H14ClN5 (251.71)) C, H, N.
X-ray Crystallography of 12e and 13a. The intensity data of
the crystals were collected using a KumaCCD diffractometer. The
crystals structures were solved with SHELXS-9751 and refined with
SHELXL-97.52 Crystallographic data for the structures 12e and 13a
are available in Supporting Information.
The animals were fed a commercial rodent chow (Labofeed-B,
Poland) and tap water, available ad libitum. Rats were anesthetized
by ip injection of thiopental (Sandoz, Austria) at a dose 70 mg/kg
body weight and maintained under anesthesia by thiopental
supplementation (30 (µg/kg)/min) during the experiment. The
animals were placed on a heated table, and body temperature was
maintained between 36 and 37 °C. Tracheostomy was performed
in all experimental groups. Catheters were inserted into the carotid
artery for pressure and heart rate monitoring, into a jugular vein
for infusions, and into the bladder for free diuresis. Blood pressure
and heart rate were constantly monitored to the end of experiment.
After all surgical procedures, a 40 min recovery period was
allowed to establish steady state. During the whole experiment, rats
were infused with isotonic saline (Fresenius Kabi, Poland) supple-
mented with thiopental at a rate of 1.2 mL/h.
Pharmacology. Radioligand Binding Assays. I1-Binding Site
Assay.Kidneyswereobtainedpost-mortemfrommaleSprague-Dawley
rats (250-280 g) and crude P2 membranes prepared according to
methods of Lione et al.53 [3H]Clonidine (3 nM, Perkin-Elmer) was
bound in the presence of 10 µM rauwolscine to preclude binding
to R2-adrenoceptors. The specific component was defined by 10
µM rilmenidine. Under these conditions the site labeled is a model
of the central I1 binding site.54 Membrane aliquots (400 µL, 0.2-0.5
mg of protein) were incubated with 11 concentrations of the test
compound over the range 0.01 µM to 100 mM. Incubations were
carried out in 50 mM Tris-HCl buffer (pH 7.4) at room temperature
for 45 min. Bound ligand and free radioactivity were separated by
rapid filtration through presoaked (0.5% polyethylamine) glass-fiber
filters (Whatman GFB). Trapped ligand was determined by liquid
scintillation counting, and data were analyzed by GraphPad Prism,
version 3.02, for Windows (GraphPad Software, San Diego, CA)
to yield IC50 values (the concentration of drug that displaces 50%
of specifically bound [3H]clonidine).
r2- and I2-Binding Site Assays. Crude P2 brain membranes were
prepared as follows. All procedures were carried out at 4 °C unless
otherwise stated. Rat brains (male Sprague-Dawley rats, 250-280
g) were taken and homogenized in 10 volumes of ice cold buffer
(50 mM Tris-HCl, 1 mM MgCl2, and 320 mM sucrose, pH 7.4).
The homogenate was centrifuged (1000g for 10 min) and the
precipitate discarded. The supernatant was centrifuged a second
time (32000g for 20 min) and the supernatant discarded, with the
remaining precipitate making up the crude P2 membrane prepara-
tion. This was washed twice in excess buffer (50 mM Tris-HCl, 1
mM MgCl2) at room temperature. An amount of 30 mL was added,
and the precipitate was resuspended and centrifuged (32000g for
20 min). The washed membrane preparations were stored at -70
°C until use. Prior to use they were thawed and washed (as above)
an additional two times. Membrane aliquots (400 µL, 0.2-0.3 mg
of protein) were incubated with 11 concentrations of the test
compound over the range 0.01 nM to 100 µM in the presence of
the selective I2 binding site ligand [3H]2BFI55 (1 nM) or the R2-
adrenoceptor antagonist [3H]RX82100244 (1 nM), to final volume
of 500 µL. Nonspecific binding was determined using 10 µM
BU224,56 I2 binding and 10 µM rauwolscine, R2-adrenoceptor
binding. Each incubation was performed in triplicate at room
temperature and allowed to reach equilibrium (45 min). Bound
radioactivity and free radioactivity were separated by rapid filtration
through presoaked (0.5% polyethyleimine) glass-fiber filters (What-
man GF/B). Filters were then washed twice with 5 mL of ice-cold
buffer, and membrane bound radioactivity remaining on the filters
was determined by liquid scintillation counting. Data were analyzed
by iterative nonlinear regression curve fitting procedures in Graph-
Pad Prism, version 3.02, for Windows (GraphPad Software, San
Diego, CA). Each experiment was analyzed individually and the
equilibrium dissociation constant (Ki) determined by the method
of Cheng and Prusoff,57 and the resulting values are given as the
mean ( SEM for three to four separate experiments.
After 40 min of saline infusion, the tested compound was
administrated as 100 µL bolus through venous catheter at a dose
of 0.1 mg/kg. The antagonist of R2-adrenoceptors (RX821002) was
given iv at a dose of 5 or 10 µg/kg 5 min before the tested
compound 13k.
Arterial blood pressure and heart rate were monitored directly
and sampled continuously at 100 Hz, as we described previously,41
using Biopac Systems, Inc., model MP 100 (Goleta, CA). The
results of measurements were elaborated with the help of the
ACQKnowledge (Goleta, CA) measurement system that is selected,
scaled, and filtered to remove accidental signal disturbances. The
recorded time domain transient data have been presented as a graphs
with the help of Excel (Microsoft).
Statistical ANOVAs of mean arterial blood pressure (MAP) and
heart rate (HR) were performed for ∆MAP and ∆HR, calculated
as the difference in MAP and in HR between sequential measure-
ments and the time of compound application (“time 0”) for each
group, as we described previously.41 This allowed for direct
comparison of responses to treatment between groups when
baselines differed. Data were analyzed by ANOVA with repeated
measures, using Statistica StatSoft software (StatSoft, Inc., Tulsa,
OK), after test compounds or vehicle treatment. When the effect
was significant, post hoc comparisons were performed using Duncan
and Fisher tests. A value of p < 0.05 was considered statistically
significant.
Acknowledgment. This research was supported in part by
the Polish Ministry of Science and Higher Education, Poland
(Grant N40500532/0458).
Supporting Information Available: General procedures for the
preparation of compounds 9d-k, 10d-k, 12a-k, 13a-k, 15a-f,
16a-f, 19a-c, and 20a-c; elemental analysis results of reported
compounds; and crystallographic data of compounds 12e and 13a.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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