New 2-Aminothiazoline derivatives lower blood pressure of spontaneously hypertensive rats (SHR) via I1-imidazoline and alpha-2 adrenergic receptors activation

Article abstract of DOI:10.1016/j.ejphar.2016.10.009

2-Aminothiazolines share an isosteric relationship with imidazolines and oxazolines with antihypertensive activity mainly mediated by the imidazoline I₁-receptor. In the present work, we have prepared five aminothiazolines, following a previously described synthetic pathway. Aminothiazolines derived from dicyclopropylmethylamine (ATZ1) and cyclohexylamine (3) are unprecedented in the literature. Competitive radioligand assay was carried out with all synthetic compounds, and the I₁receptor affinity in comparison to rilmenidine in PC12 cells was determined. Surprisingly, the rilmenidine isoster (ATZ1) showed no I₁-receptor interaction. Diethyl (ATZ4) and 2-ethyl-hexylamine (ATZ5) derivatives bind to the receptor with 11.98 and 10.94 nmol/l, respectively. These compounds were selected for in vivo experiments. Both compounds reduced the blood pressure of spontaneously hypertensive rats (SHR). The hypotensive effect of these compounds was abrogated in the presence of α₂adrenergic (yohimbine) and I₁(efaroxan) receptor antagonists suggesting that both aminothiazolines bind to the adrenergic and imidazoline receptors. Lipinski's descriptors of the synthesized aminothiazolines were calculated and are similar to the known imidazoline I₁receptor ligands. 3D-Similarity between ATZ5 and agmatine, the natural imidazoline receptor ligand, was also observed.

Full text of DOI:10.1016/j.ejphar.2016.10.009

Author’s Accepted Manuscript
New 2-Aminothiazoline derivatives lower blood
pressure of spontaneously hypertensive rats (SHR)
via I -imidazoline and alpha−2 adrenergic
1
receptors activation
Renan B. Ferreira, Mariana G. de Oliveira, Edson
Antunes, Wanda P. Almeida, Badr M. Ibrahim,
Abdel A. Abdel-Rahman
www.elsevier.com/locate/ejphar
PII:
S0014-2999(16)30650-1
http://dx.doi.org/10.1016/j.ejphar.2016.10.009
EJP70875
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 27 June 2016
Revised date: 6 October 2016
Accepted date: 7 October 2016
Cite this article as: Renan B. Ferreira, Mariana G. de Oliveira, Edson Antunes
Wanda P. Almeida, Badr M. Ibrahim and Abdel A. Abdel-Rahman, New 2
Aminothiazoline derivatives lower blood pressure of spontaneously hypertensive
rats (SHR) via I -imidazoline and alpha−2 adrenergic receptors activation
1
European
Journal
of
Pharmacology
http://dx.doi.org/10.1016/j.ejphar.2016.10.009
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1
New 2-Aminothiazoline derivatives lower blood pressure of
spontaneously hypertensive rats (SHR) via I1-imidazoline and alpha-2
adrenergic receptors activation.
Renan B. Ferreiraa,1, Mariana G. de Oliveirab, Edson Antunesb, Wanda P. Almeidac,2
Badr M. Ibrahimd, Abdel A. Abdel-Rahmand
,
*
aInstitute of Chemistry, University of Campinas, Campinas, SP, Brazil. PO Box 6194,
ZC 13083-970
bDepartment of Pharmacology, Faculty of Medical Sciences, University of Campinas,
Campinas, SP, Brazil.
cFaculty of Pharmaceutical Sciences, University of Campinas, Campinas, SP, Brazil.
PO Box 6029, ZC 13083-859
dDepartment of Pharmacology and Toxicology, The Brody School of Medicine, East
Carolina University, Greenville, NC 27834 USA
*Corresponding author: wanda.almeida@fcf.unicamp.br
Abstract
2-Aminothiazolines share an isosteric relationship with imidazolines and oxazolines
with antihypertensive activity mainly mediated by the imidazoline I1-receptor. In the
present work, we have prepared five aminothiazolines, following a previously described
synthetic pathway. Aminothiazolines derived from dicyclopropylmethylamine (ATZ1)
and cyclohexylamine (3) are unprecedented in the literature. Competitive radioligand
1 Current address: Department of Chemistry, University of Florida, Gainsville, FL. United States.
2Both authors contributed equally for the project.

2
assay was carried out with all synthetic compounds, and the I1 receptor affinity in
comparison to rilmenidine in PC12 cells was determined. Surprisingly, the rilmenidine
isoster (ATZ1) showed no I1-receptor interaction. Diethyl (ATZ4) and 2-ethyl-
hexylamine (ATZ5) derivatives bind to the receptor with 11.98 and 10.94 nmol/l,
respectively. These compounds were selected for in vivo experiments. Both compounds
reduced the blood pressure of spontaneously hypertensive rats (SHR). The hypotensive
effect of these compounds was abrogated in the presence of 2 adrenergic (yohimbine)
and I1 (efaroxan) receptor antagonists suggesting that both aminothiazolines bind to the
adrenergic and imidazoline receptors. Lipinski’s descriptors of the synthesized
aminothiazolines were calculated and are similar to the known imidazoline I1 receptor
ligands. 3D-Similarity between ATZ5 and agmatine, the natural imidazoline receptor
ligand, was also observed.
Keywords: Imidazoline Receptor, Aminothiazoline, Hypertension, PC12 Cells, Binding
Assay
1. Introduction
The activation of 2 (adrenergic) and I1 (imidazoline) receptors in the central
nervous system generates a hypotensive response, making these receptors an important
target for the development of antihypertensive drugs (Nikolic et al., 2008). Clonidine
belongs to the class of antihypertensive drugs with central action, which promotes
inhibition of the sympathetic activity. Currently, its activity is attributed both to the
stimulus of 2 adrenergic receptors in the nucleus tractus solitarius and receptors located
in the rostral ventrolateral medulla (RVLM). However, stimulation of 2 receptors
located at locus coeruleus is responsible for significant side effects, such as sedation (De

3
Sarro et al., 1987). The binding sites in the RVLM, called imidazoline receptors, are
regions that specifically recognize imidazolines or similar structures, both in the brain
and peripheral system, and some of them are involved in blood pressure regulation. As
result of the stimulus of I1 receptors in the RVLM, a decrease in the release of
norepinephrine (NE) from the storage vesicles (pre-synaptic neurons) and epinephrine
in the adrenal medulla, there is a decrease in the blood pressure (Bousquet and Feldman,
1999; Ernsberger, 2000; Zhang and Abdel-Rahman, 2005). Hence, there is a growing
interest in developing drugs that preferentially activate the the imidazoline receptors
while exhibiting low or no activity at the 2 receptors (Fellmann et al., 2013;
Wasilewska et al., 2014; Krasavin, 2015). These findings resulted in the development of
a second generation of central antihypertensive drugs, represented by the commercially
available moxonidine and rilmenidine (Van Zwieten, 1999; Bousquet et al., 1984;
Bousquet, 2001). The chemical structures of these compounds, clonidine and 2-
aminothiazolines are shown in Fig. 1A, in which the isosteric relationship of their
heterocyclic moieties is highlighted. Despite the structural similarity, thiazolines have
not been studied as potential antihypertensive agents, except for some 3-methyl-2-
thiazolidinimine derivatives (Rokach et al., 1980). Thus, we evaluated N-substituted 2-
aminothiazolines (Fig. 1B), as potential ligands for I1receptors and hypotensive agents.
Besides the 2-aminothiazoline ATZ1, a sulfur analogue of rilmenidine, we prepared
four more N-substituted-2-aminothiazolines ATZ2-5 to study their I1-receptor affinity.
Compounds that presented good affinity for the I1-receptor were selected for the in vivo
evaluation of their effects on arterial blood pressure of spontaneous hypertensives rats
(SHR).
Fig.1.
2. Materials and Methods

4
2.1. Chemistry
Compounds ATZ2, 4 and 5 were obtained as previously reported (Ferreira et al.,
2013) and are available in our laboratory. The syntheses of compounds ATZ1 and 3 are
first reported in this work and followed the same protocol of others. The synthetic
pathway to achieve these compounds involves the following steps: preparation of
isothiocyanates from the appropriated amine and carbon disulfide followed by treatment
of isothiacyanates with ethanolamine led to the corresponding thioureas, which were
converted into the desired aminothiazolines, by S-cyclization process, as detailed in the
supplementary material.
2.2. Calculations of physicochemical properties
The partition coefficient, represented by its logarithm (Log P), was calculated on
line (Tesco, 2005) by the software ALOGPS (VCCLAB, 2005). The optimization of 3D
structures of agmatine and aminothiazolines were carried out by using Gaussian 09
(Frisch et al., 2009). Polar surface area and molecular volume are obtained by
Molinspiration software.
2.3. Determination of the I1 receptor affinity for the synthesized compounds ATZ1-5 in
comparison to rilmenidine in PC12
A modified protocol for competitive radioligand binding assay previously
reported (Greney et al., 2000) was used to determine the I1 receptor affinity for the
investigated compounds using PC12 cell membranes.
2.3.1. Cell culture
PC12 cells, which express I1 receptor, were cultured in 75-cm2 flasks at 37°C
with 10% CO2 in F12 Kaighn's medium supplemented with 15% Horse Serum (HS),

5
2.5% fetal bovine serum (FBS) and 1% Anti-Anti (100x). After removing the medium,
cells at confluence were frozen in the flasks at -20°C until use to prepare membranes.
2.3.2. Membrane preparations
Frozen PC12 cells were scraped into cold Tris-HEPES buffer (5 mM Tris-
HEPES, pH 7.7, 0.5 mM EDTA, 0.5 mM EGTA, and 0.5 mM MgCl2) and
homogenized with a Potter homogenizer. After centrifugation at 35,000 rpm for 20 min,
the pellet was washed in cold Tris-HEPES buffer and centrifuged again. Pellets were re-
suspended in Tris-HEPES buffer at 4 mg protein/ml and used immediately for binding
assays.
2.3.3. Binding assay
[125I] paraiodoclonidine (PIC) was used as the radioligand to detect the
imidazoline receptors in the PC12 cell membranes (Bmax = 20 fmol/mg of protein).
Incubations were initiated by the addition of membranes (40 g of protein/80 l final
volume) and were carried out at 25°C for 30 min. For competition assay, increasing
concentrations of compounds (10-7 to 10-2 M) were added with 0.5 nM [125I] PIC
(corresponding to the KD value of the radioligand). To stop the incubation, samples
were filtered very quickly through GF/B glass fiber filters, incubated for 3 h in 0.03%
polyethylenimine with a Brandel harvester, and filters washed twice with 3 ml of 50
mM cold TrisHCl buffer, pH 7.7. Radioactivity retained on the filters was determined in
a Minaxi gamma counter. All compounds were dissolved in DMSO (100%) in stock
solution and diluted as needed to concentrations 10-7 to 10-2 M. The values of the
inhibition constants (Ki nM) are calculated from the respective IC50 values by applying

6
the Cheng-Prusoff equation using GraphPad Prism 5 software. Each value is mean of 5-
16 experiments.
2.4. Arterial blood pressure measurement
Animals were housed under a 12:12ꢀh light-dark cycle conditions with
maintained temperature (24°C). All animal procedures and the experimental protocols
were approved by the Ethical Principles in Animal Research adopted by Brazilian
College for Animal Experimentation (COBEA; No. 3341-1). Four-month-old SHRs,
provided by CEMIB-UNICAMP, were anesthetized by sodium thiopental (40 mg/kg,
i.p). The right femoral artery and left femoral vein were cannulated for the measurement
of arterial blood pressure and drug administration, respectively (Antunes et al., 1996;
El-Mas and Abdel-Rahman, 2001). Arterial pressure was measured via a pressure
transducer and the signal converted by a bridge amplifier coupled with PowerLab
(ADInstruments, Sydney, Australia). Mean arterial pressure (MAP) was recorded with
LabChart 7 software (ADInstruments). Drugs used were noradrenaline, clonidine,
yohimbine, efaroxan (Sigma, St. Louis, MO, USA), as well as compounds ATZ4 and 5.
2.4.1. Effect of combining ATZ5 with clonidine or yohimbine
Blood pressure was allowed to stabilize for at least 15 min before administration
of any drug or vehicle. All drugs were diluted in albumin/saline (0.3% v/v) solution, and
injected intravenously as a single bolus and were washed in with a further 100 µl of
saline. First, we injected the vehicle and then noradrenaline (2.5 µg/kg), followed by
ATZ5 (300 µg/kg) and clonidine (10 µg/kg). Arterial pressure was allowed to return to
the baseline level before the subsequent injections were conducted. In separate groups

7
of rats, the tested-compound was administered following α2-adrenergic blockade with
yohimbine (2.5 mg/kg) (Allard et al., 1995). Results are expressed as the mean values ±
standard error of means of 3-8 rats per group. Statistical analysis was carried out using
one-way ANOVA followed by Tukey’s post-test. In all analyses, a P-value < 0.05 was
considered significant. All procedures were performed using Graph Pad Prism 5.0
(Graph Pad Software Inc., San Diego, CA, USA).
2.4.2. Effect of efaroxan on cardiovascular effects of ATZ4 and ATZ5
In this experiment, we investigated the effect of imidazoline (I1) receptor
blockade with efaroxan (10 g/kg; Bock et al., 1999), on the cardiovascular effects of
compounds ATZ4 or 5 (300 g/kg). Each rat served as its own control and received one
of the two compounds before and after efaroxan. Adequate time was allowed to permit
full recovery of MAP and HR to baseline after the first injection. At the conclusion of
the experiment, all animals were killed with sodic thiopental anesthesia (100 mg/kg,
i.p). Results are expressed as the mean values ± S.E.M of changes in MAP after the
injections. For each compound, a number of four to six rats were used. Statistical
analysis was performed likewise as mentioned in the previous sub-item.
3. Results
Building on the availability of amines in our laboratory, we decided to study a
series of the aminothiazolines. ATZ2, 4 and 5 are available in our laboratory from a
previous work (Ferreira et al., 2013).
3.1. Preparation of compounds ATZ1 and 3

8
ATZ1 and 3 were prepared from dicyclopropylmethylamine and
cyclohexylamine, respectively. Experimental procedure of each synthetic step is
available in the supplementary material.
3.1.1. N-(Dicyclopropylmethyl)-4,5-dihydro-1,3-thiazol-2-amine (ATZ1)
White solid; Mp 123-124 °C.
IR (KBr, max): 3443, 3194, 3078, 3003, 2941, 2860, 1607, 1547, 1319, 1248, 1020,
1041, 829, 642, 608 cm-1;1H NMR (250 MHz, CDCl3):  3.94 (t, J = 7.3 Hz, 2H), 3.27
(t, J = 7.4 Hz, 2H), 2.97 (t, J = 7.1 Hz, 1H), 0.55-0.24 (m, 8H), 0.92 (st, J = 6.8, 2H);
13C NMR (62.5 MHz, CDCl3):  161.3 (C), 61.1(CH), 59.9 (CH2), 34.9(CH2),
15.5(CH), 2.8 (CH2), 1.9 (CH2); HRMS (EI TOF): Calculated for C10H16N2S: 196.1034;
Found M+: 196.1024
3.1.2. N-cyclohexyl-4,5-dihydro-1,3-thiazol-2-amine (ATZ3)
White solid; Mp: not determined due the decomposition ( 100° C)
IR (KBr, max): 3184, 2997, 2930, 2854, 1609, 1547, 1450, 1321, 1246, 1153, 1097,
1030, 891, 648, 453 cm-1; 1H NMR (500 MHz, CDCl3):  4.00 (t, J = 7.4 Hz, 2H), 3.66
(br s, 1H); 3.52 (tt, J = 3.9 and 10.5 Hz, 1H); 3.30 (t, J = 7.4 Hz, 2H); 2.03 (m, 2H);
1.71 (m. 1H); 1.59 (m, 1H); 1.36 (m, 3H); 1.17 (m, 3H); 13C NMR (62.5 MHz, CDCl3):
 160.9 (C), 60.5 (CH2), 53.9 (CH), 35.3 (CH2), 33.9 (CH2), 25.1 (CH2), 25.8(CH2).
HRMS (ESI+ TOF): Calculated for C9H17N2S+: 185.1107; Found M+: 185.1149.
3.2. Physicochemical properties
The results for calculated logarithm of the partition coefficient and Lipinski’s
descriptors (Lipinski, 1997) of the synthesized aminothiazolines and known I1-
imidazoline receptor are summarized in Table 1. Log P values of the synthesized

9
compounds ATZ1-5 range from 2.06 to 3.86, whereas for the known I1-receptor ligands
this range is -0.91 to 3.67. All synthesized compoundsATZ1-5 have three hydrogen
bond donating groups (HBD) and one acceptor (HBA). These numbers are the same of
clonidine, rilmenidine, efaroxan and 4-iodoclonidine.
Table 1. Lipinski’s descriptors of I1-Receptor Imidazoline receptor and
Aminothiazolines
Compound
Clonidine
Moxonidine
Rilmenidine
LNP 911
Log Pa
2.53
1.46
1.57
3.67
2.53
1.92
2.23
1.01
2.17
-0.91
2.71
3.55
2.06
2.39
2.20
2.15
HBDb
HBAc
MWd
230
240
180
334
196
172
153
204
216
130
182
153
196
206
184
172
1
1
1
1
1
1
1
1
1
4
1
1
1
1
1
1
3
5
3
2
2
2
1
4
3
4
2
3
3
3
3
3
Benazoline
Tracizoline
AGN 192403
Idazoxan
Efaroxan
Agmatine
Harmane
4-Iodoclonidine
ATZ 1
ATZ 2
ATZ 3
ATZ 4

10
3.86
1
3
214
ATZ 5
aAverage from ALOGPS; b Hydrogen Bond Donors;c Hydrogen Bond Acceptors; d Molecular weight
3.3. Imidazoline receptor affinity for Aminothiazolines ATZ1-5
The investigated compounds displaced the specific binding of [125I] PIC in PC12
cell membranes, except for ATZ3 as shown in the Fig. 2. Relative Ki values, compared
to rilmenidine (2.62 nM) are also presented.
Fig. 2.
Except for ATZ3, I1 receptor showed affinity for the synthesized compounds as well as
rilmenidine. CompoundsATZ4 and 5 presented the lowest Ki values: 11.98 and 10.94
nM, respectively.
3.4. Effects of yohimbine on arterial blood pressure changes by ATZ5 and clonidine
Blood pressure recordings of two different groups of SHRs are presented in Figs.
3A and B. Basal MAP in SHR was 163 ± 5.1 mmHg. Intravenous administration of
vehicle produced no significant variations in MAP. After intravenous injections of
either compound ATZ5 or clonidine there was an increase in arterial blood pressure of
6.4 ± 1.8 and 37.9 ± 5.6 mmHg (n = 5), respectively, which was followed by significant
reductions in MAP (Fig. 3A). In a separate group of SHR, the blockade of α2-adrenergic
receptor with yohimbine (2.5 mg/kg, i.v) abolished the hypotensive response caused by
ATZ5 or clonidine (Figs. 3B, C).
Fig. 3
3.5. Effects of efaroxan on arterial blood pressure changes by ATZ4 and 5
Baseline value for MAP of SHR was 174.0 ± 5.5 mmHg (n = 12). As seen in
Fig. 4, intravenous administration of vehicle had no significant effect on MAP (2.5 ±

11
0.8 mmHg). However, the administration of compound ATZ5 induced significant (P <
0.05) hypotensive response (-10.3 ± 1.0 mmHg). Similarly, ATZ4 significantly (P <
0.05) reduced MAP (-10.8 ± 3.3 mmHg). These hypotensive responses were short lived
and MAP fully recovered to the baseline within a few minutes (Fig. 4). The blockade of
imidazoline I1 receptors by efaroxan significantly (P < 0.05) attenuated the hypotensive
responses caused by ATZ4 or 5 (-3.9 ± 0.9 and -4.02 ± 1.2 mmHg, respectively; Fig. 4).
Fig. 4.
5. Conclusions
The I1-receptor affinity for the five aminothiazolines sharing a bioisosteric
relationship with rilmenidine, a known selective I1-agonist, were evaluated by a
competition experiment using the radioligand [125I]-p-iodoclonidine in PC12 cell
membranes. Except for compound ATZ3, all compounds exhibited affinity to the I1-
receptor. ATZ4 and 5 showed the lowest Ki values. To our disappointment, the
synthesized compounds were not exclusively selective for I1-receptor because the
antihypertensive effect of compound ATZ4 or 5 was blocked by efaroxan or yohimbine.
Based on these results, we concluded that the mechanism of MAP reduction involves
the activation of both 2 and I1 receptors, a biological characteristic known for clonidine
as well as rilmenidine. The limited number of compounds along with the difference in
the I1-receptor affinity by compounds sharing molecular similarity precluded
conducting a full structure-activity relationship (SAR) study.
Additional information
Authors have no conflit of interest.
Acknowledgements

12
This research was supported by São Paulo Research Foundation (Fapesp,
2008/06397-1) and Faepex-Unicamp. Authors wish to thank Prof. Claudio Tormena
(Institute of Chemistry, Unicamp) by Gaussian software license use.
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Fig. 1. (A) Structures of clonidine, rilmenidine, moxonidine and 2-aminothiazoline, highlighting the
imidazoline, oxazoline, pyrrolidine and thiazoline isosteric moieties, respectively. (B) Structures of 2-
compounds ATZ1-5.
Fig. 2. Plots of binding (CPM) versus Log of the aminothiazolines concentration (nM) and rilmenidine.
Fig. 3: Representative blood pressure recordings of the pressor response caused by noradrenaline (NOR)
and the reductions in mean arterial pressure (MAP) caused by compound ATZ5 (300 µg/kg) or clonidine
(10 µg/kg) in anesthetized spontaneously hypertensive rats. B. Pretreatment with yohimbine (2.5 mg/kg,
i.v) significantly attenuated the responses elicited by compound ATZ5 or clonidine. C. Bar graphs
showing the MAP responses in different groups. Values are expressed as mean ± S.E.M (n = 3 – 8). *P
<. .05; **P < .01 versus vehicle (one-way ANOVA, Tukey’s post-test).
Fig. 4. Representative blood pressure recordings showing the effect of compound ATZ5 (300 µg/kg; A)
or compound ATZ4 (300 µg/kg; B) before and after efaroxan (10 g/kg, i.v.). C. Changes in mean
arterial pressure (MAP) in animals treated with vehicle, ATZ4 or ATZ5 in the absence or presence of
efaroxan. Bars represent the means ± S.E.M for n = 4-6 animals. *P < 0.05 compared with vehicle; #P <
0.05 compared with ATZ 5; ϕP < 0.05 compared with ATZ 4.
Fig. 5. The most important ligands for imidazoline receptors.
Fig. 6. Hypothetical construction of the aminothiazoline 5 from the structure of agmatine.
Fig. 7. A. 3D optimized geometry of agmatine (top), ATZ5 (center) and aminothiazoline ATZ4 (bottom);
B. Superimposed structures of agmatine (magenta), aminothiazoline 5 (green).

17
Fig. 1.
A:
Cl
N
Cl
N
N
N
R
N
S
N
H
N
H
N
C
N
H
H
H2
Cl
OCH3
2-aminothiazoline
(general structure)
moxonidine
clonidine
N
O
N
H
rilmenidine
B:
N
N
N
S
N
H
S
N
S
N
H
H
ATZ 3
ATZ 2
ATZ 1
N
N
S
N
S
N
H
H
ATZ 4
ATZ 5

18
Fig.2.

19

20
Fig. 3.

21
Fig. 4.

22
Fig. 5.
Fig. 6.

23
Fig.7A

24
Fig. 7B