Thiophene/thiazole-benzene replacement on guanidine derivatives targeting α2-Adrenoceptors

Article abstract of DOI:10.1016/j.ejmech.2017.06.008

Searching for improved antagonists of α₂-adrenoceptors, a thorough theoretical study comparing the aromaticity of phenyl-, pyridinyl-, thiophenyl- and thiazolylguanidinium derivatives has been carried out [at M06-2X/6–311++G(p,d) computational level] confirming that thiophene and thiazole will be good ‘ring equivalents’ to benzene in these guanidinium systems. Based on these results, a small but chemically diverse library of guanidine derivatives (15 thiophenes and 2 thiazoles) were synthesised to explore the effect that the bioisosteric change has on affinity and activity at α₂-adrenoceptors in comparison with our previously studied phenyl derivatives. All compounds were tested for their α₂-adrenoceptor affinity and unsubstituted guanidinothiophenes displayed the strongest affinities in the same range as the phenyl analogues. In the case of cycloakyl systems, thiophenes with 6-membered rings showed the largest affinities, while for the thiazoles the 5-membered analogue presented the strongest affinity. From all the compounds tested for noradrenergic activity, only one compound exhibited agonistic activity, while two compounds showed very promising antagonism of α₂-adrenoceptors.

Full text of DOI:10.1016/j.ejmech.2017.06.008

Accepted Manuscript
Thiophene/thiazole-benzene replacement on guanidine derivatives targeting α -
2
Adrenoceptors
Aoife Flood, Cristina Trujillo, Goar Sanchez-Sanz, Brendan Kelly, Carolina Muguruza,
Luis F. Callado, Isabel Rozas
PII:
S0223-5234(17)30452-X
10.1016/j.ejmech.2017.06.008
EJMECH 9503
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2017
Revised Date: 28 May 2017
Accepted Date: 3 June 2017
Please cite this article as: A. Flood, C. Trujillo, G. Sanchez-Sanz, B. Kelly, C. Muguruza, L.F. Callado, I.
Rozas, Thiophene/thiazole-benzene replacement on guanidine derivatives targeting α -Adrenoceptors,
2
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.008.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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Thiophene/Thiazole-Benzene Replacement on
Guanidine Derivatives Targeting α -Adrenoceptors
2
a
a
b
a
c
Aoife Flood, Cristina Trujillo, Goar Sanchez-Sanz, Brendan Kelly, Carolina Muguruza,
c
,a
Luis F. Callado, and Isabel Rozas*
a
School of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin,
52-160 Pearse Street, Dublin 2, Ireland
1
b
Irish Centre of High-End Computing, Grand Canal Quay, Dublin 2, Ireland
c
Department of Pharmacology, University of the Basque Country UPV/EHU, and Centro de
Investigación Biomédica en Red de Salud Mental, CIBERSAM, Spain
*
Corresponding Author: Isabel Rozas, email: rozasi@tcd.ie; Phone: +353 1 896 3731.

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Graphical Abstract
Highlights
•
•
•
•
•
Phenyl-, thiophenyl- and thiazolylguanidinium derivatives have similar aromaticity
Thiophenyl- & thiazolylguanidines must act as phenyl-based α -adrenoceptor ligands
2
A high-diversity library of thiophenyl- and thiazolylguanidines was prepared
Their affinity for α -adrenoceptor was measured in human prefrontal cortex tissue
2
Activity on α -adrenoceptors was measured: 2 antagonists and 1 agonist were found
2

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Abstract
Searching for improved antagonists of α -adrenoceptors, a thorough theoretical study
2
comparing the aromaticity of phenyl-, pyridinyl-, thiophenyl- and thiazolylguanidinium
derivatives has been carried out [at M06-2X/6-311++G(p,d) computational level] confirming
that thiophene and thiazole will be good ‘ring equivalents’ to benzene in these guanidinium
systems. Based on these results, a small but chemically diverse library of guanidine
derivatives (15 thiophenes and 2 thiazoles) were synthesised to explore the effect that the
bioisosteric change has on affinity and activity at α -adrenoceptors in comparison with our
2
previously studied phenyl derivatives. All compounds were tested for their α -adrenoceptor
2
affinity and unsubstituted guanidinothiophenes displayed the strongest affinities in the same
range as the phenyl analogues. In the case of cycloakyl systems, thiophenes with 6-membered
rings showed the largest affinities, while for the thiazoles the 5-membered analogue
presented the strongest affinity. From all the compounds tested for noradrenergic activity,
only one compound exhibited agonistic activity, while two compounds showed very
promising antagonism of α -adrenoceptors.
2
Keywords
Bioisosterism; Aromaticity; Thiophene; Thiazole; Guanidinium; α -Adrenoceptors;
2
Antagonists; Affinity constants.
Abbreviations used
NA: noradrenaline; α2-ARs: α -adrenoceptors; PFC: human brain prefrontal cortex;
2
3
35
[
H]RX821002: 2-methoxyidazoxan; K : affinity constant; [ S]GTPγS: radioligand
i
guanosine 5'-O-[gamma-thio]triphosphate.

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1
. INTRODUCTION
Bioisosterism has been used for decades in Medicinal Chemistry as a source of
structural/molecular diversity [1]. One of the most classical bioisosteric changes is the use of
ring-equivalents and, thiophene in particular, has been used as a substitute for other aromatic
rings improving biological activity. For example, Song et al. have just reported the synthesis
of a series of thenoylhydrazide derivatives making use of the bioisosterism between
thiophene and benzene [2]. Recently, to improve the activity of a family of inhibitors of
dengue viral RNA-dependent RNA polymerase, the benzene moiety of the lead compound
was substituted by thiophene resulting in a 10-fold increase in potency [3]. Another recent
example of benzene-thiophene bioisosterism is the development of biphenyl derivatives as
bioisosteres of thienylphenyl agonists/antagonists of the AT receptor showing similar
2
affinity and functional activity [4]. Thiophene is present in range of very successful
commercial drugs such as olanzapine, raloxifen or clopidogrel, and similarly, the thiazole
ring features in well-known drugs such as ritonavir, pramipexole and famotidine [5].
Accordingly, considering that thiophene has often been utilised as a ring equivalent of
benzene in drug discovery due to its reported similar aromaticity making it an effective
mimic [6], we propose now to rigorously assess from a theoretical point of view the
aromaticity of arylguanidine derivatives of the aromatic cores previously used by our group
(benzene and pyridine) compared to those now proposed (thiophene and thiazole) in order to
accurately establish the potential similarity of their aromatic properties. With that aim we
have calculated a well-known and established type of aromaticity indicator, the Nucleus
Independent Chemical Shift (NICS) [7,8], at Density Functional Theory (DFT) level.
Moreover, continuing with our research in arylguanidine derivatives as antagonists of α2-
adrenoceptor (α2-AR) [9,10,11,12,13,14] and based on the proposed theoretical study on

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aromaticity, we also present the preparation and pharmacological evaluation of a library of
guanidinothiophenes and 2-guanidinothiazoles with high chemical diversity.
To date, we have found several structure-activity relationships (SAR) to optimise the
antagonistic activity of arylguanidine-like compounds as well as their α2-AR affinity. Thus,
we have explored the mono- (compounds 1, 2 and 4 in Figure 1) and di-substitution
(compound 3, Figure 1) of the guanidine-like system as well as the nature and position of
different substituents in 6-membered aromatic rings (mostly benzene [9-12,14] as in
compounds 1-3 and more recently pyridine [13] as in compound 4, Figure 1) with respect to
the guanidine-like moiety. In this way, we identified several lead compounds that were not
only α2-AR antagonists (in vitro and in vivo), but also showed antidepressant activity in
animal models such as the forced-swimming and tail suspension tests (e.g. compounds 1 and
2
, Figure 1) [15]. Additionally, we were able to identify that double substitution in the
guanidine system (e.g. compound 3, Figure 1) or pyridine derivatives (e.g. compound 4,
Figure 1) always results in antagonist or inverse agonist functional activity [10,11].
Figure 1.- Compounds previously prepared and tested by Rozas and collaborators as α2-AR
antagonists and general structure of the guanidinothiophenes proposed.

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. RESULTS AND DISCUSSION
2
2
.1. Comparative aromaticity study
The aromaticity of certain derivatives of furan, pyrrole and thiophene compared to that of the
corresponding azo-heterocycles (oxazole, imidazole and thiazole) has been previously
calculated by Gümüs and Türker at DFT level using B3LYP/6-31G(d,p) and B3LYP/6-
3
1++G(d,p) computational methods [16]. By considering NICS indexes they determined that
the second heteroatom substitution decreases aromaticity, but that this can be gained back to
some extent by the substitution of strong electron withdrawing groups (i.e. NO and F) at
2
position 4 [17]. In a different approach, Karadakov and coworkers [18,19] have recently
investigated the aromaticity of these 5-membered heterocycles by using isotropic magnetic
shielding distributions (HF-GIAO and MP2-GIAO isotropic shielding plots) in the regions of
space surrounding the rings. Their study indicates that aromaticity decreases in the order
thiophene > thiazole > pyrrole > imidazole > furan > oxazole suggesting that inclusion of a
second heteroatom has a detrimental effect on its aromaticity, which is small but noticeable in
thiazole and thiophene. These results are in agreement with previous studies also published
by some of the current authors [20].
In order to assess the aromatic character of the heterocyclic cores of the guanidine derivatives
proposed here, we have carried out NMR theoretical calculations of model systems, namely
benzene, phenylguanidine (5), 2- (6), 3- (7) and 4-pyridinoguanidine (8), 2- (9) and 3-
thiophenoguanidine (10) and 2-thiazologuanidine (11), at M06-2X [21] computational level
with the 6-311++G(d,p) [22] basis set, using the GIAO method at 0, 1 and 2 Å over the ring
centre of each model molecule and the results are presented in Table S1 (Supplemental
Information). Further, to avoid possible interactions with the magnetic field of the atoms,
several authors [23,24,25,26,27,28,29] have recommended extending the calculations up to 2

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Å above and below the ring plane. In Figure S1 (Supplemental Information) an explanation
and schematic representation of the NICS positions above/below the aromatic rings is given.
1
-(Pyridin-2-yl)guanidine 6 can present two orientations of the guanidine moiety with respect
to the pyridine ring; however, we have previously reported that the rotamer in which the
guanidine moiety forms an intramolecular hydrogen bond (IMHB) with the pyridine N atom
-1
is 33.9 kJ mol more stable [30] and, therefore, we have only considered that particular
rotamer to assess its aromaticity.
Similarly, 1-(thiazol-2-yl)guanidine (11) presents two different energy minima, one where the
guanidine also forms an IMHB with the N atom and another in which the guanidine is
perpendicular to the thiazole ring. We have calculated [M06-2X/6-311++G(d,p)] that the
-1
planar structure is 20.6 kJ mol more stable than the perpendicular one and, for that reason
we have studied only the aromaticity of the most stable one. As seen in Table S1, all the
compounds studied present similar magnetic field responses to that of benzene as shown by
their closely matching values of NICS(1) (variations up to 0.7 ppm) and NICS(2) (variations
up to 1.1 ppm).
However, a certain trend can be observed indicating that, although very subtle, there is a
certain aromaticity order: benzene ≈ phenylguanidine (5) ≈ 1-(pyridin-2-yl)guanidine (6) ≈ 1-
(pyridin-4-yl)guanidine (8) > 1-(pyridin-3-yl)guanidine (7) > 1-(thiophen-3-yl)guanidine (10)
>
1-(thiophen-2-yl)guanidine (9) ≈ 1-(thiazol-2-yl)guanidine (11). Moreover, we had
previously calculated the NICS values for 1-(pyridin-2-yl)guanidine (6) at a similar
computational level and the results obtained here are consistent with our reported data [31].

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Furthermore, to obtain additional information on the aromaticity of each compound, we have
calculated and plotted the NICS values on the 0.001 a.u. electron density isosurface (Figure
2
) and summarised the minima values over the ring of each surface (vdW and vdWbottom) in
up
Table S1.
Benzene
5
6
7
8
9
1
0
11
Figure 2. Bottom view of the NICS values on the 0.001 au electron density isosurface at the
M06-2X/6-311++G(d,p) computational level of benzene and compounds 5-11. NICS colour
scheme: Red > 0.0, Yellow > -2.5, Green > -5.0, Blue < -5.0

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Blue areas in Figure 2 correspond to NICS values < -5.0 ppm while red areas are those
associated to positive NICS indices. As observed in benzene, a blue (negative) area is located
over the centre of the ring denoting aromatic character. Very similar NICS patterns have been
found in all the isosurfaces plotted in Figure 2. As with the NICS(1) and NICS(2) indices,
five-membered ring compounds (9-11) present the same negative area but slightly smaller
which can be related to a small decrease of aromaticity.
Therefore, in terms of the aromatic properties of the cyclic cores carrying the guanidine
moiety, it seems that thiophene and thiazole derivatives could form the same type of
interactions (even though slightly weaker) as the phenyl and pyridine motifs and, hence,
preparing these types of analogues to target α2-AR as antagonists is merited.
2
.2. Chemistry
The only synthesis found in the literature, to the best of our knowledge, of simple guanidine-
like thiophenes is that of 3-N-(2-aminoimidazolinyl)thiophene [32]. Standard preparation of
aromatic 2-aminoimidazoline [33] and guanidine derivatives [34] generally involves the use
of aromatic amine intermediates. Since many thiophene amines are known to be unstable, this
could explain the lack of guanidine-like thiophenes reported thus far. 2-Aminothiazoles are
commercially available, and a number of thiazole guanidines have already been prepared by
other methods than our standard guanidylation approach [33,34].
To test the suitability of our standard guanidylation methodology for the synthesis of
thiophene analogues of the lead compound 1 (Figure 1) and looking for structural diversity,
we first prepared guanidine derivatives of some commercially available aminothiophenes

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such as 2-amino-3-methoxycarbonylthiophene (12) and 3-amino-2-methoxycarbonyl
thiophene (13). Moreover, since the most closely related thiophene-based compounds tested
at the α2-AR are 2- (14) or 3-thenylimidazole (15) derivatives of dexmedetomidine, which is
an α2-AR agonist with high affinity (Figure 3) [35], we also used 2-aminomethyl- (16) and 2-
aminoethylthiophenes (17) as starting materials (Scheme 1).
Figure 3.- Structure of α2-AR agonist dexmedetomidine and general structure of its
thiophene analogues 14 and 15.
Thus, syntheses of the Boc-protected guanidine derivatives of 12, 13, 16 and 17 (Scheme 1)
were carried out by the reaction of the corresponding commercial aminothiophenes with an
activated N-Boc-protected thiourea [9-14] in the presence of mercury(II) chloride and an
excess of triethylamine producing the corresponding derivatives 18-21 in moderate to
excellent yields (57, 37, 94 and 80%, respectively). As expected, aliphatic amines 16 and 17
gave significantly higher yields than their aromatic counterparts (12 and 13) because of the
higher nucleophilic nature of the amino group.
Deprotection of the Boc-protected guanidines 18 to 21 was carried out by using a TFA/DCM
solution followed by treatment with strongly basic anion-exchange resin giving the
corresponding hydrochloride salts (22, 23, 24 and 25, Scheme 1) in yields of 99, 51, 54 and

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4
3% respectively. Deprotection of 18, 19 and 21 was also carried out using a 4M
HCl/dioxane solution producing better yields (99, 90 and 95%, respectively).
Scheme 1. Preparation of thiophene guanidinium derivatives 22-25
Next, expanding the library of thienylguanidines, thiophene analogues of α2-AR antagonist 1
(
Figure 1), which shows good affinity (K = 77.6 nM [10]), were prepared. Hence, the
i
corresponding starting aminothiophenes were prepared following the Gewald synthesis and
even though the typical outcome of this synthesis is a carboxylic ester (or carbonitrile) of the
corresponding aminothiophene, we hypothesised that a subsequent decarboxylation could
produce the target thiophene. Preparation of thiophenes 26-29 was carried out using the
corresponding ketone (cyclopentanone, cyclohexanone, cycloheptanone and acetone,
respectively), sulfur powder and ethyl cyanoacetate in ethanol at 40 °C with morpholine as
catalytic base (Scheme 2) in poor to good yields (10, 79, 61 and 19%) [36].

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The corresponding Boc-protected guanidino derivatives 30, 31, 32 and 33 were obtained
using the standard guanidylation conditions, in 36, 52, 49 and 68% yields, respectively
(Scheme 2). Boc-deprotection of 30-33 using 50% TFA/DCM resulted in the formation of the
corresponding trifluoroacetate salts, which were treated directly with IRA400 Amberlyte
resin in its chloride-activated form to yield the more water-soluble hydrochloride salts 34-37
in very good yields of 90, 93, 71 and 61% (Scheme 2). Using the HCl/dioxane method, 33
was successfully converted to 37 in 93% yield.
Taking advantage of the Gewald synthesis and extending the structural diversity of our
thienylguanidine library, we prepared 2-aminothiophene-3-carbonitriles and their guanidine
derivatives. Thus, reaction of malononitrile, sulfur and the appropriate ketone
(cyclopentanone, cyclohexanone and cycloheptanone) in EtOH, with dropwise addition of
morpholine resulted in the formation of thiophene carbonitriles 38, 39 and 40 in respective
yields of 46, 51 and 43% (Scheme 2). Synthesis of the Boc-protected guanidines (41-43) was
carried out using the standard method and Boc-deprotection was carried out using the
HCl/dioxane method, yielding hydrochlorides 44-46 in reasonable yields (Scheme 2).
Scheme 2. Preparation of thiopheno guanidinium derivatives 34-37 and 44-46.

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Next, to further expand the diversity of our guanidinothiophene library, decarboxylation of
the thiophene esters was attempted. Using compound 13 as a model substrate, the conditions
described by Barker et al. [37] using oxalic acid were tested. This method involves the
hydrolysis of the corresponding thiophene ester using weakly basic conditions (2M NaOH)
and refluxing over 30 min followed by decarboxylation and subsequent acidification with
oxalic acid to pH = 3 to precipitate the ammonium salt. Following this procedure the oxalate
salt of 3-aminothiophene (Scheme 3) was obtained, which was free-based and immediately
guanidylated under the standard conditions to prevent decomposition prior to the next
reaction step.
Scheme 3. Preparation of decarboxylated guanidino thiophenes

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The desired Boc-protected guanidine product 47 (Scheme 3) was obtained in a low yield and
its deprotection was achieved by treatment with HCl/dioxane followed by reverse phase
column chromatography resulting in the clean isolation of the hydrochloride salt of 3-
thienylguanidine (10, Scheme 3) in 88% yield.
A modified version of Barker’s conditions (using KOH in 1:1 water/ethanol for the
hydrolysis step to facilitate dissolution of the starting materials) was applied to 26, 27 and 28,
and, after guanidylation, the Boc-protected guanidines 48-50 were isolated in 4, 23 and 35%
yields (Scheme 3). Deprotection with 4M HCl/dioxane followed by reverse phase column
chromatography resulted in the corresponding hydrochloride salts 51-53 in 60, 79 and 99%
yields, respectively (Scheme 3).
To complete these families of sulphur-containing heterocyclic guanidines, 2-aminothiazole
analogues of the lead compound 4 were synthesized. These derivatives are more stable than
their thiophene counterparts and many synthetic routes for the formation of thiazole and
benzothiazole guanidines have been previously described, starting from α-haloketones and 2-
imino-4-thiobiuret [38] or from 2-aminothiophenols and cyanoguanidine [39]. Since some of
the desired α-chloroketones and 2-aminothiophenols were commercially available, we
prepared the corresponding 2-guanidino cycloalkylthiazoles following Beyer’s synthesis [38].
Therefore, both 2-chloro derivatives of cyclopentanone and cyclohexanone were treated with
2
-imino-4-thiobiuret directly yielding the corresponding hydrochlorides 54 and 55 (Scheme
4
) in good yields. Even though these two compounds are reported in a patent by Actelion
Pharmaceuticals [40], no mention is made of their synthesis.

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Scheme 4. Preparation of guanidino thiazoles
2
.3. Pharmacology and SAR analysis
3
2
.3.1. [ H]RX821002 Binding Assays
The α2-AR binding affinities of all compounds were measured in human brain prefrontal
cortex (PFC) tissue by competition assays with the α2-AR selective radioligand
3
3
[
H]RX821002, which was used at a constant concentration of 2 nM. Specific [ H]RX821002
3
binding was measured in PFC membranes, which were incubated with [ H]RX821002 for 30
min at 25 °C in the absence or presence of the competing compounds at increasing
-12
-3
concentrations (10 - 10 M, ten concentrations). Thus, the specific binding was determined,
plotted as a function of the compound concentration and the affinities obtained were
expressed as affinity constants (K , nM).
i
3
The results of the [ H]RX821002 binding affinity experiments for some of the thiophene and
thiazole derivatives prepared are listed in Table 1. In general, many of the compounds tested
show low α2-AR affinity (only 6 out of 17 compounds have K < 1000 nM), but some
i
interesting trends can be observed. First, when the guanidinium group is attached to the
thiophene ring through aliphatic chains (compounds 24 and 25) relatively good α2-AR
affinity values are obtained indicating that there is room in the active site to accommodate a
linker between the guanidine and the thiophene nucleus. The best affinity (K = 751 nM) in
i
this group is attained when the chain is longer (two -CH - groups, 25).
2

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Table 1.- Binding affinity for the human brain prefrontal cortex α2-ARs expressed as K
calculated from [ H]RX821002 (≈ 2 nM) competition binding experiments.
i
3
Compound
Structure
K (nM)
i
2
2
2
3
>100,000
>100,000
2
2
4
5
3928 ±54
751 ±7
NH HCl
N NH2
S
H
34
35
36
37
44
45
46
10
30,150 ±1114
9086 ±210
>100,000
66,530 ±6339
2215 ±50
733 ±8
1198 ±58
1968 ±41
5
5
1
2
87.9 ±0.5
31.3 ±0.3
53
54
55
156 ±1
706 ±9
1205 ±25

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The K values obtained for compounds 22 and 10 indicate that the presence of an ester group
i
in position 2 of the 3-guanidinium derivatives (compound 22) completely abolishes α2-AR
affinity; moreover, when the positions of the guanidinium and carboxylic ester groups are
interchanged (compounds 22 and 23), the α2-AR affinity continues to be abolished; this
possibly indicates that these compounds are sterically or electronically disfavoured from
properly fitting into the active site. However, introduction of a methyl group in position 4
(
compound 37) partially recovers the affinity (K = 66,530 nM). It has been proposed that
i
good antagonists should occupy the pocket created towards TM5 in class A GPCRs without
directly interacting with Ser5.42 and Ser5.43 residues (which is necessary for G-protein
signalling activation) [41]. Thus, assuming that the guanidinium cation will bind the Asp
residue on TM3 that is conserved in class A GPCRs (Asp3.32), the coplanar arrangement
between thiophene and guanidinium moieties would be unfavourable for binding, but, the
introduction of the 4-methyl group would benefit affinity because it would partially occupy
that pocket near TM5.
When a methyl group is present in position 4 or an alkyl cycle is attached to positions 4 and 5
of a 2-guanidino-3-carboxylate thiophene core, the α2-AR affinity observed depends on the
size of such alkyl system. Thus, the 5-membered cycloakyl and the 4-methyl analogues (34
and 37, respectively) show worse affinity than the 6-membered ring derivative (35), but
better than the 7-membered cycloakyl analogue (36), which shows no affinity under the assay
conditions. This can be explained by the steric limits of the lipophilic pocket at TM5 that
clashes with the size of the cyclohepta alkyl ring of compound 36 but is optimal for the 6-
member ring analogue 35.

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In this series of fused cycloakyl derivatives, replacement of the ester group (34-36) by a
carbonitrile functionality (44-46) results in a significant increase in α2-AR affinity. In the
case of 44 and 45, the K values (nM) are 12-13 times better than the ester analogues 34 and
i
3
5, whereas the difference is even larger between 7-membered analogues 46 and 36, with the
K of the latter around 1198 nM while the former does not bind to the receptor under the
i
assay conditions. These results may indicate that compound 36, with both a bulky ethyl ester
group in the 3-position and a large seven membered ring fused in 4- and 5-positions, is too
large to fit the active site of the α2-AR receptor and, moreover, these bulky systems could
prevent the optimal interaction between the guanidinium and Asp3.32. However 46, with the
smaller nitrile group in the 3-position can actually fit the receptor well. In general, the 6-
membered derivatives display stronger α2-AR affinity than the 7-membered ones, whereas
the K difference between 45 and 46 (both with a CN in position 3) is not too large.
i
All evidence presented until now points to larger substituents disrupting the salt-bridge with
Asp3.32, thereby lowering affinity. Accordingly, decarboxylated derivatives 51-53 show the
best α2-AR affinity values (87.9, 31.3 and 156 nM, respectively) among all the tested
compounds in this small library. This is in agreement with the idea that the aliphatic rings of
these compounds, by occupying the pocket towards TM5 and displacing water molecules
from this region, produce an increase in affinity. Again the fused 6-membered derivative is
the best of this series whereas the 7-member analogue is the worst.
Thiazole compounds 54 and 55, which have good K values (Table 1), display the opposite
i
affinity pattern to their thiophene counterparts, with the 5-membered ring derivative (54)
showing stronger affinity than the 6-membered ring analogue (55).

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Compared with their phenyl and pyridine guanidine analogues, we found that, in the case of
(
hetero)aromatic systems only substituted with guanidine, all the Ki values are very large and
the similar aromaticity does not account for the large differences observed in α2-AR affinity
162,181 nM for pyridine-3-ylguanidine 7, 1950 nM for thiophen-3-ylguanidine 10, 1820
nM for pyridine-2-ylguanidine 6 and 646 nM for phenylguanidine 5). In the case of cycloakyl
hetero)aromatic guanidines, despite the slightly poorer aromatic character of thiophene and
(
(
thiazole, good α2-AR affinities were obtained (see Ki values for 51 and 52 in Table 1) further
improving those reported by us for 1-(2,3-dihydro-1H-inden-5-yl)guanidine (309 nM [10]),
1
-(5,6,7,8-tetrahydroisoquinolin-3-yl)guanidine 4 (468 nM [14]) or the lead compound 1
(77.6 nM [10]). Therefore, aromaticity cannot be considered as a determining factor in α2-
AR binding; however, these heteroaromatic rings may act as a scaffold for the different
substituents and, thus, the differences observed between benzene and thiophene/thiazole may
be due to changes in the placement of substituents.
3
5
2
.3.2. [ S]GTPγS Binding Assays
This assay allows for the direct evaluation of G-protein activation by determining the GTP
3
5
exchange using [ S]GTPγS in human PFC membranes. In particular, agonists increase the
nucleotide binding, inverse agonists reduce nucleotide binding and antagonists do not affect
-12
binding. Assays were incubated at 30 °C for 120 min with shaking. Ten concentrations (10
-3
-
10 M) of the different compounds were added to the assay in order to evaluate their effect
35
on [ S]GTPγS binding. Then, the plates were subjected to vacuum filtration and the
radioactivity of the filter was measured by scintillation spectrometry. The EC50 values
(concentration that provokes 50% of the maximal response) and Emax values (maximal
response in % over basal binding) were calculated for all compounds and for the well-known
α2-AR agonist UK14304. Typical potency values are 1-2 log units lower than the affinity

ACCEPTED MANUSCRIPT
values obtained in radioligand receptor binding experiments among other reasons, because
assays were performed in low-affinity receptor conditions for agonists (in the presence of G
nucleotides and sodium).
3
5
All compounds that showed an antagonist activity in the [ S]GTPγS binding assays, i.e.
3
5
those that did not stimulate binding of [ S]GTPγS by their own, were also assayed at a
-5
35
constant concentration (10 M) for [ S]GTPγS binding in the presence of increasing
-13
-4
concentrations of the α2-AR agonist UK14304 (10 -10 M). If the concentration-response
curve for UK14304 in these assays is shifted to the right, the antagonist effect of these
derivatives against the α2-ARs is confirmed. These experiments were also performed in order
to investigate the potential of the compounds to specifically modify the EC50 or the Emax
values in the UK14304 stimulation curve.
Activity at the α2-AR was examined for compounds with a K < 1000 nM (25, 45, 51, 52, 53
i
and 54), along with thiazole 55 for comparison. Interestingly, all the compounds except 53,
35
displayed antagonistic activity in the [ S]GTPγS assays, similar to the lead compound 1
phenyl core); this is likely due to the orientation of the thiophene nucleus when attached to
the guanidine group. The corresponding EC values for UK14304 in the presence of these
(
5
0
compounds are shown in Table 2. Compound 53, with a bulky fused 7-membered alkyl ring,
3
5
was determined to be an agonist since it stimulated [ S]GTPγS binding by its own and this
-5
stimulation was abolished in the presence of the α2-AR antagonist RX821002 at 10 M. This
agonistic activity may be a result of the size of the 7-membered ring, which is big enough to
alter TM5 (somehow interacting with Ser5.42 and Ser5.43) in such a way that leads to
activation of G-protein coupling.

ACCEPTED MANUSCRIPT
Addition of 25, 51, 52 and 54 to the experiment induced a very small (<5-fold) rightward
shift in the EC value for UK14304, indicating that they have only weak antagonistic effects
50
at the α2-AR. On the contrary, 45 and 55 caused a larger shift to the EC of the standard
50
agonist UK14304, characteristic of competitive antagonists. These two compounds have
average binding affinities (K = 733 and 1205 nM, respectively) suggesting that they may
i
form different interactions or access a pocket not available to the rest of the compounds,
allowing them to block activation of the receptor by UK14304.
Table 2.- EC5 and E
values obtained from the concentration-response curves for
max
0
13
-
-4
35
UK14304 (10 -10 M) stimulation of [ S]GTPγS binding in the absence or presence of the
-
5
different compounds (10 M).
EC (µM) ±SEM
Emax (%) ±SEM
132 ±2
5
0
UK14304
0.4 ±0.01
UK14304 + 25
UK14304 + 45
UK14304 + 51
UK14304 + 52
UK14304 + 53
UK14304 + 54
UK14304 + 55
1.7 ±0.06
6.6 ±0.52
0.5 ±0.04
0.8 ±0.09
10.7 ±3.7
1.5 ±0.06
117 ±4
128 ±6
111 ±2
111 ±4
124 ±9
126 ±3
11.4 ±3.40
105 ±4
This particular orientation could be explained by the presence of an IMHB in compound 55
that positions the cycloakyl moiety towards the mentioned lipophilic pocket by TM5, and the
orientation achieved by the guanidinium in the 3-CN derivative 45 which situates the
cycloakyl ring within the same pocket (Figure 4) [42].
According to this, the space allowed for the cycloakyl moiety seems to be limited to that of a
6
-member ring for optimally engaging with TM5 of the α2-AR, and, by hosting this

ACCEPTED MANUSCRIPT
cycloalkyl moiety, facilitates the appropriate orientation of the guanidinium moiety to
achieve antagonist activity.
55
45
55
45
45+55
Figure 4.- Optimised structures of compounds 55 and 45 (calculated at M062X/6-
11++G(p,d) level) and their superimposition viewed from the front (left) and a
perpendicular view (right).
3
3
. CONCLUSIONS
A comparative study of aromaticity has been carried out at M062X/6-311++G(p,d)
computational level between phenyl, pyridine, thiophene and thiazole guanidinium
derivatives by evaluating their corresponding NICS indices. The outcomes of this study
indicate that thiophene and thiazole will be good bioisosteric alternatives to benzene in these
guanidinium systems.
Accordingly, a small library of guanidine sulphur containing heterocyclic derivatives (15
thiophenes and 2 thiazoles) has been prepared, following standard guanidylation
methodologies and new synthetic routes, to explore the effect that the bioisosteric changes

ACCEPTED MANUSCRIPT
now introduced has on affinity and antagonist activity at the α2-ARs compared to previously
prepared phenyl and pyridine derivatives.
Compounds 22, 23 and 36 showed no binding affinity for the human α2-ARs in
3
[
H]RX821002 (≈ 2 nM) competition binding experiments. Compounds 34, 35 and 37 bound
poorly to the α2-AR, having K values around 9-66 ꢀM. Medium to good α2-AR affinity was
i
observed for compounds 10, 24, 25, 44, 45, 46, 53, 54 and 55, all of which have K values
i
between 3.9 ꢀM and 156 nM. Finally, compounds 51 and 52 have very good α2-ARs binding
affinities of 88 and 31 nM, respectively.
Some trends were observed with regard to engagement with the α2-ARs; thus, 6-membered
cycloalkyl rings conferred the strongest affinity (smaller K values) within each thiophene
i
series. In the case of the thiazoles the 5-membered analogue proved to be a better binder.
Thiophen-2-ylguanidines with only cycloalkyl substituents gave better binding affinities than
those with added ester or nitrile functionalities. However, in the case of the thiophen-3-
ylguanidine 10, more modest results were observed in comparison to derivatives containing
other aromatic rings.
Almost all compounds tested for activity at α2-ARs (25, 45, 51, 52, 53, 54 and 55) proved to
be antagonists with only compound 53 showing agonistic activity. The large size of the 7-
membered cycloalkyl system in 53 is likely responsible for changing the mode of binding of
this compound and hence its activity.

ACCEPTED MANUSCRIPT
In summary, even though the aromatic character of guanidinium derivatives of thiophene and
thiazole is slightly poorer than that of their phenyl or pyridine analogues, α2-AR affinities in
the nM range were observed for some of the S-containing compounds and, in particular 45
and 55, which are antagonists that displaced α2-ARs agonist binding 16 and 28 times to the
right and place the cycloakyl systems in a similar orientation, can be considered compounds
of interest for future investigations.
4
. EXPERIMENTAL SECTION
4
.1. Computational details
All calculations have been carried out using the Gaussian09 computational package [43]. All
systems have been optimized at the M06-2X [21] computational level using the 6-
3
11++G(d,p) [22] basis sets. Frequency calculations have been carried out to confirm that the
structures obtained correspond to energetic minima. Effects of water solvation have been
included by means of the SCFR–PCM approaches implemented in the Gaussian09 starting
from the gas–phase geometries and re-optimizing.
Nucleus-Independent Chemical Shifts (NICS) values [7] were calculated using the Gauge-
Including Atomic Orbital (GIAO) method [44,45] on the B3LYP/6-311++G(d,p) geometries.
To obtain the spatial distribution of the NICS, its values have been calculated on a three
dimensional (3D) cubic grid of 12 Å size following the procedure described in our previous
works [20,46]. The points in the grid are located at 0.2 Å one from other in the three spatial
directions. The result is a cube of 226,800 NICS values, which in the next step are
represented over the 0.001 a.u. electron density isosurface using the Wave Function Analysis
Surface Analysis Suite (WFA-SAS) program [47].

ACCEPTED MANUSCRIPT
4
.2. Synthesis
All commercial chemicals were obtained from Sigma-Aldrich or Fluka and used without
further purification. Deuterated solvents for NMR use were purchased from Apollo. Dry
solvents were prepared using standard procedures, according to Vogel, with distillation prior
to use. Solvents for synthesis purposes were used at GPR grade. Analytical TLC was
performed using Merck Kieselgel 60 F254 silica gel plates or Polygram Alox N/UV254
aluminium oxide plates. Visualisation was performed by UV light (254 nm). NMR spectra
were recorded on Bruker DPX–400 Avance spectrometers, operating at 400.13 MHz and
1
13
6
00.1 MHz for H NMR; 100.6 MHz and 150.9 MHz for C-NMR. Shifts are referenced to
the internal solvent signals. NMR data were processed using Bruker TOPSPIN software.
HRMS spectra were measured on a Micromass LCT electrospray TOF instrument with a
WATERS 2690 autosampler and methanol/acetonitrile as carrier solvent. Melting points were
determined using a Stuart Scientific Melting Point SMP1 apparatus and are uncorrected.
Infrared spectra were recorded on a Perkin Elmer Spectrum One FT-IR Spectrometer
equipped with a Universal ATR sampling accessory. Infrared spectra were obtained on a
Perkin-Elmer Spectrum 100 FT-IR spectrometer equipped with Universal ATR sampling
accessory. The X-ray crystallography data for crystal samples were collected on a Rigaku
Saturn 724 CCD Diffractometer. Elemental analysis was carried out at the Microanalysis
Laboratory, School of Chemistry and Chemical Biology, University College Dublin. HPLC
purity analysis was carried out using a Varian ProStar system equipped with a Varian Prostar
3
35 diode array detector and a manual injector (20 ꢀL). For purity assessment, UV detection
was performed at 245 nm and peak purity was confirmed using a purity channel. The

ACCEPTED MANUSCRIPT
stationary phase consisted of an ACE 5 C18-AR column (150 × 4.6 mm), and the mobile
-1
phase used the following gradient system, eluting at 1 mL min : aqueous formate buffer (30
mM, pH 3.0) for 10 min, linear ramp to 85% methanol buffered with the same system over 25
minutes, hold at 85% buffered methanol for 10 min. Minimum requirement for purity was set
at 95.0%.
4
.2.1. General Methods
4
.2.1.1. Method A: Preparation of 2-Amino-3-substituted Thiophenes via the Gewald
Reaction
A mixture of ketone (1.0 eq.), sulfur powder (1.0 eq.) and t-butyl cyanoacetate, ethyl
cyanoacetate or malononitrile (1.0 eq.) in EtOH (2 mL/mmol) was prepared before
morpholine (1.0 eq.) was added dropwise, ensuring that the reaction did not heat up above
6
0 °C during the addition. The mixture was then heated at 40 °C and stirred for 4-20 h.
4
.2.1.2. Method B: Synthesis of the Boc-Protected Guanidinothiophene Derivatives
A solution of the corresponding amine (1 eq.), Boc-protected S-methylthiopseudourea (1 eq.),
TEA (~3.5 eq.) in dry DCM (10-20 mL/mmol) was prepared, set at 0 °C and stirred for 20
min. Then, HgCl (1.2 or 1.5 eq.) was added and the solution stirred at 0 °C for a further 40
2
min and was then stirred for 48 h. at r.t. until the reaction had reached completion (TLC). The
reaction mixture was filtered through a pad of celite in a sintered glass funnel, and washed
with EtOAc. The filtrate was then washed with brine, dried over MgSO and the solvents
4
removed, before further purification.
4
.2.1.3. Method C: Synthesis of the Boc-Protected Guanidine Derivatives involving
Decarboxylation

ACCEPTED MANUSCRIPT
The relevant ester was refluxed in 2M NaOH (10 mL) or (5 mL) with ethanol (5 mL) for the
period of time specified. The solution was cooled, acidified to pH 3 with concentrated HCl
and the precipitate was filtered off and dissolved in acetone (12.5 or 6.5 mL). The solution
was dried (MgSO4), filtered and the solvent evaporated at 20 °C. The resulting solid was
treated with 2-propanol (3 or 1.5 mL) and anhydrous oxalic acid (1 or 0.6 g) at 38 °C for 45
min. The mixture was cooled, diluted with ether, the solid filtered off and washed with ether
and dried. The salt was dissolved in water, basified with conc. ammonia. The mixture was
extracted with DCM (3 × 5 mL), the combined extracts were dried (MgSO4) and the solvents
removed. The material was redissolved in DCM (10 mL) before an appropriate quantity of
each of the following were added sequentially, based on the mass of the crude dicationic salt:
6
2 (2.4 eq.), TEA (8.0 eq) and HgCl2 (3.0 eq.). The solution was stirred for 2 days, was
filtered through a pad of celite and washed with EtOAc. The filtrate was washed with brine
before being dried over MgSO4 and the solvents removed. The resulting material was
purified on using column chromatography on a Biotage (hexane/EtOAc 1%/CV over 20 CV).
4
.2.1.4. Method D: Preparation of the Hydrochloride Salts using TFA/DCM
A 50% (v/v) solution of TFA in DCM (10 mL) was added to the corresponding di-Boc-
protected guanidine precursor (1.0 eq.). The mixture was stirred for 4 h at r.t., and the solvent
was then removed under vacuum yielding the trifluoroacetate salt. This was dissolved in
water (10 mL) and IRA400 Amberlyte Resin in its chloride form (1.0 g/eq.) was added. The
mixture was stirred at r.t. for 24 h. The resin was then removed by filtration, the aqueous
solution was washed with DCM, and the water evaporated, yielding the pure hydrochloride
1
9
salt. Absence of the trifluoroacetate anion was checked for by F NMR.
4
.2.1.5. Method E: Preparation of the Hydrochloride Salts using HCl/Dioxane

ACCEPTED MANUSCRIPT
The di-Boc-protected guanidine precursor (1 eq.) was dissolved in 4 M HCl in dioxane (25
eq.). The reaction was stirred at 60 °C during 6 h before the solvent was removed. The
sample was purified through a 3 cm reverse phase pencil column (3 CV water, 2 CV 9:1
water:acetonitrile, 2 CV 1:1 water:acetonitrile, 2 CV acetonitrile).
4
.2.1.6. Method F: Direct Preparation of the Thiazole Hydrochloride Salts
A mixture of 2-imino-4-thiobiuret (236.3 mg; 2 mmol; 1 eq.) and the corresponding α-
chlorocycloketone (2 mmol; 1 eq.) was heated to 130 °C for 1 h. After cooling, the mixture
was filtered and washed with water and HCl (32%; 1 mL). The filtrate was then evaporated
down in vacuo yielding the corresponding salt.
4
.2.2. 1-(Thiophen-3-yl)guanidine hydrochloride (10)
Following Method E from 47 (50.0 mg; 0.146 mmol; 1 eq.) a colourless gel was obtained in a
1
8
1
1
1
1
8% yield (22.8 mg). H NMR (400 MHz, D O) δ 7.42 (dd, 1H, J = 5.1, 3.1 Hz), 7.27 (dd,
2
13
H, 4J = 0.9, 3.1 Hz), 6.95 (dd, 1H, J = 5.1, 4J = 0.9 Hz). C NMR (100 MHz, D O) δ 156.1,
2
31.5, 126.7, 124.3, 120.2. IR νmax 3301, 3108 (NH), 1664 (C=N), 1600, 1534, 1438, 1408,
-1
+
360, 1230, 1185, 1079, 837, 790 cm . HRMS (ESI) calculated, 142.0394 [M + H] ; found,
42.0364. Anal. (C H N S) C, H, N.
5
7
3
4
.2.3. Methyl 3-guanidinothiophene-2-carboxylate hydrochloride (22)
Following Method D from 18 (300.0 mg; 0.75 mmol; 1 eq.) or following Method E from 18
(100 mg; 0.25 mmol; 1 eq.) a white solid was obtained in a 99% yield (174.5 mg or 58.7 mg,
1
respectively). M.p. 170 °C, decomposes > 172 °C. H NMR (400 MHz, D O) δ 7.66 (d, 1H, J
2
1
3
=
5.5 Hz), 7.08 (d, 1H, J = 5.5 Hz), 3.74 (s, 3H). C NMR (100 MHz, D O) δ 163.5, 155.6,
2
1
38.2, 133.3, 124.4, 120.1, 52.6. IR νmax 3425 (NH), 3136, 3106 (NH), 1659 (C=O), 1601