Synthesis and biological evaluation of a new series of 2,3,5-substituted [1,2,4]-thiadiazoles as modulators of adenosine A1 receptors and their molecular mechanism of action

Article abstract of DOI:10.1021/jm049337s

We synthesized two series (7a-i and 8a-i) of 2,3,5-substituted [1,2,4]-thiadiazole analogues of SCH-202676 (7a, 2,3-diphenyl-5-N-methylimino- 2H-[1,2,4]-thiadiazole) with emphasis on the N-imino substituent. Compounds 7a-g,i and 8a-g at a final concentrat

Full text of DOI:10.1021/jm049337s

J. Med. Chem. 2005, 48, 1145-1151
1145
Synthesis and Biological Evaluation of a New Series of 2,3,5-Substituted
[1,2,4]-Thiadiazoles as Modulators of Adenosine A1 Receptors and Their
Molecular Mechanism of Action
Aniko´ Go¨blyo¨s, Henk de Vries, Johannes Brussee, and Adriaan P. IJzerman*
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, P.O. Box 9502,
2300 RA Leiden, The Netherlands
Received August 11, 2004
We synthesized two series (7a-i and 8a-i) of 2,3,5-substituted [1,2,4]-thiadiazole analogues
of SCH-202676 (7a, 2,3-diphenyl-5-N-methylimino-2H-[1,2,4]-thiadiazole) with emphasis on
the N-imino substituent. Compounds 7a-g,i and 8a-g at a final concentration of 1 µM
significantly inhibited [3H]CCPA (2-chloro-N6- cyclopentyladenosine) agonist binding to human
A1 adenosine receptors. At the same concentration, all compounds appeared to increase
[3H]DPCPX (1,3-dipropyl-8-cyclopentylxanthine) antagonist binding. Compound 7a and LUF5855
(7g) were selected for further characterization and studied in both equilibrium and kinetic
radioligand binding experiments. The results suggest a nonstoichiometric interaction with the
receptor. Further bioanalytical procedures (HPLC and MS) provided proof for an unusual
receptor interaction in which 7a and 7g upon incubation were transformed into their
corresponding thioureas 5a and 5g. We suggest that the thiadiazoles are sulfhydryl modifying
agents rather than allosteric modulators, as they appear to reversibly modify the sulfhydryl
groups of cysteine residues in cell membrane preparations.
Introduction
Many G protein-coupled receptors (GPCRs) including
adenosine receptors appear to have allosteric binding
sites, occupation of which may modify the action of the
endogenous ligand (i.e., hormone or neurotransmit-
ter).1,2 An allosteric modulator is thus best defined as a
ligand that increases or decreases the action of an
agonist or antagonist (the “orthosteric” ligand) by com-
bining with a distinct (allosteric or allotopic) site on the
receptor macromolecule.3 Allosteric drugs are able to
modulate receptor activity through conformational
changes in the receptor protein that are transmitted
from the allosteric to the orthosteric site. Allosteric
modulators may have several advantages over ortho-
steric ligands as potential therapeutic agents, including
a natural limitation of their effect (the endogenous
ligand needs to be present) and tissue selectivity (the
endogenous ligand concentrations may vary over dif-
ferent tissues).
In 1990, Bruns and co-workers reported that 2-amino-
3-benzoylthiophene derivatives are capable of enhancing
the binding and activity of reference A1 receptor ago-
nists, such as N6-cyclopentyladenosine (CPA). However,
the compounds were found to act as competitive an-
tagonists at these receptors also.4,5 PD81723 (Figure 1)
became the reference allosteric modulator among them
with the best ratio of enhancement to antagonistic
action. Other derivatives of 2-amino-3-benzoylthiophene
based on the structure of PD81723 have been synthe-
sized. Among them, several compounds appeared more
potent than PD81723.6-10
Figure 1. Chemical structures of PD81723 and compound 7a.
Recently, compound 7a (Figure 1) was found to be a
general allosteric modulator of both agonist and an-
tagonist binding to GPCRs. The compound inhibited
radioligand binding to a number of GPCRs, including
the human µ-, δ-, and κ-opioid, R- and â-adrenergic,
muscarinic M1 and M2, and dopaminergic D1 and D2
receptors.11,12 In our previous work, a number of 2,3,5-
substituted [1,2,4]-thiadiazole analogues of compound
7a, with emphasis on substituents on both phenyl
groups, were synthesized and tested as potential allo-
steric modulators of adenosine receptors.13 They inhib-
ited the binding of [3H]CCPA (2-chloro-N6- cyclopentyl-
adenosine), a radiolabeled agonist for adenosine A1
receptors, in a concentration-dependent manner (see
also ref 14). Among them, 5-N-methylimino-2-(3-meth-
ylphenyl)-3-phenyl-2H-[1,2,4]-thiadiazole (8a) proved to
be the most active with an EC50 value of 0.64 µM, 4
times more active than 7a.
*
Author to whom correspondence should be addressed. Phone:
In the present study, we prepared two series of
analogues based on 7a and 8a, in which the N-imino
+31 (0)71 527 4651. Fax: +31 (0)71 527 4565. E-mail: ijzerman@
lacdr.leidenuniv.nl.
10.1021/jm049337s CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

1146 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Go¨blyo¨s et al.
Table 1. Modulation of [3H]CCPA and [3H]DPCPX Binding to
Human A1 Adenosine Receptors by 2,3,5-Substituted
[1,2,4]-Thiadiazoles
Scheme 1a
% specific binding of
radioligand remaining
[3H]DPCPXa
10-6
122
[3H]CCPAa
compound R1
R2
M
10-6
M
10-7
M
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
H
H
H
H
H
H
Me
Et
16
20
26
27
27
23
25
66
43
23
24
29
36
29
29
35
89
66
97
99
98
101
106
97
100
104
101
96
100
97
99
97
134
135
126
127
126
123
106
124
132
125
126
114
142
121
101
107
116
Pr
Bu
3-OH-Pr
iPr
cPentyl
Ph
a Reagents: (i) SOCl2; (ii) NaNCS, R2NH2; (iii) Br2.
Bn
8a
8b
8c
8d
8e
8f
8g
8h
8i
Me Me
substituent (R2 in Scheme 1) was now varied. Intrigued
by the peculiar findings in both equilibrium and kinetic
radioligand binding experiments, we examined their
mechanism of action in more detail. We conclude that
these compounds are best described as protein modifiers
rather than allosteric modulators.
Me Et
Me Pr
Me Bu
Me 3-OH-Pr
Me iPr
Me cPentyl
Me Ph
Me Bn
100
96
96
99
Results and Discussion
a Data are expressed as means from three independent experi-
ments performed in duplicate; individual values varied less than
10% for [3H]CCPA binding and less than 35% for [3H]DPCPX
binding. The results are given as percentage specific binding of
radioligand remaining, where total control binding is 100% and
nonspecific binding is 0%.
Chemistry. To prepare the compounds, we applied
the following synthetic route.15 Benzamides 1 and 2
were used as starting materials that were converted into
benzimidoyl chlorides 3 and 4 (Scheme 1).16 Substitution
of chlorine by NCS followed by addition of an amine
afforded thioureas 5a-i and 6a-i in moderate to good
yields. Finally, an oxidation with bromine yielded the
targeted 2,3,5-substituted [1,2,4]-thiadiazoles as their
hydrobromide salts 7a-i and 8a-i.
Biology. All compounds were initially characterized
in equilibrium radioligand displacement studies on
human adenosine A1 receptors. Both a radiolabeled
agonist ([3H]CCPA) and a radiolabeled antagonist ([3H]-
DPCPX (1,3-dipropyl-8-cyclopentylxanthine)) were used.
Subsequently, selected compounds were used for further
characterization by, for example, studying their influ-
ence on association and dissociation kinetics of the
adenosine A1 receptor radioligands.
All final 2,3,5-substituted [1,2,4]-thiadiazoles 7a-i
and 8a-i were tested on human adenosine A1 receptors
(Table 1). All compounds at a final concentration of
1 µM significantly inhibited [3H]CCPA binding except
8h. In particular, 7a-g and 8a,b appeared to be most
potent with 16-36% specific binding of radioligand
remaining. At the same concentration, all compounds
appeared to increase [3H]DPCPX binding. At a lower
concentration, (0.1 µM) none of the compounds appar-
ently affected [3H]CCPA binding. Introduction of alkyl
R2 groups with an increasing number of carbon atoms,
methyl, ethyl, propyl, isopropyl, butyl, or the introduc-
tion of a 3-hydroxypropyl group or a cyclic alkyl group,
namely, cyclopentyl, did not affect the results of binding
experiments significantly. However, for compounds with
aromatic phenyl or benzyl groups as R2 substituents, a
smaller decrease in the inhibition of [3H]CCPA binding
was observed for both series of compounds 7 (for 7h 66%
and for 7i 43%) and 8 (for 8h 89% and for 8i 66% specific
binding of radioligand remaining). Comparing the com-
pounds bearing the same R2 but a different R1 substitu-
ent (compound series 7 and 8), we concluded that the
values were only marginally influenced by the R1 (3-
methyl) substituent.
One compound (7g) was analyzed in more detail,
together with 7a, for reasons of comparison. Both
inhibited [3H]CCPA binding in a concentration-depend-
ent manner. The EC50 value of 7g was 503 ( 39 nM
compared to the EC50 value of 7a (2800 ( 200 nM). The
displacement curve for 7g was steep (nH ) -2.7 ( 0.1),
although less pronounced than for 7a (nH ) -6.5 ( 2.8),
full effects being observed within 1-2 concentration log
units for both. The aberrant pseudo-Hill coefficients, not
compatible with a 1:1 stoichiometric interaction with the
receptor, prompted us to examine the compounds’
mechanism of action in more detail.
We first analyzed the behavior of 7a and 7g (at
concentrations of 1 µM) on the kinetics of the interaction
between the radiolabeled agonist [3H]CCPA and the
receptor. Both association and dissociation of [3H]CCPA
were affected by the two compounds in that the extent
of association and dissociation was inhibited (Figures
2A and 2B). For compound 7a, total radioligand as-
sociation (i.e., the asymptotic level of binding) was only
half of the control value (49% vs 93%), for 7g it was
slightly higher (71%). The dissociation process was
similarly inhibited; for 7a, the lowest value of the

2,3,5-Substituted [1,2,4]-Thiadiazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1147
Figure 2. Association (A) and dissociation kinetics (B) of [3H]-
CCPA binding to and from human adenosine A1 receptors.
Data are from a representative experiment performed in
duplicate. Radioligand binding is expressed as percent of
specific binding. Concentrations of compounds 7a (1) and 7g
(b) were 1 µM. Control (9) was without compounds.
Figure 3. HPLC chromatograms (at λ ) 270 nm). Graph A:
product from the interaction of 7a (10-5 M) with CHO cell
membranes expressing human adenosine A1 receptors. Graph
B: product from the incubation of 7a (10-5 M) with cysteine
(10-3 M).
Table 2. Association and Dissociation Kinetic Parameters of
[3H]CCPA Binding to and from Human Adenosine A1 Receptors
in the Presence or Absence of 7a and 7g (1 mM)a
association
t1/2 (min) kon (nM-1 min-1
dissociation
t1/2 (min) )
koff (min-1
perature or after incubation at 65 °C for 5 h. We then
reasoned that the compounds might react with protein
components as present during the radioligand binding
experiments. In Figure 3, graph A is the HPLC chro-
matogram of compound 7a after incubation with CHO
cell membranes expressing hA1 receptors in Tris buffer.
In fact, using membranes of wild-type CHO cells (i.e.,
not expressing the receptor), we obtained almost identi-
cal results (data not shown). The chromatogram shows
that in the presence of membranes a part of 7a (tr )
7.6 min) is transformed into its synthetic thiourea
precursor 5a (tr ) 6.8 min, also checked independently
with 5a itself). We subsequently carried out experiments
in which compounds 7a and 7g were reacted with most
of the 20 essential amino acids. Only after reaction of
7a and 7g with cysteine we found a significant change
in the respective HPLC chromatograms. In the presence
of cysteine, a full conversion of 7a into 5a was observed
(Figure 3B). Next to its tr value, the identity of 5a was
confirmed after isolation from the reaction mixture
through its UV and 1H NMR spectra. In a similar
approach, we identified 5g (now with HPLC-MS) as the
resulting product of the interaction of 7g with either
cell membranes or cysteine.
)
control 2.6 ( 0.3
0.27 ( 0.02
0.35 ( 0.01
0.25 ( 0.07
10.8 ( 2.8 0.067 ( 0.016
12.4 ( 0.4 0.056 ( 0.001
12.0 ( 0.6 0.058 ( 0.003
7a
7g
2.0 ( 0.1
2.9 ( 0.8
a The values are (SEM (n ) 3).
asymptote was 51%, for 7g 26%, compared to the control
value of 3% radioligand remaining after 60 min (Figure
2B). Interestingly, the association and dissociation
kinetic parameters (i.e., t1/2 and k values) did not change
significantly compared to the control values (Table 2).
This finding, in fact, discounts the compounds to be
allosteric modulators, which by definition influence
these parameters, for either the dissociation or the
association process or both. Rather, it seems that in the
association kinetic experiments the thiadiazoles mask
the receptors’ primary binding sites, preventing [3H]-
CCPA from binding. In a similar vein, in dissociation
kinetic experiments the radioligand seems to be locked
into the cavity of the binding site by the thiadiazoles,
preventing complete dissociation. In our former publica-
tion, other substituted thiadiazoles were similarly tested,
but at a concentration of 10 µM. Under these conditions,
the association of [3H]CCPA binding to human adeno-
sine A1 receptors was almost negligible (<5%) and could
not be characterized numerically.13
From graphs A and B in Figure 3, it appears that the
amounts of 5a formed are different. Complete conver-
sion is obtained in the presence of cysteine alone (Figure
3B), whereas this is not the case in the presence of CHO
cell membranes expressing hA1 receptors. It can be
calculated that with 20 µg of protein present and an
average cysteine content in mammalian proteins of
To shed further light on these findings, we used a
number of bioanalytical approaches. We first studied the
stability of the compounds per se. It appeared that the
1H NMR spectrum of compound 7a in DMSO did not
change either in the course of 1 month at room tem-

1148 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Go¨blyo¨s et al.
Scheme 2
2.26%17 the final cysteine concentration is approxi-
mately 10-5 M in the experimental setup that led to
graph A. This is roughly equal to the thiadiazole
concentration, whereas in graph B there is a 100-fold
molar excess of cysteine compared to the thiadiazole.
We suggest that the mechanism of action is the
following (Scheme 2). Compound 7a (and analogues)
binds to the receptor; however, it is reduced by cysteine
residues (a), present either in the binding site or in any
protein in the membrane preparation, into a covalent
thiourea adduct (b). Formation of this adduct is not the
end stage; it is either reverted to 7a or transformed into
the free thiourea 5a in the presence of a reducing agent
(e.g., another cysteine (R-SH, c)). In the latter case,
two thiol groups are oxidized to form a disulfide bond.
These disulfide bridges are supposed to be responsible
for the behavior of 7a and analogues in their capability
of noncompetitively influencing radioligand binding. We
anticipate the process to be reversible, since we13 and
Fawzi et al.11 had demonstrated that the effect of 7a
on radioligand binding could be washed out.
(NEM) and phenylmercuric chloride (PMC). It is well
established that treatment with 1 mM NEM shifts A1
adenosine receptors from a high-affinity to a low-affinity
state for agonists.24 This effect has been ascribed to the
alkylation of a sulfhydryl group of the inhibitory gua-
nine nucleotide binding protein Gi. Phenylmercuric
chloride (PMC) forms a covalent mercaptide with sulf-
hydryl groups, which can be reversed by treatment with
dithiothreitol.25-27 To this class of sulfhydryl modifiers
may now be added the highly (re)active thiadiazoles,
instead of classifying these compounds as allosteric
modulators.
Conclusion
Thiadiazoles were synthesized with different N-imino
substituents. Results of receptor-ligand binding experi-
ments and stability studies by HPLC and HPLC-MS
showed that the compounds are highly reactive sulfhy-
dryl modifying agents rather than allosteric inhibitors.
They appear to be reduced into their thiourea precursors
by thiol groups of cysteine residues. This general feature
explains their overall lack of selectivity.
The stability and reactivity of other thiadiazoles have
been studied by several authors. Pan et al. have
demonstrated that thiadiazoles bearing aryl R2 substit-
uents shown to be active as melanocortin MC4 receptor
agonists may undergo a thermal rearrangement to
2-amidinobenzothiazole in DMSO.18,19 The same authors
reduced 2-(4-methylphenyl)-3-phenyl-5-(4-methoxyphen-
ylamino)-[1,2,4]-thiadiazolium bromide to its precursor
imidoylthiourea by dithioerythritol. They suspected that
this compound also reacts with the thiol group on
cysteine residues or that it oxidizes two thiol groups to
create a disulfide bond, causing irreversible binding to
the receptor.18 Marrano et al. found this type of com-
pound to be irreversible inhibitors of guinea pig liver
transglutaminase through a mechanism of action simi-
lar to that suggested by Pan and co-workers (i.e.,
irreversible inhibition by the formation of a disulfide
bond upon opening of the thiadiazole ring).20 Others
found that 1,2,4-thiadiazoles are susceptible to nucleo-
philic attack at the sulfur atom followed by ring open-
ing.21 Moreover, 1,2,4-thiadiazole derivatives reacted
with active site thiol residues of certain cysteine pro-
teases to form covalent adducts.22 A similar mechanism
was suggested to explain how 1,2,4-thiadiazoles act as
cathepsin B inhibitors. The compounds were proven to
undergo ring opening with thiols but not with amines
and/or alcohols. The C3 substituent can be tailored to
tune the reactivity of the heterocycle, that is to increase
or decrease the rate of ring opening by thiols.23 Next to
the current thiadiazoles, other and better-known sulf-
hydryl group modifying agents are N-ethylmaleimide
Experimental Section
Chemicals. [3H]DPCPX (128 Ci/mmol) was purchased from
Amersham Pharmacia Biotech. [3H]CCPA (54.9 Ci/mmol) was
obtained from NEN. DPCPX was from Sigma-RBI. All other
chemicals, including starting materials, were from standard
sources and of the highest purity commercially available.
1
Instruments and Analysis. H NMR spectra were mea-
sured at 200 MHz with a Bruker AC 200, Bruker AV 400, or
Bruker DMX 600 spectrometer. 13C NMR spectra were mea-
sured at 50, 100, or 150 MHz. Chemical shifts for H and 13C
1
are given in ppm (δ) relative to tetramethylsilane (TMS) as
internal standard; coupling constants are given in Hz. Melting
points were determined with a Bu¨chi capillary melting point
apparatus and are uncorrected. Combustion analyses of new
target compounds (series 7 and 8) were performed by the
analytical department of the Gorlaeus Laboratories, Leiden
University (The Netherlands) and are within (0.4% of theo-
retical values unless otherwise specified.
For HPLC analysis, samples were analyzed by means of a
Gilson (Middleton, WI) solvent delivery system, equipped with
a Gilson 234 sample injector (100 µL loop) and a Spectra focus
(Thermo Separation Products, Breda, The Netherlands) diode
array detector. The column was a 125 mm × 4.0 mm Prontosil
Eurobond C18 (5.0 µm particles) from Bischoff (Leonberg,
Germany). The effluent was monitored from 210 to 360 nm.
For HPLC-MS analysis, the HPLC system was a Jasco low-
pressure system, 900 series. The column was a 4.6 mm × 150
mm Alltima C18 with a flow of 1 mL/min. The MS system was
an API 165 PE-SCJEX (Applied Biosystems), QI system.
Preparation of Benzamides and Benzimidoyl Chlo-
rides. Compound 1 is commercially available, while com-
pounds 2-4 were prepared according to known procedures.16

2,3,5-Substituted [1,2,4]-Thiadiazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1149
General Procedure for the Preparation of the Thio-
ureas.15The appropriate benzimidoyl chloride was dissolved
in acetone p.a. (1 mmol/3 mL). At -10 °C, a solution of NaNCS
(1.1 equiv) in acetone (1 mmol/2 mL) was added dropwise. The
mixture was stirred in the cooling bath until 0 °C was reached.
The precipitate (NaCl) was removed by filtration over a glass
filter with Hyflo. The filtrate was stirred on an ice bath, and
the appropriate amine (1.1 equiv) was added. The reaction
mixture was allowed to warm to room temperature (with
stirring), then the solvent was removed by evaporation, and
the residue was triturated with diethyl ether, collected on a
glass filter, washed once more, and dried in vacuo to give the
thiourea. This product was then used in the next step without
further purification.
1-Methyl-3-(phenyl-phenyliminomethyl)thiourea (5a).13
Scale: 20.9 mmol. Yield: 4.50 g (80%) of an off-white solid.
1H NMR (CDCl3): δ 3.26 (d, 3H, J ) 4.4 Hz, NCH3); 6.68 (d,
2H, J ) 8.8 Hz, Ar); 6.98 (d, 1H, J ) 9.5 Hz, Ar); 7.47 (m, 7H,
Ar); 8.06 (s, 1H, NH); 11.84 (bs, 1H, NH).
1-Ethyl-3-(phenyl-phenyliminomethyl)thiourea (5b).13
Scale: 9.2 mmol. Yield: 2.30 g (88%) of a pale yellow solid. 1H
NMR (CDCl3): δ 1.33 (t, 3H, J ) 7.3 Hz, CH3); 3.76 (m, 2H,
CH2); 6.68 (d, 2H, J ) 8.8 Hz, Ar); 6.97 (m, 1H, Ar); 7.24 (m,
7H, Ar); 8.00 (s, 1H, NH); 11.90 (bs, 1H, NH).
1-(Phenyl-phenyliminomethyl)-3-propyl-thiourea (5c).
Scale: 7.0 mmol. Yield: 1.67 g (80%). 1H NMR (CDCl3): δ 1.02
(t, 3H, J ) 7.30 Hz, CH3); 1.69-1.79 (m, 2H, CH2); 3.69 (q,
2H, J ) 6.56 Hz, CH2); 6.68 (d, 2H, J ) 7.32 Hz, Ar); 6.92 (t,
1H, J ) 7.30 Hz, Ar); 7.10-7.32 (m, 7H, Ar); 8.04 (s, 1H, NH);
12.00 (s, 1H, NH).
1-Butyl-3-(phenyl-phenyliminomethyl)thiourea (5d).
Scale: 4.0 mmol. Yield: 0.64 g (52%). 1H NMR (CDCl3): δ 0.97
(t, 3H, J ) 7.30 Hz, CH3); 1.36-1.55 (m, 2H, CH2); 1.60-1.78
(m, 2H, CH2); 3.68-3.78 (m, 2H, CH2); 6.67 (d, 2H, J ) 8.04
Hz, Ar); 6.97 (t, 1H, J ) 7.30 Hz, Ar); 7.11-7.36 (m, 7H, Ar);
7.99 (s, 1H, NH); 11.98 (s, 1H, NH).
1-(3-Hydroxypropyl)-3-(phenyl-phenyliminomethyl)-
thiourea (5e). Scale: 7.0 mmol. Yield: 1.95 g (91%). 1H NMR
(CDCl3): δ 1.83-1.93 (m, 2H, CH2); 2.72 (s, 1H, OH); 3.75 (t,
2H, J ) 7.76 Hz, CH2); 3.87-3.96 (m, 2H, CH2); 6.68 (d, 2H,
J ) 7.32 Hz, Ar); 6.97 (t, 1H, J ) 7.30 Hz, Ar); 7.11-7.33 (m,
7H, Ar); 8.11 (s, 1H, NH); 12.06 (s, 1H, NH).
1-Isopropyl-3-(phenyl-phenyliminomethyl)thiourea (5f).
Scale: 7.0 mmol. Yield: 1.90 g (91%). 1H NMR (CDCl3): δ 1.35
(d, 6H, J ) 6.58 Hz CH3); 4.47-4.64 (m, 1H, CH); 6.68 (d, 2H,
J ) 7.32 Hz, Ar); 6.97 (t, 1H, J ) 7.30 Hz, Ar); 7.11-7.39 (m,
7H, Ar); 7.93 (s, 1H, NH); 11.90 (s, 1H, NH).
7.3 Hz, Ar); 7.01 (dd, 1H, J ) 7.3, 8.0 Hz, Ar); 7.32 (m, 5H,
Ar); 7.96 (s, 1H, NH); 11.91 (bs, 1H, NH).
1-(Phenyl-3-tolyliminomethyl)-3-propyl-thiourea (6c).
Scale: 5.0 mmol. Yield: 1.52 g (97%). 1H NMR (CDCl3): δ 1.02
(t, 3H, J ) 7.30 Hz, CH3); 1.69-1.79 (m, 2H, CH2); 2.21 (s,
3H, CH3); 3.69 (q, 2H, J ) 7.30 Hz, CH2); 6.43 (d, 1H, J )
8.80 Hz, Ar); 6.54 (s, 1H, Ar); 6.78 (d, 1H, J ) 7.32 Hz, Ar);
7.00 (t, 1H, J ) 7.32 Hz, Ar); 7.21-7.39 (m, 5H, Ar); 7.99 (s,
1H, NH); 12.01 (bs, 1H, NH).
1-Butyl-3-(phenyl-3-tolyliminomethyl)thiourea (6d).
Scale: 4.0 mmol. Yield: 0.80 g (62%). 1H NMR (CDCl3): δ 0.97
(t, 3H, J ) 7.30 Hz, CH3); 1.37-1.55 (m, 2H, CH2); 1.62-1.78
(m, 2H, CH2); 2.21 (s, 3H, CH3); 3.67-3.78 (m, 2H, CH2); 6.43
(d, 1H, J ) 7.30 Hz, Ar); 6.54 (s, 1H, Ar); 6.79 (d, 1H, J ) 8.04
Hz, Ar); 7.01 (t, 1H, J ) 7.30 Hz, Ar); 7.20-7.37 (m, 5H, Ar);
7.97 (bs, 1H, NH); 12.00 (s, 1H, NH).
1-(3-Hydroxypropyl)-3-(phenyl-3-tolyliminomethyl)-
thiourea (6e). Scale: 4.0 mmol. Yield: 1.01 g (71%). 1H NMR
(CDCl3): δ 1.81-1.94 (m, 2H, CH2); 2.21 (s, 3H, CH3); 3.75 (t,
2H, J ) 5.12 Hz, CH2); 3.87-3.96 (q, 2H, J ) 5.84 Hz, CH2);
6.43 (d, 1H, J ) 8.02 Hz, Ar); 6.55 (s, 1H, Ar); 6.79 (d, 1H,
J ) 8.04 Hz, Ar); 7.01 (t, 1H, J ) 7.30 Hz, Ar); 7.20-7.38
(m, 5H, Ar); 8.08 (bs, 1H, NH); 12.10 (bs, 1H, NH).
1-Isopropyl-3-(phenyl-3-tolyliminomethyl)thiourea (6f).
Scale: 4.0 mmol. Yield: 1.05 g (84%). 1H NMR (CDCl3): δ 1.34
(d, 6H, J ) 6.58 Hz, 2CH3); 4.47-4.64 (m, 1H, CH); 6.43 (d,
1H, J ) 8.02 Hz, Ar); 6.54 (s, 1H, Ar); 6.79 (d, 1H, J ) 7.30
Hz, Ar); 7.01 (t, 1H, J ) 7.30 Hz, Ar); 7.25-7.36 (m, 5H, Ar);
7.91 (bs, 1H, NH); 11.93 (bs, 1H, NH).
1-Cyclopentyl-3-(phenyl-3-tolyliminomethyl)thio-
urea (6g). Scale: 4.0 mmol. Yield: 1.15 g (85%). 1H NMR
(CDCl3): δ 1.63-2.22 (m, 8H, 4CH2); 4.60-4.78 (m, 1H, CH);
6.44-6.52 (m, 2H, Ar); 6.79 (d, 1H, J ) 4.2 Hz, Ar); 7.00 (t,
1H, J ) 7.30 Hz, Ar); 7.24-7.32 (m, 5H, Ar); 7.93 (s, 1H, NH);
12.10 (bs, 1H, NH).
1-Phenyl-3-(phenyl-3-tolyliminomethyl)thiourea (6h).
Scale: 5.0 mmol. Yield: 1.21 g (70%). 1H NMR (CDCl3): δ 2.23
(s, 3H, CH3); 6.50 (d, 1H, J ) 8.02 Hz, Ar); 6.63 (s, 1H, Ar);
6.82 (d, 1H, J ) 7.30 Hz, Ar); 7.03 (t, 1H, J ) 7.32 Hz, Ar);
7.12-7.45 (m, 8H, Ar); 7.74 (d, 2H, J ) 8.04 Hz, Ar); 8.13 (bs,
1H, NH); 10.83 (bs, 1H, NH).
1-Benzyl-3-(phenyl-3-tolyliminomethyl)thiourea (6i).
Scale: 6.0 mmol. Yield: 2.03 g (94%). 1H NMR (CDCl3): δ 2.18
(s, 3H, CH3); 4.98 (d, 2H, J ) 5.12 Hz, CH2); 6.34-6.49 (m,
2H, Ar); 6.80 (d, 1H, J ) 10.54 Hz, Ar); 6.94 (t, 1H, J ) 7.30
Hz, Ar); 7.21-7.42 (m, 10H, Ar); 8.07 (s, 1H, NH); 12.43 (bs,
1H, NH).
General Procedure for the Preparation of [1,2,4]-
Thiadiazoles.15b The thiourea was dissolved in a minimal
amount of CH2Cl2 (usually 2-4 mL/mmol) and then diluted
with twice the volume of ethyl acetate. At room temperature,
a 0.5 M solution of bromine in ethyl acetate (2 equiv) was
added dropwise. The thiadiazole hydrobromide precipitated.
After addition of light petroleum ether, the mixture was left
to stand at 5 °C overnight. Filtering, washing (2×) of the
residue with petroleum ether, and drying in vacuo afforded
the [1,2,4]-thiadiazole. The products were recrystallized from
methanol.
2,3-Diphenyl-5-N-methylimino-2H-[1,2,4]-thiadiazole
Hydrobromide (7a).13 Scale: 16.3 mmol. Yield: 4.50 g (79%)
as a white solid. Mp: 248-250 °C. 1H NMR (MeOD-d3): δ 3.37
(s, 3H, NCH3); 7.49 (m, 10H, Ar). Anal. (C15H13N3S‚HBr) C,
H, N.
1-Cyclopentyl-3-(phenyl-phenyliminomethyl)thio-
urea (5g). Scale: 7.0 mmol. Yield: 1.96 g (87%). 1H NMR
(CDCl3): δ 1.62-1.72 (m, 6H, CH2); 2.04-2.22 (m, 2H, CH2);
4.61-4.78 (m, 1H, CH); 6.66 (d, 2H, J ) 7.30 Hz, Ar); 7.14 (t,
1H, J ) 7.30 Hz, Ar); 7.10-7.36 (m, 7H, Ar); 7.94 (s, 1H, NH);
12.08 (s, 1H, NH).
1-Phenyl-3-(phenyl-phenyliminomethyl)thiourea (5h).
Scale: 7.0 mmol. Yield: 1.98 g (85%). 1H NMR (CDCl3): δ 6.76
(d, 2H, J ) 8.04 Hz, Ar); 7.01 (t, 1H, J ) 7.30 Hz, Ar); 7.14-
7.45 (m, 10H, Ar); 7.75 (d, 2H, J ) 7.30 Hz, Ar); 8.16 (s, 1H,
NH); 10.90 (s, 1H, NH).
1-Benzyl-3-(phenyl-phenyliminomethyl)thiourea (5i).
Scale: 7.0 mmol. Yield: 2.28 g (95%). 1H NMR (CDCl3): δ 4.98
(d, 2H, J ) 5.10 Hz, CH2); 6.44 (d, 2H, J ) 8.04 Hz, Ar); 6.95
(t, 1H, J ) 7.30 Hz, Ar); 7.12 (t, 2H, J ) 7.32 Hz, Ar); 7.27-
7.40 (m, 10H, Ar); 8.10 (s, 1H, NH); 12.40 (s, 1H, NH).
1-Methyl-3-(phenyl-3-tolyliminomethyl)thiourea (6a).13
Scale: 6.9 mmol. Yield: 1.08 g (56%). 1H NMR (CDCl3): δ 2.21
(s, 3H, CH3); 3.25 (d, 3H, J ) 4.4 Hz, NCH3); 6.43 (d, 1H, J )
8.0 Hz, Ar); 6.56 (s, 1H, Ar); 6.78 (d, 1H, J ) 7.3 Hz, Ar); 7.00
(dd, 1H, J ) 7.3, 8.0 Hz, Ar); 7.28 (m, 5H, Ar); 8.03 (s, 1H,
NH); 11.83 (bs, 1H, NH).
2,3-Diphenyl-5-N-ethylimino-2H-[1,2,4]-thiadiazole Hy-
drobromide (7b).13 Scale: 8.1 mmol. Yield: 2.41 g (82%) as
1
a white solid. Mp: 240-242 °C. H NMR (MeOD-d3): δ 1.40
(t, 3H, J ) 7.3 Hz, CH3); 3.85 (q, 2H, J ) 7.3 Hz, NCH2); 7.48
(m, 10H, Ar). Anal. (C16H15N3S‚0.8 HBr‚1.3 H2O) C, H, N.
2,3-Diphenyl-5-N-propylimino-2H-[1,2,4]-thiadiazole Hy-
drobromide (7c). Scale: 5.0 mmol. Yield: 1.56 g (83%) as a
white solid. Mp: 239-240 °C. 1H NMR (CDCl3): δ 1.07 (t, 3H,
J ) 7.30 Hz, CH3); 1.70-1.91 (m, 2H, CH2); 3.72 (q, 2H, J )
7.32 Hz, CH2); 7.21-7.53 (m, 10H, Ar); 10.38 (bs, 1H, HBr).
1H NMR (CDCl3 + 1 drop of D2O): δ 1.06 (t, 3H, J ) 7.30 Hz,
1-Ethyl-3-(phenyl-3-tolyliminomethyl)thiourea (6b).13
Scale: 7.0 mmol. Yield: 1.52 g (73%). 1H NMR (CDCl3): δ 1.32
(t, 3H, J ) 7.3 Hz, CH3); 2.21 (s, 3H, CH3); 3.75 (m, 2H, CH2);
6.44 (d, 1H, J ) 7.3 Hz, Ar); 6.56 (s, 1H, Ar); 6.78 (d, 1H, J )

1150 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Go¨blyo¨s et al.
CH3); 1.71-1.89 (m, 2H, CH2); 3.72 (t, 2H, J ) 7.30 Hz, CH2);
7.21-7.52 (m, 10H, Ar). Anal. (C17H17N3S‚HBr) C, H, N.
2,3-Diphenyl-5-N-butylimino-2H-[1,2,4]-thiadiazole Hy-
drobromide (7d). Scale: 1.8 mmol. Yield: 0.37 g (54%) as a
white solid. Mp: 206-207 °C. 1H NMR (CDCl3): δ 0.96 (t, 3H,
J ) 7.30 Hz, CH3); 1.43-1.62 (m, 2H, CH2); 1.73-1.87 (m, 2H,
CH2); 3.81 (q, 2H, J ) 6.58 Hz, CH2); 7.27-7.56 (m, 10H, Ar);
9.69 (bs, 1H, HBr). Anal. (C18H19N3S‚HBr) C, H, N.
2,3-Diphenyl-5-N-(3-hydroxypropyl)imino-2H-[1,2,4]-
thiadiazole Hydrobromide (7e). Scale: 5.4 mmol. Yield:
1.75 g (83%) as a white solid. Mp: 165-167 °C. 1H NMR
(CDCl3): δ 1.96-2.05 (m, 2H, CH2); 3.80-3.99 (m, 4H, 2CH2);
7.25-7.54 (m, 10H, Ar); 10.41 (bs, 1H, HBr). Anal. (C17H17N3-
OS‚HBr‚0.3 H2O) C, H, N.
2,3-Diphenyl-5-N-isopropylimino-2H-[1,2,4]-thiadia-
zole Hydrobromide (7f). Scale: 5.7 mmol. Yield: 1.69 g
(79%) as a white solid. Mp: 250-251 °C. 1H NMR (CDCl3): δ
1.42 (d, 6H, J ) 6.58 Hz, CH3); 4.39-4.56 (m, 1H, CH); 7.21-
7.51 (m, 10H, Ar); 10.32 (bs, 1H, HBr). Anal. (C17H17N3S‚HBr‚
0.3 H2O) C, H, N.
(CDCl3): δ 1.47 (d, 6H, J ) 6.58 Hz, 2CH3); 2.43 (s, 3H, CH3);
4.47-4.62 (m, 1H, CH); 7.04-7.57 (m, 9H, Ar); 9.64 (bs, 1H,
HBr). Anal. (C18H19N3S‚HBr‚0.3 H2O) C, H, N.
5-N-Cyclopentylimino-2-(3-methylphenyl)-3-phenyl-
2H-[1,2,4]-thiadiazole Hydrobromide (8g). Scale: 4.0 mmol.
Yield: 1.05 g (63%) as a white solid. Mp: 238-239 °C. 1H NMR
(CDCl3): δ 1.70-2.15 (m, 8H, CH2); 2.38 (s, 3H, CH3); 4.52-
4.71 (m, 1H, CH); 7.02 (s, 1H, Ar); 7.16 (s, 1H, Ar); 7.27-7.40
(m, 5H, Ar); 7.54 (t, 2H, J ) 8.04 Hz, Ar); 9.91 (bs, 1H, HBr).
Anal. (C20H21N3S‚HBr) C, H, N.
5-N-Phenylimino-2-(3-methylphenyl)-3-phenyl-2H-
[1,2,4]-thiadiazole Hydrobromide (8h). Scale: 4.0 mmol.
Yield: 1.25 g (74%) as a white solid. Mp: 219-220 °C. 1H NMR
(CDCl3): δ 2.40 (s, 3H, CH3); 7.04-7.63 (m, 12H, Ar); 7.90 (d,
2H, J ) 8.04 Hz, Ar); 12.40 (bs, 1H, HBr). Anal. (C21H17N3S‚
HBr‚0.7 H2O) C, H, N.
5-N-Benzylimino-2-(3-methylphenyl)-3-phenyl-2H-
[1,2,4]-thiadiazole Hydrobromide (8i). Scale: 4.0 mmol.
Yield: 0.98 g (56%), as an off-white solid. Mp: 206-207 °C.
1H NMR (CDCl3): δ 2.38 (s, 3H, CH3); 4.97 (d, 2H, J ) 3.64
Hz, CH2); 7.05-7.57 (m, 14H, Ar); 10.13 (bs, 1H, HBr). Anal.
(C22H19N3S‚HBr‚0.1 H2O) C, H, N.
Radioligand Binding Assays. For displacement experi-
ments, membranes of CHO cells expressing recombinant
human adenosine A1 receptors (20 µg of protein for [3H]CCPA
and 10 µg of protein for [3H]DPCPX) were incubated at 25 °C
for 60 min with ∼1.0 nM of [3H]CCPA or ∼1.6 nM of
[3H]DPCPX and a fixed concentration or increasing concentra-
tions of the compounds in a final volume of 0.4 mL Tris-HCl
buffer (50 mM, pH ) 7.4 at 25 °C). Nonspecific binding was
measured in the presence of 10 µM DPCPX. Binding reactions
were terminated by dilution with ice-cold 50 mM Tris HCl
buffer. Samples were then filtered through Whatman GF/B
glass-fiber filters using a Brandell cell harvester or a Millipore
manifold. Filters were washed three times with 2-3 mL of
the same buffer. Bound radioactivity was measured in a liquid
scintillation counter (LKB Wallac) after the addition of 3.5 mL
of scintillation liquid (Emulsifier-Safe, Packard).
In kinetic studies, the association of the radiolabeled agonist
[3H]CCPA (1 nM) was started by addition of the membranes
(30 µg) to the radioligand, in the presence or absence of
allosteric modulators. To study dissociation of [3H]CCPA,
membranes were preincubated with [3H]CCPA (1 nM) at
25 °C for 60 min. Dissociation of [3H]CCPA was then initiated
by the addition of 100 µM 8-cyclopentyltheophylline (CPT) in
the presence or absence of allosteric modulators. Samples were
handled as mentioned before.
2,3-Diphenyl-5-N-cyclopentylimino-2H-[1,2,4]-thiadia-
zole Hydrobromide (7g). Scale: 5.4 mmol. Yield: 1.65 g
(76%) as a white solid. Mp: 242-243 °C. 1H NMR (CDCl3): δ
1.68-2.15 (m, 8H, 4CH2); 4.58-4.76 (m, 1H, CH); 7.27-7.56
(m, 10H, Ar); 9.53 (bs, 1H, HBr). Anal. (C19H19N3S‚HBr) C, H,
N.
2,3-Diphenyl-5-N-phenylimino-2H-[1,2,4]-thiadiazole
Hydrobromide (7h). Scale: 5.1 mmol. Yield: 0.95 g (45%)
as a light yellow solid. Mp: 227-228 °C. 1H NMR (CDCl3): δ
7.23-7.60 (m, 13H, Ar); 7.88 (d, 2H, J ) 8.02 Hz); 12.2 (bs,
1H, HBr). Anal. (C20H15N3S‚HBr‚0.3 H2O) C, H, N.
2,3-Diphenyl-5-N-benzylimino-2H-[1,2,4]-thiadiazole Hy-
drobromide (7i). Scale: 5.8 mmol. Yield: 2.21 g (90%) as a
light yellow solid. Mp: 226-227 °C. 1H NMR (CDCl3): δ 4.97
(d, 2H, J ) 5.84 Hz, CH2); 7.27-7.54 (m, 15H, Ar); 10.4 (bs,
1
1H, HBr). H NMR (CDCl3 + 1 drop of D2O): δ 4.97 (s, 2H,
CH2); 7.27-7.88 (m, 15H, Ar). Anal. (C21H17N3S‚HBr) C, H,
N.
5-N-Methylimino-2-(3-methylphenyl)-3-phenyl-2H-
[1,2,4]-thiadiazole Hydrobromide (8a).13 Scale: 3.9 mmol.
Yield: 1.25 g (89%) as a white solid. Mp: 237-238 °C. 1H NMR
(CDCl3): δ 2.37 (s, 3H, CH3); 3.35 (d, 3H, J ) 5.1 Hz, NCH3);
7.00 (m, 1H, Ar); 7.12 (s, 1H, Ar); 7.34 (m, 4H, Ar); 7.53 (m,
3H, Ar); 10.21 (bs, 1H, HBr). Anal. (C16H15N3S‚0.8 HBr‚1.2
H2O) C, H, N.
5-N-Ethylimino-2-(3-methylphenyl)-3-phenyl-2H-[1,2,4]-
thiadiazole Hydrobromide (8b).13 Scale: 5.1 mmol. Yield:
1.45 g (76%) as a white solid. Mp: 226-227 °C. 1H NMR
(CDCl3): δ 1.43 (t, 3H, J ) 7.3 Hz, CH3); 2.37 (s, 3H, CH3);
3.81 (m, 2H, NCH2); 6.98 (m, 1H, Ar); 7.13 (s, 1H, Ar); 7.33
(m, 4H, Ar); 7.53 (m, 3H, Ar); 10.33 (bs, 1H, HBr). Anal.
(C17H17N3S‚HBr‚0.3 H2O) C, H, N.
Statistical Analysis. Binding parameters were estimated
by GraphPAD Prism software (GraphPAD, San Diego, CA).
Data are expressed as mean ( SEM for the number of
experiments indicated.
HPLC Analysis. As eluent, a gradient between (A) aceto-
nitrile 10%, 10 mM acetic acid, 5 mM sodium dodecyl sulfate
in water and (B) acetonitrile 90%, 10 mM acetic acid, 5 mM
sodium dodecyl sulfate in water with a flow of 0.6 mL/min was
used. The gradient was 0 min/40% B, 1 min/40% B, 15 min/
100% B, 18 min/100% B, 18.2 min/40% B, and 20 min/40% B.
Samples were injected dissolved in Tris or eluent A. Com-
pounds were identified on the basis of their retention times
(for 5a tr ) 6.8 min, for 7a tr ) 7.6 min) and typical UV spectra
(from 210 to 360 nm). To convert compounds 7a and 7g into
their precursors, the compound (10-5 M) in Tris buffer was
incubated with cysteine (10-3 M) at room temperature for 10
min.
5-N-Propylimino-2-(3-methylphenyl)-3-phenyl-2H-
[1,2,4]-thiadiazole Hydrobromide (8c). Scale: 4.0 mmol.
Yield: 1.13 g (72%) as a white solid. Mp: 214-215 °C. 1H NMR
(CDCl3): δ 0.86 (m, 3H, CH3); 1.70-1.86 (m, 2H, CH2); 2.40
(s, 3H, CH3); 3.70-3.90 (m, 2H, CH2); 7.08-7.54 (m, 9H, Ar);
9.57 (bs, 1H, HBr). Anal. (C18H19N3S‚HBr) C, H, N.
5-N-Butylimino-2-(3-methylphenyl)-3-phenyl-2H-[1,2,4]-
thiadiazole Hydrobromide (8d). Scale: 2.5 mmol. Yield:
0.63 g (64%) as a white solid. Mp: 170-171 °C. 1H NMR
(CDCl3): δ 0.99 (t, 3H, J ) 7.30 Hz, CH3); 1.42-1.82 (m, 4H,
2CH2); 2.36 (s, 3H, CH3); 3.69-3.83 (m, 2H, CH2); 6.99-7.56
(m, 9H, Ar); 10.32 (bs, 1H, HBr). Anal. (C19H21N3S‚HBr) C, H,
N.
5-N-(3-Hydroxypropyl)imino-2-(3-methylphenyl)-3-
phenyl-2H-[1,2,4]-thiadiazole Hydrobromide (8e). Scale:
2.0 mmol. Yield: 0.95 g (96%) as a white solid. Mp: 180-181
Reaction of compound 7a with CHO cell membranes ex-
pressing hA1 receptors was investigated under the same
conditions as in radioligand binding experiments. To isolate
the product formed from the reaction of 7a and cysteine, a 20
mL solution in Tris was made, containing 10-4 M 7a and 10-3
M cysteine. After 10 min of incubation at room temperature,
the solution was extracted twice with CH2Cl2 (2 mL). The
combined organic phases were dried over Na2SO4 and evapo-
rated under reduced pressure.
1
°C. H NMR (CDCl3): δ 2.02 (s, 1H, OH); 2.36 (s, 3H, CH3);
3.65-3.94 (m, 6H, 3CH2); 7.01-7.52 (m, 9H, Ar); 10.36 (bs,
1H, HBr). Anal. (C18H19N3S‚HBr) C, H, N.
5-N-Isopropylimino-2-(3-methylphenyl)-3-phenyl-2H-
[1,2,4]-thiadiazole Hydrobromide (8f). Scale: 2.5 mmol.
Yield: 1.25 g (80%) as a white solid. Mp: 243-245 °C. 1H NMR

2,3,5-Substituted [1,2,4]-Thiadiazoles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1151
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The same samples were analyzed with an HPLC-MS system
also. As eluent, a gradient between (A) water, (B) acetonitrile,
and (C) 1% TFA in water with a flow of 1.0 mL/min was used.
The gradient was 0 min/30% B, 2 min/30% B, 17 min/90% B,
22 min/90% B, and 30 min/30% B. C was 10% throughout the
gradient. Compounds were identified on the basis of their
retention times and their masses. For 7g tr ) 11.0 min, MS )
322.0; for 5g tr ) 15.7 min, MS ) 324.0; for the product
obtained from the reaction of 7g and cysteine and for the
product obtained from the interaction of 7g with CHO cell
membranes expressing hA1 receptors tr and MS values were
identical with the values obtained for compound 5g.
Acknowledgment. We thank Hans van den Elst for
performing the HPLC-MS experiments.
Supporting Information Available: 13C NMR spectra for
5a-i, 6a-i, 7a-i, and 8a-i and elemental analyses for 7c-i
and 8c-i. This information is available free of charge via the
Internet at http://pubs.acs.org.
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