2-Aminothiazoles: A new class of agonist allosteric enhancers of A1 adenosine receptors

Article abstract of DOI:10.1016/S0960-894X(02)00236-6

This report describes the synthesis and structure-activity relationships of a new class of A₁ adenosine receptor agonist allosteric enhancers, 2-aminothiazolium salts. The EC₅₀ of compounds 6a, 6b, 7, and 8 were 0.3, 4.5, 3.8, and 1.2 μM, substantially lower than that of the 'Gold Standard' 2-amino-3-benzoyl thiophene (PD 81,723), which has an EC₅₀ of 38 μM.

Full text of DOI:10.1016/S0960-894X(02)00236-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1563–1566
2-Aminothiazoles: A New Class of Agonist Allosteric
Enhancers of A1 Adenosine Receptors
Mahendra D. Chordia,a,* Lauren J. Murphree,b Timothy L. Macdonald,a
Joel Lindenc and Ray A. Olssona
aDepartment of Chemistry, University of Virginia, Charlottesville, VA 22901, USA
bDepartment of Pharmacology, University of Virginia, Charlottesville, VA 22901, USA
cDepartment of Medicine (Cardiology), University of Virginia, Charlottesville, VA 22901, USA
Received 31January 2002; accepted 16 April 2002
Abstract—This report describes the synthesis and structure–activity relationships of a new class of A1 adenosine receptor agonist
allosteric enhancers, 2-aminothiazolium salts. The EC50 of compounds 6a, 6b, 7, and 8 were 0.3, 4.5, 3.8, and 1.2 mM, substantially
lower than that of the ‘Gold Standard’ 2-amino-3-benzoyl thiophene (PD 81,723), which has an EC50 of 38 mM. # 2002 Elsevier
Science Ltd. All rights reserved.
The four kinds of G protein-coupled adenosine recep-
tors [ARs] (A1, A2A, A2B, and A3) are important physio-
logical regulators in many tissues.1ꢀ3 The A1AR couples
to several effectors, inhibiting adenylate cyclase,
opening K+ channels and stimulating phospholipases.
A1AR density is high in the CNS but lower in kidney
and heart.4 A1AR activation decreases excitatory neu-
rotransmitter release in brain, suppresses pacemaker
activity and AV node conduction in the heart, mediates
preconditioning in heart, brain and other organs, mod-
ulates tubulo-glomerular feedback in the kidney and
increases glucose uptake in adipocytes.
The present study shows that 2-aminothiazole derivatives
are a new class of AE at the human A1AR.
A one-step synthesis7 (Scheme 1) yielded the 2-amino-
thiazolium salts from their respective ketones, thiourea,
and iodine. Workup consisted of concentrated, washed
with ether and treated with water to yield a yellow to
red-brown solid for characterization by 1H and 13C
NMR. The notes8 report general procedure for their
synthesis and spectral characterization data for some of
the new compounds.
Routine synthetic procedures (Scheme 2) afforded some
indanone and tetralone derivatives not commercially
An agonist allosteric enhancer [AE] binds to a site on the
receptor distinct from the orthosteric site that binds the
natural ligand and amplifies the action of the agonist by
stabilizing the agonist-receptor–G protein ternary com-
plex.5 Because the activity of an AE depends on the
presence of the natural ligand, it is tissue- and event-
specific. Bruns6 found that 2-amino-3-aroylthiophenes,
particularly PD 81,273 (Fig. 1), allosterically enhance
binding of agonists to the A1AR. These compounds
were selective for the A1AR, with little or no effect on
agonist binding to A2A-adenosine, M2-muscarinic or a2-
adrenergic receptors. Since hypoxic or ischemic tissues
produce adenosine, a protective metabolite, an AE
might be useful clinically when it is desirable to enhance
the action of endogenous adenosine.
available. Those ketones were characterized by 1H and 13
NMR and their purity was analyzed by TLC and GCMS.
C
DMSO was the solvent for stock solutions of the
thiazolium salts. Binding assays employed membranes
from CHO-K1cells stably expressing recombinant
human A1ARs. Figure 2 summarizes the assay method.9
The first phase consisted of incubating the radiolabeled
agonist, 125I-ABA, with membranes for 120 min. The
second phase consisted of the addition of the candidate
AE and incubation for 5 min, a time sufficient to pro-
duce allosteric enhancement without disturbing equili-
brium 125I-ABA binding. Dissociation of 125I-ABA was
initiated by addition of 50 mM 8-CPT (antagonist) and
50 mM GTPgS. After 10 min, the assay was stopped by
rapid filtration of membranes over glass fiber filters.
*Corresponding author. E-mail: mdc3x@virginia.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00236-6

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M. D. Chordia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1563–1566
Table 1. Structure, yields, and biological data for 2-aminothiazolium
salts
b
Compd
Yield (%)
% AE activitya EC50
1
a. n=2, 70
b. n=3, 72
c. n=8, 72
19ꢁ3
NS
19ꢁ4
ND
ND
ND
Figure 1. Structures of PD 81,723 and 125I-ABA (agonist).
2
3
4
a. n=1, 76
b. n=2, 82
c. n=3, 64
91ꢁ3
80ꢁ6
NS
38
17
ND
a. n=1, 93
b. n=2, 80
NS
NS
ND
ND
Scheme 1. General synthesis of 2-aminothiazoliums.
a. n=1, 72
b. n=2, 71
66ꢁ4
78ꢁ3
50
8.7
5
6
a. R=C5H11, 76
b. R=C8H17, 79
c. R=C5H5, 70
41ꢁ20*
45ꢁ19*
NS*
ND
ND
ND
Scheme 2. Derivatization of ketones. (a) NaH/RBr, THF, reflux;
(b) RCOCl, NEt3, RT: R0=R or COR.
a. R=CH3, 52
b. R=C5H9, 82
c. R=C6H11, 89
74ꢁ1*
85ꢁ1
NS
0.3
4.5
ND
7
8
9
a. R=CH3, 67
b. R=C6H5, 62
95ꢁ10
54ꢁ2*
3.8
ND
78
76
.278ꢁ11
24ꢁ3
ND
Figure 2. Determination of
% score AE activities. Score for
10
11
12
13
14
80
77
69
60
77
60ꢁ5*
72ꢁ2
81ꢁ3
NS
17
AE(X)=%(AE(X)ꢀNo AE)/(No AntagonistꢀNo AE) measured 10
min after the initiation of dissociation, and ranges from 0 to 100%. In
the examples shown B slows dissociation more than A and has a
higher enhancer score.
4.2
Specific 125I-ABA bound to the A1AR (Fig. 2) is the
difference obtained by subtracting unspecific equili-
brium binding in membranes treated with 8-CPT and
GTPgS but not AE, from residual radioactivity on the
filter. AE activity is the ratio of specific binding at the
end of dissociation to specific binding at the end of 120
min of equilibration, expressed as per centum. % AE
activity values vary from 0%, reflecting no enhance-
ment, to 100%, corresponding to complete prevention
of agonist dissociation.
ND
ND
62ꢁ1
5
ND
a% Score values are means of 2–3 experiments, ꢁSEM, concentration
of AE 50 mM and in some cases where * is mentioned concentration is
5 mM (NS=not significantly active).
Table 1summarizes the chemical and biological data.
Figure 3 compares dose–response curves for two of the
thiazolium salts with that of PD 81,273.
bEC50 values are in mM concentrations; ND, not done.

M. D. Chordia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1563–1566
1565
(15), 29 (3.1)%, respectively. These compounds are not
yet tested for AE activities against other receptor types
such as adrenergic and muscarinic. Further studies are
progressing in that direction will be reported in due
course.
2-Aminothiazolium salts are a new class of allosteric
enhancers of A1ARs distinct from the 2-amino-3-aroyl
thiophenes. Some of these compounds are more potent
than PD 81,723.
Acknowledgement
Figure 3. Typical dose–response curves.
Supported by grant number RO1-HL56111 from the
National Institutes of Health.
An established synthesis gave new thiazolium salts 4a,
5a–b, 6a–c, 9, 10, 11, and 12 for this investigation. The
new compounds have simple structures, contain a vari-
ety of functional groups, and can be easily transformed
into a variety of analogues. The only structural feature
that these 2-amino thiazolium salts share with PD
81,723 is a five-membered aromatic ring containing sul-
fur. The 2-aminothiazolium salts lack the 3-aroyl moi-
ety thought to be important for the AE activity of
2-amino-3-aroyl thiophenes6 and yet were potent allo-
steric enhancers at the A1AR. Indeed, some were sub-
stantially more potent than PD 81,723. Examples are:
4b, 8.7 mM; 6a, 0.3mM; 6b, 4.5mM; 7a, 3.8 mM; 8, 1 .2
mM, and 12, 4.2 mM.
References and Notes
1. Poulsen, S. E.; Quinn, R. J. Bioorg. Med. Chem. 1998, 6,
619.
2. Stehle, J. H.; Rivkees, S. A.; Lee, J. J; Weaver, D. R.;
Deeds, J. D.; Reppert, S. M. Mol. Endocrinol. 1992, 6, 384.
3. Romano, F. D.; Dobson, J. G. Life Sci. 1996, 58, 493.
4. Fredholm, B. B.; Arslan, G.; Halldner, L.; Kull, J.; Schulte,
G.; Wasserman, W. Naunyn-Schmiedeberg’s Arch. Pharmacol.
2000, 362, 364.
5. Henis, Y. I.; Kloog, Y.; Sokolovsky, M. In The Muscarinic
Receptors; Brown, J. H., Ed.; Humana: Clifton, 1989; p 377.
6. Bruns, R. F.; Fergus, J. H. Mol. Pharmocol. 1990, 38, 939.
7. King, L. C.; Hlavacek, R. J. J. Am. Chem. Soc. 1950, 72,
3722.
The structure–activity profile of the 2-aminothiazolium
salts indicates that bicyclic 2-aminothiazolium deriva-
tives have less AE activity than tricyclic salts (compare
1a–c with 2a–c). Among the tricyclic derivatives the
potency order of allosteric enhancement is 6:5:5 (2a)
ꢂ6:6:5 (2b)>6:7:5 (2c).
8. General procedure for synthesis of 2-aminothiazolium salts:
Thiourea (3–4 mmol) and iodine (1.1 mmol) were added to a
solution of ketone (1.0 mmol) in absolute ethanol (2 mL). The
mixture was heated for 2–3 h in an open vessel on oil bath at
100 ꢃC. Heating evaporated the ethanol. The crude residue was
washed with ether (3ꢄ5 mL) and recrystallized from hot
water. Drying under reduced pressure yielded yellow to red-
brown solids. A few of these compounds in free amine form
are reported in literature and their structural characterization
was not carried out thoroughly for example compounds 1a–b,
2a–c, 3a–b, 4a, 8 and 14. 1H and 13C-spectral data for selected
compounds: Compound 4a, 5-methoxy-8H-indeno[1,2-d]thia-
zol-2-ylamine hydroiodide: 1H NMR (DMSO-d6) d 3.70 (s,
2H, CH2), 3.76 (s, 3H, OCH3), 6.82 (dd, J=2.4, 7.8 Hz, 1H),
7.37 (d, J=2.4 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 9.5 (br s).
Compound 8, 5,6-dimethoxy-8H-indeno[1,2,-d]thiazole-2-yla-
Thus, planarity of the overall skeleton and the dihedral
angle between the thiazole ring and the aromatic ring
might be important. Exchanging the positions of nitro-
gen and sulfur as in case of derivatives 2a–b and 3a–b
has a marked effect on enhancer activity, suggesting that
the disposition of the nitrogen is important for mole-
cular recognition. Steric hindrance exerted by the
phenyl groups of 3a–b is an alternative explanation for
the difference in AE activity. An electron donor group
on the aromatic ring improves allosteric enhancer
activity (compare 2b with 4b).
1
mine hydroiodide: H NMR (DMSO-d6) d 3.68 (s, 2H, CH2),
3.77 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 7.20 (1H, CH, ArH),
7.25 (s, 1H, ArH), 9.00 (br s); 13C NMR (DMSO-d6) d 33.6,
55.6, 55.8, 102.4, 109.8, 119.1, 137.8, 147.6, 148.0, 173.7.
Compound 9, 5,6,7-trimethoxy-8H-indeno[1,2-d]thiazol-2-yla-
Compounds 6a, 7a, and 8 are interesting as lead com-
pounds for additional AE’s. Although more informa-
tion is needed to define the structure–activity rules more
precisely, the discovery of AE activity in 2-aminothi-
azoles advances our understanding of these rules.
1
mine hydroiodide: H NMR (DMSO-d6) d 3.73 (s, 2H, CH2),
3.76 (s, 3H, OCH3), 3.81(s, 3H, OCH ), 3.93 (s, 3H, OCH3),
3
7.11 (s, 1H, ArH), 8.72 (br s); 13C NMR (DMSO-d6) d 34.0,
56.3, 60.6, 61.5, 106.3, 118.5, 119.2, 140.2, 141.2, 144.7, 152.7,
174.4. Compound 10, 4,5,6-trimethoxy-8H-indeno[1,2-d]thia-
zol-2-ylamine hydroiodide: 1H NMR (DMSO-d6) d 3.73 (s,
2H, CH2), 3.76 (s, 3H, OCH3), 3.81(s, 3H, OCH 3), 3.93 (s, 3H,
OCH3), 7.11 (s, 1H, ArH), 8.72 (br s); 13C NMR (DMSO-d6) d
31.4, 56.1, 60.2, 60.6, 98.6, 121.2, 128.2, 128.8, 139.6, 143.1,
149.5, 153.2, 173.8. Compound 11, 6-methyl-8H-indeno[1,2-
d]thiazol-2-ylamine hydroiodide: 1H NMR (DMSO-d6) d 2.36 (s,
3H), 3.73 (s, 2H, CH2), 7.07 (dd, 1H, J=1.5, 7.8 Hz, ArH), 7.38
(d, 1H, J=1.5 Hz, 7.42 (d, 1H, J=7.8 Hz, ArH), 9.03 (br s);
A few of these compounds were tested for their subtype
selectivities within adenosine receptors subfamily and
were found to be more potent and highly efficacious on
A1 compared to their A2A and A3 AR counterparts. For
example compounds 4b, 6a, 7a, and 8 displayed AE
maximal scores (pEC50 value) at A1 94 (8.7), 80 (0.3), 92
(3.8), 99 (1.2)% as against at A2A 18 (30), 19 (1.8), 24
(23), 36 (12)% and at A3 8 (not determined), 22 (1.8), 30

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M. D. Chordia et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1563–1566
13C NMR (DMSO-d6) d 21.1, 33.6, 118.8, 122.1, 124.8, 126.4,
132.9, 136.1, 142.3, 142.6, 173.9. Compond 12, 4-methyl-8H-
Hz, ArH), 9.02 (br s); 13C NMR (DMSO-d6) d 18.0, 32.8, 116.0,
121.6, 127.0, 127.2, 132.4, 133.9, 143.2, 143.8, 173.8.
indeno[1,2-d]thiazol-2-ylamine hydroiodide: (DMSO-d6)
2.33 (s, 3H, CH3), 3.70 (s, 2H, CH2), 7.09 (d, J=7.8 Hz, 1H,
ArH), 7.27 (t, 1H, J=7.5, 7.8 Hz, ArH), 7.40 (d, 1H, J=7.5
d
9. Tranberg, C. E.; Zickgraf, A.; Giunta, B. N.; Luetjens, H.;
Figler, H.; Murphree, L. J.; Falke, R.; Fleischer, H.; Linden, J.;
Scammells, P. J.; Olsson, R. A. J. Med. Chem. 2002, 45, 382.