A new orally bioavailable dual adenosine A2B/A3 receptor antagonist with therapeutic potential

Article abstract of DOI:10.1016/j.bmcl.2005.04.021

The synthesis and SAR of 5-heterocycle-substituted aminothiazole adenosine receptor antagonists is described. Several compounds show high affinity and selectivity for the A_2B and A₃ receptors. One compound (5f) shows good ADME properties in the rat and as such may be an important new compound in testing the current hypotheses proposing a therapeutic role for a dual A_2B/A₃ antagonist in allergic diseases.

Full text of DOI:10.1016/j.bmcl.2005.04.021

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3081–3085
A new orally bioavailable dual adenosine A2B/A3
receptor antagonist with therapeutic potential
Neil J. Press,a,* Roger J. Taylor,a Joseph D. Fullerton,a Pamela Tranter,a Clive McCarthy,b
Thomas H. Keller,c Lyndon Brown,a Robert Cheung,a Julie Christie,a Sandra Haberthuer,a
Julia D. I. Hatto,a Mark Keenan,a Mark K. Mercer,a Nicola E. Press,a Helene Sahri,a
Andrew R. Tuffnell,a Morris Tweeda and John R. Fozardb
aNovartis Institutes for Biomedical Research, Respiratory Diseases Area, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK
bNovartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4056 Basel, Switzerland
cNovartis Institute for Tropical Diseases Pte Ltd, 10, Biopolis Road, #05-01Chromos, Singapore 138670, Singapore
Received 20 December 2004; revised 30 March 2005; accepted 14 April 2005
Available online 3 May 2005
Abstract—The synthesis and SAR of 5-heterocycle-substituted aminothiazole adenosine receptor antagonists is described. Several
compounds show high affinity and selectivity for the A2B and A3 receptors. One compound (5f) shows good ADME properties
in the rat and as such may be an important new compound in testing the current hypotheses proposing a therapeutic role for a dual
A
2B/A3 antagonist in allergic diseases.
Ó 2005 Elsevier Ltd. All rights reserved.
Receptors for adenosine are currently of great interest as
targets for therapeutic intervention due to their ubiqui-
tous distribution throughout the body and their impor-
tant modulatory effects on cell function.1–6 Four distinct
G-protein coupled receptors A1, A2A, A2B and A3 have
been characterised and cloned,7–13 and while A1 and
A2A receptor pharmacology is well established, the role
of the A2B and A3 receptors in human disease remains
unclear. As a consequence, there has been an intensive
effort to identify selective tools to facilitate pharmaco-
logical studies in vitro and in vivo.
involved in asthma and their potential role in the main-
tenance of the inflammatory response remains unclear.
Current theory would suggest that an adenosine A2B
and/or A3 receptor antagonist would be the appropriate
molecular modifier to provide a therapy for asthma-
tics.18,26–30 A2B receptors mediate the synergistic effects
of adenosine and allergen on human mast cells, which
are believed to be involved in adenosine-induced bron-
choconstriction in asthmatics. The role of the A3 recep-
tor is less clear, and a case could be argued for either an
agonist or antagonist being beneficial in treating
inflammation.18,31
We are particularly interested in the role of adenosine as
a mediator in allergic asthma—an area which is cur-
rently receiving renewed attention.5,14–24 The inhalation
of adenosine causes bronchoconstriction in asthmatics,
but not in normal subjects, and there is a significantly
higher concentration of adenosine in the bronchoalveo-
lar lavage fluid of asthma sufferers compared to nor-
mals.25 However, the adenosine receptor subtype(s)
We have previously described the design and synthesis
of aminothiazoles as highly potent and selective adeno-
sine A3 receptor antagonists, which have been used to
probe the role of adenosine in inflammation.32 Investi-
gation of the structure–activity relationship (SAR) pro-
file of adenosine receptor antagonists revealed a
pharmacophore hypothesis that enabled the production
of compounds selective for the A3 receptor.
As previously described,32 we identified compound
(1) (Fig. 1) as a lead from high throughput screening
(HTS) of an in-house library against the A3 recep-
tor. Subsequent structural optimisation led to the
Keywords: Adenosine; A2B; A3; Adenosine receptor antagonist;
Asthma; Aminothiazole.
*
Corresponding author. Tel.: +44 (0)1403 323819; fax: +44 (0)1403
323837; e-mail: neil.press@novartis.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.021

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N. J. Press et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3081–3085
the aim of achieving greater selectivity and affinity for
the A2B and A3 receptors.
As illustrated in Scheme 1, starting from commercially
available acetophenones, bromination followed by Han-
tzsch-type cyclisation with thioureas (obtained by amine
condensation with benzoylisothiocyanate followed by
hydrolysis, as shown in Scheme 2) quickly yielded
aminothiazoles unsubstituted at the 5-position. Treat-
ment with bromine in acetic acid gave the 5-bromoami-
nothiazole as a stable solid. We then found that these
compounds were able to undergo nucleophilic substitu-
tion on the bromine, which allowed replacement of the
5-pyridyl moiety present in 1 and 2 with smaller ring het-
erocycles such as imidazoles and triazoles, linked via a
nitrogen–carbon bond. This is the first example, of
which we are aware, of direct substitution of bromine
on a 2-aminothiazole with aromatic nitrogen heterocy-
cles. While the yield of the final step precluded the use
of this route for large scale synthesis, by this process a
large number of novel aminothiazole compounds was
synthesised and screened for their affinity as adenosine
receptor antagonists. The results observed with some
of the more interesting compounds in our biological as-
says are shown in Table 1.33
Figure 1. Structures of aminothiazole-based adenosine receptor
antagonists.
synthesis of (2), an orally active, selective adenosine A3
receptor antagonist. A series of 4-phenyl-5-pyridyl-
aminothiazoles was synthesised, however, within this
series it was not possible to obtain good potency at
both A2B and A3 receptors whilst maintaining selec-
tivity against A1 and A2A—which is expected to be
necessary in order to achieve a compound with a low
side effects profile. In this communication, we disclose
the continuation of this work, which led to the synthesis
and evaluation of a dual adenosine A2B/A3 receptor
antagonist.
We chose to replace the 2-aminoacetamide moiety of
previously described compounds with pyridine deriva-
tives. This was found to confer improved selectivity
against the A1 and A2A receptors than had been previ-
ously seen (compare 3a with 3b–f). Our pharmacophore
model suggested that a hydrogen bond acceptor on the
amino group was important for A2B and A3 activity,
and consistent with this, all pyridyl derivatives showed
good affinity for these receptor subtypes. Increasing
the basicity of the pyridyl nitrogen by incorporating
an adjacent methyl group further improved both
The aminothiazole moiety provides a scaffold allowing a
range of different substitution patterns at positions 2, 4
and 5. We found that regardless of the groups at the
2- and 5-positions, the 3-cyanobenzene substituent at
the 4-position generally bestowed the highest affinity
for the A2B and A3 receptors, while giving some selecti-
vity against A1 and A2A. This was in line with our previ-
ously described pharmacophore model.32 During the
course of this study, we expanded our SAR by varying
the substitution at positions 2 and 5 on the ring with
Scheme 1. Synthetic scheme for the preparation of compound 3e. Other aminothiazoles are made by analogous routes.
Scheme 2. Example of a thiourea synthesis.

N. J. Press et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3081–3085
Table 1. In vitro activity of antagonists in human adenosine receptor assaysa
3083
Compounds
hA1 Ki (nM)
2840
hA2A Ki(nM)
5610
hA2B KB (nM)
365
hA3 Ki (nM)
3a R = Acetate
3b R = 2-Py
134
24
1760
1530
2020
2570
53
86
62
20
12
3c R = 3-Py
698
304
37
3d R = 4-Py
3780
4160
3e R = 6-Me-2Py
3f R = Pyrazine
>10,000
2090
>10,000
3580
16
4c R = 3-Py
560
8
2700
>10,000
349
92
5
124
44
4e R = 6-Me-2Py
4f R = Pyrazine
46
7
11
5e R = 6-Me-2Py
5f R = Pyrazine
224
175
10
3d
419
10e
197b
1670c
a The data represent the mean of at least two separate experiments, each performed in duplicate.
b SEM = 37 nM (n = 7).
c SEM = 460 nM (n = 6).
d SEM = 0.1 nM (n = 3).
e SEM = 0.3 nM (n = 6).
selectivity and potency (compare 3e with 3b). However,
replacement of pyridine with the substantially less basic
pyrazine (3f) was not notably detrimental to either po-
tency or selectivity.
Three of the compounds exemplified in Table 1 showed
our desired selectivity and affinity profile at the adeno-
sine receptors (3e, 3f and 5f), and were screened further
for appropriate drug-like properties. In this regard, the
mesylate salt of compound 5f, 3-[5-(2-methylimidazol-
1-yl)-2-(pyrazin-2-ylamino)-thiazol-4-yl]-benzonitrile,
was found to be the superior compound. As well as
being inactive against other receptor and enzyme targets
in a broad screening panel, compound 5f had a good in
vivo pharmacokinetic profile in the rat, as shown in
Table 2. The in vitro binding studies were carried out
on human adenosine receptors, and it should be consid-
ered that the affinities and selectivity profile in rat recep-
The SAR around the 5-position on the aminothiazole
ring was rather less well defined, and affinity for the dif-
ferent receptors was very substrate dependent. However,
in general it was seen that affinity to A2B and A3 recep-
tors was maintained with triazole or (methyl) imidazole,
but that the 5-triazole compounds showed greater selec-
tivity against A1 and A2A receptors (compare 3f with 5f
and 4f; and 3e with 4e and 5e).
Table 2. In vivo pharmacokinetics of compound 5f (mesylate salt) in the Wistar rat
T1/2 (min)a
Clearance (mL/min/kg)a
Vss (L/kg)a
Cmax (lM)c
Tmax (min)c
T1/2 (min)c
BAV (%)c
Mean
SEM
29.0
3.13b
94.4
12.3b
3.02
0.64b
0.56
0.23d
39.3
15.0d
191
90.2d
30.2
7.78d
a Compound dosed iv in 2:1 PEG200/0.1 M phosphate buffer (pH 7.4) at 5 lmol/mL (2.28 mg/mL). Dosed at 1 mL/kg.
b n = 3.
c Compound dosed po in water (pH 4) at 15 lmol/mL (6.83 mg/mL). Dosed at 1 mL/kg.
d n = 7.

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N. J. Press et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3081–3085
Scheme 3. Method for the large scale (up to 20 g) synthesis of 5f.
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with compounds in this series that the profile in rat is
maintained in vivo.32
While the synthetic scheme detailed in Scheme 1 was
appropriate for the small-scale production of a number
of aminothiazole analogues, it was necessary to improve
the process in order to synthesise enough material for
this more advanced in vivo screening. To this end the
synthesis detailed in Scheme 3 was elucidated. Thus, it
was found that imidazole addition to 3-(2-bromoacet-
yl)benzonitrile occurred at 45 °C to give the cyclisation
precursor. We found that direct bromination a- to the
ketone followed by reaction with the thiourea was
unsuccessful, however the use of iodine and the thiourea
in pyridine did give the desired product in acceptable
yield. We believe that this reaction probably proceeds
via iodine-mediated oxidative dimerisation of the thio-
urea, followed by addition to the enolate. Salt formation
was achieved in the normal way. By means of this route
we were able to produce up to 20 g of final compound in
three simple steps.
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In conclusion, we have expanded upon the SAR of
aminothiazoles as adenosine receptor antagonists. By
means of novel chemistry, we have synthesised 5-hetero-
cyclic substituted aminothiazoles, which have shown im-
proved selectivity and affinity for the A2B and A3
receptors. One of these compounds, 5f, has a good in
vivo ADME profile when given orally in the rat, and
as a potent, dual A2B and A3 antagonist, will be used
to further develop the hypotheses regarding the role of
these receptors in allergic disease.
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3085
33. Biological assay procedures. (i) A1 receptor assay: A
radioligand binding assay was used with the ligand
[3H]DPCPX and membranes from CHO cells transfected
with the human A1 receptor; (ii) A2A receptor assay: A
radioligand binding assay was used with the ligand [3H]-
ZM241385 and membranes from HEK 293 cells transfec-
ted with the human A2A receptor; (iii) A2B receptor assay:
A reporter gene assay using CHO cells transfected both
with the human A2B receptor and a luciferase-expressing
reporter plasmid was used. Activation of the A2B receptor
by NECA induces cAMP formation which, when bound
to cAMP binding protein, elicits luciferase production
from the reporter plasmid; (iv) A3 receptor assay: A
scintillation proximity assay was used with the ligand
[
125I]-AB-MECA and CHO cells expressing the human
adenosine A3 receptor.