Design, synthesis and evaluation of N6-substituted 2-aminoadenosine-5′-N-methylcarboxamides as A3 adenosine receptor agonists

Article abstract of DOI:10.1039/c3md00364g

A series of N⁶-substituted 2-aminoadenosine-5′-N- methylcarboxamides were synthesized from the versatile intermediate, O ⁶-(benzotriazol-1-yl)-2-amino-2′,3′-O- isopropylideneinosine-5′-N-methylcarboxamide (1). These compounds were evaluated as A₃ adenosine receptor agonists in terms of their potency and receptor sub-type selectivity in a cAMP accumulation assay and a number of potent and selective compounds were identified. One such compound, 2-amino-N⁶-(3-chlorobenzyl)adenosine-5′-N-methylcarboxamide (3i), was over 500-fold selective for the A₃AR over the other three adenosine receptor subtypes. This represents a significantly greater selectivity profile compared with the prototypical IB-MECA.

Full text of DOI:10.1039/c3md00364g

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CONCISE ARTICLE
6
Design, synthesis and evaluation of N -substituted
0
2
-aminoadenosine-5 -N-methylcarboxamides as A
3
Cite this: Med. Chem. Commun., 2014,
, 192
5
adenosine receptor agonists†
a
b
a
Shane M. Devine, Lauren T. May and Peter J. Scammells*
6
0
A series of N -substituted 2-aminoadenosine-5 -N-methylcarboxamides were synthesized from the versatile
6
0
0
0
intermediate, O -(benzotriazol-1-yl)-2-amino-2 ,3 -O-isopropylideneinosine-5 -N-methylcarboxamide (1).
These compounds were evaluated as A adenosine receptor agonists in terms of their potency and
receptor sub-type selectivity in a cAMP accumulation assay and a number of potent and selective
3
Received 29th November 2013
Accepted 18th December 2013
6
0
compounds were identified. One such compound, 2-amino-N -(3-chlorobenzyl)adenosine-5 -N-
methylcarboxamide (3i), was over 500-fold selective for the A AR over the other three adenosine
3
DOI: 10.1039/c3md00364g
receptor subtypes. This represents a significantly greater selectivity profile compared with the
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prototypical IB-MECA.
crystal structure with a bound antagonist (ZM241385) has been
reported.
Increasingly, this has resulted in greater insights aimed at
Introduction
11
Nitrogen-containing heterocycles are ubiquitous in nature,
including purine nucleosides such as adenosine and guano- exploiting this information through chemical modulation of
sine. Adenosine is involved in a host of physiological processes putative small molecules targeting this receptor, by the identi-
through interaction with adenosine receptors, of which there cation of selective A2AAR agonists, such as UK-432097
1
2,13
are four distinct receptor subtypes, namely A
1
, A2A, A2B and A
3
(Fig. 1).
A common feature of these molecules is the incor-
1,2
adenosine receptors (ARs). This family A or rhodopsin-like poration of bulky groups substituted through the 2-position,
purinergic G protein-coupled receptor (GPCR) mediates a typically with retention of the N -amino group present in the
number of important biological functions and agonists acting parent molecule, adenosine, as seen by CGS-21680. However,
6
14
specically at the A AR have been shown to be involved in retention of a 2-amino group, such as that present in guanosine,
3
3
cancer, inammation and cardioprotection. Many pharmaco- has not been explored to determine the selectivity prole and
logically relevant A
range of potential therapeutic applications. Selected examples sine receptor subtypes. We envisaged retaining an N -bulky
are shown in Fig. 1. IB-MECA is currently in clinical trials for substituent known to give rise to A AR selectivity, whilst the
psoriasis, rheumatoid arthritis, dry eye disease and glaucoma. added 2-amino group present could potentially lead to the
3
AR agonists are known and have a diverse affinity for this series of molecules at the four distinct adeno-
6
3
The structurally related Cl-IB-MECA has potential indications identication of an A
3
AR selective molecule. Another modi-
4
for hepatitis and liver cancer. Meanwhile, CP-532,903 has anti- cation tolerated in known A2AAR agonists is that of the incor-
5
0
12,13
inammatory effects and is involved in myocardial ischemia
and reducing superoxide production in damaged tissues.
poration of an 5 -ethylcarboxamido group,
whereas tuning
6
the selectivity towards the A
3
AR might be achieved by adapta-
0
With the recent emergence of GPCR crystal structure deter- tion of the 5 -position. From these scaffolds and other known
mination and the realisation that the seven transmembrane
7TM) topology is common throughout many family A GPCRs, in A
A
3
AR agonists, a few key qualities demonstrate improvements
AR selectivity over the other subtypes. A signicant feature
such as the CXCR4 chemokine, D3 dopamine, and b1 and b2 that imparts selectivity towards A AR specicity is the inclusion
(
3
7
8
3
9
,10
0
adrenergic receptors, a new frontier has opened up in GPCR of a 5 -N-methylcarboxamido moiety, as demonstrated in Fig. 1.
6
drug discovery. Highlighting this paradigm shi, the A AR Incorporation of an N -substitution comprising benzyl func-
2
A
3
tionality imbued with a 3-halo moiety gives enhanced A AR
selectivity as well. The addition of a 2-substituted halogen,
typically a chloro group engenders even greater activity at the
A3AR, however anything bulkier such as pyrazolo or alkyne
linked groups as demonstrated by Regadenoson and Apadeno-
a
Medicinal Chemistry, Monash University, Parkville, Victoria 3052, Australia. E-mail:
peter.scammells@monash.edu
b
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash
University, 381 Royal Parade, Parkville, Victoria 3052, Australia
15–17
son impart A AR selectivity.
Whilst the effect of a range of
2
A
†
Electronic supplementary information (ESI) available: Experimental methods,
2-substituents on adenosine receptor affinity has been
adenosine receptor agonist synthesis, cAMP assay. See DOI: 10.1039/c3md00364g
192 | Med. Chem. Commun., 2014, 5, 192–196
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Fig. 1 Pharmacologically relevant AR agonists.
18
0
0
studied, there is a paucity of information on the inuence of 2- 5 -carboxylic acid. The 5 -methylcarboxamide was subsequently
amino and substituted 2-amino groups. Our study addresses achieved via esterication with thionyl chloride in methanol
this shortfall in understanding.
2
followed by nucleophilic displacement with MeNH . Finally,
reaction with BOP, in the presence of DBU introduced the
6
desired O -benzotriazol-1-yl moiety (compound 1). This versa-
Chemistry
0
tile intermediate retains the highly A
3
AR selective 5 -N-methyl-
As part of our ongoing program to develop potent and selective carboxamido functionality and is set up for nucleophilic attack
6
6
adenosine agonists, we recently described the synthesis of O - at the 6-position with its incorporation of a labile O -(bentriazol-
0
0
0
(benzotriazol-1-yl)-2-amino-2 ,3 -O-isopropylideneinosine-5 -N-
1-yl) motif.
A host of nitrogen-bearing nucleophiles were selected to
demonstrate the pharmacological character of this class of
methylcarboxamide (1) using a phosphonium-mediated coupling
19,20
approach via C–OH activation of guanosine over 5 steps.
0
0
6
0
Firstly, the 2 ,3 -diol moiety present in guanosine was pro- N -substituted, 2-aminoadenosine-5 -N-methylcarboxamides.
tected as the corresponding 2 ,3 -isopropylidene group with Firstly, N -substitution occurred by reaction of 1 with various
acetone, p-tosic acid and dimethoxypropane, greatly improving amines in the presence of a non-nucleophilic organic tertiary
solubility for further chemical manipulation (Scheme 1). amine base (DIPEA) in t-BuOH to give the 2 ,3 -O-isopropylidene
Oxidation using TEMPO and BAIB afforded the corresponding protected N -substituted adenosine-5 -N-methylcarboxamides
0
0
6
0
0
6
0
ꢀ
19
ꢀ
19
ꢀ
ꢀ
19
Scheme 1 (CH
iv) 2.0 M MeNH
2
3
)
2
C(OCH
3
)
2
, 25 C, 16 h; 94%; (ii) TEMPO, BAIB MeCN/H
2
O (1 : 1), 25 C, 16 h, 72%; (iii) SOCl
2
20
, MeOH 0 C / 25 C, 16 h, 61%;
ꢀ
20
ꢀ
ꢀ
(
2
in THF, MeOH/DMF (9 : 1), 100 C, MW, 2 h, 100%; (v) BOP, DBU, MeCN, 25 C, 16 h, 92%; (vi) R–NH
h; (vii) MeCN/1 M HCl (4 : 1), 60 C, 1 h, 58–78%.
2
, t-BuOH, DIPEA, 80 C,
ꢀ
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2a–l). Secondly, deprotection of the diol took place under hot
Concise Article
(
aqueous acidic conditions; aided by the inclusion of MeCN, to
6
facilitate solubilisation of the reagents. This generated N -
0
substituted 2-aminoadenosine-5 -N-methylcarboxamides (3a–l)
in moderate to good yields (ranging from 58–78% over 2 steps).
These compounds were subsequently evaluated in a cAMP
functional assay (Table 1).
A small series of N-alkyl and N,N-dialkylamino analogs
were prepared in order to assess the inuence of these
substituents on AR potency and receptor subtype selectivity.
6
These compounds were prepared from O -(benzotriazol-1-yl)-
2
-uoroinosine derivative 4 (Scheme 2). Installation of the
6
6
N -cyclopentyl group was achieved by reaction of O -(benzo-
0
0
0
triazol-1-yl)-2-uoro-2 ,3 -O-isopropylideneinosine-5 -N-methyl-
carboxamide (4) with cyclopentyl amine in t-BuOH with DIPEA.
A second substitution at the 2-position was introduced by
heating the required amine at 150 C under microwave
conditions, displacing the uorine. Deprotection of the 2 ,3 -O-
isopropylidene was achieved with 1 M HCl and MeCN to Scheme 2 Reagents and conditions: (i) cyclopentylNH
ꢀ
0
0
2
, t-BuOH,
DIPEA, 80 C; (ii) R NH or RNH , t-BuOH, 150 C (iii) MeCN/1 M HCl
ꢀ
ꢀ
6
0
2
2
give N -(cyclopentyl), 2-N-substituted adenosine-5 -N-methyl-
carboxamides (7).
ꢀ
(4 : 1), 60 C, 48–72%.
Table 1 pEC50 data (cAMP accumulation assay) of compounds 3a–l at all AR subtypes
R
pEC50
A
1
AR
pEC50
A
2AAR
pEC50
A
2BAR
pEC50
A
3
AR
A
3
/A
0
1
A
3
/A2A
A
3
/A2B
1
NECA
IB-MECA
CGS21680
—
—
—
–Me
–Et
–Pr
–Bu
8.9 ꢁ 0.2
7.5 ꢁ 0.2
6.3 ꢁ 0.2
5.4 ꢁ 0.3
5.6 ꢁ 0.2
6.5 ꢁ 0.1
6.1 ꢁ 0.2
7.9 ꢁ 0.2
7.1 ꢁ 0.1
7.9 ꢁ 0.1
<5
<5
<5
8.5 ꢁ 0.3
7.5 ꢁ 0.2
7.4 ꢁ 0.2
5.8 ꢁ 0.4
5.8 ꢁ 0.3
5.3 ꢁ 0.1
5.9 ꢁ 0.3
8.5 ꢁ 0.2
9.3 ꢁ 0.2
8.0 ꢁ 0.3
8.3 ꢁ 0.2
8.0 ꢁ 0.2
8.0 ꢁ 0.2
7.4 ꢁ 0.2
4
158
1
63
50
794
251
32
63
4
316
158
501
32
3
3
3
3
a
b
c
>1995
>1000
>1000
>251
d
<5
20
3
e
f
7.2 ꢁ 0.1
5.6 ꢁ 0.3
5.9 ꢁ 0.2
6.7 ꢁ 0.3
8.3 ꢁ 0.2
13
501
40
3
7.4 ꢁ 0.1
6.1 ꢁ 0.3
7.8 ꢁ 0.4
3
79
50
3
3
g
7.2 ꢁ 0.1
5.2 ꢁ 0.2
6.1 ꢁ 0.2
5.5 ꢁ 0.2
5.6 ꢁ 0.2
6.9 ꢁ 0.2
6.9 ꢁ 0.2
7.5 ꢁ 0.4
1
6
20
4
h
200
100
3
3
i
j
6.5 ꢁ 0.1
6.4 ꢁ 0.2
6.4 ꢁ 0.3
9.2 ꢁ 0.4
501
158
631
158
631
100
6.6 ꢁ 0.2
6.6 ꢁ 0.3
6.8 ꢁ 0.3
8.8 ꢁ 0.3
3
3
k
l
–CH
–CH
2
CH
CHPh
2
Ph
6.5 ꢁ 0.2
6.4 ꢁ 0.2
7.1 ꢁ 0.1
6.1 ꢁ 0.2
6.8 ꢁ 0.3
6.7 ꢁ 0.2
8.4 ꢁ 0.3
8.3 ꢁ 0.3
79
79
20
158
40
40
2
2
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with IB-MECA's activity. Interestingly, the A
escalated signicantly with 501-, 631- and 631-fold selectivity
, A2A and A2BAR respectively. This also equates to an
AR selectivity (Table 1). In some 8-, 4- and 10-fold increase at the A , A2A and A2BAR, respectively,
cases, large selectivity increases were seen, with four over IB-MECA, thus representing a highly selective molecule.
compounds showing more than 100-fold selectivity for the A
AR The 3-iodobenzyl derivative (3j), was also highly A AR selective
over all of the other receptor subtypes (namely compounds 3a, and more selective than IB-MECA with a slight reduction in
b, 3i and 3j).
potency (pEC50 ¼ 8.8).
The 2-aminoadenosine-5 -N-methylcarboxamides with alkyl
Bulkier substituents attached at the N -position, as exhibited
3
AR selectivity
Pharmacology
6
0
In general the N -substituted 2-aminoadenosine-5 -N-methyl- over the A
1
carboxamides demonstrated A
3
1
3
3
3
0
6
6
groups in the N -position (compounds 3a–d) had relatively by the phenethyl (3k) and diphenylethyl (3l) compounds had
6
similar potencies with pEC50's ranging from 7.4–8.3. N -methyl similar potency to NECA (pEC50 ¼ 8.4, for 3k and pEC50 ¼ 8.3,
containing 3a, exhibited ꢂ800 fold selectivity for A
AR over the for 3l, compared with NECA, pEC50 ¼ 8.5). Unlike NECA
3
A
1
AR and 300-fold selectivity over A2BAR, exhibiting the highest however, 3k and 3l were 40-fold and 79-fold selective over A
1
and
potency of the alkyl chain derivatives with a pEC50 of 8.3.
A
2BAR for the A AR. A2AAR selectivity was even greater for 3l,
3
Increasing the carbon chain from methyl to butyl (3a–d), demonstrating a ꢂ150-fold selectivity prole, compared with
6
maintained activity, but decreased the A AR character of these 20-fold for 3k. This series of N -substituted, 2-aminoadenosine-
3
0
molecules resulting in a reduction in selectivity.
5 -N-methylcarboxamides, personied by 3i are highly effica-
6
In terms of the saturated cycloalkyl N -substituents (3e–g), cious A
the cyclobutyl (3e), had a marked effect in terms of A AR over
To determine the effect of the 2-amino group on potency and
2AAR selectivity, with a 500-fold increase and a pEC50 of 8.3, selectivity a small series of N -substitued molecules were
AR selective molecules, worthy of further study.
3
3
2
A
2
comparable with the best of the n-alkyl derivatives, namely 3a. prepared (Scheme 2). Installation of a substituent at the N -
As ring size increased from cyclobutyl to cyclohexyl, A
AR potency position greatly reduced activity in comparison to the parent 3f,
decreased (pEC50 for 3e, 3f and 3g ¼ 8.3, 7.8 and 6.9, respectively). indicating the absolute necessity for retention of the 2-amino
A corresponding reduction in A AR selectivity versus all other group for A AR selectivity. The N-methyl (7a) and N-ethyl (7c)
receptor subtypes was generally observed with increasing ring had very similar affinities at the A AR and the A AR, but only
AR selectivity. The N,N-dimethyl (7b) and N,N-diethyl
AR, as N -cyclo- (7d) had further reduced activity (Table 2).
AR selective molecule.
However, 3f did show a striking difference in A2AAR and A2BAR
3
3
3
1
3
6
size. Unsurprisingly, the N -cyclopentyl group in 3f did not modest A
3
6
display great selectivity between A
3 1
AR and A
pentyladenosine (CPA) is a potent A
1
Conclusion
affinity, with 79-fold and 50-fold A
3
AR selectivity, respectively.
6
The inclusion of N -benzyl (3h) demonstrated the typical
6
0
A
series of N -substituted 2-aminoadenosine-5 -N-methyl-
increased A AR selectivity prole expected, known when benzyl
3
carboxamides were designed and synthesised and their phar-
macological properties were evaluated in a cAMP accumulation
assay. A number of these compounds proved to be potent,
6
moieties are incorporated into adenosine compounds at the N -
position. This however, was coupled with relatively modest
6
activity with a measured pEC50 of 7.5. 2-Amino-N -(3-chlor-
6
selective A AR agonists and in particular 2-amino-N -(3-chlor-
3
0
obenzyl)adenosine-5 -N-methylcarboxamide (3i) showed
a
0
obenzyl)adenosine-5 -N-methylcarboxamide (3i) and 2-amino-
substantial increase in pEC50 to 9.2, which is commensurate
6
0
N -(3-iodobenzyl)adenosine-5 -N-methylcarboxamide
(3j)
6
0
Table 2 pEC50 (cAMP accumulation assay) of 2-N-substituted N -(cyclopentyl)adenosine-5 -N-methylcarboxamides at all AR subtypes
1
2
R
R
pEC50
A
1
AR
pEC50
A2AAR
pEC50
A2BAR
pEC50
A
3
AR
A
3
/A
1
A
3
/A2A
A
3
/A2B
NECA
IB-MECA
CGS21680
—
—
—
H
H
Me
H
—
—
—
H
Me
Me
Et
8.9 ꢁ 0.2
7.5 ꢁ 0.2
6.3 ꢁ 0.2
7.4 ꢁ 0.1
6.4 ꢁ 0.1
<5
7.9 ꢁ 0.2
7.1 ꢁ 0.1
7.9 ꢁ 0.1
5.9 ꢁ 0.2
5.4 ꢁ 0.2
5.3 ꢁ 0.4
6.3 ꢁ 0.1
<5
8.5 ꢁ 0.3
7.5 ꢁ 0.2
7.4 ꢁ 0.2
6.1 ꢁ 0.3
<5
<5
<5
<5
8.5 ꢁ 0.2
9.3 ꢁ 0.2
8.0 ꢁ 0.3
7.8 ꢁ 0.4
7.0 ꢁ 0.2
6.2 ꢁ 0.3
7.4 ꢁ 0.2
5.6 ꢁ 0.3
0
4
158
1
79
40
8
1
63
4
63
50
3
4
>16
8
3
7
7
7
7
f
50
a
b
c
d
>100
>16
>251
>4
6.3 ꢁ 0.2
8
>4
Et
Et
<5
>4
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showed massive selectivity increases over the other three
receptor subtypes. These compounds also had similar potency,
but much greater selectivity over IB-MECA, the classic selective
A3AR agonist.
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Acknowledgements
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