Development of polar adenosine A2A receptor agonists for inflammatory bowel disease: Synergism with A2B antagonists

Article abstract of DOI:10.1021/ml200189u

Adenosine A_2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects. 4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5- (hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments. Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid and showed synergism with an A_2B-selective antagonist. Thus, nonabsorbable, locally active A_2A agonists, as a monotherapy or in combination with an A_2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of known A _2A agonists.

Full text of DOI:10.1021/ml200189u

Letter
pubs.acs.org/acsmedchemlett
Development of Polar Adenosine A2A Receptor Agonists for
Inflammatory Bowel Disease: Synergism with A2B Antagonists
Ali El-Tayeb,†,§ Sebastian Michael,‡ Aliaa Abdelrahman,† Andrea Behrenswerth,† Sabrina Gollos,†
Karen Nieber,‡ and Christa E. Muller*,†
̈
†PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121
Bonn, Germany
‡University of Leipzig, Institute of Pharmacy, Talstrasse 33, D-04109 Leipzig, Germany
S
* Supporting Information
ABSTRACT: Adenosine A2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and
synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects.
4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)-
benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments.
Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid
and showed synergism with an A2B-selective antagonist. Thus, nonabsorbable, locally active A2A agonists, as a monotherapy or in
combination with an A2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of
known A2A agonists.
KEYWORDS: A2A receptor agonist, inflammatory bowel disease, anti-inflammatory drug, nonabsorbable A2A receptor agonist,
A2B receptor antagonist
denosine receptors (AR) are G protein-coupled receptors.
A
Four subtypes have been cloned and characterized, A1, A2A,
A2B, and A3.1 In brain, A2AAR are expressed in high density in
the striatum, while in the periphery, A2AAR are highly expressed
in the intestinal mucosa, enteric neurons, hepatocytes, and a
variety of immune cells. In the intestine, A2AAR are expressed in
the jejunum, ileum, and cecum. They are coupled to Gs
proteins and thereby activate adenylate cyclase.1,2 Crystal
structures of the human A2A receptor in complex with agonists,
including adenosine (1) and NECA (2),3,4 and also with an
antagonist5 have recently been published. A2A-selective AR
Figure 1. Structures of selected A2A adenosine receptor agonists and a
selective A2B antagonist.
agonists [for example, 3 and 4 (Figure 1)] typically possess a
large substituent at position 2 of the adenosine core.
including Crohn’s disease, colitis, and irritable bowel disease, as
well as other inflammatory conditions.8−11
However, the systemic use of A2A adenosine receptor
agonists as anti-inflammatory drugs is limited by their potent
hypotensive activity caused by the activation of A2AAR
expressed in heart and blood vessels.1,7
Activation of the A2AAR on a variety of inflammatory cell
types leads to anti-inflammatory effects that can attenuate
injury as a result of mucosal inflammation, ischemia, or sepsis.
Adenosine, being part of the innate immune system, is one of
the strongest endogenous immunosuppressive agents. There-
fore, A2AAR agonists have been suggested as novel anti-
inflammatory drugs.2,6,7 Inflammatory bowel disease (IBD) is
an inflammatory condition in the gastrointestinal tract. A2AAR
agonists have had beneficial effects on the development of
intestinal inflammation in a variety of animal models of IBD,
Received: August 8, 2011
Accepted: October 10, 2011
Published: October 10, 2011
© 2011 American Chemical Society
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a
Scheme 1. Synthesis of the Target Adenosine Derivatives
a
Reagents and conditions: (a) three steps, (1) H2O2, CH3COOH; (2) 5 N aq. NaOH; (3) CS2, MeOH, H2O, 120 °C autoclave, 5 h; (b) 4-(2-
bromoethyl)benzenesulfonic acid for compound 7, 4-(2-bromoethyl)benzoic acid for compound 9, and bromoacetic acid for compound 10, H2O/
NaOH, rt, 4−9 h; (c) m-chloroperbenzoic acid, ethanol, 0−5 °C → room temperature, 12 h.
A recent strategy for achieving a certain drug targeting effect
has been the preparation of phosphorylated adenosine-derived
A2AAR agonists (AMP derivatives), which would be preferably
dephosphorylated in inflamed tissues with a high level of
expression of ecto-5′-nucleotidase.12 This is thought to allow a
separation of anti-inflammatory and hypotensive effects because
the concentration of the active drug, the A2AAR agonist, would
be achieved only locally at the sites of inflammation with high
ecto-5′-nucleotidase activity.12 An additional approach to
preventing side effects has been local inhalation therapy for
the treatment of chronic obstructive pulmonary disease.13
In this study, we pursued yet another approach, the development
of highly polar, perorally nonabsorbable A2A-selective agonists. Such
compounds would serve as a local therapy, e.g., of inflammatory
bowel disease, avoiding the hypotensive effects of centrally acting
anti-inflammatory A2A agonists. Besides, such polar, highly water-
soluble A2AAR agonists would be suitable for parenteral application
(e.g., inhalation and injection), as well. Therefore, we introduced an
acidic function such as a sulfonate or carboxylate moiety into 2-
(ar)alkylthio-substituted adenosine derivatives and evaluated their
To obtain our target compounds, we started from 2-
thioadenosine (6), which was synthesized from adenosine (1)
according to published procedures (Scheme 1; for details, see
the Supporting Information).12,18 2-Alkylated derivatives 7, 9,
and 10 were obtained by reaction of 6 with the corresponding
(ar)alkyl bromide in water in the presence of NaOH as a base
(Scheme 1).12,19 Sulfone derivative 8 was prepared by oxidation
of compound 7 using m-chloroperoxybenzoic acid in ethanol.20
The synthesized 2-substituted (ar)alkylthioadenosine deriv-
atives were investigated in radioligand binding studies of human
and rat A2AAR using the agonist radioligand [3H]CGS21680.21
Selectivity versus other adenosine receptor subtypes was
assessed by performing radioligand binding studies with
human and rat A1AR using [3H]CCPA,22 human A2BAR
using [3H]PSB-603,16 and human A3AR using [3H]PSB-1123
and/or [3H]NECA24 as radioligands (see Table 1).
Analysis of the structure−activity relationships of the
synthesized 2-substituted adenosine derivatives showed that
introduction of a carboxymethyl moiety directly linked to 2-
thioadenosine in compound 10 led to a compound with only
low affinity for A2AAR. The introduction of an acidic moiety
into the para position of the phenyl ring of the previously
described 2-(phenylethylthio)adenosine [Ki(rat A2A) =
18.7 nM]12,25 decreased A2A affinity by only 2-fold in the case
of introduction of a sulfonate moiety [compound 7; Ki(rat A2A) =
44.4 nM] and by 8-fold in case of introduction of a carboxylate
function (compound 9; Ki = 152 nM). The selectivity of both
compounds versus A1AR was markedly increased by the acidic
function [Ki(rat A1) values of ≥10000 nM for 7 and >1000 nM
for 9] in comparison to that of 2-(phenylethylthio)adenosine
[Ki(rat A1) = 180 nM].12
A2A agonistic activity as well as the ability of a selected compound to
reduce the inflammation in an inflamed rat ileum/jejunum
preparation. Because of the low pKa value of free sulfonic acid
groups (pKa < 1), the A2AAR agonists bearing a sulfonate function
will be deprotonated under physiological conditions and expected to
be nonabsorbable;14 therefore, they would constitute a local therapy
for IBD. In contrast to A2A receptors, A2BAR have been shown to
exhibit proinflammatory effects in several organs, including lung and
the intestine.1,2,6,7,15 A2BAR are predominantly expressed in colonic
epithelial cells. They are upregulated in models of intestinal
inflammatory disease,15,16 and A2BAR antagonists have been
suggested as anti-inflammatory drugs.2,7,15 In this study, we therefore
wanted to investigate the possibility of synergism with regard to the
anti-inflammatory effects of a highly polar, not perorally absorbable
A2A-selective agonist in combination with A2BAR antagonist PSB-
601 [5 (Figure 1); Ki(hA2BAR) = 3.6 nM]17 and to determine
their ability to reduce inflammation in ex vivo experiments in an
inflamed rat ileum/jejunum preparation.
Species differences exist as compound 7 exhibited a lower
affinity for human A2A receptors (Ki = 360 nM) compared with
that for rat brain striatal membrane A2A receptors (Ki =
44.4 nM). Surprisingly, compound 9, which was active at rat
A2AAR (Ki = 152 nM), lost its affinity for human A2A receptors
(Table 1). The results were reversed in the case of A1 receptors.
Compound 7, which showed no activity with rat brain cortical
membrane AR (Ki ≥ 10000 nM), was well tolerated at human
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Table 1. Adenosine Receptor Affinities of Adenosine Derivatives Bearing Acidic Functions at the 2-Substituent
Ki ± SEM (nM) (n = 3)
A2B receptor
A3 receptor
[3H]PSB-11
a
A1 receptor [3H]CCPA
A2A receptor [3H]CGS21680
[3H]PSB-603
rat
human
recombinant
rat brain
striatum
human
recombinant
human
recombinant
human
recombinant
g
nucleoside
brain cortex
pKa, log P, and log D (pH 7.4)
2 (NECA)
5.11
13.622
2891
1521
201
271
189016
6.222
log P = −2.00
log D = −2.00
pKa = 4.72
4 (CGS21680) 18001
1821
>1000016
11426
b
,
log P = 0.25
log D = −2.15
pKa = −2.35
log P = 0.31
log D = −2.07
pKa = −2.60
log P = 1.23
log D = −3.60
pKa = 4.23
d
7 (PSB-0777)
≥10000
541 ± 167
545 ± 183
404 ± 197
44.4 ± 2.4
>10000
152 ± 10
360 ± 30
>10000
≫10000, >1000
c
e
8
1800 ± 300
1010 ± 180
1930 ± 612
>10000
nd
>10000
c
d
9
≥10000
>10000
>10000, >1000
log P = 0.78
log D = −2.16
pKa = 3.30
e
f
c
e
10
nd
>10000
>10000
nd
>10000
log P = 1.62
log D = −4.88
a
b
c
d
Antagonist radioligand because an agonist radioligand for A2BAR does not exist. [125I]I-AB-MECA used as a radioligand. n = 2. Versus agonist
e
f
g
radioligand [3H]NECA. Not determined. Versus antagonist radioligand [3H]MSX-2. Calculated by the MarvinSketch program from ChemAxon,
online version; log P was calculated for the nonionic species of the compounds.
Figure 2. (A) Competition curves of compounds 7 and 9 vs 10 nM [3H]CGS-21680 in rat brain striatal membranes. Ki values of 44.4 ± 2.4 nM (7)
and 152 ± 10 nM (9) were determined. (B) Concentration−response curves of 2 and 7 in cAMP accumulation assays using CHO-K1 cells
expressing the human A2AAR (n = 3). EC50 values of 17.6 ± 14 nM (2) and 117 ± 10 nM (7) were determined. All data represent means ± SEM of
three separate experiments performed in triplicate.
A1 receptors with a Ki value of 541 nM. Likewise, the affinity of
compound 9 for human A1 receptors was enhanced (Ki = 404 nM)
compared with that for rat brain A1AR (Ki = 1010 nM).
Oxidation of the sulfur atom in the thioether linkage of
compound 7 to form the corresponding sulfone derivative 8 led
to a loss of affinity for the A2AAR, while the affinity for human
A1AR was retained (Ki = 545 nM). All tested compounds
showed no or negligible affinity for human A2B and A3AR.
As shown in Table 1, the best results were obtained with
compound 7 (PSB-0777), which showed high affinity for the
A2AAR with a Ki value of 44.4 nM and high selectivity (>225-
fold) versus the other AR subtypes. It was superior to the
corresponding carboxylate 9. Selected concentration−inhibition
curves are shown in Figure 2A. Compound 7 showed affinity
for both human and rat A2AAR. Therefore, 7 was selected as a
pharmacological tool for further evaluation to perform proof-of-
principle studies in a model of inflammation. Functional
properties of the new ligand 7 were assessed by adenylate
cyclase assays measuring accumulation of cAMP in CHO cells
stably expressing the human A2AAR. For comparison, the full
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agonist NECA (2) was investigated under the same condition.
Concentration−response curves of 2 and 7 were obtained
showing that 7 acted as an agonist at A2AAR. For NECA, an
EC50 value of 17.6 nM was determined, while 7 showed an
EC50 value of 117 nM (Figure 2B). Thus, 7 exhibited an ∼6-
fold lower potency for human A2AAR expressed in CHO cells
compared with that of NECA. The efficacy of 7 was not
significantly different from that of NECA, indicating that 7 is a
full agonist of A2AAR.
Compound 7 (PSB-0777) was further evaluated in untreated
and inflamed rat ileum/jejunum preparations in ex vivo
experiments.15,27,28 Acetylcholine (Ach, 1 mM)-induced
contractions were assessed in the absence of A2AAR agonist 7
(set at 100%) and in its presence. Agonist 7 increased
concentration-dependently Ach contractions (see the Support-
ing Information). A statistically significant increase of 17.5 ±
5.7% compared to the control (P < 0.05; n = 12) was found at a
concentration of 7 of 10 μM. The increase was prevented by
the A2A antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine
(CSC, 0.2 μM; 89.6 ± 5.2% of control; P > 0.05; n = 12).
Thereafter, 7 (0.1−10 μM) was preincubated simultaneously
with 2,4,6-trinitrobenzenesulfonic acid (TNBS, 10 mM), which
induced acute inflammation. The A2A agonist restored
concentration-dependently the TNBS-induced inhibition
(41.6 ± 3.7%) of the Ach contractions; the effects were
significant at concentrations of 1 and 10 μM [62.7 ± 3.8 and
78.9 ± 3.5% of control, respectively; n = 9 (Figure 3A)].
Comparable experiments were performed with A2B antago-
nist 5 (Figure 3B). Compound 5 (1−100 μM) was without
effect on the Ach contractions in untreated preparations.
However, it reversed concentration-dependently the TNBS-
induced reduction (35.2 ± 2.9%) of the Ach-induced con-
tractions to 53.3 ± 5.7% (10 μM) and 86.1 ± 4.7% (100 μM)
of the control, and this effect was statistically significant.
The combination of 7 (0.1 μM) and 5 (1 μM) each at a
concentration without a significant effect alone was tested in
further experiments. It significantly reduced the TNBS-induced
impairment of Ach contractions (43.6 ± 8.3%) to 65.7 ± 3.8%
of the control (Figure 4; P < 0.05; n = 9).
Figure 4. Effect of the combined preincubation of 7 (0.01 μM) and 5
(1.0 μM) on the TNBS-induced decrease in the 1 mM Ach-induced
contractions in rat ileum/jejunum segments. Means ± SEM of 12
experiments. *P < 0.05 vs control.
In conclusion, we have successfully developed polar A2AAR
agonists. They have been proven to be promising drugs for the
local treatment of inflammatory intestinal diseases and can be
expected to be devoid of hypotensive side effects. Furthermore,
additivity and even potential synergism between the A2A agonist
and A2B antagonist were observed in an ex vivo model.
EXPERIMENTAL PROCEDURES
■
For syntheses, the synthesized 2-thioadenosine12,18 (6, 1 mmol) was
dissolved in 20 mL of water, and 5 mL of sodium hydroxide (0.5 N)
was added to the reaction mixture followed by the addition of 4-(2-
bromoethyl)benzenesulfonic acid for compound 7, 4-(2-bromoethyl)-
benzoic acid for compound 9, or bromoacetic acid for compound 10
(1.2 mmol) 10 min later. The reaction mixture was stirred for 4−9 h at
room temperature, and the completion of the reaction was assessed by
TLC (2:1:1 n-butanol/CH3COOH/H2O). The reaction mixture was
evaporated to dryness under reduced pressure, and the crude product
was crystallized first several times from methanol and then from
ethanol to afford after drying the pure products as a white powder.
For the synthesis of sulfone derivative 8, compound 7 (1 mmol) was
dissolved in ethanol (10 mL) and cooled in an ice bath (0−5 °C), to
which a solution of m-chloroperoxybenzoic acid (4 mmol) was slowly
added. The reaction mixture was then stirred for 12 h at room
temperature. After the completion of the reaction, the solvent was
evaporated under reduced pressure and the residue was recrystallized
several times from methanol to afford the pure product, compound 8.
Purity was confirmed by ESI-LC/MS spectra and found to be >98%
for all final products.
Figure 3. Effects of A2AAR agonist 7 and A2BAR antagonist 5 on the
TNBS-induced reduction of the 1 mM Ach-induced contractions in rat
ileum/jejunum segments. (A) Concentration-dependent effect of 7
(0.1−10 μM) on the TNBS-induced attenuation of the 1 mM Ach-
induced contractions. (B) Concentration-dependent effect of 5 (1.0−
100 μM) on the TNBS-induced attenuation of the 1 mM Ach-induced
contractions. Means ± SEM of nine or seven experiments. *P < 0.05
4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid
1
(7): H NMR (500 MHz, DMSO-d6) δ 2.93−2.95 (m, 2H), 3.20−
+
vs control; P < 0.05 vs TNBS-reduced Ach contraction.
3.27 (m, 2H), 3.51−3.64 (m, 2H), 3.92−3.94 (q, 1H, J = 3.67 Hz),
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4.12−4.13 (q, 1H, J = 2.41 Hz), 4.58−4.60 (t, 1H, J = 5.51 Hz), 5.0
(m, 1H), 5.20 (m, 1H), 5.39 (m, 1H), 5.86−5.88 (d, 1H, J = 5.99 Hz),
7.25−7.54 (m, 6H), 8.22 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ
31.87, 35.40, 61.67, 70.58, 73.51, 85.60, 87.29, 117.05, 125.79, 128.0,
138.69, 140.91, 146.67, 150.40, 155.73, 163.62; LC/ESI-MS negative
mode 482 ([M − H]−), positive mode 484 ([M + H]+).
Biological evaluation of rat intestinal preparations was performed as
described previously15,27,28 (for details, see the Supporting Informa-
tion).
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ASSOCIATED CONTENT
■
(12) El-Tayeb, A.; Iqbal, J.; Behrenswerth, A.; Romio, M.; Schneider,
S
* Supporting Information
1
Synthetic procedures, H and 13C NMR spectral data, HPLC−
M.; Zimmermann, H.; Schrader, J.; Muller, C. E. Nucleoside-5′-
̈
monophosphates as prodrugs of adenosine A2A receptor agonists
activated by ecto-5′-nucleotidase. J. Med. Chem. 2009, 52, 7669−7677.
(13) Mantell, S. J.; Stephenson, P. T.; Monaghan, S. M.; Maw, G. N.;
Trevethick, M. A.; Yeadon, M.; Walker, D. K.; Selby, M. D.; Batchelor,
D. V.; Rozze, S.; Chavaroche, H.; Lemaitre, A.; Wright, K. N.;
Whitlock, L.; Stuart, E. F.; Wright, P. A.; Macintyre, F. SAR of a series
of inhaled A2A agonists and comparison of inhaled pharmacokinetics in
a preclinical model with clinical pharmacokinetic data. Bioorg. Med.
Chem. Lett. 2009, 19, 4471−4475.
MS purity data, and a description of pharmacological
experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Pharmazeutisches Institut, An der Immenburg 4, D-53121
Bonn, Germany. Phone: +49-228-73-2301. Fax: +49-228-73-
2567. E-mail: christa.mueller@uni-bonn.de.
(14) Yan, L.; Muller, C. E. Preparation, properties, reactions, and
̈
adenosine receptor affinities of sulfophenylxanthine nitrophenyl esters:
Toward the development of sulfonic acid prodrugs with peroral
bioavailability. J. Med. Chem. 2004, 47, 1031−1043.
Notes
§On leave from the University of Al-Azhar, Assiut, Egypt.
(15) Michael, S.; Warstat, C.; Michel, F.; Yan, L.; Muller, C. E.;
̈
ABBREVIATIONS
Nieber, K. Adenosine A2A agonist and A2B antagonist mediate an
inhibition of inflammation-induced contractile disturbance of a rat
gastrointestinal preparation. Purinergic Signalling 2010, 6, 117−124.
(16) Borrmann, T.; Hinz, S.; Bertarelli, D. C.; Li, W.; Florin, N. C.;
■
A2AAR, A2A adenosine receptors; A2BAR, A2B adenosine receptors;
Ach, acetylcholine; CSC, 1,3,7-trimethyl-8-(3-chlorostyryl)-
xanthine; IBD, inflammatory bowel disease; PSB-601, 8-[4-(4-
benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine; PSB-0777,
4-(2-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)-
benzenesulfonic acid; SEM, standard error of the mean; TNBS,
2,4,6-trinitrobenzenesulfonic acid
Scheiff, A. B.; Muller, C. E. 1-Alkyl-8-(piperazine-1-sulfonyl)-
̈
phenylxanthines: Development and characterization of adenosine A2B
receptor antagonists and a new radioligand with subnanomolar affinity
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K.-N.; Muller, C. E. A new synthesis of sulfonamides by aminolysis of
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