A2B Adenosine Receptor Antagonists with Picomolar Potency

Article abstract of DOI:10.1021/acs.jmedchem.9b00071

The A_2B adenosine receptor (A_2BAR) was proposed as a novel target for the (immuno)therapy of cancer since A_2BAR blockade results in antiproliferative, antiangiogenic, antimetastatic, and immunostimulatory effects. In this study, we explored the structure-activity relationships of xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents attached to the sulfonamide residue. A new, convergent strategy was established, which facilitated the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar affinity for the A_2BAR combined with high selectivity. Functional groups were introduced, which will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-1-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A_2B-antagonist (K_i 0.0835 nM, K_B 0.0598 nM, human A_2BAR) with >10 000-fold selectivity versus all other AR subtypes. It was similarly potent and selective at the mouse A_2BAR, making it a promising tool for preclinical studies. Computational studies predicted halogen bonding to contribute to the outstanding potency of 34.

Full text of DOI:10.1021/acs.jmedchem.9b00071

Subscriber access provided by UNIV OF TEXAS DALLAS
Article
A2B Adenosine Receptor Antagonists with Picomolar Potency
Jie Jiang, Catharina Seel, Ahmed Temirak, Vigneshwaran Namasivayam, Antonella Arridu,
Jakub Schabikowski, Younis Baqi, Sonja Hinz, Jörg Hockemeyer, and Christa E Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00071 • Publication Date (Web): 05 Mar 2019
Downloaded from http://pubs.acs.org on March 5, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A Adenosine Receptor Antagonists with Picomolar Potency
2B
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Jie Jiang,1,& Catharina Julia Seel, Ahmed Temirak, Vigneshwaran Namasivayam, Antonella
1
1
1
1
1,#
2
1
1
Arridu, Jakub Schabikowski, Younis Baqi, Sonja Hinz, Jörg Hockemeyer, Christa E.
Müller*1
1PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der Immenburg
4
, D-53121 Bonn, Germany.
2Department of Chemistry, Faculty of Science, Sultan Qaboos University, PO Box 36, Postal
Code 123, Muscat, Oman.
&Present address: Key Laboratory of Colloid and Interface Chemistry & Key Laboratory of Special
Aggregated Materials, Shandong University, Ministry of Education, Jinan 250100, P. R. China
#J.S. was on leave from the Department of Technology and Biotechnology of Drugs, Faculty of
Pharmacy, Jagiellonian University Medical College, Kraków, Poland
KEYWORDS
Adenosine, adenosine A2B receptor, antagonist, cancer, halogen bonding, immunotherapy,
molecular modeling, structure-activity relationships, sulfonamide synthesis, xanthine
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
The A2B adenosine receptor (A2BAR) was proposed as a novel target for the (immuno)therapy of
cancer since A2BAR blockade results in antiproliferative, anti-angiogenic, anti-metastatic, and
immuno-stimulatory effects. In this study, we explored the structure-activity relationships of
xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents
attached to the sulfonamide residue. A new, convergent strategy was established which facilitated
the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar
affinity for the A2BAR combined with high selectivity. Functional groups were introduced which
will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A2B-antagonist (K
i
0
.0835 nM, K 0.0598 nM, human A2BAR) with >10,000-fold selectivity versus all other AR
B
subtypes. It was similarly potent and selective at the mouse A2BAR, making it a promising tool for
preclinical studies. Computational studies predicted halogen bonding to contribute to the
outstanding potency of 34.
2
ACS Paragon Plus Environment

Page 3 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
G protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins
in the human genome and constitute the most important class of drug targets.1, 2 Among the class
A, rhodopsin-like GPCRs, adenosine-activated receptors (ARs) are an important subfamily, which
is involved in a variety of physiological and pathological functions.3, 4 Four subtypes of ARs exist
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
which are termed A
1
, A2A, A2B and A . The A2BAR shows the lowest affinity for adenosine among
3
all four subtypes.2, 5 Thus, the A2BAR is thought to remain silent under normal physiological
3
conditions where extracellular adenosine levels range between 30 to 300 nM. However, the
receptor becomes activated under pathological conditions such as hypoxia, inflammation and
cancer, where extracellular adenosine levels are increased up to micromolar concentrations.
While A2BAR expression is ubiquitously found, typically at low levels, higher expression is
6
detected in human cecum, large intestine, mast cells, and hematopoietic cells. Importantly, A2BAR
expression can be upregulated by disease; significant upregulation is observed by hypoxia
7
inducible factor 1-α (HIF-1α), in many cancers, and under inflammatory conditions. Both, increase
8
in extracellular adenosine concentrations, and upregulation of A2BAR expression under
pathological conditions9, 10 indicate an important role of the A2BAR in disease. A2A and A2BARs
are the most closely related AR subtypes, which are frequently co-expressed on cells. We recently
demonstrated the formation of heteromeric A2A-A2BAR complexes and revealed that ligand
recognition and signaling of the A2AAR is blocked by the A2BAR, which leads to significantly
altered pharmacology of A2AARs.11
A2BAR antagonists have been proposed as drugs for the treatment of asthma, inflammation, pain,
diabetes, and Alzheimer’s disease.12, 13 Recent findings further revealed that A2BAR antagonists
can directly influence the growth and migration of cancerous cells of bladder, breast, colon and
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
prostate.14-17 In addition, adenosine was found to be favorable for cancerous cell survival due to its
immuno-suppressive effects.18, 19 Adenosine inhibits cytotoxic effector functions of natural killer
and T cells mainly through A2AAR signaling, which results in tumor immune escape and evasion.
Preclinical studies have confirmed that the blockade of A2AAR activation markedly promotes
antitumor immunity.20 On the other hand, adenosine polarizes myeloid cells to develop into
immunosuppressive M2 macrophages and tolerogenic dendritic cells by A2A and A2BAR
stimulation. There is growing evidence that tumor progression can also be delayed by A2BAR
blockade through inhibiting tumor myeloid-derived suppressor cell accumulation and by restoring
efficient antitumor T cells.21, 22 These results implicate that the A2BAR acts as an important
promoter of cancer growth and cancer immune evasion, and therefore antagonists of this receptor
are regarded as promising new cancer (immuno)therapeutics, which not only activate the immune
system, but, in addition display direct anti-proliferative effects, and, moreover, can reduce pain.13
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The non-selective AR antagonists caffeine and theophylline, which block human A
AR at micromolar concentrations, are used as central stimulants (caffeine), for the treatment of
sleep apnoea and for improving lung functions in pre-term babies (caffeine), as anti-asthmatics
theophylline), and for the treatment of pain in combination with analgesics (caffeine). The
1
, A2A, A2B and
A
3
(
analgesic effect of caffeine, and its synergism with paracetamol and non-steroidal anti-
23
inflammatory drugs, are due to the blockade of A2BARs. Moreover, caffeine has recently been
shown to have anti-cancer effects in animal studies due to the blockade of both A2A and A2BAR.24-
2
6
Several epidemiological studies indicated that caffeine may protect humans from some cancer
types, but other studies were not conclusive.24, 27
4
ACS Paragon Plus Environment

Page 5 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
O
H
Cl
HN
O
N
N
N
N
O
CH3
N
N
N
O
N
N
N
O
O
CH3
N
N
N
N
N
N
N
CH3
F
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1 LAS101057
2
3 ISAM140
4
human
human
human
= 3.49 nM
> 10000 nM
> 10000 nM
> 10000 nM
human
=
>
>
24 nM
2500 nM
10000 nM
A2B
A2A
A2B
A2A
A1
A3
=
20 nM
A2B
A2A
A1
A3
A2B
A2A
A1
A3
=
3.10 nM
=
800 nM
> 10000 nM
> 10000 nM
= 1100 nM
A1
=
300 nM
> 1000 nM
A3 >10000 nM
values of known non-xanthine A2BAR antagonists.28-31
Figure 1. Structures and K
i
Considerable efforts have been made in recent years to develop selective high-affinity antagonists
for A2BARs. These compounds can be divided into non-xanthine derivatives (Fig. 1), for example
2
8
29
2
-aminopyrazines (e.g. 1), pyrazolotriazolopyrimidines (e.g. 2), pyrimidobenzimidazoles (e.g.
3
0
31
3
), triazinobenzimidazolones (e.g. 4), and xanthine derivatives related to the natural products
caffeine and theophylline (Fig. 2).
O
O
N
O
HN1 6 5
H
N
H
N
CF
3
H
H
3
C
H C
3
N
N
N
N
N
7
9
O
O
8
2
4
N
3
O
N
N
N
O
O
N
HN
CN
H
CH3
CH3
5 MRS1754
rat
Xanthine scaffold
6 CVT-6883
human
mouse
3.12 nM
human
A2B
=
1.97 nM A2B
=
=
=
12.8 nM A2B
=
A2B
A2A
=
22 nM
=
=
=
503 nM
403 nM
570 nM
612 nM
16.8 nM
>10000 nM
= 3280 nM
A2A
A1
A3
A2A
A1
A2A
A1
A3
=
1.45 nM
A1 = 1940 nM
= 1070 nM
A3
>10000 nM
> 10000 nM
A3
O
O
H C
3
H
N
H
N
H C
H
3
C
O
3
N
N
N
N
O
N
O
S
H
N
H
N
H
3
C
N
N
N
O
N
O
O
N
O
H
SO H
3
O
N
N
O
N
O
H
CH3
Cl
7
MRE-2029-F20
human
8 PSB-1115
9 PSB-603
human
53.4 nM A2B
rat
mouse
human
0.553 nM A
rat
mouse
= 0.265 nM
2B
A2B
=
5.5 nM
A2B
A2A
=
=
3140 nM A2B = 1940 nM
A2B
=
=
0.355 nM A
2B
= 3790 nM
= 24000 nM
>10000 nM
591 nM
>10000 nM
> 10000 nM
> 10000 nM
>10000 nM
A2A >1000 nM
A1 = 200 nM
A2A
A2A
A2A
A2A
A2A
A1
A3
=
=
42.4 nM
A1 >10000 nM A1
=
2200 nM A1
A1 > 10000 nM
A3 > 10000 nM
A1 > 10000 nM
> 10000 nM
A3
>
1000 nM
>10000 nM
> 10000 nM
>10000 nM
A3
A3
A3
A3
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. Selected xanthine-based A2BARs antagonists and their K
values at AR subtypes.2, 32
i
Some selected xanthine-based A2BAR antagonists are shown in Figure 2, e.g. MRS1754 (5)33,
3
4
35
36
37
CVT-6883 (GS6201, 6), MRE2029-F20 (7), PSB-1115 (8), and PSB-603 (9) . Compound 5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was the first potent, A2BAR-selective antagonist, for which a Th1-suppressive effect in autoimmune
38
diseases was shown. It significantly inhibited colon carcinoma cell growth in a dose-dependent
manner, and induced anti-angiogenesis in microvascular endothelial cells.39, 40 Compound 6 was
clinically evaluated in a phase I trial with the aim to develop it as a drug for the treatment of lung
remodeling, pulmonary hypertension and asthma.2, 34, 41, 42 Further studies found that 6 led to a more
favorable cardiac remodeling and reduced ventricular dysfunction and ventricular arrhythmias after
acute myocardial infarction in the mouse or rat.43, 44
Our group has been interested in improving the potency, selectivity and/or water-solubility of
xanthine-based A2BAR antagonists, and a series of sulfonic acid and sulfonamide derivatives has
previously been developed. For example, the water-soluble sulfophenylxanthine derivate 8 (PSB-
1115) is a selective A2BAR antagonist in humans, that displayed dose-dependent antinociceptive
effects and could potently diminish inflammatory pain in mice, showing synergism with other
analgesics including morphine, non-steroidal anti-inflammatory drugs, and pegylated adenosine
deaminase (PEG-ADA, an FDA approved drug).23, 45, 46 Recent research further revealed that 8
improved intestinal barrier function in colon inflammation, and under hypoxic/ischemic conditions
4
7
and reperfusion injury. In addition, 8 reduced the immuno-suppression in a tumor environment
and inhibited tumor angiogenesis with increasing T cells numbers in a mouse melanoma model.13,
21, 48The sulfonate function of 8 confers high water-solubility, however, the deprotonation of this
free sulfonic acid group under physiological conditions (pK < 1) likely prevents peroral absorption
a
and central nervous system (CNS) penetration.
6
ACS Paragon Plus Environment

Page 7 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A sulfonamide group was introduced instead to solve the drawbacks of the sulfonate moiety.
Compound 9 (PSB-603) was then developed as one of the most potent and selective A2BAR
antagonists in all three species, human, rat, and mouse.32, 37 Compound 9 was found to inhibit the
growth of prostate cancer cells and to attenuate cell proliferation of neuroendocrine tumours.7, 15, 49
Compound 8 and 9 were also found to be useful for treating infectious diseases due to their
immunostimulatory effects, and to prevent A2BAR-induced opening of the blood-brain barrier.50, 51
All of these findings confirmed that A2BAR antagonists have a great potential as future drugs and
are particularly promising for the immunotherapy of cancer and infectious diseases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
A tritium-labeled derivative of 9, [ H]PSB-603, has been prepared, and a radioligand binding assay
was established for the labeling of A2BARs.1, 37 Recently, the first potent and selective fluorescent
A
2BAR ligands were reported.52
Homology modeling based on the X-ray structures of the related A2AAR and site-directed
mutagenesis studies have provided fundamental information of the A2BAR and its orthosteric
binding site.On the basis of the most potent A2BAR antagonist, compound 9, we established a
pharmacophore model (Fig. 3), in which the xanthine scaffold represents a flat core, that offers a
hydrogen bond acceptor (the C6-carbonyl function) - hydrogen bond donor (N7-hydrogen) motif.
A benzenesulfonamide spacer, which features hydrogen bond acceptor groups, was used to connect
the flat core with another aromatic residue. Propyl substitution at position 1 was optimal and found
to increase A2BAR affinity via hydrophobic interactions with the amino acid residue Trp2476.48. A
free NH at position 3 enhanced potency at the A2BAR as well as selectivity versus the A
1
, A2A, and
A
3
AR subtypes. The hydrophobic interactions between Val2506.51 and the xanthine core, as well
as Leu813.28 and the aromatic ring that is connected to the xanthine core, appear to be crucial for
high A2BAR binding affinity of antagonist 9 36, 55, 56 However, interactions of the sulfonamide
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
residue have not been clearly elucidated so far. In the present study, we varied the spacers and the
substituted aromatic ring attached to the xanthine-8-phenylsulfonate via sulfonamide formation.
Our aim was (i) to explore the structure-activity relationships (SARs) for obtaining highly potent
and selective A2BAR antagonists, (ii) to modulate the compounds’ physicochemical properties, (iii)
to develop ligands for potential further labeling with fluorescent dyes or other reporter groups, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(iv) to explore the possibility of obtaining dual A2A/A2B AR antagonists. Computational studies
were performed to rationalize the SARs for these sulfonamidophenyl-xanthine derivatives.
-
Leu813.28
HA HD
HA
O
SARs?
H
H C
O
3
N
N
O
S
N
N
O
N
H
N
6.48
Trp247
Val2506.51
Flat core
Cl
Long spacer
Aromatic ring
HA: Hydrogen bond acceptor
HD: Hydrogen bond donor
Figure 3. Features of A2BAR antagonist 9 that are important for binding to the receptor.
RESULTS and DISCUSSION
Chemistry. The synthetic routes for the target compounds are described in Scheme 1 and 2. 3-
Propyl-substituted 5,6-diaminouracil derivatives 10a-c were obtained according to previously
reported procedures.57-59 Detailed synthetic information for compound 11 and some of the required
amines (58, 60, 62-64, 81, 82, 85-87 and 89) is provided in the Supporting Information.36
Compounds 10a-c and 11 were subsequently condensed in the presence of N-(3-
(dimethylamino)propyl)-N´-ethylcarbodiimide (EDC) to yield amides 12a-c (see Scheme 1). After
ring closure of 12a-c using various condensation methods (see Scheme 1) yielding xanthines 13a-
8
ACS Paragon Plus Environment

Page 9 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
c, different piperazine derivatives (55-84 and 88-90) or other amines (91, 92) were introduced to
obtain the corresponding xanthine sulfonamide derivatives 14-44 and 50-54 (see Scheme 1).
Aminolysis was performed using optimized conditions in dry dimethyl sulfoxide (DMSO) under
3
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
an argon atmosphere, instead of dimethylformamide (DMF) which had been used before. Dry
DMSO prevented ester hydrolysis observed in the presence of water, as well as side-reactions with
dimethylamine which resulted from DMF decomposition yielding compound 53. In this modified
nucleophilic substitution reaction, pure sulfonamide products were obtained for reactions of acyclic
amines 91 and 92 yielding 53 and 54, while side-products were obtained for sterically hindered
piperazine derivatives. These resulted from the competing pathway by attack at the electron-
deficient carbon atom of the nitro phenyl ester.
A new synthetic route was designed for the preparation of some of the final products as depicted
in Scheme 2. The sulfonamide-substituted benzoic acid derivatives 85b-87b were prepared from
piperazine derivatives 85-87 and 4-(chlorosulfonyl)benzoic acid (11a), and subsequently coupled
with 10a yielding 85c-87c. The final ring closure reaction was performed with P
2
5
O for 10 min to
yield xanthines 45-47. The methoxyphenyl derivatives 46 and 47 were further subjected to
demethylation to furnish the phenol derivatives 48 and 49. This new method to prepare the targeted
xanthine derivatives was found to be more economic and convenient compared with the previously
described procedure.
The final products were purified by reversed-phase high performance liquid chromatography (RP-
HPLC) or directly recrystallized from acetonitrile. Newly synthesized xanthine-8-yl-
1
13
benzenesulfonamides are listed in Table 1. All products were characterized by H and C NMR
spectroscopy as well as mass spectrometry (MS) employing electrospray ionization (ESI).
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. Preparation of sulfonamides 14-44 and 50-54.a,b
NO2
O O
S
O
O
O
H C
NH2
NH2
O
H
3
N
H C
N
O
3
H OOC
S
O
(a)
N
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NO
O
O
N
2
O
N
R1
NH2
R1
1
1
1
0a: R = H
12a: R = H
= methyl
=
methyl
0b: R1
11
12b: R1
1
1
1
10c: R = ethyl
12c: R = ethyl
R3
O
N
R2 or HN
HN
HN
N
N Ph or
H
N
R4
H C
O
3
N
O
S
O
55-84, 88, 89
(b)
90
91, 92
N
NO2
O
R1
(c) or (d) or
e) or (f)
(
1
1
3a: R = H
= methyl, R4 = methyl
= methyl, R4 = phenethyl
91: R3
2: R3
=
methyl
13b: R1
9
1
13c: R = ethyl
O
O
O
H
H
H C
O
H
N
3
N
N
H
3
C
O
H
3
C
N
O
O
N
S
N
O
O
S
S
N
N
O
N
R1
R3
N
N
N
N
N
O
N
H
O
H
R4
N
N
R2
1
1
1
1
4: R = methyl
= methyl, R4 = methyl
= methyl, R4 = phenethyl
53: R3
54: R3
= ethyl
5: R1
6-44, 50, 51: R1
52
=
H
a
Reagents and conditions: (a) EDC, MeOH, rt, 4 h; (b) polyphosphoric acid trimethylsilyl ester
(PPSE), 170 °C, 5 h; (c) method A: DMF; (d) method B: dry DMSO, Ar, 3h, 150 °C; (e) method
C: analogous to method A with consecutive reaction; (f) method D: dimethylamine in 33 %
b
2
ethanolic solution, rt, 16h. For R see Table 1.
10
ACS Paragon Plus Environment

Page 11 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 2. New strategy for the preparation of compounds 45-49.
Cl
Cl
Cl
H
H
O
a
H N
R1
HN
N
R1
b
O
N
2
HOOC
S
N
R2
OCH
R2
R2 = F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
= F
= OCH
N
8
8
8
5a, R1
6a, R1
7a, R1
3, R
2
85
, R
1
= OCH
85b
, R1
, R1
2
2
= F
3,
3
, R
, R
= OCH
= Cl
= OCH R2
= OCH
2
86 R1
,
, R
= Cl
86b
87b
= Cl
3
3,
3
= Cl, R = OCH
= Cl, R2 = OCH
= OCH
2
, R1
1
= Cl, R2
3
87
3
, R
3
R1
R2
O
O
O
S
N
H C
NH2
3
c
N
O
O
N
d
85b-87b
O
N
NH2
H C
NH
3
R1
H
N
R2
10a
O
N
NH2
H
=
OCH
= F
8
8
8
5c, R1
3, R2
= OCH
= Cl
6c, R1
, R2
3
1
2
7c, R = Cl, R = OCH
3
O
H
N
O
H
H C
O
S
3
N
O
H C
O
3
N
N
O
S
N
N
O
N
e
N
N
H
46 or 47
O
N
H
N
N
=
F
= OH, R2
1
2
48, R1
49, R1
4
5, R = OCH , R
= Cl
= Cl, R2 = OH
3
=
OCH
= Cl
1
1
, R2
4
6, R
3
2
R1
R2
R1
R2
47, R = Cl, R = OCH
3
Reagents and conditions: (a) diethyleneglycol monomethylether, 150 °C, 20 h, 47-62 %; (b) 4-
chlorosulfonyl)benzoic acid (11a), N,N-diisopropylethylamine (DIPEA), dichloromethane
(DCM), rt, 36 h, 72-87 %; (c) EDC*HCl, DMF, rt, 18 h, 50-90 %; (d) P , DMF, reflux, 10 min,
0-58 %; (e) 1M BBr , DCM, rt, 24 h, 13-25 %.
(
2
O
5
1
3
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Isolated yields, purities and calculated physicochemical properties of newly synthesized
sulfonamidophenylxanthine derivatives.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd.a
M.W. c
Clog P d
PSA d
Purity
%) b
(
Piperazine derivatives
2
1
1
1
1
4
5
6
7
R = 4-chlorophenyl
98.1
97.4
98.8
100.0
97.3
98.6
99.0
98.0
97.6
543.0
557.1
587.5
587.5
538.6
522.6
549.6
552.6
582.7
3.5
3.9
4.3
4.3
3.1
3.8
4.0
1.2
2.5
109.9
109.9
118.7
118.7
127.9
118.7
148.1
156.0
148.2
2
R = 4-chlorophenyl
2
R = 4-bromobenzyl
2
R = 3-bromobenzyl
2
18
R = 3-methoxybenzyl
2
1
9
R = 3-methylbenzyl
20 e
1 f
22
R = 4-azidobenzyl
2
2
2
R = benzyl-4-carboxylate
2
R = 4-(2-
hydroxyethoxy)benzyl
2
2
3
R = 4-(2-(2-
95.1
626.7
3.1
146.4
methoxyethoxy)ethoxybenzy
l
2
2
2
2
4
5
6
R = 3-fluoro-4-
97.8
98.5
556.6
556.6
556.6
605.5
3.4
3.4
3.4
4.5
127.9
127.9
127.9
118.7
methoxybenzyl
2
R = 4-fluoro-3-
methoxybenzyl
2
R = 3-fluoro-5-
100.0
98.0
methoxybenzyl
2
27
R = 3-fluoro-4-
bromobenzyl
2
2
2
3
3
8
9
0
1
R = phenyl
95.6
98.3
95.5
97.1
12
494.6
512.6
512.6
512.6
3.7
3.9
3.9
3.9
118.7
118.7
118.7
118.7
2
R = 4-fluorophenyl
2
R = 3-fluorophenyl
2
R = 2-fluorophenyl
ACS Paragon Plus Environment

Page 13 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
3
3
3
3
3
3
2 g
3 g
4
R = 3-chlorophenyl
98.2
95.2
99.1
98.3
97.8
100.0
97.6
98.1
98.5
98.4
98.0
95.8
99.1
95.8
529.0
4.3
4.3
4.5
4.5
4.5
4.6
3.6
4.2
4.2
3.4
2.9
3.7
1.6
3.7
118.7
118.7
118.7
118.7
118.7
118.7
127.9
118.7
118.7
138.9
135.8
118.7
118.7
127.9
2
R = 2- chlorophenyl
529.0
573.5
573.5
573.5
620.5
524.6
508.6
508.6
510.6
522.6
522.6
432.5
542.6
2
R = 4-bromophenyl
2
5
R = 3-bromophenyl
2
6
R = 2-bromophenyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
7
R = 4-iodophenyl
2
38
R = 3-methoxyphenyl
2
3
9
R = 4-methylphenyl
2
40
R = 2-methylphenyl
2
4
1 e
R = 4-hydroxyphenyl
2
42
R = benzoyl
2
4
4
4
3
R = 1-phenylethyl
2
4
R = methyl
5 g,
R = 3-fluoro-4-
2
methoxyphenyl
2
4
4
4
6 g
7 g
8 g
R = 3-chloro-4-
96.6
96.6
99.0
95.0
97.4
98.4
95.9
559.0
559.0
545.0
545.0
547.0
554.6
508.6
4.2
4.2
4.0
4.0
4.5
2.9
3.4
127.9
127.9
138.9
138.9
118.7
148.2
118.7
methoxyphenyl
2
R = 4-chloro-3-
methoxyphenyl
2
R = 3-chloro-4-
hydroxyphenyl
49 g
R = 4-chloro-3-
2
hydroxyphenyl
2
5
5
5
0
1
2
R = 4-chloro-2-
fluorophenyl
2
R = 4-(2-hydroxy-
ethoxy)phenyl
See structure above
Acyclic amino derivatives
53 h
R = methyl
3
95.9
99.1
377.4
467.5
2.0
4.0
115.5
115.5
4
R = methyl
3
5
4
R = methyl
4
R = phenethyl
a
b
All final products were synthesized according to method B (see Scheme 1) unless otherwise noted.
c
Purity was determined by HPLC-UV-MS at 254 nm; molecular weight was calculated by the
d
e
Chemdraw software; cLogP and polar surface area were calculated by ChemAxon; synthesized
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
f
g
according to method A ; synthesized according to method C. synthesized according to method
h
D; synthesized according to method E.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
ACS Paragon Plus Environment

Page 15 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Calculation of Physicochemical Properties and Solubility Determination of Selected Products
Several physicochemical parameters were calculated to estimate the potential suitability of the final products as
tool compounds or drugs, including polar surface area (PSA) and lipophilicity (clog P value). PSA values were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
in all cases, except for compounds 20-23, and 51, lower than 140 Å , indicating that they might be intestinally
6
0
absorbable. All of the compounds showed a clog P value lower than 5, which is consistent with Lipinski’s
6
1
rule for drugs exhibiting pharmacokinetic properties that are suitable for peroral application. Benzylpiperazine
derivatives showed lower clog P values but the same PSA values as the corresponding phenylpiperazine
derivatives (compare compounds 16/34, 18/38, and 19/39). Compounds 14 and 15 which are substituted at the
xanthine N3-position and therefore lack the corresponding hydrogen bond donor function display lower PSA
values; modification at this position may allow modulation of the physicochemical parameters of the
compounds. It should be kept in mind that these values were not experimentally determined and therefore
provide only estimations. The water-solubility of p-chlorophenylpiperazine derivative 9 was determined in
comparison to that of the analogous p-chlorobenzylpiperazine derivative 98 using a shaking flask method. The
latter compound, which is more basic, displayed significantly higher solubility (3.2 µM) compared to 9 (0.2
µM). However, the solubility of the compounds should be further improved for use as therapeutics.
Biological Evaluation
Radioligand binding assays were performed to determine the affinities of the products at human
and/or mouse A
1
, A2A, A2B, and A
3
AR subtypes. Human and mouse adenosine receptors
recombinantly expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney
3
6
(HEK) cells were employed. The following radioligands were employed: [ H]2-chloro-N -
3
3
cyclopentyladenosine ([ H]CCPA), [ H](E)-3-(3-hydroxypropyl)-8-(2-(m-methoxyphenyl)vinyl)-
3
[3H]8-(4-(4-(4-
7
-methyl-1-prop-2-ynyl-3,7-dihydropurine-2,6-dione
([ H]MSX-2),
3
chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propyl-3,7-dihydropurine-2,6-dione
([ H]PSB-
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6
03), and [ H]2-phenyl-8-ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one
[3H]PSB-11) for human and mouse A
, A2A, A2B, and human A
AR. [ H]5’-N-
3
(
1
3
Ethylcarboxamidoadenosine ([³H]NECA) was used as a radioligand for assays at the mouse A AR
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
due to the low potency of [ H]PSB-11 at the rodent receptors. Biological data for human ARs are
summarized in Tables 2−6 and Figure 4−7. Selected data of published compounds are included for
comparison. Concentration-inhibition curves of selected compounds are depicted in Figure 8.
These compounds were further explored at the mouse AR subtypes to investigate potential species
differences. In addition, for selected compounds cAMP accumulation assays were performed in
recombinant A2BAR-expressing CHO cells to demonstrate their functional properties as
antagonists (see below).
Structure-Activity Relationships
The previously developed sulfonate 8 exhibits high water-solubility and has been used in in vivo
studies; however, its A2BAR affinity is moderate, its selectivity, especially in rodents, is low, and it
is too polar for oral absorption.32, 36, 55Aprodrug concept had been developed to overcome the latter
issue. 36 Later on, sulfonamide derivatives of 8, such as xanthine 9, were synthesized. Although
37
A
2BAR affinity and selectivity of 9 are already high, a broad exploration of the SARs of this class
of compounds is required. This will provide highly useful knowledge allowing to fine-tune the
compounds’ properties in subsequent multi-dimensional drug development efforts. Moreover,
comprehensive exploration of SARs is needed as a basis for molecular modeling studies to explore
the ligand binding site, since X-ray structures of the A2BAR are still lacking. Therefore, taking
compounds 8 and 9 as lead structures, a variety of 8-phenylxanthine derivatives bearing a broad
range of p-sulfonamido-substituents was synthesized in this study and optimized as A2BAR
antagonists. Most of the investigated compounds showed high affinity for theA2BAR with K values
i
16
ACS Paragon Plus Environment

Page 17 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in the nanomolar or even subnanomolar range, very high selectivity versus A
1
and A ARs, and
3
different degrees of selectivity towards the A2AAR.
Table 2. Adenosine receptors affinities of N3-substituted compounds compared with PSB-603.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compd.
R
K
i
± SEM (nM) (or % inhibition of radioligand binding at
Selectivity
e
indicated concentration)
Index
human A2Ba
0.553
human A2Ab
human A c
human A d
1
3
9 PSB-603 37
H
>10000
>10000
>1000 (22)f
>1000 (24)f
3
>10000
>1000 (39)f
>1000 (28)f
d
>18000
>520
75
1
4
Methyl
Ethyl
1.91 ± 0.32
4.31 ± 1.16
3
>1000 (33)f
322 ± 157
c
15
a
3
b
3
vs. [ H]PSB-603 (n = 3); vs. [ H]MSX-2 (n = 3); vs. [ H]CCPA (n = 3); vs. [ H]PSB-11 (n =
e
3); selectivity index was calculated by dividing the second lowest K
i
value by the A2BAR K value;
i
f
percent inhibition of radioligand binding at 1 μM.
In our previous study, 1-propyl substitution had been proven to be optimal conferring high A2BAR
affinity combined with high selectivity versus all other AR subtypes (see compound 9, Figure 2).
3
7
A free NH function at position 7 is known to be crucial as a hydrogen bond donor for interacting
with the A2BAR and to allow the 8-phenyl ring to be coplanar with the xanthine core structure. 13
Even though 3-substituted compounds are not preferred based on previous results, we introduced
small residues, methyl and ethyl, to potentially modulate the physicochemical properties of the
xanthine derivatives by removing the N3-H donor function. The rank order of A2BAR affinities for
these compounds was as follows: H (9, 0.553 nM) > methyl (14, 1.91 nM, p = 0.0157 *) ≥ ethyl
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
15, 4.31 nM). The selectivity was also decreased in the same order. But affinity and selectivity
14, >524-fold selective; 15, ≥75-fold) of both N3-substituted xanthine derivatives were still high
(
enough for potential application as A2B-selective drugs (see Table 2). Nevertheless, compound 15
also displayed significant affinity for the A2AAR indicating that the development of dual A2A/A2B
antagonists via substitution of the N3-position would be feasible. Furthermore, the increased
lipophilicity might increase the chances for brain penetration.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Adenosine receptor affinities of 8-sulfonamidoxanthines with variations of residues on
the sulfonamido function.
Compd.
28
R
K
i
± SEM (nM) (or % inhibition of radioligand binding at
Selectivity
Indexe
indicated concentration
human A2Ba
human
human
1
human
3
b
c
d
A
2A
A
A
0.643
0.035
±
122 ± 32f
364 ± 57f
>1000
(27%)g
190
3
N
N
N
N
48.2 ± 10.9f
334 ± 109f
>1000 (7%)g
4
5
5
4
3
4
140 ± 91
52.7 ± 12.2
357 ± 75f
18.6 ± 0.9
190 ± 54f
1220 ± 220
3
N
1.69 ± 0.73f
1036 ± 384f >1000 (4%)g
210
N
93 56
H
N
3.62
769
183
≥10000
50
18
ACS Paragon Plus Environment

Page 19 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
3
b
3
c
3
d
3
vs. [ H]PSB-603 (n = 3); vs. [ H]MSX-2 (n = 3); vs. [ H]CCPA (n = 3); vs. [ H]PSB-11 (n =
e
3
); selectivity index was calculated by dividing the second lowest K
i
value by the A2BAR K value;
i
f
g
extrapolated curve; percent inhibition of radioligand binding at 1 μM.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Our main focus was the introduction of a variety of substituents on the sulfonamido function.
Removal of the para-chloro substituent in lead structure 9 (Fig. 2) resulted in 28, which showed
similar potency, but lower selectivity. Replacing the terminal phenyl ring of 28 by a methyl group
(
in 44) strongly reduced A2B affinity (by 76-fold) without much change at the other AR subtypes.
Replacement of the piperazine ring by the small dimethylamine (in 53) yielded a potent A2B
antagonist (K 18.6 nM), that showed, however, only moderate selectivity especially versus A2A (3-
fold) and A (10-fold) receptors. Introducing a larger phenethyl residue (93) increased potency
again (K 3.62 nM) as well as selectivity. Additional N-methylation leading to 54 retained A2BAR
affinity and increased selectivity, particularly versus the A AR subtype. These data indicated that
i
1
i
1
an aromatic residue was favorable for high A2BAR affinity and also for selectivity (compare 28/44,
p = 0.0021**, and 53/54, p < 0.0001 ***). Moreover, disubstitution of the sulfonamide N-atom
appears to contribute to high A2BAR selectivity, and a piperazine residue is advantageous. Based
on these findings, we concentrated our efforts on piperazine derivatives containing a terminal
aromatic residue, and a large series of piperazine derivatives was designed and synthesized (Figure
4
). SARs were further explored regarding the following aspects: (i) the type of linker between the
piperazine and the aromatic ring; (ii) substitution of the aromatic ring (type of substituent, position,
mono- and di-substitution, and functional groups for further attachment of moieties, e.g. for future
fluorescent labeling).
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
H
H C
3
N
O O
S
N
N
N
O
N
H
N
linker
R1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
substituents
R2
Figure 4. Structures of targeted piperazinyl-substituted sulfonamide derivatives.
Linker modification
A methylene or an ethylene group between the piperazine ring and the aromatic residue of the
phenylpiperazine derivative 28, was previously found to be tolerated (see benzylpiperazine 94 and
phenethylpiperazine 95).55 However, the phenylpiperazine was 6-fold more potent than the
corresponding benzylpiperazine derivative (compare 28/94, K
more potent than the phenethyl derivative 95 (K 7.51 nM). The rank order of A2BAR affinities and
selectivities vs. A2AARs among compounds with different linker length, i.e. number of CH groups,
was as follows: (CH (28, 0.643 nM, 190-fold vs. A2AAR) > (CH (94, 3.6 nM, 134-fold vs.
2AAR, p = 0.0002 ***) > (CH (95, 7.51 nM, 36-fold vs. A2AAR, p = 0.0228 *), indicating that
2BAR potency improved with decreasing linker length of the alkyl chain between the piperazine
i
0.634 nM vs. 3.6 nM) and 12-fold
i
2
2
)
0
2 1
)
A
A
2 2
)
residue and the aromatic ring.
As alternative linkers carbonyl (42, K
i
5.69 nM), and methylbenzyl (43, K 8.34 nM) were explored,
i
both of which were tolerated, but also resulted in decreased potency compared to 28. In compound
2, the phenyl ring was attached to the 3-position of the piperazine ring combined with N-
methylation. This modification was also tolerated (K 7.15 nM) showing again that an aromatic ring
5
i
increases potency (compare 44 (without phenyl substitution)/52), but it was 11-fold less potent than
benzylpiperazine derivative 28.
20
ACS Paragon Plus Environment

Page 21 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Direct attachment of the phenyl ring to the piperazine N-atom resulted in the highest A2B affinity
(
sub-nanomolar K value) combined with the highest selectivity index. However, modulation is
i
possible and may be used for the future fine-tuning. Especially a benzyl or a 1-methylbenzyl
residue (compounds 94 and 43) still yield potent and selective A2BAR antagonists which feature
increased basicity as compared to the aniline derivative 28 and might therefore display somewhat
increased solubility. On the other hand, a carbonyl linker as in benzoylpiperazine 42, resulted in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
increased A2AAR affinity (K
2BAR, but this class of compounds may be suitable for future optimization aimed at obtaining
dual A2A/A2B antagonists.
i
A2A 143 nM, K
i
A2B 5.69 nM), still with a 30-fold selectivity for the
A
Table 4. Adenosine receptors affinities of xanthine-8-yl-benzenesulfonamide derivatives with
linker modification.
Compd.
Linker
K
i
± SEM (nM) (or % inhibition of radio ligand binding at
Selectivity
Indexe
indicated concentration)
human A2Ba human A2Ab
human A c
human A d
1
3
0.643
0.035
±
122 ± 32f
143 ± 18f
364 ± 57f
>1000
190
30
2
4
8
2
N
N
N
N
27%)g
(
5.69 ± 0.35
>1000
>1000
(
15%)g
26%)g
(
O
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
≥1000 (42)g
≥1000
4
5
3
2
8.34 ± 1.51
7.15 ± 2.35f
>1000
>120
42%)g
(
0)g
(
N
N
N
N
CH3
293 ± 55f
>1000
>1000
(-2%)g
40
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(15%)g
CH3
9
9
4 PSB-60156
3.6
484
2067
>1000
130
40
N
N
N
N
537
7.51
271
>10000
>1000
a
3
b
3
c
3
d
3
vs. [ H]PSB-603 (n = 3); vs. [ H]MSX-2 (n = 3); vs. [ H]CCPA (n = 3); vs. [ H]PSB-11 (n =
e
3
); selectivity index was calculated by dividing the second lowest K
i
value by the A2BAR K value;
i
f
g
extrapolated curve; percent inhibition of radioligand binding at 1 μM.
Substitution of the aromatic ring
A large variety of substitutions on the aromatic ring were then explored for both benzyl- and
phenyl- piperazine derivatives, ranging from lipophilic to polar residues.
Mono-substitution on the aromatic ring
A lipophilic substituent on the o-, m-, or p-position of the phenyl ring, such as F (29, 30, 31, 96,
97), Cl (9, 32, 33, 98, 99), Br (16, 17, 34, 35, 36), I (37), Me (19, 39, 40, 101), OMe (18, 38, 100,
1
04), N
3
(20) or CF (102, 103) was tolerated in benzyl- and phenyl-substituted piperazine
3
derivatives (see Table 5 and Fig. 5A). On the other hand, hydrophilic functional groups (COOH or
22
ACS Paragon Plus Environment

Page 23 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
OH) at the p-position, as in compounds 21 and 41, led to dramatically decreased potency at the
A2BAR, also resulting in lower selectivity. In Fig. 5, it is clearly demonstrated that lipophilic
substituents are preferred for high A2BAR affinity as compared to more polar ones, which is likely
attributed to the existence of a lipophilic binding pocket in the A2BAR, in which the phenyl ring is
accomodated, with bromine leading to the highest potency. It can also be observed, that the more
rigid phenylpiperazines in combination with p-substituents that are bulker than F, e.g. Br, Cl, Me,
OMe, were significantly more selective than the correspoinding benzylpiperazine derivatives (Fig.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
B).
Compounds 20, 21, and 41 containing N
3
, COOH and OH residues respectively, were designed as
functionalized congeners that could be coupled to fluorescent dyes, small peptides, proteins,
nucleotides, or other moieties for labeling. Even though the potency was moderate for the polar
compounds 21 (COOH) and 41 (OH), the functionalized products will be less polar, and these
functionalized compounds are therefore still expected to be very promising for further
derivatization. Compounds 22, 23, and 51, which are bearing a longer terminal chain, were found
to be better tolerated by the A2BAR than 41 with a free phenolic group, confirming that
functionalization on the tail was feasible.
Substitution at the m- or p-position of either benzyl- or phenyl-piperazine derivatives was favorable
in comparison with substitution at the o-position. Interestingly, we found that the rank order of
potency for p-halogen-substituted phenylpiperazine derivatives was as follows: Br (34, 0.0835 nM)
≥
I (37, 0.159 nM) ≥ Cl (9, 0.553 nM) ≥ F (29, 0.644 nM). A similar rank order was noticed for
benzylpiperazine derivatives: Br (16, 0.153 nM) > Cl (98, 0.393 nM, p = 0.0081 **) ≥ F (96, 0.595
nM). This phenomenon indicates the existence of a halogen bonding interaction. In fact, according
to homology modeling and docking studies, a halogen bond with a carbonyl function of a backbone
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
amino acid residue in transmembrane domain1 (TM1, near the N-terminal region), is likely, which
will be discussed in detail below. Bromophenyl derivative 34 exhibited an outstanding potency
with a K
i
value of 0.0835 nM (pK = 10.08) and over 10000-fold selectivity versus all other AR
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
subtypes. The affinity of this compound is almost one order of magnitude higher than that of the
lead structure PSB-603 (p = 0.0017 **) and may be the most potent A2BAR antagonist known to
date.
Table 5. Adenosine receptor affinities of xanthine-8-yl-benzenesulfonamide derivatives with
mono-substitution of the phenyl/benzyl ring.
Compd.
R1
K
i
± SEM (nM) (or % inhibition of radioligand binding at
Selectivity
Indexe
indicated concentration)
human A2Ba
human A2Ab
human A c
human A d
1
3
Substitutions on the benzyl ring
9
9
456
637
H
3.6
484
2067
>1000
758
130
410
4-F
0.595
244 ± 3 f, g
9090 ±
f, g
1
260
9
737
3-F
0.446
0.393
278
333
2300
2240
>1000
>1000
620
850
98
4-Cl
PSB-0788)37
(
9
1
1
937
6
3-Cl
4-Br
3-Br
0.782
161
1090
24200
210
510
620
77.8 ± 11.7f
>1000 (31%)h
>1000 (28%)h
0.153 ± 0.004
0.148 ± 0.039
928 ± 231f
>1000 (23%)
7
92.4 ± 12.0
h
10037
4-OMe
3-OMe
328
>10000
>10000
350
100
0.944
1.28 ± 0.21f
131 ± 15f
205 ± 13f
>1000 (27%)h
1
8
24
ACS Paragon Plus Environment

Page 25 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
2
2
2
0137
9
4-Me
3-Me
0.858
212 ± 10f, g
189 ± 31
145
398 ± 152f, g
156
180
480
480
400
850
10
0.397 ± 0.119
0.303
795 ± 201f
>1000 (17%)h
02 37
03 37
0
4-CF
3-CF
5630
>10000
3
3
0.775
306
2610
30000
492 ± 89f
>1000 (32%)h
4-N
3
0.244 ± 0.122
96.7 ± 14.4
12.2 ± 4.0
208 ± 51
1160 ± 160
241 ± 49
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4-COOH
>1000 (12%)h >1000 (-7%)h
1
OH
560 ± 13f
>1000 (17%)h
2
20
O
O
7.02 ± 1.1
314 ± 74
1010 ± 250
>1000 (30%)h
50
2
3
O
2
Substitutions on the phenyl ring
H
0.643 ± 0.035
0.644 ± 0.154
122 ± 32f
108 ± 25
364 ± 57h
>1000 (27%)h
190
170
28
4-F
3-F
>1000 (14%)h >1000 (38%)h
>1000 (34%)h >1000 (44%)h
2
9
30
0.632 ± 0.178 >1000
>1580
(38%)h
>1000 (13%)h
>1000 (9%)h
3
9
1
2-F
3.22 ± 1.39
>1000
>310
>18080
160
27%)h
(
4-Cl
3-Cl
0.553
>10000
>10000
>10000
(
PSB-603) 37
3
2
1000
0
.342 ± 0.077
>1000 (21%)h
53.5 ± 13.7
(
42%)h
2-Cl
4-Br
0.403 ± 0.089
204 ± 62
>1000(25%)h >1000(32%)h
510
33
3
4
0.0835
.0327
±
±
>1000
>1000 (13%)h >1000 (29%)h
>11980
0
(33%)h
(
PSB-1901)
>1000 (-1%)h
>1000 (27%)h
3
5
3-Br
0.234
>1000
>4270
f
(24%)h
0
.069
2-Br
4-I
0.137 ± 0.033
0.159 ± 0.066
89.5 ± 5.4
2990 ±
>1000 (28%)h >1000 (20%)h
>1000 (24%)h >1000 (36%)h
650
3
6
37
>6290
f
1133
1
0437
4-OMe
3-OMe
1.99
3720
>10000
>1000
>500
320
0.616 ± 0.092
197 ± 45f
>1000 (-3%)h >1000 (-1%)h
>1000 (12%)h >1000 (25%)h
38
3
9
4-CH
3
0.706 ± 0.141 >1000
>1420
34%)h
(
2-CH
3
0.432 ± 0.116
4.30 ± 0.81
6.08 ± 1.23
140 ± 26
96.7 ± 3.9
168 ± 5
419 ± 62f
>1000 (25%)h
320
20
4
4
5
0
1
1
356 ± 92f
>1000 (21%)h
4-OH
OH
>1000 (24%)h >1000 (23%)h
30
O
a
3
b
3
c
3
d
3
vs. [ H]PSB-603 (n = 3); vs. [ H]MSX-2 (n = 3); vs. [ H]CCPA (n = 3); vs. [ H]PSB-11 (n =
e
3); the selectivity index was calculated by dividing the second lowest K
i
value by the A2BAR K
i
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
f
g
h
value; extrapolated curve; data obtained in this study; percent inhibition of radioligand binding
at 1 μM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
ACS Paragon Plus Environment

Page 27 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A.
Affinity
phenylpiperazine derivatives
benzylpiperazine derivatives
*compound not synthesized
1
1
1.0
0.5
10.0
.5
.0
*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
9
8
8
*
* *
* *
.5
.0
)
1
9
)
3
7)
20)
41)
2
34)
39)
F
29)
H
28)
(
103)
(
104)
H
(
(
I
l (98,
7
.5
.0
6.5
(
N 3
94,
3
16,
96,
O
(
101,
(
(
CF
r
C
100,
e (
OOH
B
C
e (
7
M
M
O
B.
2
5000
0000
5000
0000
Selectivity
phenylpiperazine derivatives
benzylpiperazine derivatives
compound not synthesized
2
*
*
1
1
500
0
37)*
*
*
20)
(
41)
**
2
)
1
9)
34)
39)
F
29)
H
28)
(
103)
104)
H
(
(
I
l (98,
(
3
3
N
96,
94,
O
016,
101,
e (
(
(
CF
(
C
100,
e (
OOH
r
C
B
M
M
O
Figure 5. Effects of substituents in the p-position of phenylpiperazine (red bars) and
benzylpiperazine (blue bars) derivatives. A. Affinities of compounds at human A2BARs determined
in radioligand binding assays are given as pK
i
values. B. A2BAR selectivity versus the A2AAR.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 80
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Di-substitution on the aromatic ring
Next, we investigated combination of two substituents on the aromatic ring in benzyl- and
phenylpiperazines (Table 6). Di-substitution with small residues was found to be very well
tolerated in different positions, e.g. F and OMe in compounds 24-26 and 45, Cl and OMe in
compounds 46 and 47, F and Cl or Br in compounds 50 and 27. However, combinations were not
synergistic, and the combination of two favourable substituents did not appear to increase affinity
over that of mono-substituted analog (compare, for example, 16/77). However, some 3,4-di-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
substitutions resulted in increased A2A, and in some cases also A
selectivity, e.g. 105 (3,4-methylenedioxy; K 2B 1.06 nM, K 2AAR 112 nM), 48 (3-Cl, 4-OH; K
2B 3.55 nM, A2A 93.8 nM) and 49 (4-Cl, 3-OH; K 2B 0.215 nM, A2A 35.3 nM, A 113 nM).
1
affinities, and reduced A2B
i
A
i
A
i
A
i
A
1
Potency comparisons between di-substituted compounds and each of the respective mono-
substitued compounds are depicted in Fig. 6. In most cases, affinities of the di-substituted
derivatives with small substituents were similar to those of the mono-substituted analogs, while the
selectivity indices were interdependently differing.
Table 6. Adenosine receptors affinities of xanthine-8-yl-benzenesulfonamide derivatives with di-
substitution of the phenyl/benzyl ring.
Compd.
R1
R2
K
i
± SEM (nM) (or % inhibition of radioligand binding at indicated
Selectivity
Indexe
concentration)
human A2Ba
human A2Ab
human A c
human A d
1
3
28
ACS Paragon Plus Environment

Page 29 of 80
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Substitutions on the benzyl ring
>1000 (31%) f
>1000 (12%)f
2
2
2
2
1
6
4
5
7
3-F
3-F
4-F
3-F
5-OMe
4-OMe
3-OMe
4-Br
1.02 ± 0.35
160 ± 45
245 ± 35
135 ± 36
403 ± 6
112
160
270
220
690
110
0.894 ± 0.173g
0.629 ± 0.031
0.473 ± 0.076
>1000 (24%) f >1000 (15%)f
>1000 (25%) f
>1000 (-5%)f
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
326 ± 49g
443
>1000 (27%)f
17600
05 37 3,4-methylenedioxy 1.06
Substitutions on the phenyl ring
3-F
4-OMe
4-OMe
3-OMe
4-OH
3-OH
4-Cl
1.24 ± 0.03
>1000 (30%)f
>1000 (31%)f
35.3 ± 8.2
>1000 (34%) f >1000 (21%)f
>1000 (23%) f >1000 (1%) f
>800
>2840
160
4
5
46
3-Cl
4-Cl
3-Cl
4-Cl
2-F
0.352 ± 0.072
0.215 ± 0.067
3.55 ± 0.77
535 ± 250
>1000 (37%) f >1000 (41%)f
>1000 (46%)f
4
7
93.8 ± 17.7 g
40.7 ± 10.4
>1000 (6%)f
>30
48
>1000 (31%)f
4
5
9
0
0.421± 0.041
1.91 ± 0.47
113 ± 39
100
>1000 (9%)f
>1000 (14%)f
>500
a
3
b
3
c
3
d
3
vs. [ H]PSB-603 (n = 3); vs. [ H]MSX-2 (n = 3); vs. [ H]CCPA (n = 3); vs. [ H]PSB-11 (n =
e
3
); the selectivity index was calculated by dividing the second lowest K
i
value by the A2BAR K
i
f
g
value; percent inhibition of radioligand binding at 1 μM; extrapolated curve.
29
ACS Paragon Plus Environment