Development of Potent and Selective Antagonists for the UTP-Activated P2Y4 Receptor

Article abstract of DOI:10.1021/acs.jmedchem.7b00030

P2Y₄ is a G_q protein-coupled receptor activated by uridine-5′-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y₄ receptor antagonist has been described so far. Therefore, we developed and optimized P2Y₄ receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescence-based assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y₄ receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4-dimethylphenylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, 61) exhibited an IC₅₀ value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand-receptor interactions. Compound 64 (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most selective P2Y₄ receptor antagonist known to date. Compounds 61 and 64 are therefore anticipated to become useful tools for studying this scarcely investigated receptor.

Full text of DOI:10.1021/acs.jmedchem.7b00030

Subscriber access provided by University of Colorado Boulder
Article
Development of Potent and Selective
4
Antagonists for the UTP-Activated P2Y Receptor
Muhammad Rafehi, Enas Malik, Alexander Neumann, Aliaa Abdelrahman,
Theodor Hanck, Vigneshwaran Namasivayam, Christa E Müller, and Younis Baqi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00030 • Publication Date (Web): 17 Mar 2017
Downloaded from http://pubs.acs.org on March 18, 2017
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Development of Potent and Selective Antagonists for
the UTPꢀActivated P2Y Receptor
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†,#
†,#
†
†
Muhammad Rafehi, Enas M. Malik, Alexander Neumann, Aliaa Abdelrahman, Theodor
†
†
,†
,‡
Hanck, Vigneshwaran Namasivayam, Christa E. Müller,* and Younis Baqi*
†
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical
‡
Sciences Bonn (PSB), University of Bonn, An der Immenburg 4, Dꢀ53121 Bonn, Department
of Chemistry, Faculty of Science, Sultan Qaboos University, PO Box 36, Postal Code 123,
Muscat, Oman
#
These authors contributed equally to this work
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 68
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. P2Y
4
is a G
q
proteinꢀcoupled receptor activated by uridineꢀ5’ꢀtriphosphate
(
UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective
P2Y
P2Y
4
4
receptor antagonist has been described so far. Therefore, we developed and optimized
antagonists based on an anthraquinone scaffold. Potency was assessed by a
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
fluorescenceꢀbased assay measuring inhibition of UTPꢀinduced intracellular calcium release in
1321N1 astrocytoma cells stably transfected with the human P2Y receptor. The most potent
compound of the present series, sodium 1ꢀaminoꢀ4ꢀ[4ꢀ(2,4ꢀdimethylphenylthio)phenylamino]ꢀ
4
9
,10ꢀdioxoꢀ9,10ꢀdihydroanthraceneꢀ2ꢀsulfonate (PSBꢀ16133, 61) exhibited an IC50 value of
2
33 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric
antagonist. A receptor homology model was built and docking studies were performed to
analyze ligandꢀreceptor interactions. Compound 64 represents the most potent and selective
P2Y receptor antagonist known to date and is therefore anticipated to become a useful tool
4
for studying this scarcely investigated receptor.
KEYWORDS. anthraquinone, cystic fibrosis, infectious diarrhea, homology modeling,
4
neurodegenerative diseases, P2Y antagonist, Reactive Blue 2, selectivity, synthesis
ACS Paragon Plus Environment

Page 3 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
The G proteinꢀcoupled P2Y receptors represent, together with the ATPꢀgated ion channels
known as P2X receptors, the nucleotideꢀactivated P2 receptor family. Eight subtypes of P2Y
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
receptors are known to exist: P2Y
1 2 4 6 1
, P2Y , P2Y , P2Y , and P2Y11 belonging to the P2Y ꢀlike
subgroup, and P2Y , P2Y , and P2Y that are members of the P2Y ꢀlike subgroup of P2Y
1
2
13
14
12
1
,2
receptors.
P2Y receptors are present in almost all human tissues, where they exert various biological
functions. They are of great interest as (potential) therapeutic targets for several indications,
3,4
including neurodegenerative disorders, pain, cancer, and cardiovascular diseases. In fact,
several antithrombotic drugs on the market, namely clopidogrel, prasugrel and ticagrelor,
5
–7
achieve their effect through inhibiting the human platelet P2Y12 receptor.
generally strong interest in the P2Y receptor family, relatively little is known regarding the
P2Y subtype. This uridineꢀ5'ꢀtriphosphate (UTP)–activated receptor shows a wide
Despite a
4
distribution in the body, including heart, gastrointestinal tract (GIT), central nervous system
8–12
(CNS), skin, and ear (cochlea).
4
In the GIT, P2Y receptors were demonstrated to mediate
−
chloride (Cl ) secretion in the jejunal epithelium, and therefore represent possible targets for
4 4
the treatment of cystic fibrosis (P2Y receptor agonists) and diarrhea (P2Y receptor
13–16
antagonists).
P2Y
4
receptors also appear to be involved in the regulation of amyloid
17
precursor protein (APP) production and its release in the brain. Since APP accumulation is
associated with the progression of Alzheimer’s disease, P2Y
useful as therapeutic agents for this debilitating and yet incurable disorder. In the heart,
P2Y receptors are expressed on cardiac endothelial cells; they were found to be important for
4
receptor antagonists might be
1
7
4
cardiac endothelial cell growth and migration, as well as secretion of plateletꢀderived growth
factor B (PDGFꢀB). Moreover, P2Y receptorꢀknockout mice showed defective postꢀnatal
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 68
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
cardiac development, including reduced heart weight. It has thus been postulated that P2Y
4
receptors could be possible targets to modulate angiogenesis, and regulate cardiac remodeling
18
and postꢀischemic revascularization. In the cochlea, P2Y
4
receptors are expressed in the
+
epithelial cells of Reissner’s membrane, where they regulate Na absorption upon noise
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
8
+
exposure, and in the apical membrane, where they are involved in K secretion across strial
9
marginal cell epithelium during stimulation of the cochlea by sound.
1
So far, no selective antagonists for the P2Y
4
receptors have been described. However, such a
compound is required as a pharmacological tool for receptor characterization and for studies
19
aimed at target validation. Mouse and rat P2Y
4
1
receptors, which share 51 % and 83 %
2,20
sequence identity with the human P2Y
4
receptor,
are nonꢀcompetitively antagonized by
PPADS (pyridoxalphosphateꢀ6ꢀazophenylꢀ2',4'ꢀdisulfonic acid, 1, Figure 1), and also blocked
2
1,22
by Reactive Blue 2 (RBꢀ2, 2, Figure 1).
were found to be weakly antagonized by suramin (3, Figure 1). On the other hand, human
P2Y receptors expressed in 1321N1 cells were ineffectively inhibited by PPADS and were
insensitive to suramin, but were inhibited by RBꢀ2 at micromolar concentrations.
Rat P2Y receptors, expressed in Xenopus oocytes,
4
2
3
4
2
3
24
RBꢀ2 is a wellꢀknown nonꢀselective P2 receptor antagonist that has served as an important
2
5,26
pharmacological tool in the field of purinergic signaling.
Our laboratory has been
extensively working on the development of anthraquinone derivatives structurally related to
2
4,27–36
RBꢀ2 as potent and selective antagonists of purine receptors and ectonucleotidases.
For
30
example, we have previously developed PSBꢀ0739 (4, Figure 1), an extremely potent and
3
7
2
selective, competitive P2Y12 receptor antagonist (pA of 9.8), and PSBꢀ1011 (5, Figure 1), a
3
1
competitive inhibitor of the rat P2X2 receptor with a K value of 79 nM.
i
ACS Paragon Plus Environment

Page 5 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CHO
O
P
O
B
NH2
C
HO
SO3Na
ONa
O
A
ONa
H C
3
N
N
Cl
O
HN
N
N
D
E
N
F
SO Na
3
N
H
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NaO S
3
SO Na
3
(m, p)
H
NaO S
3
1
, PPADS
2, Reactive Blue 2 (RBꢀ2)
SO Na
SO Na
3
3
NaO S
SO Na
3
3
NaO S HN
O
O
NH SO Na
3
3
O
O
H
H
N
N
N
N
H
H
CH3
O
, Suramin
CH3
3
O
O
NH2
O
NH2
SO Na
SO Na
3
3
HN
O
HN
N
H
SO Na
3
NaO S
HN
N
Cl
3
N
N
4
, PSBꢀ0739
5, PSBꢀ1011
P2X2ꢀselective
antagonist
P2Y ꢀselective
12
Cl
antagonist
Figure 1. Structures of the classical P2 receptor antagonists PPADS, Reactive Blue 2 (RBꢀ2),
suramin, and receptor subtypeꢀselective tool compounds derived from RBꢀ2.
High potency and selectivity could be attained, for example, by optimizing the residue at the
4
ꢀposition of the anthraquinone core. Structureꢀactivity relationships (SARs) were found to be
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 68
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
very different at the different purine receptor subtypes, and the anthraquinone core structure
2
6
can thus be described as a ‘privileged structure’ in medicinal chemistry.
The aim of this study was to specifically design and develop the first potent and selective
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
P2Y
4
receptor antagonist. To this end, a library of RBꢀ2ꢀrelated anthraquinone derivatives was
12–15
synthesized using recently developed methods,
and the compounds were tested for their
2
+
16
potency to block P2Y
4
receptors using a Ca ꢀmobilization assay. This data served as a basis
for subsequent structureꢀactivity relationship analyses and receptor docking studies aimed at
enhancing our knowledge with respect to P2Y receptor ligand preferences and for further
4
optimizing the structures. Modifications of the substituents at the 4ꢀposition of the
4
anthraquinone core led to an increase in potency at the P2Y receptor and resulted in
selectivity for P2Y over other P2Y receptor subtypes.
4
Results and discussion
Chemistry. The target compounds were synthesized as depicted in Schemes 1–3. The
24,27–30,33,36,38–40
syntheses of several compounds had been previously described.
Some of the
known anthraquinone derivatives (19–21, 24, and 33–35 (Table 1)) have now been obtained
by improved synthetic procedures. In addition to known compounds, a series of 21 new
1
compounds was prepared. Condensation of bromaminic acid (R = SO H (6a)) or 1ꢀaminoꢀ4ꢀ
3
1
bromoꢀ2ꢀmethylanthraquinone (R = CH (6b)) with the appropriate amine yielded the target
3
compounds in good to excellent isolated yields (Scheme 1). Reactions were conducted in
0
phosphate buffer (pH 6ꢀ7) in the presence of copper powder (Cu ) under microwave
irradiation at 80ꢀ120 °C (in case of 6a as a starting compound) or in methanol in the presence
0
of potassium acetate and a mixture of Cu and copper(II) acetate monohydrate under
microwave irradiation at 95 °C (in case of 6b).
ACS Paragon Plus Environment

Page 7 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Scheme 1. General Synthesis of 4ꢀSubstituted Anthraquinone Derivatives (7–53 and 67)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
2
0
Reagents and conditions: (i) R ꢀNH
C, 5ꢀ24 min; (ii) 2ꢀaminoꢀ4ꢀfluorobenzoic acid, methanol, potassium acetate, Cu , Cu(II)
acetate, microwave, 95 °C, 70 min.
2
, phosphate buffer (pH 6–7), Cu , microwave, 80ꢀ120
0
°
Compounds 65 and 66 were synthesized from 1ꢀaminoꢀ4ꢀbromoꢀ2ꢀmethylanthraquinone (6b)
by treatment with an excess of the appropriate aniline derivative (15 equiv.) under argon in the
presence of potassium acetate and a catalytic amount of copper(I) acetate at 110 °C for 6ꢀ15 h
(Scheme 2).
a
Scheme 2. Synthesis of Anthraquinone Derivatives 65 and 66
a
Reagents and conditions: (i) CuOAc, KOAc, 110 °C, argon, 6–15 h.
Three anilinoanthraquinone derivatives, 12, 27, and 46, which bear a primary amino group in
the 1ꢀposition of the anthraquinone moiety, were treated with sodium nitrite in hydrochloric
acid solution (1 M) at 0 °C for 5 min, then allowed to warm up to room temperature followed
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 68
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
9
by the addition of ethanol and an excess of zinc powder (10 equiv.) to achieve deamination
affording the desired products 68–70 (Scheme 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
a
Scheme 3. Synthesis of Deaminated Anthraquinone Derivatives 68–70
a
Reagents and conditions: (i) NaNO
ethanol, rt, 30 s.
2
, HCl (1 M), 0–5 °C, 5 min, (ii) Zn (10 equiv.),
ACS Paragon Plus Environment

Page 9 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Yields, Molecular Weights, UV Absorption, Color, and Purity of the Newly
Synthesized Anthraquinone Derivatives
O
O
NH2
O
O
NH
2
O
O
R1
R1
R1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R2
HN
HN
HN
R2
R2
X = O, S
X
Structure A
Structure B
Structure C
1
2
Compd
R
R (A, C)
Yield
MW Absorption Color
Purity by
2
a
or R -X (B)
(%)
(g/mol) λmax (nm)
LC-MS/UV
b
(%)
Structure A
14
19
20
21
24
25
33
34
35
37
SO
SO
SO
SO
SO
SO
SO
SO
SO
SO
3
3
3
3
3
3
3
3
3
3
Na 2ꢀchloro
36
80
70
72
64
35
49
60
50
46
450.83
430.41
430.41
430.41
444.44
458.46
444.44
444.44
444.44
476.43
590
624
626
626
626
628
624
624
624
624
Blue
Blue
Blue
Blue
Blue
Blue
Blue
Blue
Blue
Blue
99.0
99.0
99.1
100.0
99.0
97.7
99.0
99.0
99.5
98.5
Na 2ꢀmethyl
Na 3ꢀmethyl
Na 4ꢀmethyl
Na 4ꢀethyl
Na 3ꢀpropyl
Na 2,3ꢀdimethyl
Na 2,4ꢀdimethyl
Na 2,5ꢀdimethyl
Na 3,4ꢀdimethoxy
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 68
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
39
40
41
42
43
44
49
52
53
SO
SO
SO
SO
SO
SO
SO
SO
SO
3
3
3
3
3
3
3
3
3
Na 3ꢀmethoxyꢀ4ꢀmethyl
Na 4ꢀchloroꢀ2ꢀmethyl
Na 4ꢀchloroꢀ3ꢀmethyl
Na 4ꢀhydroxyꢀ3ꢀmethyl
Na 4ꢀfluoroꢀ3ꢀmethoxy
Na 4ꢀchloroꢀ3ꢀmethoxy
Na 2ꢀcarboxyꢀ3ꢀfluoro
Na 2ꢀcarboxyꢀ4ꢀhydroxy
Na 2ꢀcarboxyꢀ4ꢀnitro
44
36
40
40
43
40
40
68
21
460.43
464.85
464.85
446.41
464.40
480.85
478.38
476.39
505.39
626
626
624
626
624
624
594
630
584
Blue
Blue
Blue
Blue
Blue
Blue
Blue
Blue
Blue
98.3
99.0
99.0
96.0
99.3
99.7
98.0
100.0
95.7
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
Structure B
58
59
60
61
62
63
64
SO
SO
SO
SO
SO
SO
SO
3
3
3
3
3
3
3
Na 4ꢀfluorophenoxy
41
25
17
10
7
526.47
542.92
587.38
552.59
552.59
552.59
539.56
630
630
625
640
640
640
640
Blue
Blue
Blue
Blue
Blue
Blue
Blue
99.5
98.6
96.1
96.1
98.0
95.9
98.6
Na 4ꢀchlorophenoxy
Na 4ꢀbromophenoxy
Na 2,4ꢀdimethylphenylthio
Na 2,5ꢀdimethylphenylthio
Na 3,4ꢀdimethylphenylthio
Na 3ꢀpyridylmethylthio
7
14
Structure C
ACS Paragon Plus Environment

Page 11 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
68
69
SO
SO
3
3
H
H
fluoro
97
38
397.38
409.41
500
506
Purple
Purple
98.5
95.2
methoxy
8
9
a
b
Isolated yield; Purity of the compounds was determined using LCꢀMS coupled to a diode array
detector (220ꢀ900 nm).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Purification of Reactive Blue 2. RBꢀ2 has been used since decades as a valuable
pharmacological tool for studying nucleotide receptors. Most studies relied on commercially
available products. However, there has always been some doubt regarding the identity and
purity of commercial products, making it difficult to obtain reliable pharmacological
4
1–43
results.
RBꢀ2 is a mixture of two isomers bearing a sulfonate group either in position 3 or
4
of ring F (compound 2, Figure 1). Thus, we decided to investigate the purity of
commercially available RBꢀ2. The purity of the investigated RBꢀ2 was declared to be 88.2 %
as determined by HPLCꢀUV. However, the employed technique is not suitable to detect
inorganic impurities, e.g. inorganic salts. Therefore, we purified commercially available RBꢀ2
by reversed phaseꢀ18 flash column chromatography (RPꢀ18 FCC, for details see experimental
section). Subsequent analysis showed that the commercial product only contained 54 % of
RBꢀ2, while the main organic contaminant was found to be the precursor of RBꢀ2, namely
disodium
9,10ꢀdioxoꢀ9,10ꢀdihydroanthraceneꢀ2ꢀsulfonate (ca. 12 %). Moreover, it was found to be
contaminated with ca. 34 % of inorganic salts, presumably NaCl and Na PO , which were
washed out during the purification step using RPꢀ18 column chromatography.
1ꢀaminoꢀ4ꢀ[4ꢀ(4,6ꢀdichloroꢀ[1,3,5]triazinꢀ2ꢀylamino)ꢀ3ꢀsulfonatophenylamino]ꢀ
3
4
Biological assays. The potencies of the compounds were determined using a fluorescence–
2
+
based Ca ꢀmobilization assay according to the following principle: monoclonal colonies of
4
1321N1 astrocytoma cells stably transfected with the human P2Y receptor were loaded with
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 68
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
4
the acetoxymethyl ester of the calciumꢀsensitive fluorescent dye fluoꢀ4. The P2Y receptor is
coupled to G protein. Thus, receptor activation with the endogenous ligand UTP will lead to
q
an increase in the intracellular calcium ion concentration and a rise in the intensity of
4
fluorescent light emitted by the dye. Test compounds that act as P2Y receptor antagonists
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
inhibit the increase in fluorescence intensity, which is monitored using a microplate
fluorescence reader. Prior to evaluating the antagonistic activity of the anthraquinone
derivatives, a concentrationꢀresponse curve of the agonist UTP at the human P2Y
4
receptor
was obtained (Figure 2). The EC of UTP at this cell line was determined to be 565 nM. A
8
0
UTP concentration of 500 nM, close to its EC80 value, was subsequently used for receptor
activation for the determination of antagonist potency using the calcium mobilization assay.
A total of 64 synthesized anthraquinone derivatives including 21 new compounds not
previously described in literature along with the commercially obtained and the purified RBꢀ2
4
were tested for their inhibitory activity at the human P2Y receptor. The IC50 values are
summarized in Tables 2ꢀ4.
Structure–activity relationships. Purified RBꢀ2 (2’) displayed an IC50 value of 0.625 µM
and hence was more potent than the commercially available RBꢀ2 (IC50 = 1.14 µM, Figure 3
and Table 1). This is very reasonable, since the dye content of commercially available RBꢀ2
was found to be only 54 %. While the hill slope of the concentrationꢀinhibition curve for
purified RBꢀ2 (2’) was found to be close to unity, crude RBꢀ2 had a much steeper hill slope of
ꢀ2.20. This might be due to allosteric modulatory effects of the contaminants present in the
4
4
nonꢀpurified material, e.g., inorganic salts.
ACS Paragon Plus Environment

Page 13 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
25
00
1
7
5
2
5
0
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-25
10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 4
UTP], M
[
Figure 2. Concentrationꢀresponse curve of the agonist UTP determined by calciumꢀ
mobilization assays on recombinant 1321N1 astrocytoma cells stably expressing the human
4
P2Y receptor. Data points shown are the mean values of eight independent experiments, each
performed in duplicates. The determined EC50 of UTP is 203 ± 41 nM. The calculated EC80
value is 565 nM.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 68
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
1
25
00
2
(RBꢀ2)
2' (purified RBꢀ2)
1
7
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
50
2
5
0
-25
10-9
10-8
10-7
10-6
10-5
[
Compound], M
Figure 3. Concentrationꢀresponse curves of the commercially available RBꢀ2 (2) and purified
RBꢀ2 (2’), determined using the calciumꢀmobilization assay on recombinant 1321N1
4
astrocytoma cells stably expressing the human P2Y receptor. UTP at a concentration of 500
nM (~EC80) was used for receptor activation. Data points shown are the mean values of at
least three independent experiments, each performed in duplicates. The IC50 values are 1.14 ±
0
.31 for the commercially available RBꢀ2 (2), Hill slope ꢀ2.20, and 0.625 ± 0.198 for the
purified RBꢀ2 (2’), Hill slope ꢀ0.80.
Regarding the synthesized compounds, the structurally simple 1ꢀaminoꢀ4ꢀphenylaminoꢀ2ꢀ
sulfoanthraquinone, Acid Blueꢀ25 (ABꢀ25, 7), showed inhibitory activity at low micromolar
concentrations (IC50 = 3.10 µM), being almost as potent as the much larger RBꢀ2 (IC50 = 1.14
µM). Replacing the phenylamino ring by benzylamino, phenethylamino, or αꢀnaphthylamino
(
8–10) decreased the activity by more than twoꢀfold (Table 2).
ACS Paragon Plus Environment

Page 15 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2. Antagonistic Activity of Anthraquinone Derivatives at the Human P2Y
4
Receptor –
Part I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Compound
7
R
IC ± SEM (µM)
5
0
3.10 ± 0.35
14.4 ± 3.50
7.18 ± 0.80
7.42 ± 2.77
8
9
1
0
2 (RBꢀ2)
For structure see Figure 1
For structure see Figure 1
1.14 ± 0.31
2’ (RBꢀ2 purified)
0.625 ± 0.198
a
Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP
EC ) in 1321N1 astrocytoma cells stably transfected with the human P2Y receptor.
(
8
0
4
These results suggested that a phenylamino substituent at the 4ꢀposition of the anthraquinone
core structure is preferred for P2Y receptor inhibitory potency over a benzyl or phenethyl
4
residue. Therefore, a library consisting of 60 derivatives of lead structure 7 with various
monoꢀ and diꢀsubstitutions on the phenyl ring attached to the 4ꢀamino group was investigated
4
for inhibitory potency at the P2Y receptor (see Table 3).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 68
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
4
Table 3. Antagonistic Activity of Anthraquinone Derivatives at the Human P2Y Receptor –
Part II
O
B
NH2
C
O
B
NH2
C
SO Na
SO Na
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
A
A
R1
O
HN
R2
O
HN
D
D
R3
S
11ꢀ63
64
E
R4
N
1
2
3
4
a
Compound
R
R
R
R
IC ± SEM (µM)
5
0
(or % inhibition at
indicated concentration)
1.97 ± 0.42
1.39 ± 0.36
4.04 ± 1.01
9.34 ± 2.07
2.34 ± 0.71
3.03 ± 0.66
6.96 ± 0.73
3.30 ± 0.09
3.49 ± 0.29
2.22 ± 0.31
2.82 ± 0.92
3.53 ± 1.21
1.51 ± 0.48
3.21 ± 0.91
2.99 ± 0.12
3.98 ± 1.39
1.70 ± 0.24
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
F
H
F
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
CH
H
H
H
H
H
H
Cl
H
Cl
H
Br
H
Br
H
3
H
CH
H
H
3
CH
H
3
C
2
H
5
H
H
H
H
C
2
H
H
5
7
H
H
2 5
C H
C
3
H
H
H
OMe
H
H
OMe
ACS Paragon Plus Environment

Page 17 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
H
H
OMe
OH
H
H
2.59 ± 0.72
3.16 ± 0.58
6.10 ± 1.72
23.4 ± 3.6
H
H
H
CO
H
2
H
H
H
CO
H
2
3
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
b
H
CO
H
2
3
H
H
≈ 30 (57 %)
2.63 ± 0.59
3.58 ± 0.25
11.2 ± 1.20
6.45 ± 2.22
4.98 ± 0.99
CH
CH
CH
H
CH
H
H
3
3
3
CH
H
H
H
CH
CH
H
3
CH
3
H
3
H
OMe
OMe
OMe
H
OMe
H
H
OMe 9.25 ± 4.56
H
CH
Cl
3
H
H
H
H
H
H
F
4.13 ± 0.99
2.20 ± 0.52
1.65 ± 0.37
CH
H
3
CH
CH
3
3
Cl
H
OH
F
0.746 ± 0.076
1.54 ± 0.66
3.37 ± 1.09
12.5 ± 2.30
H
OMe
OMe
F
H
Cl
H
H
14
c
CO
CO
CO
CO
CO
CO
CO
CO
H
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
F
H
H
H
H
F
> 100 (17 %)
c
47
48
49
50
51
52
53
54
55
H
Cl
Br
H
> 100 (31 %)
b
H
> 100 (30 %)
c
F
> 100 (10 %)
c
H
H
> 100 (33 %)
H
H
Cl
H
H
H
H
2.76 ± 0.75
21.1 ± 7.1
H
OH
NO
2
2
b
H
2
> 30 (30 %)
H
benzyl
3.45 ± 0.41
1.94 ± 0.54
H
H
phenoxy
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 68
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
56
57
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
phenylamino
H
H
H
H
H
H
H
H
1.91 ± 0.28
phenylthio
1.18 ± 0.09
58
59
60
61
62
63
64
4ꢀfluorophenoxy
4ꢀchlorophenoxy
4ꢀbromophenoxy
2,4ꢀdimethylphenylthio
2,5ꢀdimethylphenylthio
3,4ꢀdimethylphenylthio
1.69 ± 0.38
0.373 ± 0.112
1.76 ± 0.49
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
0.233 ± 0.079
0.395 ± 0.082
0.482 ± 0.137
0.409 ± 0.138
see structure above
a
Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP (EC80
in 1321N1 astrocytoma cells stably transfected with the human P2Y receptor. % Inhibition at
0 µM. % Inhibition at 100 µM.
)
b
4
c
3
Introducing different lipophilic substituents in the orthoꢀ, metaꢀ, and paraꢀpositions in ring
D, e.g. a halogen atom (F, Cl, Br), an alkyl (CH , C H , C H ), or a methoxy group, did not
3
2
5
3
7
alter the activity, which ranged between 1 and 9 µM (compounds 11ꢀ28, Table 3). On the
other hand, introducing hydrophilic moieties led to a reduction in potency by over a tenꢀfold in
the case of metaꢀ and paraꢀcarboxy, while orthoꢀcarboxy and paraꢀhydroxy substitutions were
tolerated (compounds 29ꢀ32, Table 3).
In the next step, we assessed diꢀsubstitution in ring D (Table 3): diꢀsubstitution with
lipophilic residues did not alter the compounds’ potency (compounds 33ꢀ41 and 43ꢀ45, Table
3
). However, a combination of lipophilic and hydrophilic substituents affected the activity in
three different directions: 3ꢀmethylꢀ4ꢀhydroxy (42; IC50 = 0.746) enhanced the antagonistic
activity by over fourꢀfold, 2ꢀcarboxyꢀ5ꢀchloro (51; IC50 = 2.76) was tolerated, whereas any
other combination, 2ꢀcarboxyꢀ4ꢀfluoro (46), 2ꢀcarboxyꢀ4ꢀchloro (47), 2ꢀcarboxyꢀ4ꢀbromo
(
48), 2ꢀcarboxyꢀ3ꢀfluoro (49), 2ꢀcarboxyꢀ5ꢀfluoro (50), 2ꢀcarboxyꢀ3ꢀhydroxy (52), and 2ꢀ
carboxyꢀ3ꢀnitro (53), dramatically decreased the inhibitory activity up to over a hundredꢀfold
Table 3).
(
ACS Paragon Plus Environment

Page 19 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In order to investigate the effect of the presence of an additional ring E, eleven new
compounds (54ꢀ64) were synthesized. In the first step, different bridges (CH , O, NH, S) in
2
the paraꢀposition of ring D were investigated. All four linkers were tolerated (compounds 54ꢀ
5
7, IC50 = 3.45, 1.94, 1.91, and 1.18 µM, respectively, Table 3). In the next step, different
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
substituent were introduced into ring E, including halogen (F, Cl, Br) and methyl groups. 4ꢀ
fluorophenoxy and 4ꢀbromophenoxy residues were found to have no influence on the
antagonistic activity (compounds 58 and 60, IC50 = 1.69 and 1.76, respectively, Table 3),
while 4ꢀchlorophenoxy substitution increased the potency by > eightꢀfold (59, IC50 = 0.373).
Diꢀmethylation in ring E with a sulfide linker between ring D and E improved potency by
more than 13ꢀfold (compounds 61-64, IC = 0.233, 0.395, 0.482, and 0.409 µM, respectively,
5
0
Table 3 and Figure 4). Indeed, compound 61 (IC50 = 0.233 µM) can be considered as the most
potent P2Y receptor antagonist known to date.
4
As a next step, we were interested in investigating the effects of other substituents on the
anthraquinone core, namely the amino group in the 1ꢀposition and the sulfonate group in the
2ꢀposition. Therefore, we developed six new analogs (65-70, Table 4) of selected
anthraquinone derivatives (12, 27, and 46). No improvement in the activity of compound 46
(IC50 > 100, Table 3) was observed upon replacement of the sulfonate group with a methyl
group (compound 67) or upon deamination at the 1ꢀposition (compound 70). Moreover,
replacement of the 2ꢀsulfonate group of compounds 12 and 27, (IC50 = 1.39 and 1.70 µM,
respectively, Table 3) with a methyl group completely abolished the activity (compounds 65
and 66, respectively, Table 4). Similar results were obtained with the deꢀaminated compounds
6
8 and 69 (Table 4). These results indicate that the presence of an amino group in the 1ꢀ
position and a sulfonate function in the 2ꢀposition of the anthraquinone core are essential for
antagonistic activity at the P2Y receptor.
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 68
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
7
6
6
6
1
25
00
1
2
3
1
7
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
50
2
5
0
-25
10- 9 10- 8 10- 7 10- 6 10- 5 10- 4
Compound], M
[
Figure 4. Concentrationꢀresponse curves of selected anthraquinone derivatives, determined
using the calcium mobilization assay with recombinant 1321N1 astrocytoma cells stably
4
expressing the human P2Y receptor. UTP at a concentration of 500 nM (EC80) was used for
receptor activation. Data points shown are the mean values of at least three independent
experiments, each performed in duplicates. The IC50 values are found in Tables 2 and 3.
ACS Paragon Plus Environment

Page 21 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 4. Inhibitory Potencies at the Human P2Y
Derivatives Modified in the 1ꢀ and 2ꢀPositions.
4
Receptor of Selected Anthraquinone
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
3
a
Compound
R
R
R
IC ± SEM (µM)
5
0
(
or % inhibition at indicated
concentration)
Structure A
12
27
46
H
H
F
H
H
F
1.39 ± 0.36
1.70 ± 0.28
OMe
H
c
CO
2
2
2
H
H
H
> 100 (17 %)
Structure B
Structure C
b
65
66
67
H
F
H
H
F
> 30 (ꢀ4 %)
b
H
OMe
H
> 30 (38 %)
c
CO
≈ 100 (57 %)
b
68
69
70
H
F
H
H
F
≈ 30 (49 %)
b
H
OMe
H
> 10 (77 %)
c
CO
> 100 (25 %)
a
Potency to inhibit calcium mobilization following receptor activation with 500 nM UTP (EC )
in 1321N1 astrocytoma cells stably transfected with the human P2Y
0 µM; % Inhibition at 100 µM
8
0
b
4
receptor; % Inhibition at
c
3
Comparison of structure-activity relationships of anthraquinone derivatives at different
targets. An overview of the structureꢀactivity relationships (SARs) of the investigated
4
anthraquinone derivatives at the human P2Y receptor is depicted in Figure 5. The ABCDE
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 68
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
aromatic ring system proved to be important for high inhibitory potency. The amino function
in the 1ꢀposition and the sulfonate group in the 2ꢀposition of the anthraquinone scaffold were
found to be essential for the activity. This is in accordance with previous studies on the SARs
of RBꢀ2 and related dyes, which showed that ABꢀ25 is the minimum structural requirement
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
4,29,30,33,45–52
for biological activity of this class of compounds.
Moreover, the activity on the
P2Y receptor was strongly affected by the substituents present on ring E, e.g. a lipophilic
4
substitution in the orthoꢀ, metaꢀ, or paraꢀposition increased the activity, while an
unsubstituted ring E with an ꢀSCH ꢀlinker was also tolerated.
2
Figure 5. Summary of structureꢀactivity relationships of anthraquinone derivatives as P2Y4
receptor antagonists.
It is interesting to compare the results of the SAR analysis at the human P2Y receptor with
4
that at other purinergic targets (Figure 53, Supplementary material). The main difference
between the human P2Y
4 1
receptor and both the human P2Y12 receptor and P2Y ꢀlike
receptors of the guineaꢀpig taenia coli (receptor was not cloned but found to be
4
1
1
pharmacologically similar to the recombinant P2Y ꢀreceptor subtype) is that substitution of
ACS Paragon Plus Environment

Page 23 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
9,37
ring D with a sulfonate group at the metaꢀposition (for P2Y12 receptors)
and at the orthoꢀ
position (for the P2Y ꢀlike receptors) was essential for the inhibitory activity of the
1
32,42
compounds. An additional ring F was also important in case of the P2Y
1
ꢀlike receptor.
For
activity at the AMPꢀhydrolyzing enzyme ectoꢀ5’ꢀnucleotidase, ring system ABCD was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4
sufficient for the inhibitory activity. In contrast, a triazine or pyrimidine as ring E was
3
1
preferred by the P2X2 receptor. On the other hand, development of anthraquinone
2
+
+
compounds as large conductance Ca ꢀactivated K channel openers showed a preference for a
53
bulky and hydrophobic ring D. Interestingly, the amino group at ring C was not essential for
53
activity at that target.
These differences in SARs emphasize the influence of the nature of substituents at the 4ꢀ
position of the anthraquinone ring on the potency and targetꢀselectivity of the compounds, and
allow the development of receptor (subtype)ꢀselective ligands.
Selectivity. To gain insights into the selectivity of these compounds, we assessed the most
potent compounds of this series, 61ꢀ64, on the receptor subtypes that share the greatest
sequence homology with the P2Y
the subgroup of G proteinꢀcoupled P2Y
selected compounds at the P2Y12 receptor that had previously been shown to be potently
4
receptor, namely P2Y
1 2 6
, P2Y , and P2Y , which belong to
q
1
ꢀlike P2Y receptors. In addition, we investigated
3
0
blocked by anthraquinone derivatives. Table 5 and Figure 6 summarize the results. Of this
series of compounds, 64 is the most selective one, with particularly high selectivity versus the
P2Y
1 6
and P2Y receptors, where it showed only low potency. Selectivity against the P2Y12
receptor was also observed. It can therefore be concluded that these compounds display P2Y
4
receptor selectivity, and it is not only the potency, but also the selectivity of lead structure 7
that has been significantly enhanced (see Figure 6).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 68
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
7
.0
P2Y1
P2Y2
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
6.5
6.0
P2Y4
P2Y6
P2Y12
5.5
5.0
4.5
7
61
62
63
64
Compound
.
Figure 6. Potency of selected anthraquinone derivatives on different human P2Y receptors
determined using the calcium mobilization assay (P2Y1ꢀ6 receptors) or βꢀarrestin translocation
assay (P2Y12 receptor). Shown are the pIC50 values of at least three independent experiments
performed in duplicates.
ACS Paragon Plus Environment

Page 25 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. Selectivity of the Most Potent P2Y
4
Receptor Antagonists versus Other P2Y
a
Receptor Subtypes
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IC50 ± SEM (µM) or (% Inhibition at 50 µM)
Compd
R
hP2Y
4
hP2Y
1
hP2Y
2
hP2Y
6
hP2Y12
7
4
H
3.10 ± 0.35
17.6 ± 6.5
17.1 ± 6.3
2.79 ± 0.17 3.12 ± 1.17
4.75 ± 1.49 0.0604 ±
2
3ꢀmethylꢀ4ꢀhydroxy
0.746 ± 0.076
7.65 ± 1.90 16.4 ± 4.5
0.0147
6
6
1
2
2,4ꢀdimethyl
0.233 ± 0.079
0.395 ± 0.082
0.482 ± 0.137
0.409 ± 0.138
5.48 ± 0.34 8.54 ± 1.45 12.5 ± 3.9
2.41 ± 0.45
2,5ꢀdimethyl
2.72 ± 0.59 6.52 ± 1.04 2.67 ± 0.91 2.96 ± 0.24
63
64
3,4ꢀdimethyl
4.55 ± 0.42 10.2 ± 1.5
4.36 ± 0.60 2.34 ± 0.10
see structure above
~ 25
13.1 ± 2.6
>> 100
3.59 ± 0.38
(61 ± 4 %)
(12 ± 19
%)
a
Potency to inhibit calcium mobilization in 1321N1 astrocytoma cells recombinantly expressing
human P2Y recetors following receptor activation with 500 nM ADP (P2Y ), 500 nM UTP
P2Y , P2Y ), or 750 nM UDP (P2Y ). Potency at the G ꢀcoupled P2Y receptor was determined
1
(
2
4
6
i
12
using a βꢀarrestin translocation assay.
The binding mode of anthraquinone derivatives at the P2Y
4
receptor. The mode of
antagonism, competitive or nonꢀcompetitive, at the P2Y receptor was determined using the
4
most potent compound (61) of the present series. Concentrationꢀresponse curves of the
endogenous ligand UTP after a preꢀincubation with fixed concentrations of 61 were obtained.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 68
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
For a competitive antagonist, a parallel rightward shift of the curves towards higher
concentrations of UTP would be expected with increasing concentrations of 61. The upper
plateaus of the curves, corresponding to the maximum receptor activation, should remain
unaltered. A corresponding Schild plot would show a straight line with a slope of
approximately 1. Nonꢀcompetitive antagonism, on the other hand, is characterized by a
suppression of the maximum receptor activation with increasing antagonist concentrations,
while the EC50 remains largely unaffected. Our results, summarized in Figure 7, show a
significant depression of the upper plateaus of the UTP curves with increasing concentrations
of 61. The reduction in the maximum receptor activation is successively more pronounced
with rising concentrations of 61, and a complete blockade of UTPꢀmediated receptor
activation is observed at a concentration of 15 µM of 61. In the calcium mobilization assay,
the cells were first incubated with the antagonist 61 for 30 min. The agonist UTP was
subsequently injected into the cell suspension and the assay signal was read immediately
thereafter. Since there is no simultaneous incubation with both agonist and antagonist and the
measurement of the signal is very fast, the assay conditions are likely to be under nonꢀ
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
equilibrium conditions. Nevertheless, our data indicate that 61 may achieve P2Y receptor
antagonism via a nonꢀcompetitive mechanism, but competitive antagonism cannot be
completely ruled out at present. Interestingly, on the P2Y12 receptor, we previously found that
3
0
anthraquinone derivatives of this series act as competitive antagonists. However, the
percentage of sequence shared by the P2Y and P2Y12 receptors is only 25 % and,
4
consequently, significant differences exist between these two receptors (also with respect to
orthosteric ligand preferences and G protein coupling).
In order to get more insight into the interaction of the anthraquinone derivatives with the
P2Y
4
receptor on a molecular level we decided to perform docking studies.
ACS Paragon Plus Environment

Page 27 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
25
00
UTP
UTP + 61:
1
0.15 ꢁM
0.50 ꢁM
7
5
2
5
0
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
.50 ꢁM
5.00 ꢁM
5.0 ꢁM
1
-
25
0-11
10-9
10-7
UTP], M
10-5
10-3
1
[
Figure 7. Concentrationꢀresponse curves of UTP on the P2Y
with fixed concentrations of 61. The results were obtained using the calciumꢀmobilization
assay with 1321N1 astrocytoma cells recombinantly expressing the human P2Y receptor.
4
receptor after preꢀincubation
4
EC50 values are not significantly different from each other (p > 0.05, unpaired tꢀtest). The
EC50 values and maximum receptor activation are shown in the Supplementary material.
Analysis of the human P2Y
distantly related P2Y12 and the more closely related P2Y
Xꢀray structures were obtained in complex with agonists (P2Y ) and antagonists (P2Y ,
4
receptor model. Two P2Y receptor subtypes, the more
1
receptors, had been crystallized and
1
2
1
5
4–56
P2Y12).
Based on these structures, the molecular mechanism of activation of the human
P2Y
1
receptor was recently analyzed by molecular dynamics simulations, which explained the
essential role of the amino acids Asp204 in extracellular loop 2 (ECL2) and Arg310 in the
57
transmembrane region 7 (TM7). The two amino acids Asp204 and Arg310 form an ionic
1
lock and are stabilized in the inactive state by P2Y antagonists. The ionic lock is broken
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 68
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
during the activation of the receptor by the agonist adenosine diphosphate (ADP). We
constructed a homology model of the P2Y receptor based on the P2Y receptor. In our
4
1
inactive state homology model of the human P2Y
4
receptor, an ionic lock between Asp187
and Arg292 was formed, suggesting a similar mechanism of activation as in the human P2Y
receptor.
1
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
Two ligand binding sites located towards the extracellular regions of the P2Y
4
receptor were
identified in the homology model (Figure 8). The binding site denoted as ‘A’ with a volume of
190 ų was observed upon the downward movement of the ECL2. This binding site ‘A’ is
6
comparable to the binding site of the nonꢀcompetitive antagonist 2ꢀiodoꢀN ꢀmethylꢀ(N)ꢀ
methanocarbaꢀ2’ꢀdeoxyadenosineꢀ3’,5’ꢀbisphosphate (MRS2500, 72) in the human P2Y
1
4
receptor with a size of 214 ų. The second binding site in the homology model of the P2Y
receptor, denoted as ‘O’, is formed in the region comparable to the orthosteric binding site
identified in the crystal structure of the human P2Y12 receptor in complex with the agonist 2ꢀ
methylthioadenosineꢀ5’ꢀdiphosphate (2MeSADP, 71), and in the molecular dynamics
5
6,57
simulation study of the human P2Y receptor with the agonist ADP.
In our inactive state
1
model of the human P2Y
4
receptor, the binding site ‘O’ is limited in size due to the downward
movement of the ECL2. However, after its upward movement, it could accommodate the
endogenous agonists UTP and ATP, respectively. The experimental data for the P2Y receptor
4
antagonists developed in this study suggested a nonꢀcompetitive mode of inhibition. Hence,
we selected binding site ‘A’ for compound docking to probe whether 61 and analogs may
1
display a similar mode of receptor inactivation as MRS2500 (72) in the P2Y receptor.
ACS Paragon Plus Environment

Page 29 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. (a) Crystal structure of the human P2Y receptor (light red) in complex with the
1
2
agonist 2ꢀmethylthioꢀADP (2MeSADP, 71): (b) homology model of the human P2Y
4
receptor
(light green) with the schematic representation of the orthosteric binding site (denoted ‘O’)
and the allosteric binding site (denoted ‘A’); (c) Crystal structure of the human P2Y
1
receptor
6
(
light blue) in complex with the nonꢀcompetitive antagonist 2ꢀiodoꢀN ꢀmethylꢀ(N)ꢀ
methanocarbaꢀ2’ꢀdeoxyadenosineꢀ3’,5’ꢀbisphosphate (MRS2500, 72). The receptors are
represented as cartoon models and the small molecules 71 and 72 are represented as space fill
models.
ACS Paragon Plus Environment