Discovery of potent competitive antagonists and positive modulators of the P2X2 receptor

Article abstract of DOI:10.1021/jm1012193

Evaluation and optimization of anthraquinone derivatives related to Reactive Blue 2 at P2X2 receptors yielded the first potent and selective P2X2 receptor antagonists. The compounds were tested for inhibition of ATP (10 μM) mediated currents in Xenopus oocytes expressing the rat P2X2 receptor. The most potent antagonists were sodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2- ylamino)phenylamino]- 9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63, PSB-10211, IC₅₀ 8 6 nM) and disodium 1-amino- 4-[3-(4,6-dichloro[1,3, 5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene- 2-sulfonate (57, PSB-1011, IC₅₀ 79 nM). Compound 57 exhibited a competitive mechanism of action (pA₂ 7.49). It was >100-fold selective versus P2X4, P2X7, and several investigated P2Y receptor subtypes (P2Y₂₄₆₁₂); selectivity versus P2X1 and P2X3 receptors was moderate (>5-fold). Compound 57 was >13-fold more potent at the homomeric P2X2 than at the heteromeric P2X2/3 receptor. Several anthraquinone derivatives were found to act as positive modulators of ATP effects at P2X2 receptors, for example, sodium 1-amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10- dihydroanthracene-2-sulfonate (51, PSB-10129, EC₅₀ 489 nM), which led to about a 3-fold increase in the ATP-elicited current.

Full text of DOI:10.1021/jm1012193

J. Med. Chem. 2011, 54, 817–830 817
DOI: 10.1021/jm1012193
Discovery of Potent Competitive Antagonists and Positive Modulators of the P2X2 Receptor
‡
§
‡
,†
Younis Baqi,†,# Ralf Hausmann,‡,# Christiane Rosefort, Jurgen Rettinger, Gunther Schmalzing, and Christa E. Muller*
€
€
€
†PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), University of Bonn, An der
Immenburg 4, D-53121 Bonn, Germany, ‡Department of Molecular Pharmacology, RWTH Aachen University, Wendlingweg 2, D-52074 Aachen,
Germany, and §MultiChannelSystems MCS GmbH, Aspenhausstrasse 21, D-72770 Reutlingen, Germany. # These authors contributed equally.
Received September 19, 2010
Evaluation and optimization of anthraquinone derivatives related to Reactive Blue 2 at P2X2 receptors
yielded the first potent and selective P2X2 receptor antagonists. The compounds were tested for
inhibition of ATP (10 μM) mediated currents in Xenopus oocytes expressing the rat P2X2 receptor. The
most potent antagonists were sodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)phenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63, PSB-10211, IC50 86 nM) and disodium 1-amino-
4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-
2-sulfonate (57, PSB-1011, IC50 79 nM). Compound 57 exhibited a competitive mechanism of action
(pA2 7.49). It was >100-fold selective versus P2X4, P2X7, and several investigated P2Y receptor sub-
types (P2Y2,4,6,12); selectivity versus P2X1 and P2X3 receptors was moderate (>5-fold). Compound 57
was >13-fold more potent at the homomeric P2X2 than at the heteromeric P2X2/3 receptor. Several
anthraquinone derivatives were found to act as positive modulators of ATP effects at P2X2 receptors,
for example, sodium 1-amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
(51, PSB-10129, EC50 489 nM), which led to about a 3-fold increase in the ATP-elicited current.
˚
Introduction
∼45 A from the ion channel domain, in a deep cleft, inducing
conformational changes within and between subunits. These
changes, in turn, may be propagated to the ion channel by
conserved residues located at the transmembrane domain-
extracellular domain interface.5
Purinergic receptors are a family of plasma membrane
proteins, activated either by the nucleoside adenosine (P1 or
adenosine receptors), or by nucleotide derivatives, such as
ATP, ADP, UTP, and UDP (P2 or nucleotides receptors).1 In
addition, the nucleobase adenine has recently also been iden-
tified as a signaling molecule activating specific G protein-
coupled receptors termed adenine or P0 receptors.2
The P2 purinergic receptors are further divided into two
main subfamilies: ligand-gated ion channels or ionotropic
receptors, termed P2X,3a and G protein-coupled or metabo-
tropic receptors, designated P2Y.3b So far, seven subtypes in
the P2X family (P2X1-7) and eight subtypes in the P2Y family
(P2Y1,2,4,6,11,12,13,14) have been identified.3
P2X2 receptors (P2X2Rs)a are widely distributed through-
out the peripheral and central nervous system and on many
non-neuronal cell types, where they play a role in sensory
transmission and modulation of synaptic function. For in-
stance, homomeric P2X2Rs facilitate excitatory transmission
onto interneurons6 and mediate sensory transmission from
taste buds7 and ventilatory responses.8 P2X2R exhibit slow
desensitization kinetics and are the only homomeric P2X
subtype potentiated by acidic conditions; they are also poten-
tiated by Zn2þ but inhibited by other divalent cations at high
concentrations.9 P2X2 subunits may form heterotrimeric
channels with P2X3 or P2X6 subunits. P2X2R knockout mice
have shown a reduced pain-related response in the second
phase after intraplantar injection of formalin indicating po-
tential effects of P2X2 antagonists on pain.10 Inoue et al.
showed that P2X2 antisense prevented mechanical allodynia
induced by intraplantar injection of the P2X agonist R,β-
methylene-ATP.11 Furthermore, P2X2 knockout mice had
reduced bladder distension reflexes, and antagonists acting
at P2X2R might be useful for the treatment of urinary
incontinence.10
P2X receptors are membrane cation channels gated by
extracellular ATP and are composed of homomeric or hetero-
meric assemblies of three subunits.4 Recently, a crystal struc-
ture of the ATP-gated zebrafish P2X4 receptor channel
˚
was published in a 3 A resolution in the closed state without
ligand.5 ATP has been proposed to bind to an intersubunit site
€
*Address correspondence to Dr. Christa E. Muller, Pharma-
zeutisches Institut Pharmazeutische Chemie I An der Immenburg 4,
D-53121 Bonn, Germany. Phone: þ49-228-73-2301. Fax: þ49-228-73-
2567. E-mail: christa.mueller@uni-bonn.de.
a Abbreviations: AB-25, Acid Blue 25; eN, ecto-50-nucleotidase; ESI,
electrospray ionization; FCC, flash column chromatography; HPLC,
high performance liquid chromatography; LC-MS, liquid chromatog-
raphy-mass spectrometry; NMR, nuclear magnetic resonance; NPP(s),
nucleotide pyrophosphatase(s); NTPDase(s), nucleoside triphos-
phate diphosphohydrolase(s); P2X2R(s), P2X2 receptor(s); rP2X re-
ceptor, rat P2X receptor; PSB, Pharmaceutical Sciences Bonn; RB-2,
Reactive Blue 2; RB-4, Reactive Blue 4; RP-TLC, reversed phase thin
layer chromatography; SAR(s), structure-activity relationship(s);
TLC, thin layer chromatography; wt, wildtype.
In order to investigate the (patho)physiological role of
homomeric P2X2Rs and to explore their potential as drug
targets, potent and selective P2X2R antagonists are required.12
While potent antagonists for P2X1, P2X3, and P2X7 receptor
subtypeshaverecently been developed12,13 nohighly potent or
selective P2X2R antagonists have been described to date.
r
2011 American Chemical Society
Published on Web 01/05/2011
pubs.acs.org/jmc

818 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
Figure 1. Structures of P2X2R antagonists.
PPADS (1), Reactive Blue 2 (RB-2, 2), and TNP-ATP (3) (for
structures see Figure 1) are approximately equipotent inhibi-
tors of ATP-evoked currents through human or rat P2X2R
(pIC50’s range from 5.4 to 6.4), but much less potent than at
the homomeric P2X1 or P2X3 channels.14
Suramin (4) has been reported to be of similar potency
(pIC50 = 5.4-6.0) at P2X2R in some studies,15 while others
have reported suramin as having a 3-10-fold lower potency
(pIC50 = 4.5-5.0) than these antagonists.14a,b In our assay,
suramin inhibited oocyte-expressed rat P2X2R with a pIC50
value of 6.31 ( 0.01 (Supporting Information, Figure 1). The
dihydropyridine derivative nicardipine (5), a calcium channel
blocker, was shown to be an antagonist at recombinant rat
P2X2R with an IC50 value of 25 μM and an about 10-fold
lower inhibitory potency at P2X4 receptors.16
In addition to the moderate inhibitory potency of the
anthraquinone dye RB-2 (2, Figure 1) at P2X2R (IC50:
4 μM, see Table 3), it has been reported to act as an antagonist
at other nucleotide receptor subtypes as well (P2X1, P2X4,
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors).16-31
Furthermore, 2 is a nonselective inhibitor of ectonucleoti-
dases, including ecto-50-nucleotidase (eN),32 nucleoside triphos-
phate diphosphohydrolases (NTPDases),33 and nucleotide pyro-
phosphatases (NPPs).34
It had previously been shown that the substitution pattern
in the 4-position of the anthraquinone moiety plays an
important role in the ability of the compounds to antagonize
certain P2 receptor subtypes, such as P2X1 and P2Y1-like,35
P2Y2,29 and P2Y12 receptors,30,31 and to inhibit eN,32 and
NTPDase isoenzymes,33 The modification of this substituent
has, for example, led to the development of the most potent
non-nucleotide-derived P2Y12 antagonists known to date,
which exhibit high selectivity versus other P2 receptors as well
as ecto-nucleotidases.30,31,36 Also this concept led to the deve-
lopment of potent and selective inhibitors of eN32 endowed
with high selectivity versus other ecto-nucleotidases as well as
P2 receptors.32 Thus, the anthraquinone scaffold appears to
behave as a privileged structure in medicinal chemistry for
nucleotide-binding protein targets.32,37 Although 2 itself be-
haves as a so-called “frequent hitter” interacting with many
different proteins, careful structural modification can success-
fully result in high selectivity for a single target. In the present
study, we identified and developed the first potent and sele-
ctive P2X2R antagonists starting from 2 as a lead structure.
Results and Discussion
Chemistry. Compounds were synthesized as depicted in
Scheme 1. The preparation of compounds 7, 10-14, 17-24,
27, 29-31, 33-49, and 52-56 has previously been des-
cribed.29,32,33,38 The synthesis of products 8, 9, and 26 had
been described in the literature29,33 but has now been im-
proved. Like the new compounds 15, 16, 25, 28, 32, 50, and
51, they were obtained by a recently developed variant of the
Ullmann coupling reaction.28,39 The coupling of 4-bromo-
substituted anthraquinone derivatives 6a or 6b36 with the
appropriate amine in phosphate buffer (pH 6-7) in the pres-
ence of Cu0 under microwave irradiation at 80-120 °C for
5-20 min yielded the target compounds 7-56 (Scheme 1).
Good to excellent isolated yields were obtained. The anilino-
anthraquinone derivatives 39, 40, and 45-47, which bear a
primary amino group in the m- or p-position of the N4-
phenyl ring, were subsequently treated with cyanuric acid
chloride (Scheme 1) to afford the desired products 57, 58, 60,
61, and 63. Aniline derivatives 40 and 46 were further reacted
with 2-bromopyrimidine yielding compounds 59 and 62
(Scheme 1).30 Compound 58 was further treated with metha-
nol at 60 °C for 30 min to yield 64 (Scheme 1).30 Two of the
dichlorotriazinyl derivatives (57, 61) were hydrolyzed with
water at 90 °C for 1 h to produce the corresponding diol
derivatives 65 and 66 (Scheme 1).
The compounds were purified by flash column chroma-
tography on reversed phase material. The purity of the com-
pounds was determined by HPLC-MS/UV methods and a
purity of at least 95% was confirmed. In Table 1 reaction
times, isolated yields, and information on purity of the new
compounds are collected.
Purification of Reactive Blue 4. Curiously, one of the most
potent P2X2 antagonists of the present series of anthraqui-
none derivatives identified in this study, compound 57 (IC50
0.079 μM, see below), is commercially available and known
as Reactive Blue 4 (RB-4, CAS 13324-20-4). However, nei-
ther synthetic protocols nor spectral data for this compound
have been available in the literature. Commercial RB-4 is not
a pure compound and sold with a declared dye content of

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 819
Scheme 1. Syntheses of Target Compoundsa
a Reagents and conditions: (i) amine, phosphate buffer, Cu0, microwave, 80-120 °C, 5-20 min, yield 30-96%; (ii) cyanuric acid chloride, acetone:
water (1:2), 0-5 °C, 1-6 h, yield 80-95%; (iii) 2-bromopyrimidine, acetone/water (1:2), 0-5 °C 1 h, then reflux, 1-3 days, yield 90-97%; (iv) MeOH,
60 °C, 30 min, yield 96%; (v) water, Na2CO3, 90 °C, 1 h, yield 95-97%. b For R and R1 see Tables 1-3.
only 35% (Sigma-Aldrich), or ca. 40% (Alfa Aesar), respec-
tively. Since 57 could be a very useful tool we developed - in
addition to the total synthesis - a highly efficient and simple
purification procedure of commercial RB-4 by reversed
phase-18 flash column chromatography (RP-18 FCC, for
details see Experimental Section). The chemical structure
and purity were confirmed by LC-MS (96%), NMR, and
RP-TLC and found to be identical to that of synthesized
compound 57. However, we discovered that the dye content
of the commercial product had only been about 24%, since it
was contaminated by its hydrolysis products, the dihydroxy-
triazinyl derivative 66 (ca. 3%), and the correspond-
ing monohydroxy-monochlorotriazinyl derivative (ca. 1%).
Furthermore, it contained 72% of inorganic salts such as
NaCl and sodium phosphate. Nevertheless, the purification
is economically advantageous compared to the total synthe-
sis of 57, especially if larger amounts of the compound are
required.
Biological Assays. The effect of the compounds on rat
P2X2 and P2X4 receptor-mediated currents was analyzed by
using two-electrode voltage-clamp electrophysiology in Xe-
nopus oocytes transiently expressing the respective receptors
as described before.42 Further information about this func-
tional assay is given in the Experimental Section. The rat
rather than the human P2X receptors were used because rats
or other rodents are frequently used in preclinical studies.
The most active compound 57 was additionally investigated
at rat P2X1, P2X3, P2X7, and heteromeric P2X2/3 receptors
using the same methodology. In order to assess selectivity
versus P2Y receptor subtypes as well, a series of compounds
was investigated at recombinant P2Y2, P2Y4, P2Y6 receptors
in calcium mobilization assays using 1321N1 astrocytoma
cells, and at P2Y12 receptors in radioligand bindings studies
at human platelet membrane preparations as previously
described.29-31,44,45
Structure-Activity Relationships. RB-2 (2) is a negatively
charged anthraquinone dye with three sulfonate groups and
a molecular weight of 840.10 g/mol that binds to several
P2 receptor subtypes and to various ecto-nucleotidases
typically with micromolar affinity.32,33 In the literature, it
has been reported to antagonize P2X2R with an IC50-value
of 4 μM.14a,d We found that Acid Blue 25 (7), a much smaller
molecule (416.38 g/mol) bearing only one sulfonate function
was similarly potent (Table 2). In order to get more informa-
tion on the structure-activity relationships (SAR) of this
class of compounds as ligands of P2X2Rs and the related
P2X4 subtype, the effects of a large series of alkyl-
(aryl)amino-anthraquinone derivatives, structurally related
to 2, on P2X2 and P2X4 receptor-mediated currents were
analyzed.42
AB-25 (7) is the simplest structure of the present series, in
which ring D (phenyl ring on N4 of the 4-aminoanthraquinone
derivative) is not substituted, and the anthraquinone ring system
bears a sulfonate group in the 2-position; when this group was
replaced by a methyl group as in compounds 8 and 9 the
inhibitory activity was also lost (Table 2). We therefore focused
our attention on 2-sulfoanthraquinone derivatives. From initial
screening of 49 different alkyl(aryl)amino-anthraquinone

820 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
Table 1. Reaction Times, Isolated Yields, Molecular Weights and Purity of Newly Synthesized Anthraquinone Derivatives
compd
R1
R2
reaction time (min)
Structure A
yield (%)a
MW (g/mol)
purity by LC-MS/UV(%)b
8
CH3
CH3
H
CH3
5
75
80
90
91
83
80
96
75
65
60
328.4
342.4
484.5
444.4
476.5
460.5
434.4
452.4
506.5
508.5
95.3
95.5
98.2
99.7
99.5
98.5
99.9
99.2
99.2
97.4
9
5
15
16
25
26
28
32
50
51
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
3-trifluoromethyl
3-ethyl
5
5
3,5-dimethoxy
2-ethoxy
3-fluoro
5
5
5
3,5-difluoro
3-benzyl
3-phenoxy
5
10
10
Structure B
57c
63e
65
SO3Na
H
Cl
Cl
15 þ 30
5 þ 15
55d
57d
61d
53d
681.4
623.4
542.5
644.5
95.1
97.1
98.4
98.4
H
SO3Na
OH
OH
5 þ 120 þ 60
15 þ 30 þ 60
66f
a Isolated yields. b A sample (10 μL) was injected into an HPLC column and elution was performed with a gradient of water/methanol (containing 2
mM NH4CH3COO) from 90:10 to 0:100 for 30 min at a flow rate of 250 μL/min, starting the gradient after 10 min. UV absorption was detected from 200
to 950 nm using a diode array detector. Purity of the compounds was determined at 254 nm. c Known as Reactive Blue 4 (RB-4) with a dye content of
about 35-40%; neither synthesis nor spectral data have been published. d Total yield over two or three steps, respectively. e Compound was described in
one publication as a coloring dye for poly(vinylalcohol); neither synthesis nor spectral data have been published.40 f Compound described in the
literature as a dye,41 but no synthetic procedure nor spectral data have been provided.
derivatives at P2X2 and P2X4 receptors, it appeared that the
SARs are restricted. All tested compounds were inactive (at
10 μM) at the P2X4 receptor. Most of the tested compounds
exhibited moderate activity at the P2X2R in the range of
1-10 μM. A lipophilic substituent at the o- and/or p-position,
such as fluoro (29), methyl (12, 14), methoxy (22, 24), ethoxy
(26)), di-methyl (17, 18) or tri-methyl (48), was tolerated. In
addition, introducing a hydrophilic function (carboxy) in the
o-, m-, or p-position (35-37), a sulfonate at the p-position
(34) and an amino function at the o- or m-position (38, 39)
was also tolerated. Moreover, a combination of negatively
charged residues (sulfonate, carboxy) with amino (45-47) or
hydroxy (54) was tolerated as well. Introducing a large sub-
stituent in the p-position (52, 53) did not improve the activity.
Interestingly, the R-naphthyl- (11), o-sulpho- (33), and
p-amino-substituted (40) derivatives were the only compounds
which showed no activity at the P2X2R in the present series
(Table 2). However, acetylation of the p-amino group in
40 yielding compound 41 resulted in a more than 15-fold
increase in P2X2-inhibitory potency (IC50 0.69 μM).
donor with a methyl (p-methoxy, 24) or phenyl (p-phenoxy,
27) group decreased the activity. Introducing a carboxy
group in the m-position in the presence of a p-hydroxy subs-
titutent (54), decreased the activity as well (Table 2).
Interestingly, four compounds of the present study
showed potentiation of the ATP induced P2X2 channel res-
ponses rather than inhibition of the channel. Three of these
activators bear a lipophilic substituent in the m-position (19,
50, and 51), while one compound (56) has a m-sulfonate
group on ring D and a phenylamino group in the p-position
(56); see Table 2. Moderate effects were already seen at low
concentrations of 100 nM of the test compound (106-132%
increase, ATP effect set at 100%), while much higher effects
could be observed at higher concentrations (see also Figure 6).
According to these SARs, a lipophilic substituent in the
m-position of the N4-phenyl ring may enhance inhibitory or
modulatory activity, and a hydrophilic substituent (OH) in
the p-position is favorable for good inhibitory activity as
well. Therefore, several arylamino-anthraquinone derivatives
having a lipophilic substituent in the m-position were synthe-
sized and evaluated as antagonists at P2X2Rs (Table 3). For
a systematic direct comparison, lipophilic p-substituted ana-
logues were synthesized as well and evaluated at P2X2 and
P2X4 receptors. Introducing a dichlorotriazinylamino moi-
ety in the m-position in the presence of a sulfonate group in
the p-position (57) led to the first potent and selective P2X2
antagonist (IC50 79 nM) known so far.
On the other hand, introducing a lipophilic substituent in
the m-position, for example, methyl, methoxy, or chloro (13,
23, 30) increased the activity to the sub-micromolar range
(IC50 < 1 μM), while trifluoromethyl, ethyl, bromo, and
nitro in the m-position (15, 16, 31, 42) and di-substition in the
m-position with 3,5-di-methoxy or 3,5-di-fluoro residues (25,
32) did not alter the activity.
A hydroxy substituent in the p-position (21) increased the
activity (IC50 0.588 μM) as well. A hydrogen-donating
hydroxy (OH) group in the p-position appeared to be
important for the activity, because blocking of this hydrogen
A carboxy group in the m-position (in the presence of
m-dichlorotriazinylamino substitution, 60) also led to a potent
and selective antagonist (IC50 293 nM). A dichlorotriazinylamino
substituent in the p-position in the presence of a sulfonate

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 821
Table 2. Pharmacological Evaluation of Selected Anthraquinone Derivatives at Rat P2X2 and P2X4 Receptors
IC50
rP2X2a (μM)
IC50
rP2X4a (μM)
compd
R1
R2
R3
R4
R5
7 (AB-25)
H
H
H
H
H
H
∼1-10
>10
>10
>10
>10
>10
>10
>10
>10
n.d.d
n.d.d
>10
>10
n.d.d
8
see structure above
>10
9
see structure above
>10
10
11
12
13
14
15
16
17
18
19
see structure above
∼1-10
see structure above
>10
CH3
H
H
H
H
H
H
H
H
H
H
H
∼1-10
CH3
H
H
H
∼0.8
H
CH3
H
H
∼1-10
H
CF3
CH2CH3
CH3
H
H
>1
H
H
H
>1
CH3
CH3
H
H
H
∼1-10
CH3
H
H
∼1-10
CH3
CH3
potentiating effect
106% at 100 nMc
∼1-10
20
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
>10
>10
>10
>10
>10
n.d.d
>10
>10
n.d.d
>10
n.d.d
>10
n.d.d
>10
>10
>10
>10
>10
>10
>10
>10
>10
n.d.d
>10
>10
>10
>10
>10
>10
>10
n.d.d
21
22 (PSB-716)49
H
OH
H
0.588 (0.511-0.677)b
∼2
OCH3
H
H
H
H
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
OCH3
H
H
H
0.623 (0.540-0.719)b
1.73 (1.38-2.17)b
>1
H
OCH3
H
H
OCH3
H
H
OCH3
H
OC2H5
H
H
∼1-10
H
OC6H5
H
∼1-10
H
F
H
H
>1
H
H
F
H
∼1-10
H
Cl
H
H
∼0.1-0.3
∼1-10
H
Br
H
H
H
F
H
F
>1
SO3Na
H
H
H
H
>10
H
SO3Na
H
∼1-10
CO2H
H
H
H
H
∼1-10
CO2H
H
H
H
∼1-10
H
CO2H
H
∼1-10
NH2
H
H
H
H
∼1-10
NH2
H
H
H
∼1-10
H
NH2
H
>10
H
H
NHC(O)CH3
H
0.69
H
NO2
H
H
H
>1
CO2H
CO2H
H
Cl
H
∼1-10
H
H
Cl
H
∼1-10
NH2
SO3Na
CO2H
H
SO3Na
NH2
H
∼1-10
H
H
∼1-10
H
NH2
H
∼1-10
CH3
CH3
H
CH3
CH3
H
∼1-10
NH2
CH2C6H5
H
∼1-10
H
potentiating effect
120% at 100 nMc
potentiating effect
132% at 100 nMc
∼1-10
51
H
OC6H5
H
H
H
n.d.d
52
53
54
55
56
H
H
CH2CO2H
H
H
H
H
H
H
H
H
H
H
>10
>10
>10
>10
n.d.d
H
H
CH2P(=O)(OEt)2
∼1-10
H
CO2H
NH2
SO3Na
OH
∼1-10
CH3
H
H
∼1-10
NHC6H5
potentiating effect
110% at 100 nMc
a Potencies of anthraquinone derivatives at rat P2X receptors recombinantly expressed in Xenopus laevis oocytes (n g 5). b 95% confidence interval
(CI). c Percent potentiation of the ATP-induced current at indicated concentration of compound. d n.d. = not determined.

822 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
Table 3. Potency of Compounds Bearing Two Aromatic Rings (D and E) Connected to the Antraquinone Core Structure at Rat P2X2 and P2X4
Receptors
a Potencies of aryl(alkyl)anthraquinone derivatives at rat P2X receptors recombinantly expressed in Xenopus laevis oocytes (n g 3). b 95% confidence
interval (CI). c n.d. = not determined.
group in the m-position yielded 58, which exhibited an IC50
value of 1.16 μM. Formal removing of the sulfonate group in
the m-position of 58 providing 61 improved the activity by
10-fold (IC50 117 nM), indicating that a sulfonate group on
ring D was not required and even detrimental. Replacing a
chlorine atom in 58 by a methoxy group (64) slightly im-
proved the potency (0.582 μM). Hydrolysis of both chlorine
atoms of compounds 61 and 57 to the corresponding dihydroxy-
triazinyl derivatives 65 and 66 decreased the activity by ca.
2-fold (compare 61/65), and >4-fold (compare 57/66).
Introducing a pyrimidine ring instead of a dichlorotriazi-
nyl residue in the presence or absence of a sulfonate group in
the m-position (59, 62) decreased the activity. A direct ana-
logue of 57 without a sulfonate group on ring D was synthe-
sized (compound 63) and evaluated at P2X2R. As expected it
showed a very good inhibitory activity (IC50 0.086 μM, see

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 823
Figure 4. Potencies of compound 57 at different rP2X receptor
isoforms as assessed from stationary current (steady state) measure-
ments. Concentration-inhibition curves were generated from
agonist-induced current measurements in the presence of incremen-
tally larger concentrations of 57. The extent of inhibition was judged
from the ratio of the maximal and minimal stationary current in
the absence and in the presence of 57, respectively. Green square,
rP2X2-X1 (rP2X1); black diamond, rP2X2; red triangle, rP2X2/3;
blue triangle, rP2X2-X3 (rP2X3); The continuous lines represent
nonlinear curve fits by the Hill equation to the pooled data points
from n = 5-7 experiments. For convenience, all IC50 values of 57
are summarized in Table 4.
Figure 2. Concentration-response curves of selected P2X2R an-
tagonists as assessed from stationary current (steady state) mea-
surements. Concentration-inhibition curves were generated from
agonist-induced current measurements in the presence of incremen-
tally larger concentrations of the indicated antagonists. The extent
of inhibition was judged from the ratio of the maximal and minimal
stationary current in the absence and presence of antagonist,
respectively. The continuous lines represent nonlinear curve fits
by the Hill equation to the pooled data points from n = 5-7
experiments.
Table 4. Potencies of 57 at Recombinant P2X Receptor Subtypes
IC50, nMa
(95% confidence interval)
selectivity
vs P2X2
rP2X2-X1 (P2X1)
wt-rP2X2
422 (340-525)
79.2 (47-133)
494 (339-721)
1042 (810-1339)
>10,000
5.3
rP2X2-X3 (P2X3)
wt-rP2X2/3
wt-rP2X4
6.2
13.2
.120
.120
wt-rP2X7
>10,000
a IC50 values were calculated from data of 5-7 independent experi-
ments. For experimental details see Figure 4 and Experimental Section.
Figure 3. Structure-activity relationships of anthraquinone deri-
vatives at rat P2X2Rs.
the P2X2Rs, while compounds substituted in the p-position
were found to be less potent in most cases. Previously, we
have shown that the binding site of the P2Y12 receptor
Table 3 and Figure 2) and was actually - besides compound
57 - the most potent inhibitor of the present series indicating
that a sulfonate group on ring D was not required for high
inhibitory potency.
-
required a negative charge (SO3 or CO2-) in the meta-
position besides an aromatic substituent in the para-position
of ring D30,31,36 indicating that SARs for P2Y12 and P2X2Rs
are very different. Recently, we have developed novel ecto-50-
nucleotidase inhibitors based on an anthraquinone scaffold.32
The characteristics of the SARs for this enzyme target showed
the requirement of a big lipophilic substituent (naphthyl or
anthracenyl) attached directly to N4 of the anthraquinone
moiety, indicating the existence of a big lipophilic pocket in
the active site of the enzyme.32 Thus, all three targets are
showing very different characteristic properties regarding the
binding site for anthraquinone derivatives. This enables us to
develop selective antagonists and inhibitors.
Selectivity. To determine the P2X receptor subtype selec-
tivity of the most potent P2X2R-blocking compound 57 in
this series, concentration inhibition curves were generated at
the homomeric rP2X1, rP2X2, rP2X3, rP2X4, and rP2X7
receptors, as well as the heteromeric rP2X2/3 receptor
subtype (Figure 4). P2X5 receptors were not studied because
the P2X5 subunit occurs in humans predominantly as an
To date, there are no known selective or highly potent
P2X2R antagonists. So far, only PPADS (1), RB-2 (2), TNP-
ATP (3), and suramin (4) were reported as moderately potent
antagonists (pIC50 = 5.4-4.4, IC50 = 4-40 μM) at P2X2Rs.
These compounds are well-known to block almost all P2X
receptor subtypes, and some are even blocking P2Y receptor
subtypes as well. We now identified the first potent and
selective rat P2X2R antagonist with nanomolar potency,
compound 57 (IC50 P2X2 = 79 nM). It is highly selective
versus the P2Y12 receptor (>200-fold) and the P2X4 recep-
tor subtype (>125-fold). Also, we have discovered that the
direct analogue of 57, compound 63, shows a very similar
potency at the P2X2R with an IC50 value of 86 nM. Figure 3
summarizes the SARs of the compounds as P2X2R antago-
nists. A substituent in the m-position of phenyl ring D is well
accepted. Especially lipophilic substituents in the m-position
increased the affinity. An aromatic ring (E) in the meta-
position was accommodated well in the binding pockets of

824 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
Table 5. Selectivity of the Most Potent P2X2R Ligands vs Other P2 Receptor Subtypes
IC50 (μM)
Ki ( SEM (μM) vs [3H]PSB-0413
b,30,31
P2Y12
a
b,29,32
b,32
a,32
compd
P2X2
4.026-29
P2Y2
P2Y4
P2Y6
2
1.85 ( 0.39
11.0 ( 1.0
>10
9.79 ( 3.91
25.8 ( 14.3
>10
4.34 ( 0.89
84.6 ( 9.0d
3.92 ( 1.28
5.30 ( 1.00
.10
0.68 ( 0.26
9.83 ( 2.43
∼10
7
∼1-10
∼0.8
13
21
23
30
50
0.588 ( 0.083
0.623 ( 0.090
∼0.1-0.3
4.63 ( 0.53
∼10
2.22 ( 1.12d
∼10
∼10
∼10
24.5 ( 10.4
.10
21.8 ( 2.3
>10
.10
.10
6.76 ( 2.07
n.d.c
potentiating effect
120% at 100 nMe
potentiating effect
132% at 100 nMe
0.079 ( 0.011
0.293 ( 0.034
0.117 ( 0.012
0.086 ( 0.011
0.582 ( 0.156
0.197 ( 0.023
51
∼10
n.d.c
4.56 ( 0.18
n.d.c
57
60
61
63
64
65
∼10
>10
1.45 ( 0.38
∼10
.10
17.1 ( 7.8
3.76 ( 1.03
1.90 ( 0.24
n.d.c
3.27 ( 0.76
12.1 ( 1.4
24.3 ( 3.5
24.2 ( 3.5
∼30
2.55 ( 1.33
∼10
∼10
>10
.10
>10
∼10
0.66 ( 0.12
n.d.c
n.d.c
a Rat receptor. b Human receptors. c n.d. = not determined. d n = 2. e Percent potentiation of the ATP-induced current at indicated concentration of
compound.
assembly deficient natural deletion mutant.46 P2X6 recep-
tors were omitted because P2X6 does not form functional
homomeric channels under most circumstances.9 To assess
inhibition of P2X1 and P2X3 receptors also under steady-
state conditions, non-desensitizing rP2X2-X1 and rP2X2-X3
receptor chimeras were used, in which the N-terminal tail
including the first transmembrane domain is replaced by the
complementary portion of the rP2X2 subunit. Since the
ligand-binding ectodomain originates entirely from the
rP2X1 or the rP2X3 subunit, these chimeras can be reliably
used as non-desensitizing substitutes of wt-rP2X1 and rP2X3
receptors.13e ATP served as an agonist for all receptors, with
the exception of non-desensitizing heteromeric rP2X2/3
receptors that were selectively activated by R,β-methylene-
ATP (R,β-meATP, 1 μM) to avoid activation of coex-
pressed homomeric rP2X2Rs that respond to ATP, but
are virtually resistant to activation by R,β-meATP (for
Figure 5. Assessment of the mechanism of inhibition of rP2X2Rs
details, see Experimental Section).9 Concentration-inhibi-
induced by 57. ATP concentration-response curves were estab-
tion curves and IC50 values were derived from nonlinear
least-squares fits of the Hill equation to the pooled data
points and yielded the following rank order of potencies
(Figure 4) for compound 57: wt-rP2X2 > non-desensitizing
mutant of rP2X1 g non-desensitizing mutant of rP2X3 >
wt-rP2X2/3 > > wt-rP2X4, wt-rP2X7. Compound 57 was
virtually ineffective in blocking rP2X4 or rP2X7 receptor
mediated currents (<10% inhibition at 10 μM of inhibitor,
data not shown). As depicted in Figure 4, low concentrations
(10-100 nM) of compound 57 slightly potentiated P2X1 and
P2X3 receptor mediated currents. However, in the absence
of ATP compound 57 elicited no P2X1 or P2X3 receptor
responses.
Thus, compound 57 was highly selective for P2X2R versus
P2X4 and P2X7 receptor subtypes (>120-fold), while it was
moderately selective versus P2X1 and P2X3 receptors (5-6-
fold). Interestingly, 57 was 13-fold selective for the homo-
meric P2X2 receptor compared with the heteromeric P2X2/3
receptor and may therefore be used to distinguish between
effects mediated by the homomeric and those induced by the
heteromeric P2X2-subunits containing subtypes. The signifi-
cantly (>10-fold) different potency of 57 at heteromeric
P2X2/3 receptors compared to P2X2 receptors is not totally
unexpected as the known subunit composition of two P2X3
and one P2X2 subunit combined with the intersubunit ligand
lished by stimulating oocytes expressing rP2X2Rs with incremen-
tally larger concentrations of ATP in the absence (black square,
EC50 = 13.8 ( 0.9 μM, nH = 1.8 ( 0.1, n = 5) and in the presence of
30 nM 57 (red triangle, EC50 = 26.9 ( 2.1 μM, nH = 1.5 ( 0.1, n =
5) and 100 nM 57 (blue triangle, EC50 = 35.7 ( 3.4 μM, nH = 1.5 (
0.1, n = 5). Currents were normalized to those elicited by a saturat-
ing concentration of 1 mM ATP. A simultaneous (global) fit yielded
a pA2 value of 7.49. In noninjected control oocytes, ATP elicits no
current both in the absence and presence of up to 3 μM compound
57 (data not shown).
binding site implies that no pure “P2X2 binding site” is
present in these heteromeric channels.
In addition selected, potent compounds were further in-
vestigated at some P2Y receptor subtypes (Table 5) as pre-
viously described.29-32
The initial lead compound 2 (RB-2) exhibited similar
inhibitory potency at the P2X2 receptor as at the investigated
P2Y receptor subtypes in the low micromolar range. The
much smaller anthraquinone derivative 7 (sodium 1-amino-
4-phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate,
AB-25) was similarly potent at P2X2R, but somewhat weak-
er (3-20-fold) at the P2Y receptors, thus showing increased
P2X2 selectivity. The same was true for several simple, small
anthraquinone derivatives, including 13, 21, 23, and 30.
Selectivity appeared to be even more pronounced for most

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 825
Figure 6. Potentiating effect of 51 on ATP-elicited rP2X2R mediated currents. (A) ATP concentration-response analysis in the absence and
presence of 100 nM 51. ATP concentration-response curves were established by stimulating oocytes expressing the rP2X2R with incrementally
larger concentrations of ATP in the absence of 51 (black circle, EC50 = 22.2 ( 2.4 μM, nH = 1.8 ( 0.1, n = 6) or in the presence of 100 nM 51
(red triangle, EC50 = 20.6 ( 2.7 μM, nH = 1.7 ( 0.2, n = 6). Currents were normalized to those elicited by a saturating concentration of 1 mM
ATP in the absence of 51. The continuous lines represent nonlinear curve fits by the Hill equation to the pooled data points from n = 6
experiments. (B) Concentration-response analysis of 51 in the presence of 10 μM ATP. Currents were elicited by repetitive application of
10 μM ATP in the presence of incrementally larger concentrations of compound 51. Currents were normalized to those elicited by 10 μM ATP
in the absence of 51. The continuous line represents a nonlinear curve fit by the Hill equation to the pooled data points from n = 5 experiments.
An EC50 value of 489 ( 90 nM (nH = 1.4 ( 0.4, n = 5) was determined.
of the larger, more potent anthraquinone derivatives bearing
an additional ring E (e.g., 57, 60, 61, 63, 65). The most potent
compound of the present series, 57, was more than 120-fold
selective versus the investigated P2Y2, P2Y4, P2Y6, and
P2Y12 receptor subtypes.
of 489 ( 90 nM for potentiation of ATP-elicited currents
(Figure 6B). The increase in efficacy without effect on the
potency of ATP may be compatible with an allosteric
modulatory effect. On the other hand, the 3-fold increase
by 1 μM of compound 51 in the current amplitude elicited by
10 μM ATP is difficult to reconcile with a purely allosteric
mechanism. Therefore, further experiments are needed to
fully resolve the specific mechanism of action of these posi-
tive modulatory compounds.
Mechanism of Action. To delineate whether compound 57
acts as a competitive rP2X2R antagonist, ATP concentration-
response curves were established in the absence and in the
presence of 57. In these experimental series ATP activated
rP2X2R mediated inward currents with an EC50 value
of 13.8 ( 1.5 μM. In noninjected control oocytes ATP
elicits no current both in the absence and presence of up to
3 μM compound 57 (data not shown). Increasing concentra-
tions of compound 57 progressively shifted ATP concentration-
response curves parallel to the right without changing the
maximal response (Figure 5). These data are in agree-
ment with a competitive mechanism of action of compound
57 at the rP2X2R. However, we cannot completely exclude
an allosteric modulation of the ATP affinity by 57 because
the rightward shift decreases at the higher concentration of
57. A global fit of the concentration response data yielded a
pA2 value of 7.49 as an estimate of the antagonistic potency
independent of any particular mechanism.
Compounds with Positive Modulatory Action at rP2X2Rs.
As shown in Table 2 coapplication of a low concentration of
100 nM of compounds 19, 50, 51, and 56 showed increased
ATP elicited current amplitudes (106-132% of control current
(application of ATP alone, 100%)). To further characterize
this effect, we determined concentration-response curves
for the most active modulator 51. Compound 51 (up to 1 μM)
showed no agonistic effect of its own if applied without ATP
(data not shown). As shown in Figure 6A, the ATP potency
in this experimental series in the presence of 100 nM 51
(EC50 = 20.6 ( 2.7 μM, nH = 1.7 ( 0.2, n = 6) was not
significantly altered as compared to the ATP potency in the
absence of 51 (EC50 = 22.2 ( 2.4 μM, nH = 1.8 ( 0.1, n = 6).
However, 100 nM of 51 led to a significant increase in the
maximum current amplitudes. Repetitive activation of
rP2X2Rs by 10 μM ATP in the presence of incrementally
larger concentrations of compound 51 yielded an EC50 value
Conclusions
In conclusion, a series of anthraquinone derivatives was
investigated at P2X2 receptors. Analysis of structure-activity
relationships and compound optimization yielded the first
potent P2X2 receptor antagonists. Among the 59 investigated
compounds, 11 are new compounds not previously described
in the literature, while several others, including 57, 63, and 66,
can be found in databases as dyes; however, neither synthetic
procedures nor spectral data have been published, and they
have probably not been obtained in high purity required for
pharmacological testing. Most of the synthesized compounds
(52 compounds) were potent P2X2R antagonist, 33 showing
IC50 values in the low micromolar range (ca. 1-10 μM), and
11 even in the moderate to low nanomolar range (0.079-
0.83 μM). This study led to the discovery of compound 57,
which is the most potent P2X2R antagonist known to date
showing a competitive mechanism of action (IC50 = 79 nM,
pA2 = 7.49). In fact, 57 is the first highly potent antagonist for
this channel showing high (>100-fold) selective versus the
closely related P2X4 and P2X7 receptor ion channels and
versus several investigated P2Y receptor subtypes (P2Y2,4,6,12).
Moderate selectivity of 57 versus P2X1 and P2X3 receptors
(>5-fold) was also observed. The P2X2 antagonist 57 was
considerably (>13-fold) more potent at the homomeric as
compared to the heteromeric P2X2/3 receptor. Furthermore,
several anthraquinone derivatives were discovered to act as
positive modulators of ATP effects at P2X2 receptors, the
most potent being 51, which led to an about 3-fold increase in
the maximal ATP-elicited current with an EC50 value of 489 nM.

826 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
The new P2X2 receptor modulators will be useful as pharmaco-
logical tools for studying P2X2R and the potential of P2X2R
antagonists and enhancers as novel drugs.
cooled to rt, and the product was purified using the following
procedure. The contents of the vial were filtered to remove the
elemental copper. Then ca. 200 mL of water was added to the
filtrate and the aqueous solution was extracted with dichlor-
omethane (200 mL). The extraction procedure was repeated
until the dichloromethane layer became colorless (2-3 times).
Then the aqueous layer was reduced by rotary evaporation to a
volume of 10-20 mL, which was subsequently submitted to
flash column chromatography using RP-18 silica gel and water
as an eluent. The polarity of the eluent was then gradually
decreased by the addition of methanol or acetone in the follow-
ing steps: 5, 10, 20, 40, 60, and 80%. Fractions containing blue
product were collected. For some compounds, the last step of
purification (RP-18 flash chromatography) had to be repeated
two to three times to obtain pure product (g95% purity
as determined by LC-MS). The pooled product-containing
fractions were evaporated under a vacuum to remove the metha-
nol or acetone, and the remaining water was subsequently
removed by lyophilization to yield (up to 96%) as blue powders
(Scheme 1 and Table 2).
General Procedure B. Coupling reaction of cyanuric acid
chloride with the amino function in phenyl ring D: synthesis
of ABCDE ring anthraquinone derivatives 57, 58, 60, 61, and
63 was achieved analogous to the previously described proce-
dure.30 An ice-cooled solution of cyanuric acid chloride
(1.0 mmol) in water (25 mL) and acetone (25 mL) was added
to a stirred solution of amine-containing compound (39, 40, and
45-47) (0.5 mmol) in water (25 mL) at 0-5 °C. The resulting
mixture was stirred for 1 h at 0-5 °C, then allowed to warm up
to rt and kept stirring at rt for 1-2 h. The formation of product
was monitored by RP-TLC using a mobile phase of acetone/
water (2:3). After completion of the reaction, the solvents were
evaporated and the residue was purified by flash column
chromatography on RP-18 silica gel using acetone/water as an
eluent to obtain the desired products (57, 58, 60, 61, and 63)
(Scheme 1).
General Procedure C. The coupling reaction of 2-bromopyr-
imidine with the amino function in phenyl ring D: synthesis of
ABCDE ring anthraquinone derivatives 59 and 62 was achieved
as previously described.30 In brief: an ice-cooled solution of
2-bromopyrimidine (1.0 mmol) in water (25 mL) and acetone
(25 mL) was added to a stirred solution of the respective
compound (39 or 46) (0.5 mmol) in water (25 mL) at 0-5 °C.
Then the temperature was gradually increased to refluxing at
120 °C for 1-3 days. After completion of the reaction, the
solvents were evaporated and the residue was purified by flash
column chromatography on RP-18 silica to obtain the desired
products 59, and 62, in excellent (90-97%) isolated yield
(Scheme 1).
General Procedure D. Hydrolysis of the dichlorotriazine
moiety to the corresponding dihydroxytriazine group. A 0.05
mmol of dichlorotriazine containing compounds (57 or 61) was
dissolved in water (15 mL) then heated to 90 °C, followed by the
addition of 10 mg of Na2CO3 (2 equiv) and stirring at that
temperature for 1 h. After completion of the reaction, RP-TLC
control, the mixture was subjected to flash column chromatog-
raphy using RP-18 silica to obtain the desired products 65 or 66,
in excellent (95-97%) isolated yield (Scheme 1).
Experimental Section
Chemistry. Material and Methods. All materials were used as
purchased (Acros, Alfa Aesar or Sigma-Aldrich, Germany).
Thin-layer chromatography was performed using TLC alumi-
num sheets silica gel 60 F254, or TLC aluminum sheets RP silica
gel 18 F254 (Merck, Darmstadt, Germany). Colored compounds
were visible at daylight; other compounds were visualized under
UV light (254 nm). Flash chromatography was performed on
€
a Buchi system using silica gel RP-18 (Merck, Darmstadt,
Germany). 1H- and 13C NMR data were collected on a Bruker
Avance 500 MHz NMR spectrometer at 500 MHz (1H), or
126 MHz (13C), respectively. DMSO-d6 was used as a solvent.
Chemical shifts are reported in parts per million (ppm) relative
to the deuterated solvent; that is, DMSO, δ 1H: 2.49 ppm; 13C:
39.7 ppm, coupling constants J are given in Hertz and spin
multiplicities are given as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br (broad).
The purities of isolated products were determined by ESI-
mass spectra obtained on an LCMS instrument (Applied Bio-
systems API 2000 LCMS/MS, HPLC Agilent 1100) using the
following procedure: the compounds were dissolved at a con-
centration of 0.5 mg/mL in H2O: MeOH = 1:1, containing 2
mM NH4CH3COO. Then, 10 μL of the sample was injected into
an HPLC column (Phenomenex Luna 3 μ C18, 50 ꢀ 2.00 mm).
Elution was performed with a gradient of water/methanol
(containing 2 mM NH4CH3COO) from 90:10 to 0:100 for
30 min at a flow rate of 250 μL/min, starting the gradient after
10 min. UV absorption was detected from 200 to 950 nm using a
diode array detector. Purity of all compounds was determined at
254 nm. A second HPLC method was used to confirm purity as
previously described.35 For microwave reactions a CEM Fo-
cused Microwave Synthesis type Discover apparatus was used.
A freeze-dryer (CHRIST ALPHA 1-4 LSC) was used for
lyophilization. The purity of the compounds proved to be
g95%. The syntheses of compounds 7, 10-14, 17-24, 27,
29-31, 33-49, and 52-58, 60, 61, and 63 has previously been
described.29,32,33,38 All other compounds were newly prepared in
analogy to described methods.30,38,39
Purification of Reactive Blue 4 (RB-4). RB-4 was obtained
from Alfa Aesar (A12911) and subsequently purified. Initially
an RP-TLC was obtained using 40% aqueous acetone as eluent;
the Rf value of the main blue spot was 0.5. In a second step,
250 mg of RB-4 was dissolved in ca. 10 mL of water and injected
onto a Sepacore Glass Column C-690 (ID 26 ꢀ 460 mm) three-
quarter filled with reversed phase-18 silica gel (40-63 μm,
Merck) and purified by flash column chromatography. Initially,
water was used as an eluent and the polarity was gradually
decreased by increasing the concentration of acetone form 0%
to 5, 10, 20, and finally to 40%. The collected main blue-colored
fractions corresponding to the blue band that appeared last on
the column were pooled and evaporated under vacuum at 25 °C
to remove the acetone. The remaining water was subsequently
removed by lyophilization yielding 60 mg (yield: 24%) of pure
57 as a blue powder.
Synthesis and Spectral Data of the Key Compounds. Sodium
1-Amino-4-(3-benzylphenylamino)-9,10-dioxo-9,10-dihydroan-
thracene-2-sulfonate (50). Reaction conditions: According to the
general procedures A: 5 min, 120 °C, 100 W; pressure up to 10
bar. Analytical data: mp > 300 °C, blue powder. 1H NMR: δ
3.96 (s, 2H, CH2), 7.04 (d, 1H, 40-H), 7.10 (d, 1H, 60-H), 7.15 (br,
1H, 20-H), 7.18 (m, 1H, 400-H), 7.29 (m, 4H, 200-H, 300-H, 500-H,
600-H), 7.84 (m, 2H, 6-H, 7-H), 8.05 (s, 1H, 3-H), 8.26 (m, 2H,
5-H, 8-H), 10.10 (br, 2H, 1-NH2), 12.03 (s, 1H, 4-NH). 13C
NMR: δ 41.1 (CH2), 109.3 (C-9a), 111.5 (C-4a), 120.6, 122.9,
123.5, 125.0, 126.08, 126.13, 126.2, 128.7, 129.0, 129.8, 132.9 (C-
20, C-60, C-3, C-5, C-8, C-40, C-50, C100, C-200, C-300, C-400, C-500,
General Procedures for the Preparation of 4-Substituted An-
thraquinones (7-56). General Procedure A. To a 5 mL micro-
wave reaction vial equipped with a magnetic stirring bar were
added bromo-substituted compounds (bromaminic acid sodium
salt 6a, or the methyl, 6b) (0.20 mmol) and the appropriate
aniline and/or amine derivative (0.40 mmol, 2 equiv), followed
by a buffer solution of Na2HPO4 (pH 9.6) (4.5 mL) and NaH2-
PO4 (pH 4.2) (0.5 mL). A catalytic amount (ca. 0.002-0.003 g)
of finely powdered elemental copper was added. The mixture
was capped and irradiated in the microwave oven (80-100 W)
for 5-20 min at 80-120 °C. Then the reaction mixture was

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 827
C-600), 133.3, 133.7, 134.3, 139.4 (C-8a, C-10a, C-6, C-7), 140.9
(C-10), 143.0 (C-4), 143.2 (C-2), 144.5 (C-1), 181.9 (C-9), 182.6
(C-10). LC-MS (m/z): 502 [M-Na þ NH4þ]þ, 485 [M - Na]þ,
483 [M - Na]-. Purity by HPLC-UV (254 nm)-ESI-MS: 99.2%.
Sodium 1-Amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10-
dihydroanthracene-2-sulfonate (51). Reaction conditions: Ac-
cording to the general procedures A: 5 min, 120 °C, 100 W;
pressure up to 10 bar. Analytical data: mp >300 °C, blue
1-amino-4-[4-(4,6-dichloro-[1,3,5]triazine-2-ylamino)phenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (61) was dissolved in
water (15 mL) then heated to 90 °C, followed by the addition of
Na2CO3 (10 mg, 2 equiv.) and let to stirr at 90 °C for 1 h. After
completion of the reaction, RP-TLC control using a mobile phase of
acetone:water (2:3), the mixture was subjected to flash column
chromatography on RP-18 silica gel using acetone:water as an eluent
yielded 26 mg (yield 95%) of a blue powder, mp >300 °C. 1HNMR:
δ 7.09 (d, 2H, 20-H, 60-H), 7.83 (m, 2H, 6-H, 7-H), 7.85 (d, 2H, 30-H,
50-H),7.94(s,1H,3-H),8.27(m,2H,5-H,8-H),8.6(br,1H,OH),8.8
(br, 1H, OH), 10.17 (br, 2H, 1-NH2), 12.16 (s, 1H, 4-NH). 13C
NMR: δ 109.0 (C-9a), 110.2 (C-4a), 120.0 (C-20, C-60), 122.8 (C-5),
124.1 (C-30, C-50), 126.0, 126.1, 131.2, 132.7, 133.0 (C-3, C-6, C-7,
C-8, C-40), 133.9 (C-10a), 134.3 (C-8a), 139.4, 142.6, 143.2, 144.3
(C-1, C-2, C-4, C-10), 160.9 (C-100), 167.0 (C-300, C-500), 181.7 (C-9,
C-10). LC-MS (m/z): 538 [M - Na þ NH4þ]þ, 520 [M - Na]þ, 518
[M - Na]-. Purity by HPLC-UV (254 nm)-ESI-MS: 95.5%.
Disodium 1-Amino-4-[3-(4,6-dihydroxy-[1,3,5]triazine-2-ylamino)-
4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-
sulfonate (66). According to the general procedure D: 34 mg 0.05
mmol of Disodium 1-amino-4-[3-(4,6-dichloro-[1,3,5]triazine-
2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroan-
thracene-2-sulfonate (57) was dissolved in water (15 mL) then
heated to 90 °C, followed by the addition of Na2CO3 (10 mg,
2 equiv.) and let to stirr at 90 °C for 1 h. After completion of the
reaction, RP-TLC control using a mobile phase of acetone:
water (2:3), the mixture was subjected to flash column chro-
matography on RP-18 silica gel using acetone:water as an
eluent yielded 31 mg (yield 97%) of a blue powder, mp >
300 °C. 1H NMR: δ 7.02 (dd, 1H, 60-H), 7.70 (d, 1H, 50-H), 7.85
(m, 3H, 6-H, 7-H, 20-H), 8.03 (s, 1H, 3-H), 8.27 (m, 2H, 5-H,
8-H), 10.75 (br, 1H, 30-NH), 11.99 (br, 1H, 4-NH). 13C NMR:
δ 109.4 (C-9a), 112.1 (C-4a), 117.2 (C-20), 118.2 (C-50), 122.9
(C-3), 126.16 (C-60), 126.22 (C-5, C-8), 128.0 (C-40), 132.9
(C-6), 133.4 (C-7), 133.7 (C-10a), 134.0 (C-8a), 134.3 (C-10),
134.8 (C-30), 140.0 (C-4), 142.8 (C-2), 144.6 (C1), 153.4 (C-100),
154.6 (C-300, C-500), 182.1 (C-9), 183.0 (C-10). LC-MS (m/z):
601 [M - 2Na]þ, 599 [M - 2Na]-, 299 [M - 2Na]2-. Purity by
HPLC-UV (254 nm)-ESI-MS: 98.4%.
1
powder. H NMR: δ 6.79 (dd, 1H, 40-H), 6.88 (dd, 1H, 20-H),
7.03 (dd, 1H, 60-H), 7.12 (m, 3H, 200-H, 400-H, 600-H), 7.42 (m,
3H,50-H, 300-H, 500-H), 7.84 (m, 2H, 6-H, 7-H), 8.06 (s, 1H, 3-H),
8.25 (m, 2H, 5-H, 8-H), 10.01 (br, 2H, 1-NH2), 11.86 (s, 1H, 4-NH).
13C NMR: δ 109.4 (C-9a), 112.2 (C-4a), 112.5, 114.1, 117.5, 119.2,
123.1, 123.9, 126.1, 126.2, 130.3, 131.1 (C-3, C-5, C-8, C-20, C-60,
C-40, C-50, C-200, C-300, C-400, C-500, C-600), 133.0, 133.4, 133.6, 134.3
(C-8a, C-10a, C-6, C-7), 140.1 (C-10), 141.2 (C-4), 142.8 (C-2), 144.6
(C-1), 156.1, 158.0 (C-30, C100), 182.1 (C-9), 182.9 (C-10). LC-MS
(m/z): 504 [M - Na þ NH4þ]þ, 487 [M - Na]þ, 485 [M - Na]-.
Purity by HPLC-UV (254 nm)-ESI-MS: 97.4%.
Disodium 1-Amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-
4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-
sulfonate (57). According to the general procedure B: An ice-cooled
solution of cyanuric acid chloride (184.4 mg, 1.0 mmol) in water
(25 mL) and acetone (25 mL) was added to a stirred solution of
disodium 1-amino-4-(3-amino-4-sulfophenylamino)-9,10-dioxo-9,
10-dihydroanthracene 2-sulfonate (45) (256 mg, 0.5 mmol) in
water (25 mL) at 0-5 °C. The resulting mixture was stirred for
1 h at 0-5 °C, then allowed to warm up to rt and kept stirring at
rt for 2 h. The formation of product was monitored by RP-TLC
using a mobile phase of acetone/water (2:3). After completion of
the reaction the solvents were evaporated and the residue was
purified by flash column chromatography on RP-18 silica
gel using acetone:water as an eluent yielded 289 mg (yield
1
85%) of a blue powder, mp >300 °C. H NMR: δ 7.04 (dd,
1H, 60-H), 7.70 (d, 1H, 50-H), 7.84 (m, 3H, 6-H, 7-H, 20-H), 8.04
(s, 1H, 3-H), 8.27 (m, 2H, 5-H, 8-H), 10.82 (br, 2H, 1-NH2),
11.98 (br, 1H, 4-NH). 13C NMR: δ 109.4 (C-9a), 112.2 (C-4a),
117.5 (C-20), 118.3 (C-30), 123.0 (C-60), 126.17 (C-8), 126.21
(C-5), 128.1 (C-50), 133.0 (C-40), 133.4 (C-6), 133.6 (C-7), 134.2
(C-10a), 134.3 (C-8a), 134.5 (C-10), 140.0 (C-30), 140.1 (C-4),
142.8 (C-2), 144.6 (C1), 153.0 (C-100), 154.4 (C-300, C-500), 182.1
(C-9), 183.0 (C-10). LC-MS (m/z): 654 [M - 2Na þ NH4þ]þ, 637
[M - 2Na]þ, 635 [M - 2Na]-. Purity by HPLC-UV (254 nm)-
ESI-MS: 96.0%.
Biological Assays
Expression of P2X Receptors in Xenopus laevis Oocytes.
Oocyte expression plasmids encoding wild type and hexahistidyl-
tagged rat P2X subunits (rP2X2, rP2X3, rP2X4, rP2X7)
were available from previous studies.13e Chimeric constructs
rP2X21-47X148-399 and rP2X21-47X342-397 encoding amino
acids Met1-Val47 of the rP2X2 subunit joined in frame with
48Val-399Ser of the 399-amino acids rP2X1 subunit or 42Val-397-
His of the 397-amino acids rP2 ꢀ 3 subunit, respectively, have
also been described previously.13e For expression of P2X receptors
capped cRNAs were synthesized and injected into collagenase-
defolliculated Xenopus laevis oocytes using a Nanoliter 2000
injector (WPI) as described previously.13e For expression of
the heteromeric rP2X2/3 receptor, a 1:2 (w/w) ratio of rP2X2
and rP2X3cRNA was injected. Oocytes were cultured at 19°C
in sterile oocyte Ringer’s solution (ORi: 90 mM NaCl, 1 mM
KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes, pH 7.4)
supplemented with 50 μg/mL of gentamycin.
Two-Electrode Voltage-Clamp Electrophysiology. Two to
three days after cRNA injection, current responses were
evoked by ATP (rP2X1, rP2X2, rP2X3, rP2X4, and rP2X7)
or R,β-meATP (rP2X2/3) as indicated. Recordings were per-
formed at ambient temperature (21-24 °C) by a conventional
two-electrode voltage-clamp (TEVC) with a Turbo TEC-05
amplifier (npi Electronics) or by the Roboocyte automated
electrophysiology system47 (MultiChannelSystems MCS GmbH,
Reutlingen, Germany) at a holding potential of -60 mV as
Sodium 1-Amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-
phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63).
According to the general procedure B: An ice-cooled solution of
cyanuric acid chloride (184.4 mg, 1.0 mmol) in water (25 mL) and
acetone (25 mL) was added to a stirred solution of sodium
1-amino-4-(3-aminophenylamino)-9,10-dioxo-9,10-dihydroanthra-
cene 2-sulfonate (39) (216 mg, 0.5 mmol) in water (25 mL) at
0-5 °C. The resulting mixture was stirred for 15 min at 0-5 °C.
The formation of product was monitored by RP-TLC using a
mobile phase of acetone:water (2:3). After completion of the
reaction thesolventswereevaporatedand the residue was purified
by flash column chromatography on RP-18 silica gel using
acetone:water as an eluent yielded 275 mg (yield 95%) of a blue
powder, mp >300 °C. 1H NMR: δ 7.07 (m, 1H, H-50), 7.40 (m,
2H, H-40, H-60), 7.47 (m, 1H, H-20), 7.84 (m, 2H, H-6, H-7), 8.01
(s, 1H, H-3), 8.27 (m, 2H, H-5, H-8), 10.82 (br, 1H, NH-3), 12.01
(br, 1H, NH-4). 13C NMR: δ 109.4 (C-9a), 111.8 (C-4a), 115.8
(C-20), 117.4 (C-50), 118.8 (C-3), 122.9 (C-60), 126.2 (C-5, C-8),
130.1 (C-40), 133.0 (C-6), 133.4 (C-7), 133.7 (C-10a), 134.3 (C-8a),
138.7 (C-10), 140.0 (C-30), 140.6 (C-4), 142.9 (C-2), 144.5 (C1),
152.8 (C-100), 154.1 (C-300, C-500), 182.0 (C-9), 182.8 (C-10).
LC-MS (m/z): 575 [M - Na þ NH4þ]þ, 555 [M - Na]-, 557
[M - Na]þ. Purity by HPLC-UV (254 nm)-ESI-MS: 97.1%.
Sodium 1-Amino-4-[4-(4,6-dihydroxy[1,3,5]triazine-2-ylamino)-
phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (65).
According to the general procedure D: 29 mg 0.05 mmol of Sodium

828 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
described previously.13e Oocytes were continuously perfused
by gravity flow (5-10 mL/min) in a small flow-through cham-
ber (volume about 10 μL) or in a round-bottom well of a
96-well plate, respectively, with a nominally calcium-free ORi
solution (designated Mg-ORi), in which CaCl2 was replaced
by equimolar MgCl2 to avoid a contribution of endogenous
Ca2þ-dependent Cl- channels to the ATP response. Dilu-
tions of agonists and antagonists in Mg-ORi were prepared
daily and applied by bath perfusion. Switching between bath
solutions was controlled by a set of magnetic valves, enabling
computer-controlled applications of compounds (CellWork
Lite 5.1 software, NPI Electronics; Roboocyte system, Mul-
tiChannelSystems). As wt-rP2X1 and wt-rP2X3 receptors,
once activated, desensitize rapidly in the continued presence
of agonist, the current transient is too short to allow reaching
a binding equilibrium between the coapplied compounds. To
assess inhibition of P2X1 and P2X3 receptors also reliable
under steady-state conditions, non-desensitizing rP2X2-X1
and rP2X2-X3 receptor chimeras were used, in which the
N-terminal tail including the first transmembrane domain is
replaced by the complementary portion of the rP2X2
subunit.13e Since the ligand-binding ectodomain originates
entirely from the rP2X1 or the rP2X3 subunit, these chimeras
can be reliably used as non-desensitizing substitutes of
wt-rP2X1 and rP2X3 receptors. For concentration-inhibition
analysis of nondesensitizing P2X2-X1, P2X2, P2X2/3, P2X2-
X3, and P2X7 receptors or partial desensitizing P2X4 recep-
tors, oocytes expressing the specified P2X receptor were
superfused with agonist-containing (P2X2-X1 and P2X2-
X3, 10 nM ATP; P2X2 and P2X4, 10 μM ATP; P2X7,
100 μM ATP) bath solution until a steady-state current
was obtained. Then, the superfusion solution was switched
to one containing both antagonist and the same concentra-
tion of agonist as before. The superfusion was maintained
until a steady-state level of inhibition was reached, followed
by switching to bath solution containing the agonist alone
for washout of test compounds. Figure 7 shows representa-
tive original current traces of oocytes expressing homomeric
rP2X2 receptors to illustrate this steady state protocol
exemplarily. The percentage of the control response was
calculated as the steady-state currents in the absence and
presence of antagonist. For analysis of heteromeric P2X2/3
receptors the same steady-state protocol was used but with
1 μM R,β-meATP as agonist. Coexpression of rP2X2 and
rP2X3 subunits results in a mixed population of heteroge-
neous receptors; these include both the homomeric rP2X2
and rP2X3 receptors as well as the heteromeric rP2X2/3
receptor. To exclude a contribution of the homomeric rP2X2
receptor to the current response, 1 μM R,β-meATP was used
as an agonist, which activates the heteromeric rP2X2/3
receptor with >100-fold higher potency than the homomeric
rP2X2 receptor. Control experiments with oocytes injected
solely with cRNA for the rP2X2 subunit confirmed that
>30 μM R,β-meATP was needed to evoke significant inward
currents in these cells (data not shown). Any contribution of
the homomeric rP2X3 receptor to the R,β-meATP-induced
inward current was suppressed by repetitive R,β-meATP
applications that hold the P2X3 receptor in the fully desen-
sitized state.
Figure 7. Steady state protocol for the assessment of the inhibitory
potency of test compounds from stationary current measurements.
A representative original current trace of an oocyte expressing
rP2X2 receptors is shown. After a stationary current was elicited
by application of 10 μM ATP (black bars), an incrementally larger
concentration of the test compound (0.1-3 μM, gray bars) was
coapplied with 10 μM ATP. The current declined until a new steady
state was reached, indicating that inhibition by the test compound
was fully developed. Then 10 μM ATP alone was applied for
washout of the test compound until a new steady state was reached.
The extent of inhibition was judged for each concentration of the
test compound from the ratio of the maximal and minimal sta-
tionary current in the absence and presence of the test compound,
respectively.
maximal current value obtained with a saturating ATP con-
centration. Concentration-response curves and half-maximal
effective agonist concentrations (EC50) were obtained from
curves by iteratively fitting the Hill equation (eq 1) to the
normalized data points pooled from several (n) oocytes
nH
I=Imax ¼ 1=ð1 þ ð½EC50=AꢁÞ
Þ
ð1Þ
where I is the current evoked by agonist concentration A,
Imax the maximal current response, and nH the Hill coeffi-
cient.
Concentration-inhibition curves and IC50 values were
derived from nonlinear least-squares fits of eq 2 to the pooled
data points
nH
IAnt=Imax ¼ 1=ð1 þ ð½Antꢁ=IC50Þ
Þ
ð2Þ
where Imax is the current response in absence of antagonist
(Ant), IAnt is the current response at the respective antagonist
concentration, and IC50 the antagonist concentration caus-
ing 50% inhibition of the current elicited by a given agonist
concentration.
Experimental Procedures for Testing of Compounds at
P2Y2, P2Y4, and P2Y6 Receptors. Astrocytoma cell lines
stably transfected with either the human P2Y2, the human
P2Y4, or the rat P2Y6 receptor were used.29,43,44 Test com-
pounds were investigated by measuring their inhibition of
P2Y2, P2Y4, or P2Y6 receptor-mediated intracellular calcium
mobilization as previously described.29,44,45
Acknowledgment. Y.B. was supported by a DAAD
(Deutscher Akademischer Austauschdienst) scholarship. C.E.M.
and Y.B. are grateful for support by the Deutsche Forschungsge-
meinschaft (DFG, GRK804). Meike Welz, Natalie Poryo, Karen
Schmeling, and Dr. Anja B. Scheiff are acknowledged for testing
selected compounds at P2Y receptors.
Data Analysis. Data were plotted and fitted using Prism4
(GraphPAD Software Inc., San Diego, CA) and Origin 6.0.
Agonist concentration-response curves were produced by
measuring the maximal current induced by increasing con-
centrations of agonist, which were then normalized to the
Supporting Information Available: Concentration-response
curve for suramin at P2X2R, synthesis and spectral data of new

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 829
compounds as well as 1H NMR and 13C NMR spectra of newly
synthesized anthraquinone derivatives. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Rettinger, J.; Gerevich, Z.; Meis, S.; Kassack, M. U.; Illes, P.;
Lambrecht, G.; Schmalzing, G. The suramin analog 4,40,400,4000
-
(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-
benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype
selectivity is determined by location of sulfonic acid groups. Mol.
Pharmacol. 2006, 69, 2058–2067. (f) Stokes, L.; Jiang, L.-H.; Alcaraz,
L.; Bent, J.; Bowers, K.; Fagura, M.; Furber, M.; Mortimore, M.;
Lawson, M.; Theaker, J.; Laurent, C.; Braddock, M.; Surprenant, M.
Characterization of a selective and potent antagonist of human P2X7
receptors, AZ11645373. Br. J. Pharmacol. 2006, 149, 880–887.
(g) King, B. F. Novel P2X7 receptor antagonists ease the pain. Br.
J. Pharmacol. 2007, 151, 565–567. (h) Baxter, A.; Bent, J.; Bowers, K.;
Braddock, M.; Brough, S.; Fagura, M.; Lawson, M.; McInally, T.;
Mortimore, M.; Robertson, M.; Weaver, R.; Webborn, P. Hit-to-Lead
studies: The discovery of potent adamantane amide P2X7 receptor
antagonists. Bioorg. Med. Chem. Lett. 2003, 13, 4047–4050.
(14) (a) Bianchi, B. R.; Lynch, K. J.; Touma, E.; Niforatos, W.;
Burgard, E. C.; Alexander, K. M.; Park, H. S.; Yu, H.; Metzger,
R.; Kowaluk, E.; Jarvis, M. F.; van Biesen, T. Pharmacological
characterization of recombinant human and rat P2X receptor
subtypes. Eur. J. Pharmacol. 1999, 376, 127–138. (b) King, B. F.;
Wildman, S. S.; Ziganshina, L. E.; Pintor, J.; Burnstock, G. Effects of
extracellular pH on agonism and antagonism at a recombinant P2X2
receptor. Br. J. Pharmacol. 1997, 121, 1445–1453. (c) Lynch, K. J.;
Touma, E.; Niforatos, W.; Kage, K. L.; Burgard, E. C.; van Biesen, T.;
Kowaluk, E. A.; Jarvis, M. F. Molecular and functional characteriza-
tion of human P2X(2) receptors. Mol. Pharmacol. 1999, 56, 1171–
1181. (d) Virginio, C.; Robertson, G.; Surprenant, A.; North, R. A.
Trinitrophenyl-substituted nucleotides are potent antagonists selective
for P2X1, P2X3, and heteromeric P2X2/3 receptors. Mol. Pharmacol.
1998, 53, 969–973.
(15) (a) Evans, R. J.; Lewis, C.; Buell, G.; Valera, S.; North, R. A.;
Surprenant, A. Pharmacological characterization of heterolo-
gously expressed ATP-gated cation channels (P2X purinoceptors).
Mol. Pharmacol. 1995, 48, 178–183. (b) Pintor, J.; King, B. F.; Miras-
Portugal, M. T.; Burnstock, G. Selectivity and activity of adenine
dinucleotides at recombinant P2X2 and P2Y1 purinoceptors. Br.
J. Pharmacol. 1996, 119, 1006–1012. (c) Wildman, S. S.; King,
B. F.; Burnstock, G. Zn2þ modulation of ATP-responses at recombinant
P2X2 receptors and its dependence on extracellular pH. Br. J. Phar-
macol. 1998, 123, 1214–1220.
(16) Jacobson, K. A.; Kim, Y.-C.; King, B. F. In search of selective P2
receptor ligands: interaction of dihydropyridine derivatives at
recombinant rat P2X2 receptors. J. Auto. Nerv. Syst. 2000, 81,
152–157.
References
(1) (a) Burnstock, G. Physiology and pathophysiology of purinergic
neurotransmission. Physiol. Rev. 2007, 87, 659–797. (b) Brunschweiger,
A.; M€uller, C. E. P2 receptors activated by uracil nucleotides-an update.
Curr. Med. Chem. 2006, 13, 289–312.
(2) Borrmann, T.; Abdelrahman, A.; Volpini, R.; Lambertucci, C.;
Alksnis, E.; Gorzalka, S.; Knospe, M.; Schiedel, A. C.; Cristalli, G.;
€
Muller, C. E. Structure-activity relationships of adenine and
deazaadenine derivatives as ligands for adenine receptors, a new
purinergic receptor family. J. Med. Chem. 2009, 52, 5974–5989.
(3) (a) Khakh, B. S.; Burnstock, G.; Kennedy, C.; King, B. F.; North,
ꢀ
ꢀ
R. A.; Seguela, P.; Voigt, M.; Humphrey, P. P. International Union
of Pharmacology. XXIV. Current status of the nomenclature and
properties of P2X receptors and their subunits. Pharmacol. Rev.
2001, 53, 107–118. (b) Abbracchio, M. P.; Burnstock, G.; Boeynaems,
J. M.; Barnard, E. M.; Boyer, J. L.; Kennedy, C.; Knight, G. E.; Fumagalli,
M.; Gachet, C.; Jacobson, K. A.; Weisman, G. A. International Union
of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide
receptors: from molecular mechanisms and pathophysiology to therapy.
Pharmacol. Rev. 2006, 58, 281–341.
€
(4) (a) Nicke, A.; Baumert, H. G.; Rettinger, J.; Eichele, A.;
Lambrecht, G.; Mutschler, E.; Schmalzing, G. P2X1 and P2X3
receptors form stable trimers: a novel structural motif of ligand-
gated ion channels. EMBO J. 1998, 17, 3016–3028. (b) Benjamin
Marquez-Klaka, B.; Rettinger, J.; Nicke, A. Inter-subunit disulfide
cross-linking in homomeric and heteromeric P2X receptors. Eur.
Biophys. J. 2009, 38, 329–338.
(5) Kawate, T.; Michel, J. C.; Birdsong, W. B.; Gouaux, E. Crystal
structure of the ATP-gated P2X4 ion channel in the closed state.
Nature 2009, 592–598.
(6) Khakh, B. S.; Gittermann, D.; Cockayne, D. A.; Jones, A. ATP
modulation of excitatory synapses onto interneurons. J. Neurosci.
2003, 23, 7426–7437.
(7) Finger, T. E.; Danilova, V.; Barrows, J.; Bartel, D. L.; Vigers, A. J.;
Stone, L.; Hellekant, G.; Kinnamon, S. C. ATP signaling is crucial
for communication from taste buds to gustatory nerves. Science
2005, 310, 1495–1499.
(8) Rong, W.; Gourine, A. V.; Cockayne, D. A.; Xiang, Z.; Ford, A. P.;
Spyer, K. M.; Burnstock, G. Pivotal role of nucleotide P2X2
receptor subunit of the ATP-gated ion channel mediating ventila-
tory responses to hypoxia. J. Neurosci. 2003, 23, 11315–11321.
(9) Gever, J. R.; Cockayne, D. A.; Dillon, M. P.; Burnstock, J.; Ford,
A. P. D. W. Pharmacology of P2X channels. Pflugers Arch. - Eur.
J. Physiol. 2006, 452, 513–537.
(17) Bo, X.; Fischer, B.; Maillard, M.; Jacobson, K. A.; Burnstock, G.
Comparative studies on the affinities of ATP derivatives for P2X-
purinoceptors in rat urinary bladder. Br. J. Pharmacol. 1994, 112,
1151–1159.
€
(18) Bultmann, R.; Starke, K. P2-purinoceptor antagonists discrimi-
nate three contraction mediating receptors for ATP in rat vas
deferens. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1994, 349,
74–80.
(10) Cockayne, D. A.; Dunn, P. M.; Zhong, Y.; Rong, W.; Hamilton,
S. G.; Knight, G. E.; Ruan, H.-Z.; Ma, B.; Yip, P.; Nunn, P.;
McMahon, S. P.; Burnstock, G.; Ford, A. P. D. W. P2X2 knockout
mice and P2X2/P2X3 double knockout mice reveal a role for the
P2X2 receptor subunit in mediating multiple sensory effects of
ATP. J. Physiol. 2005, 567, 621–639.
(19) Brake, A. J.; Wagenbach, M. J.; Julius, D. New structural motif for
ligand-gated ion channels defined by an ionotropic ATP receptor.
Nature 1994, 371, 519–523.
(20) Simon, J.; Webb, T. E.; King, B. F.; Burnstock, G.; Barnard,
E. A. Characterisation of a recombinant P2Y purinoceptor. Eur.
J. Pharmacol. 1995, 291, 281–289.
(21) Chang, K.; Hanoka, K.; Kumada, M.; Takuwa, Y. Molecular
cloning and functional analysis of a novel P2 nucleotide receptor.
J. Biol. Chem. 1995, 270, 26152–26158.
(11) Inoue, K.; Tsuda, M.; Koizumi, S. ATP induced three types of pain
behaviors, including allodynia. Drug Dev. Res. 2003, 59, 56–63.
(12) (a) Jarvis, M. F.; Khakh, B. S. ATP-gated P2X cation-channels.
Neuropharmacology 2009, 56, 208–215. (b) Burnstock, G. Purinergic
signalling and disorders of the central nervous system. Nat. Rev. Drug
Discovery 2008, 7, 575–590.
(22) Communi, D.; Parmentier, M.; Boeynaems, J.-M. Cloning, func-
tional expression and tissue distribution of the human P2Y6
receptor. Biochem. Biophys. Res. Commun. 1996, 222, 303–308.
(23) Communi, D.; Robaye, B.; Boeynaems, J.-M. Pharmacological
characterization of the human P2Y11 receptor. Br. J. Pharmacol.
1999, 128, 1199–1206.
(24) Michel, A. D.; Grahames, C. B. A.; Humphrey, P. P. A. Functional
characterization of P2 purinoceptors in PC12 cells by measurement
of radiolabelled calcium influx. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 1996, 354, 562–571.
(25) Seguela, P.; Haghigi, A.; Soghommonian, J.-J.; Cooper, E. A novel
neuronal P2X ATP receptor ion channel with widespread distribu-
tion in the brain. J. Neurosci. 1996, 15, 448–455.
(26) Webb, T. E.; Feolde, E.; Vigne, P.; Neary, J. T.; Runberg, A.;
Frelin, C.; Barnard, E. A. The P2Y purinoceptor in rat brain
microvascular endothelial cells couple to inhibition of adenylate
cyclase. Br. J. Pharmacol. 1996, 119, 1385–1392.
(13) (a) Volpini, R.; Mishra, R. C.; Kachare, D. D.; Ben, D. D.;
Lambertucci, C.; Antonini, I.; Vittori, S.; Marucci, G.; Sokolova,
E.; Nistri, A.; Cristalli, G. Adenine-Based Acyclic Nucleotides as
Novel P2X3 Receptor Ligands. J. Med. Chem. 2009, 52, 4596–4603.
(b) Braun, K.; Rettinger, J.; Ganso, M.; Kassack, M.; Hildebrandt, C.;
Ullmann, H.; Nickel, P.; Schmalzing, G.; Lambrecht, G. NF449: a
subnanomolar potency antagonist at recombinant rat P2X1 receptors.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 2001, 364, 285–290.
(c) Jarvis, M. F.; Burgard, E. C.; McGaraughty, S.; Honore, P.; Lynch,
K.; Brennan, T. J .; Subieta, A.; van Biesen, T.; Cartmell, J.; Bianchi, B.;
Niforatos, W.; Kage, K.; Yu, H.; Mikusa, J.; Wismer, C. T.; Zhu, C. Z.;
Chu, K.; Lee, C.-H.; Stewart, A. O.; Polakowski, J.; Cox, B. F.;
Kowaluk, E.; Williams, M.; Sullivan, J.; Faltynek, C. A-317491, a
novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3
receptors, reduces chronic inflammatory and neuropathic pain in the
rat. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 17179–17184.
(d) Honore, P.; Mikusa, J.; Bianchi, B.; McDonald, H.; Cartmell, J.;
Faltynek, C.; Jarvis, M. F. TNP-ATP, a potent P2X3 receptor antagonist,
blocks acetic acid-induced abdominal constriction in mice: comparison
with reference analgesics. Pain 2002, 96, 99–105. (e) Hausmann, R.;
€
€
(27) Glanzel, M.; Bultmann, R.; Starke, K.; Frahm, A. W. Structure-
activity relationships of novel P2-receptor antagonists structurally
related to Reactive Blue 2. Eur. J. Med. Chem. 2005, 40, 1262–1276.

830 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Baqi et al.
€
€
(28) Glanzel, M.; Bultmann, R.; Starke, K.; Frahm, A. W. Constitu-
tional isomers of Reactive Blue 2 - selective P2Y-receptor antago-
nists? Eur. J. Med. Chem. 2003, 38, 30–12.
(38) Daly, J. W. Caffeine analogs: biomedical impact. Cell. Mol. Life
Sci. 2007, 64, 2153–2169.
€
(39) Baqi, Y.; Muller, C. E. Rapid and efficient microwave-assisted
(29) Weyler, S.; Baqi, Y.; Hillmann, P.; Kaulich, M.; Hunder, A. M.;
copper(0)-catalyzed Ullmann coupling reaction: general access to
anilinoanthraquinone derivatives. Org. Lett. 2007, 9, 1271–1274.
(40) Kalontarov, I. Ya.; Kiseleva, N. N.; Zhuravleva, I. V. Study of the
thermal and oxidative thermal degradation of poly(vinyl alcohol)
dyed with reactive dyes. IATOAN (Izvestiya Akademii Nauk Tadz-
hikskoi SSR, Otdelinie Fiziko-Matematicheskikh i Geologo-Khimi-
cheskikh Nauk) 1978, 1, 97–102.
(41) Greaves, A. J.; Churchley, J. H.; Hutchings, M. G.; Phillips,
D. A. S.; Taylor, J. A. A chemometric approach to understanding
the bioelimination of anionic, water-soluble dyes by a biomass
using empirical and semi-empirical molecular descriptors. Wat.
Res. 2001, 35, 1225–1239.
€
€
Muller, I. A.; Muller, C. E. Combinatorial synthesis of anilinoanthra-
quinone derivatives and evaluation as non nucleotide-derived P2Y2
receptor antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 223–227.
€
€
€
(30) Baqi, Y.; Atzler, K.; Kose, M.; Glanzel, M.; Muller, C. E. High-
affinity, non nucleotide-derived competitive antagonists of platelet
P2Y12 receptors. J. Med. Chem. 2009, 52, 3784–3793.
€
€
(31) Hoffmann, K.; Baqi, Y.; Morena, M. S.; Glanzel, M.; Muller,
€
C. E.; von Kugelgen, I. Interaction of new, very potent non-
nucleotide antagonists with Arg256 of the human platelet P2Y12
-
receptor. J. Pharmacol. Exp. Ther. 2009, 331, 648–655.
(32) Baqi, Y.; Lee, S.-Y.; Iqbal, J.; Ripphausen, P.; Lehr, A.; Scheiff,
(42) Rettinger, J.; Braun, K.; Hochmann, H.; Kassack, M. U.; Ull-
mann, H.; Nickel, P.; Schmalzing, G.; Lambrecht, G. Profiling at
recombinant homomeric and heteromeric rat P2X receptors iden-
tifies the suramin analogue NF449 as a highly potent P2X1 receptor
antagonist. Neuropharmacology 2005, 48, 461–468.
(43) Charlton, S. J.; Brown, C. A.; Weisman, G. A.; Turner, J. T.; Erb,
L.; Boarder, M. R. Cloned and transfected P2Y4 receptors: char-
acterization of a suramin and PPADS-insensitive response to UTP.
Br. J. Pharmacol. 1996, 119, 1301–1303.
€
A. B.; Zimmermann, H.; Bajorath, J.; Muller, C. E. Development
of potent and selective inhibitors of ecto-50-nucleotidase based on
an anthraquinone scaffold. J. Med. Chem. 2010, 53, 2076–2086.
(33) Baqi, Y.; Weyler, S.; Iqbal, J.; Zimmermann, H.; Muller, C. E.
€
Structure-activity relationships of anthraquinone derivatives
derived from bromaminic acid as inhibitors of ectonucleoside
triphosphate diphosphohydrolases (E-NTPDases). Purinergic
Signalling 2009, 5, 91–106.
ꢀ
ꢀ
€
(34) Iqbal, J.; Levesque, S. A.; Sevigny, J.; Muller, C. E. A highly
sensitive CE-UV method with dynamic coating of silica-fused
capillaries for the monitoring of nucleotide pyrophosphatase/
phosphodiesterase reactions. Electrophoresis 2008, 29, 3685–3693.
(35) Tuluc, F.; Bultmann, R.; Glanzel, M.; Frahm, A. W.; Starke, K.
P2-receptor antagonists: IV. Blockade of P2-receptor subtypes
and ecto-nucleotidases by compounds related to reactive blue 2.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998, 357, 111–120.
€
(44) Hillmann, P.; Ko, G.-Y.; Spinrath, A.; Raulf, A.; von Kugelgen, I.;
€
€
Wolff, S. C.; Nicholas, R. A.; Kostenis, E.; Holtje, H.-D.; Muller,
C. E. Key determinants of nucleotide-activated G protein-coupled
P2Y2 receptor function revealed by chemical and pharmacological
experiments, mutagenesis and homology modeling. J. Med. Chem.
2009, 52, 2762–2775.
€
€
€
€
(45) Kaulich, M.; Streicher, F.; Mayer, R.; Muller, I.; Muller, C. E.
Flavonoids - novel lead compounds for the development of P2Y2
receptor antagonists. Drug Dev. Res. 2003, 59, 72–81.
(46) Duckwitz, W.; Hausmann, R.; Aschrafi, A.; Schmalzing, G. P2X5
subunit assembly requires scaffolding by the second transmem-
brane domain and a conserved aspartate. J. Biol. Chem. 2006, 51,
39561–39572.
€
(36) (a) Baqi, Y.; Muller, C. E. Synthesis of alkyl- and aryl-amino-
substituted anthraquinone derivatives by microwave-assisted
copper(0)-catalyzed Ullmann coupling reactions. Nat. Protoc.
2010, 5, 945–953. (b) Baqi, Y.; Mu€ller, C. E. Catalyst-free micro-
wave-assisted amination of 2-chloro-5-nitrobenzoic acid. J. Org.
Chem. 2007, 72, 5908–5911.
€
€
(37) Glanzel, M.; Bultmann, R.; Starke, K.; Frahm, A. W. Members of
the acid blue 129 family as potent and selective P2Y-receptor
antagonists. Drug Dev. Res. 2003, 59, 64–71.
(47) Leisgen, C.; Kuester, M.; Methfessel, C. The roboocyte: automated
electrophysiology based on Xenopus oocytes. Methods Mol. Biol.
2007, 403, 87–109.