High-affinity, non-nucleotide-derived competitive antagonists of platelet P2Y12 receptors

Article abstract of DOI:10.1021/jm9003297

Anthraquinone derivatives related to the moderately potent, nonselective P2Y₁₂ receptor antagonist reactive blue 2 (6) have been synthesized and optimized with respect to P2Y₁₂ receptor affinity. A radioligand binding assay utilizing human blood platelet membranes and the P2Y₁₂ receptor-selective antagonist radioligand [³H]2-propylthioadenosine- 5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was applied for compound testing. 1-Amino-2- sulfoanthraquinone derivatives bearing a (p-phenylamino) anilino substitution in the 4-position and an additional acidic function in the meta-position of the aniline ring showed high P2Y₁₂ receptor affinity. These new anthraquinone derivatives became accessible by a recently developed copper(0)-catalyzed Ullmann coupling reaction of 1-amino-4-bromoanthraquinone derivatives with anilines in phosphate buffer under microwave irradiation. The most potent compounds exhibited K_i values of 24.9 nM (1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene- 2-sulfonate, PSB-0739, 39), and 21.0 nM (1-amino-4-[4-phenylamino-3- carboxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0702, 41), respectively. 1-Amino-2-sulfo-4-anilinoanthraquinone derivatives appeared to be noncytotoxic, as shown for selected derivatives at two human cell lines (melanoma and astrocytoma). Compounds 39 and 41 represent new lead structures for the development of antithrombotic drugs.

Full text of DOI:10.1021/jm9003297

3784
J. Med. Chem. 2009, 52, 3784–3793
High-Affinity, Non-Nucleotide-Derived Competitive Antagonists of Platelet P2Y12 Receptors
Younis Baqi,† Kerstin Atzler,† Meryem Ko¨se,† Markus Gla¨nzel,§,‡ and Christa E. Mu¨ller*,†
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), UniVersity of Bonn, An der
Immenburg 4, D-53121 Bonn, Germany, Department of Experimental and Clinical Pharmacology and Toxicology, UniVersity of Freiburg,
Albertstraꢀe 25, D-79104 Freiburg, Germany
ReceiVed March 16, 2009
Anthraquinone derivatives related to the moderately potent, nonselective P2Y12 receptor antagonist reactive
blue 2 (6) have been synthesized and optimized with respect to P2Y12 receptor affinity. A radioligand binding
assay utilizing human blood platelet membranes and the P2Y12 receptor-selective antagonist radioligand
[3H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([3H]PSB-
0413) was applied for compound testing. 1-Amino-2-sulfoanthraquinone derivatives bearing a (p-
phenylamino)anilino substitution in the 4-position and an additional acidic function in the meta-position of
the aniline ring showed high P2Y12 receptor affinity. These new anthraquinone derivatives became accessible
by a recently developed copper(0)-catalyzed Ullmann coupling reaction of 1-amino-4-bromoanthraquinone
derivatives with anilines in phosphate buffer under microwave irradiation. The most potent compounds
exhibited Ki values of 24.9 nM (1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihy-
droanthracene-2-sulfonate, PSB-0739, 39), and 21.0 nM (1-amino-4-[4-phenylamino-3-carboxyphenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, PSB-0702, 41), respectively. 1-Amino-2-sulfo-4-anilinoan-
thraquinone derivatives appeared to be noncytotoxic, as shown for selected derivatives at two human cell
lines (melanoma and astrocytoma). Compounds 39 and 41 represent new lead structures for the development
of antithrombotic drugs.
Introduction
mortality in the Western world.5 The P2Y12 receptor is expressed
in very high density on blood platelets.6 Apart from that, it
shows a limited expression pattern with no or only low
expression in peripheral tissues and cells and some expression
in the brain, making it an ideal drug target for selective
therapeutic intervention.7 The P2Y12 receptor on blood platelets
is the target of the thienotetrahydropyridine antithrombotic drugs
ticlopidine (5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-
c]pyridine), clopidogrel (1, Figure 1), and the newly developed
analogue prasugrel (5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxo-
ethyl]-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate),8 rep-
resenting platelet aggregation inhibitors that are effective in the
prevention and treatment of arterial thrombosis. These com-
pounds are not direct P2Y12 antagonists. Clopidogrel, for
example, has to be bioactivated by oxidation via cytochrome
P450 (CYP3A4, CYP3A5, and CYP2C19) enzymes followed
by ring-opening to the corresponding highly unstable thiol or
sulfenic acid derivatives, which are believed to covalently bind
to the receptor protein forming a disulfide bond and thus
presumably act as covalent, possibly allosteric antagonists at
P2Y12 receptors9 (see Supporting Information, Scheme 1). Major
drawbacks of clopidogrel and related thienotetrahydropyridine
derivatives are: (i) slow onset of action (up to several days)
due to the required metabolism, (ii) long duration of action due
to irreversible inhibition, (iii) “drug resistance” in a high
percentage of patients (up to 30%), (iv) moderate potency
(therefore high doses are required), and (v) difficulties in steering
and controlling the effects.
The P2 purinergic receptors are a major class of receptors in
the human body.1 They are subdivided into two main families:
G protein-coupled or metabotropic P2 receptors, designated P2Y,
and ligand-gated ion channels or ionotropic receptors, termed
P2X.2 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. Blood platelets express one P2X receptor subtype,
P2X1, activated by ATP, and two P2Y receptor subtypes, P2Y1
and P2Y12, both of which are activated by the nucleotide ADP,
which induces platelet aggregation.3 The combined action of
both P2Y receptor subtypes on thrombocytes is required for a
full aggregation response following stimulation by ADP. P2Y1,
coupled via the heterotrimeric GTP-binding protein Gq to
phospholipase Cꢀ is responsible for the mobilization of calcium
ions from internal stores mediating platelet shape change and
the initial wave of rapidly reversible platelet aggregation induced
by ADP. P2Y12, on the other hand, is coupled to inhibition of
adenylate cyclase through Gi protein and mediates a progressive
and sustained aggregation not preceded by shape change. The
latter receptor also plays an important role in the potentiation
of platelet secretion induced by several agonists, and its
congenital deficiency has been shown to result in a lifelong
bleeding disorder.4 Modulation of P2 receptors in platelets
appears to be of paramount importance in regulating platelet
function and, as a consequence, in controlling thrombotic
diseases, which are the most common cause of morbidity and
* To whom correspondence should be addressed. Phone: +49-228-73-
2301. Fax: +49-228-73-2567. E-mail: christa.mueller@uni-bonn.de.
Therefore, it is highly desirable to develop P2Y12 antagonists
that are lacking the drawbacks associated with the standard
P2Y12 antagonists such as clopidogrel and other thienotetrahy-
dropyridine derivatives. Several groups have recently been
developing competitive, reversible P2Y12 antagonists that may
be superior to clopidogrel and related drugs. Most approaches
started from the adenine nucleotides as lead structures, ADP,
†
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chem-
istry I, Pharmaceutical Sciences Bonn (PSB), University of Bonn.
‡
Department of Experimental and Clinical Pharmacology and Toxicol-
ogy, University of Freiburg.
§
Present address: Elsevier Pharma Biotech Group, Elsevier Information
Systems GmbH, Theodor-Heuss-Allee 108, D-60486 Frankfurt (Main),
Germany.
10.1021/jm9003297 CCC: $40.75
2009 American Chemical Society
Published on Web 05/22/2009

Antagonists of Platelet P2Y12 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3785
Figure 1. Structures of P2Y12 receptor antagonists with antithrombotic activity.
Table 2. Affinity of Anthraquinone Derivatives for the Human Platelet
P2Y12 Receptor: Part II
Scheme 1. General Synthesis of 4-Substituted Anthraquinones
(8-30, 35-41)a b
,
a Reagents and conditions: (i) amine, phosphate buffer, Cu0, microwave,
b
80-120 °C, 5-20 min. For R1, R2, see Tables 1-4.
Ki ( SEM (µM)
vs [3H]PSB-0413
(% inhibition at
Table 1. Affinity of Anthraquinone Derivatives for the Human Platelet
P2Y12 Receptor: Part I
compd
R1
R2
R3
R4
R5
10 µM)
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
NH2
H
H
H
H
CO2H
H
OH
H
H
H
H
H
Cl
H
NO2
H
H
H
H
H
NH2
SO3Na NH2
CO2H
H
H
H
H
NH2
H
Cl
H
H
SO3Na
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
NH2
H
H
Cl
H
H
H
>10 (37 ( 1%)
>10 (30 ( 3%)
6.76 ( 2.07
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7.35 ( 1.72
.10 (17 ( 2%)
25.1 ( 7.0
3.13 ( 0.39
≈10 (45 ( v5%)
≈10 (50 ( 5%)
.10 (4 ( 14%)
2.10 ( 0.47
2.39 ( 0.65
7.07 ( 1.70
19.74 ( 8.27
12.3 ( 1.7
12.2 ( 0.3
NHC(dO)CH3
SO3Na
H
H
H
NH2
Cl
H
CH3
CH3
SO3Na
CO2H
CO2H
CH3
CH3
H
NH2
CH3 >10 (30 ( 7%)
CH3 >10 (35 ( 5%)
C67085MX (2b) (Figure 1).10 These compounds are very polar
because they are negatively charged at a physiologic pH value
of 7.4 and therefore cannot be perorally applied. In addition,
they are metabolically unstable due to hydrolysis by nucleotide
pyrophosphatases and therefore exhibit only a short half-life in
vivo.11 Furthermore, they are not easy to purify in large amounts
as required for use as drugs. Compound 2b was reported to
possess high selectivity (>104-fold) for P2Y12 receptors versus
other P2Y and P2X receptor subtypes,10a but its selectivity
versus other nucleotide binding sites is unclear.
the physiological agonist, or ATP, which is an antagonist at
the P2Y12 receptor. Astra-Zeneca developed a series of ATP
analogues, including cangrelor (AR-C69931MX, 2a) and AR-
Inspire Pharmaceuticals Inc. developed nucleoside 5′-mono-
phosphates as P2Y12 antagonists for intravenous application, e.g.,

3786 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Baqi et al.
Table 3. Affinity of Anthraquinone Derivatives for the Human Platelet
P2Y12 Receptor: Part III
Figure 2. Structure of reactive blue 2 (RB-2).
Scheme 2. Synthesis of Pyrimidylamino-Substituted
Anilinoanthraquinones 42 and 43a
Ki ( SEM [µM] vs
compd
31
R1
R2
[3H]PSB0413
3.76 ( 1.03
2.45 ( 1.00
1.90 ( 0.24
0.66 ( 0.12
0.68 ( 0.26
32
33
34
SO3Na Cl
H
Cl
SO3Na OMe
6 (RB-2) SO3Na m-/p-sulfophenylamino (1:2)
a Reagents and conditions: (i) 2-bromopyrimidine, acetone:water (1:2),
0-5 °C 1 h, then reflux, 1-3 d, yield 90-97%.
Table 4. Affinity of Anthraquinone Derivatives for the Human Platelet
P2Y12 Receptor: Part IV
proteins in the human body, including a number of different
P2 receptor subtypes and ectonucleotidases.19,20 RB-2 was
described to antagonize P2Y12 receptor activation and may thus
be acting as a direct P2Y12 receptor antagonist.21
Some RB-2 derivatives have been synthesized and investi-
gated in functional studies at P2Y1 and P2X receptors.22
However, no data have been published on their potency at the
P2Y12 receptor subtype. In a recent review article, RB-2 was
described as a P2Y12 antagonist with a Ki value of 1.3 µM.2b In
our laboratory, we determined a similar Ki value of 0.68 µM.23
Although one anthraquinone derivative, RB-2, has shown
promise as a P2Y12 antagonist, a systematic evaluation of this
class of compounds is still lacking, which may result in
enhanced potency and selectivity.
Novel P2Y12 receptor antagonists, particularly those with high
affinity and stability, chemically and enzymatically, are very
interesting not only due to their antithrombotic properties.
Because the P2Y12 receptor is also expressed in brain, e.g., in
microglial cells,24 specific P2Y12 antagonists may in addition
be used to prevent or treat CNS diseases, including neuroin-
flammatory conditions and neurodegenerative disorders, e.g.,
Alzheimer’s and Parkinson’s disease.
Ki ( SEM [µM] vs
[3H]PSB0413
compd
R1
R2
X
Y
(% inhibition at 10 µM)
35
36
37
38
39
40
41
42
43
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
CH3
CO2H
SO3Na
SO3Na
H
H
H
CH2
NH
S
C
C
C
C
C
C
C
N
N
0.614 ( 0.095
1.85 ( 0.57
0.884 ( 0.525
0.063 ( 0.009
0.0249 ( 0.0032
0.541 ( 0.085
0.021 ( 0.003
0.0507 ( 0.0160
∼10 (57 ( 4%)
SO3Na
SO3Na
SO3Na
SO3Na
SO3Na
H
O
NH
NH
NH
NH
NH
for blood product transfusion.12 INS50589 (3, Figure 1) has been
evaluated in clinical trials.13 These compounds including 3 are
also not perorally applicable and have very short half-lives due
to enzymatic dephosphorylation, leading to inactive nucleosides
or nucleoside analogues.11 The development of 3 has meanwhile
been discontinued.14
Furthermore, 2-substituted AMP, ADP, and ATP derivatives
have been evaluated for their effects on platelets, and 2-phe-
nylethynyladenosine di- and triphosphates, but not the corre-
sponding monophosphates, inhibited ADP-induced platelet
aggregation.15
Results and Discussion
Chemistry. The anthraquinone derivatives were synthesized
as depicted in Schemes 1-3. The synthesis of compounds 8,
10-30, 32-34, 36, and 42 has previously been described.25
The new compounds (9, 35, and 37-41) and the precursors of
the new compounds (31 and 43) were obtained in analogy to
the published new method by microwave-assisted Ullmann
reaction.26 Coupling of 4-bromo-substituted anthraquinone
derivatives (bromaminic acid: R1 ) SO3H; 1-amino-4-bromo-
2-carboxyanthraquinone: R1 ) CO2H; 1-amino-4-bromo-2-
methylanthraquinone: R1 ) CH3) with the appropriate amine
in phosphate buffer (pH 6-7) in the presence of Cu0 under
microwave irradiation at 80-120 °C for 5-20 min yielded the
target compounds in good to excellent yields (Scheme 1).
Two of the compounds, 14 and 24, which bear a primary
amino group in the para-position of the N4-phenyl substituent,
were further reacted with 2-bromopyrimidine yielding com-
pounds 42 and 43 (Scheme 2).
An important progress was achieved by the development of
the nucleoside analogue ticagrelor (AZD6140, 4, Figure 1) and
related compounds, which are perorally active P2Y12 antagonists,
and 4 is currently developed for clinical application as an
antithrombotic agent.16 However, the drug molecule is stereo-
chemically sophisticated and requires a demanding, multistep
synthesis. Furthermore, recent studies have indicated dyspnoea
and bradycardia as potential side effects of 4, possibly in part
due to the formation of adenosine receptor-activating metabo-
lites.17 Very recently, the quinoline derivative 5a was described
as a novel competitive P2Y12 receptor antagonist and its ethyl
ester prodrug 5b was shown to be orally bioavailable.18
The anthraquinone derivative reactive blue 2 (RB-2, 6, Figure
2) has been found to interact with a variety of nucleotide-binding

Antagonists of Platelet P2Y12 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3787
Scheme 3. Synthesis of the Dichlorotriazinylaminoanthraquinone
Derivatives 31-33a
Figure 3. Competition curves of selected anthraquinone derivatives
vs 5 nM [3H]PSB-0413 at human platelet P2Y12 receptors.
Determined Ki values at human platelet P2Y12 receptors are
collected in Tables 1-4. Selected concentration-inhibition curves
are shown in Figure 3. It was found that the simple 1-amino-4-
phenylamino-2-sulfoanthraquinone, known as acid blue 25 (AB25,
8), showed some affinity for the P2Y12 receptor (Ki ) 9.83 µM).
Because it is relatively small (MW ) 416.38) as compared to 6
(MW ) 840.10), we selected 8 as the new lead structure. Replacing
the phenylamino ring by benzylamino, phenethylamino, or 3′,4′-
dimethoxyphenethylamino (9-11) dramatically decreased the
affinity. An R-naphthylamino group in the 4-position (12) was also
poorly tolerated (Table 1).
These results suggested that a phenylamino substituent at the
4-position is essential for P2Y12 affinity. Therefore, a small
library consisting of 18 derivatives of lead structure 8 bearing
various substituents on the phenyl ring in the N4-position was
investigated next (see Table 2).
Introducing a hydrophilic group (OH) at the ortho- or para-
position of the phenyl ring was tolerated, as shown for 20 and
21 (Table 2), while introducing an amino group (NH2) in the
meta- or para-position as for 13 and 14 was less favorable.
Acetylation of the para-amino group (22) or introducing a nitro
group in the meta-position (17) abolished the affinity. Interest-
ingly, introducing a more lipophilic, polar function (chlorine)
in the meta- or para-position increased the potency somewhat
(15, 16). A negatively charged group (sulfonate) in the para-
position (19) increased affinity by 3-fold, while a carboxylate
in the ortho-position (18) decreased the activity by 2.5-fold
compared to 8.
a Reagents and conditions: (i) cyanuric acid chloride, acetone:water (1:
2), 0-5 °C, 1-6 h, yield 80-95%, (ii) MeOH, 60 °C, 30 min, yield 96%.
Three anilinoanthraquinone derivatives, 14, 24, and 25, which
bear a primary amino group in the meta- or para-position of
the N4-phenyl ring, were treated with cyanuric acid chloride
(Scheme 3) to afford the desired compounds 31-33. Compound
32 was treated with methanol at 60 °C for 30 min to yield
product 34 (Scheme 3).
Purification and Analysis of the Synthesized Anthraquinone
Derivatives. The synthesized anthraquinone derivatives were
relatively polar compounds. Their purification was achieved by
flash column chromatography using reversed-phase C18 mate-
rial. A typical purification procedure is described in the
Experimental Section and shown in Figure 2 of the Supporting
Information. The structures of the synthesized anthraquinone
derivatives were confirmed by MS and 1H and 13C NMR spectra.
The purity of the compounds was determined by HPLC analysis
coupled with UV detection at 254 nm. The purity of all products
was at least 95% (see Experimental Section and Figure 3 in the
Supporting Information).
Biological Assays. We have recently developed the first
P2Y12-selective radioligand, a [3H]-labeled form of 2b named
[3H]PSB-04136 and developed a P2Y12 receptor radioligand
binding assay at human platelet membranes.6 This allows the
fast screening of potential P2Y12 receptor ligands.
Disubstitution of the phenyl ring appeared to be even
somewhat more promising than mono-substitution. Especially
the combination of a sulfonate and an amino group in the meta-
and para-position, respectively (23, 24), increased the affinity
by 4.5-fold as compared to the lead structure (8). The ortho-
sulfonate isomer 26 showed decreased affinity by more than
2-fold. Compound 25 (meta-amino, meta-carboxy-substituted)
was also quite potent (Table 2). A combination of para- or meta-
chloro-substitution with a carboxylate group in the ortho-position
was tolerated (27, 28), whereas introducing lipophilic substit-
uents such as 2,4,6-trimethyl-phenylamino or 2,4,6-trimethyl-
3-amino-phenylamino (29, 30) abolished the affinity (Table 2).
In the next step, compounds with two aromatic rings attached
to the anthraquinone 4-position connected via a nitrogen (NH)
linker were investigated. The dichlorotriazinyl derivatives of
arylaminoanthraquinones exhibiting an ABCDE ring system
(31-33) were found to be more active than the ABCD ring
compounds (14, 24, and 25). Interestingly, introduction of the
dichlorotriazinyl residue into the para-position (32 and 33)
All anthraquinone derivatives were investigated in this assay
initially at a concentration of 10 µM. For compounds that
showed at least 50% inhibition of radioligand binding, full
concentration-inhibition curves were obtained and Ki values
(n ) 3) were determined.
Selected compounds were investigated for their functionality
in GTP shift experiments.27 Radioligand binding studies as
described above were performed in the presence and absence
of GTP (100 µM). Agonists, such as ADP, show a significant
right-shift of the concentration-inhibition curve, while antago-
nists do not.
Two anthraquinone derivatives were investigated in cell
proliferation assays at human 1539 melanoma cells and at human
1321N1 astrocytoma cells using the MTT (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide) assay in order to
assess their cytotoxic effects.28
Structure-Activity Relationships. A series of 36 anthraquino-
ne derivatives was tested in radioligand binding assay (Tables 1-4).

3788 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Baqi et al.
Figure 4. Summary of structure-activity relationships of anthraquino-
ne derivatives as high-affinity P2Y12 antagonists.
resulted in higher affinity (2-8-fold) than in the meta-position
(31). Replacement of one chlorine in compound 32 by a
methoxy group (34) led to a similarly potent compound as 6,
indicating that it was unnecessary to introduce an extra ring
(ring F) (Table 3 and Figure 2) to obtain high P2Y12 affinity.
Replacing the triazinyl ring by a phenyl ring (ring E) in the
para-position of the phenyl ring D led to potent compounds with
affinities reaching the nanomolar range (Table 4). Different
bridging atoms between rings D and E (methylene (CH2), amino
(NH), or thioether (S) (35-37)) resulted in similarly high affinity
(Table 4), with the methylene bridge being slightly superior to
the S, and 3-fold better than NH. Introducing a sulfonate group
in the meta-position of ring D (compare 36 and 39 (PSB0739)29)
dramatically increased the affinity by ca. 75-fold, leading to a
compound with a Ki value in the low nanomolar range (Table
4 and Figure 3).
Figure 5. Selected marketed anthraquinone, naphthoquinone, and
quinone derivatives Tables 1-4.
prototypical 39, has recently been further characterized in
functional studies at human P2Y12 receptors recombinantly
expressed in astrocytoma cells by von Ku¨gelgen et al. (pre-
liminary results have been published as a conference abstract).30
Compound 39 caused a parallel shift of the concentration-
response curve of the agonist to the right with a corresponding
apparent pKB value of 9.4 (KB value ca. 0.4 nM). These results
confirmed a competitive mechanism of inhibition and very high
potency. To our knowledge, this is one of the most potent non-
nucleotide antagonists at the human P2Y12 receptor reported so
far.
In the next step, the analogue of the most potent compound
so far (39, Ki ) 24.9 nM), in which the sulfonate at position
C-2 in the anthraquinone moiety was replaced by a methyl group
(40), was synthesized. The affinity decreased dramatically by
24-fold, indicating the importance of a negatively charged group,
such as sulfonate, at the C-2 position.
Initial results indicate that the most potent compounds of this
series are also quite selective versus other P2 receptor subtypes.
For example, 42 was found to be at least 100-fold selective versus
human P2Y1, P2Y2, P2Y4, P2X2, and P2X4 receptors (data not
shown). Although the lead structures 6 and 8 are promiscuous,
interacting with many different targets,25 it appears that the right
substitution pattern on the anthraquinone scaffold can impart
selectivity for a certain target such as the P2Y12 receptor.
Cytotoxicity. Anthraquinones are an important class of
natural products found for example in medicinal plants used as
laxatives, such as senna, frangula, cascara, and rheum. A
synthetic anthraquinone derivative bearing basic substituents,
mitoxanthrone (44), is used in cancer therapy (Figure 5). It is
derived from the anthracyclinesm, which are natural products
from Streptomyces strains, which also show cytotoxic properties.
On the other hand, C.I. pigment red 177 (45), an anthraquinone
dye that is widely used for coloration of plastics and industrial
paints, has been reported as not being acutely toxic by the oral
route (LD50 value >5000 mg kg-1, Figure 5).31
Another quinone derivative, seratrodast (46), is therapeutically
applied as an antiasthmatic drug (Figure 5).32 Another example
for nontoxic quinone derivatives are the naphthoquinones
belonging to the family of vitamin K derivatives (47a-c, Figure
5). Coenzyme Q10 (also known as ubiquinone, 48), a benzo-
quinone (Figure 5) that is present in most eukaryotic cells,
primarily in the mitochondria as a component of the electron
transport chain.33,34 Some studies indicate that coenzyme Q10
protects the brain from neurodegenerative disease such as
Parkinson’s due to its antioxidative properties.35 Interestingly,
The most potent compound of the present series was 41
(PSB0702),29 in which the sulfonate function in the 2-position
of the anthraquinone core was replaced by a carboxylate group,
exhibiting a Ki value of 21 nM in binding studies. This result
shows that the sulfonate can be replaced by a carboxylate
without any reduction in affinity.
Figure 4 summarizes the structure-activity relationships
(SAR) of the investigated anthraquinone derivatives at human
platelet P2Y12 receptors. The ABCDE aromatic ring system
proved to be very important for the affinity, and acidic groups
such as sulfonate or carboxylate in the 2 position of the
anthraquinone scaffold and in the meta-position of ring D play
a crucial role because they are required for high affinity.
Selected anthraquinone derivatives, namely compounds 6 and
42, were investigated in GTP shift assays in order to investigate
whether they behaved as agonists or antagonists at P2Y12
receptors. For the agonist ADP, a statistically significant, 2.8-
fold increase of the IC50 value was detected in the presence of
GTP (100 µM) in radioligand binding studies at human platelet
membranes versus [3H]PSB-0413. In contrast, GTP did not show
any significant effect on the competition curves of compounds
6 and 42. The GTP shift values defined as IC50 value in the
presence of 100 µM GTP divided by IC50 without GTP added
were as follows: 0.95 ( 0.22 for 6 and 0.76 ( 0.11 for 42.
Statistical analysis revealed that the shifts were not significantly
different from 1. These results indicated that the compounds
behave as antagonists rather than agonists at P2Y12 receptors.
One of the most potent compounds of the present series, the

Antagonists of Platelet P2Y12 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3789
in coenzyme Q10 as well as in seratrodast, the R,ꢀ-positions of
the quinone or naphthoquinone, respectively, are blocked by
alkylation (methyl or long alkyl chain). Similarly the R,ꢀ-
positions of anthraquinone are blocked by two benzene rings
that prevent Michael addition, which is the main cause of cellular
damage that occurs through alkylation in related molecules
containing a free ꢀ-position.
pounds will be useful as pharmacological tools for studying the
role of peripheral as well as brain P2Y12 receptors. Several
members of this class of compounds have been identified, e.g.,
39 and 41, which constitute novel lead structures for the
development of antithrombotic drugs.
Experimental Section
Chemistry
To further investigate the toxicity of the anthraquinone
scaffold (1-amino-4-anilino-9,10-dioxo-9,10-dihydroanthracene-
2-sulfonate) present as a partial structure in most of our newly
developed P2Y12 antagonists, we selected two structurally simple
derivatives: compound 16 (N4-p-chlorophenyl-substituted), which
had shown some P2Y12 affinity, and compound 12 (N4-R-
napththyl-substituted), which was inactive. Both compounds
were investigated in the MTT assay at two cell lines, human
1539 melanoma cells and human 1321N1 astrocytoma cells.
The colorimetric MTT assay can be taken as a measure of
mitochondrial activity and cell toxicity. The cytotoxic anticancer
drug 5-fluorouracil was included as a control. While 5-fluoro-
uracil inhibited the proliferation of both cell lines, with IC50
values of 49 ( 14 µM (astrocytoma cells) and 5.7 ( 0.8 µM
(melanoma cells) after 72 h of incubation (similar values were
obtained after longer incubation times of up to 144 h), both
anthraquinone derivatives did not show any effects at the tested
concentrations of 0.1, 1, and 100 µM on both cell lines (for
data see Supporting Information). This indicates that the
anthraquinone scaffold present in the described class of com-
pounds is not cytotoxic; the compounds had no effects on cell
proliferation and appeared not to inhibit mitochondrial activity.
Interaction of Therapeutically Used Anthraquinone
Drugs with P2Y12 Receptors. As a next step, we investigated
whether therapeutically used natural and synthetic products with
anthraquinone structure interact with platelet P2Y12 receptors.
Frangulin B, an anthraquinone glycoside (emodin-6-O-ꢀ-D-
apiofuranoside) had previously been found to inhibit collagen-
induced platelet aggregation.36 Thus, several anthraquinone
derivatives present in medicinal plants were tested, including
aloe emodin (1,8-dihydroxy-3-(hydroxymethyl)anthraquinone),
emodin (6-methyl-1,3,8-trihydroxyanthraquinone), chrysophanol
(1,8-dihydroxy-3-methylanthraxquinone), frangulin (mixture of
isomers A and B: emodin-6-O-R-L-rhamnopyranoside (A) and
emodin-6-O-ꢀ-D-apiofuranoside (B)), physcion (1,8-dihydroxy-
3-methoxy-6-methylanthraquinone), and rhein methyl ester (1,3-
dihydroxy-anthraquinone-3-carboxylic acid methyl ester). In
addition, the related anthrone derivatives aloin (1,8-dihydroxy-
10-(ꢀ-D-glucopyranosyl)-3-(hydroxymethyl)-9(10H)-anthra-
cenone) and emodin anthrone were investigated. None of these
compounds showed a significant inhibition of [3H]PSB-0413
binding to human platelet membranes at a test concentration of
10 µM. The cytotoxic anthracyclines mitoxantrone (44) and
doxorubicin ((8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahy-
dro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione) as well as
the related tetracyclines, tetracycline and chlorotetracycline, also
showed no effect in P2Y12 receptor binding assays at 10 µM
concentration (for structures and data see Supporting Informa-
tion).
Material and Methods. All commercially available chemicals
and drugs were obtained from Sigma-Aldrich or Acros, Germany,
and used as purchased. Thin-layer chromatography was performed
using TLC aluminum sheets with silica gel 60 F254, or TLC
aluminum sheets RP silica gel 18 F254 (Merck, Darmstadt, Ger-
many). Colored compounds were visible at daylight; other com-
pounds were visualized under UV light (254 nm). Flash chroma-
tography was performed on a Bu¨chi 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)
1
relative to the deuterated solvent, i.e., DMSO, δ H, 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), or br (broad).
The purities of isolated products were determined by HPLC-
UV obtained on an LCMS instrument (Applied Biosystems API
2000 LCMS/MS, HPLC Agilent 1100) using the following proce-
dure: the compounds were dissolved at a concentration 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 (Phenom-
enix Luna 3 µ C18, 50 mm × 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 the compounds was
determined at 254 nm and proved to be g95%. A second HPLC
method was developed using a Beckman Coulter HPLC for the
determination of the synthesized anthraquinone derivatives’ purity
as follows: the compounds were dissolved at a concentration of
0.5 mg/mL in H2O. Then, 10 µL of the sample was injected into
an HPLC column (Ultrasphere 5 µm Spherical 80 Å Pore C-18,
250 mm × 4.6 mm). Elution was performed with a gradient of
water:acetonitrile from 90:10 to 0:100 for 30-40 min at a flow
rate of 500 µL/min, starting the gradient after 10 min. UV
absorption was detected at 254 nm using a UV detector. The purity
of the compounds proved to be g95%. For a typical HPLC
spectrum see Figure 3 in the Supporting Information. For microwave
reactions a CEM Focused Microwave Synthesis type Discover
apparatus was used. A freeze-dryer (CHRIST ALPHA 1-4 LSC)
was used for lyophilization. The syntheses of compounds 8, 10-30,
32-34, and 42 were previously described.20,22,26,37
General Procedures for the Preparation of 4-Substituted
Anthraquinones (8-30, 35-41): General Procedure A. To a 5
mL microwave reaction vial equipped with a magnetic stirring bar
were added halogen-substituted compounds 7 (bromaminic acid,
sodium salt, carboxy isomer, or the methyl isomer) (0.20 mmol)
and the appropriate aniline and/or amine derivative (0.40 mmol),
followed by a buffer solution of Na2HPO4 (pH 9.6) (4 mL) and
NaH2PO4 (pH 4.2) (1 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 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 dichloromethane (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
Conclusions
In conclusion, we have developed a new class of highly
potent, competitive non-nucleotide derived P2Y12 antagonists.
The most potent compounds exhibited Ki values for the human
platelet P2Y12 receptor in the lower nanomolar range and
selectivity versus other P2 receptor subtypes. The new com-

3790 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Baqi et al.
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 in the
following steps: 5, 10, 20, 40, 60, 80, and 100%. 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 vacuum to remove the methanol, and the
remaining water was subsequently removed by lyophilization to
yield (up to 90%) as blue powders.
General Procedure B. Coupling reaction of cyanuric acid
chloride with the amino function in phenyl ring D: synthesis of
ABCDE ring anthraquinone derivatives 31-33. An ice-cooled
solution of cyanuric acid chloride (0.5-1.0 mmol) in water (25
mL) and acetone (25 mL) was added to a stirred solution of amine-
containing compound (14, 24, and 25) (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 to rt and kept stirring at rt for 4-6 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 (31-33) (Scheme 3). It
should be noted that the reaction did not require the addition of
sodium carbonate to keep the solution basic as described in the
literature.22,37 Interestingly, a higher yield was obtained without
adding the basic salt.
General Procedure C. Coupling reaction of 2-bromopyrimidine
with the amino function in phenyl ring D: synthesis of ABCDE
ring anthraquinone derivatives 42 and 43. An ice-cooled solution
of 2-bromopyrimidine (0.5-1.0 mmol) in water (25 mL) and
acetone (25 mL) was added to a stirred solution of the respective
compound (14 or 24) (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 d. The formation of product was monitored by RP-
TLC using a mobile phase of methanol:water (2:3). After comple-
tion of the reaction, the solvents were evaporated and the residue
was purified by flash column chromatography on RP-18 silica gel
using a methanol:water eluent to obtain the desired products 42
and 43. Following the previously described synthesis22b was not
successful in our hands because it failed to yield the desired product.
It is worth mentioning that the yield of 42 was very strongly
improved (97%) in comparison to that reported in the literature
(47%), while 43 has not been previously described.
166.66 (C-3′′, C-5′′), 182.20 (C-9), 183.22 (C-10), 183.30 (COOH).
LC-MS (m/z): 618 [M - Na + NH4+]+, 599 [M - Na]-. Purity by
HPLC-UV (254 nm) ESI-MS: 95%.
Sodium 1-Amino-4-(4-benzylphenylamino)-9,10-dioxo-9,10-
dihydroanthracene-2-sulfonate (35). According to general pro-
cedure A, bromaminic acid (0.2 mmol) and 4-benzylaniline (3 equiv,
0.6 mmol) were heated in the microwave oven at 120 °C for 5
1
min. Analytical data: yield 51%, mp >300 °C, blue powder. H
NMR: δ 3.97 (s, 2H, CH2), 7.20, 7.29 (2 m, 6H and 3H, 2′-H,
3′-H, 5′-H, 6′-H, 2′′-H, 3′′-H, 4′′-H, 5′′-H, 6′′-H), 7.83 (m, 2H,
6-H, 7-H), 7.98 (s, 1H, 3-H), 8.26 (m, 2H, 5-H, 8-H), 12.05 (s,
1H, 4-NH). 13C NMR: δ 40.6 (CH2), 109.2 (C-9a), 111.2 (C-2′,
C-6′), 122.7 (C-4a), 123.4, 126.05, 126.1, 128.6, 128.9, 130.0 (C-
3, C-5, C-8, C-4′, C-3′, C-5′, C-4′′), 132.9, 133.2, 133.7, 134.3,
137.2, 137.8 (C-8a, C-10a, C-6, C-7, C-2′′, C-6′′, C-3′′, C-5′′), 141.3
(C-1′), 143.0 (C-4), 144.4 (C-2, C-1), 181.9 (C-9), 182.4 (C-10).
LC-MS (m/z): 502 [M - Na + NH4+]+, 485 [M - Na]+, 483 [M
- Na]-. Purity by HPLC-UV (254 nm) ESI-MS: 99.8%.
Sodium 1-Amino-4-(4-phenylthiophenylamino)-9,10-dioxo-
9,10-dihydroanthracene-2-sulfonate (37). According to general
procedure A, bromaminic acid (0.2 mmol) and 4-phenylthioaniline
(3 equiv, 0.6 mmol) were heated in the microwave oven at 120 °C
for 10 min. Analytical data: yield 48%, mp >300 °C, blue powder.
1H NMR: δ 7.31 (m, 5H, 2′′-H, 3′′-H, 4′′-H, 5′′-H, 6′′-H), 7.36 (d,
2H, 2′-H, 6′-H), 7.41 (d, 2H, 3′-H, 5′-H), 7.85 (m, 2H, 6-H, 7-H),
8.05 (s, 1H, 3-H), 8.26 (m, 2H, 5-H, 8-H), 10.1 (br, 2H, 1-NH2),
11.92 (s, 1H, 4-NH). 13C NMR: δ 109.5 (C-9a), 112.5 (C-4a), 123.1
(C-2′, C-6′), 123.3, 126.1, 126.2 127.1, 128.6, 129.6, 129.9 (C-3,
C-5, C-8, C-4′, C-5′, C-3′, C-2′′, C-4′′, C-6′′), 133.0, 133.3, 133.5,
133.6, 134.3, 136.0 (C-8a, C-10a, C-6, C-7, C-3′′, C-5′′, C-1′′),
139.6 (C-1′), 139.7 (C-4), 142.7, (C-2), 144.7 (C-1), 182.1 (C-9),
183.0 (C-10). LC-MS (m/z): 520 [M - Na + NH4+]+, 502 [M -
Na]+, 501 [M - Na]-. Purity by HPLC-UV (254 nm) ESI-MS:
99.5%.
Disodium 1-Amino-4-[4-phenoxy-3-sulfophenylamino]-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (38). According to
general procedure A, bromaminic acid (0.2 mmol) and 4-phenoxy-
3-sulfoaniline (2 equiv, 0.4 mmol) were heated in the microwave
oven at 120 °C for 5 min. Analytical data: yield 35%, mp >300
1
°C, blue powder. H NMR: δ 6.84 (d, 1H, 5′-H), 6.98 (d, 2H, 2′′-
H, 6′′-H), 7.06 (dd, 1H, 4′′-H), 7.21 (dd, 1H, 6′-H), 7.34 (dd, 2H,
3′′-H, 5′′-H), 7.63 (d, 1H, 2′-H), 7.84 (m, 2H, 5-H, 8-H), 7.91 (s,
1H, 3-H), 8.27 (m, 2H, 6-H, 7-H), 10.15 (br, 2H, 1-NH2), 12.07
(br, 1H, 4-NH). 13C NMR: δ 109.22, 111.20, 119.01, 121.31,
122.61, 122.72, 124.26, 125.18, 126.14, 129.63, 132.87, 133.22,
133.80, 134.28, 140.86, 141.60, 143.14, 144.44, 150.71, 158.12,
Sodium 1-Amino-4-(benzylamino)-9,10-dioxo-9,10-dihydroan-
thracene-2-sulfonate (9). According to general procedure A,
bromaminic acid (0.2 mmol) and benzylamine (3 equiv, 0.6 mmol)
were heated in the microwave oven at 120 °C for 10 min. Analytical
181.91, 182.47. LC-MS (m/z): 565 [M - 2Na]-, 282 [M - 2Na]2-
,
584 [M - 2Na + NH4+]+. Purity by HPLC-UV (254 nm) ESI-
MS: 99%.
1
data: yield 30%, mp >300 °C, blue powder. H NMR: δ 4.67 (s,
Disodium 1-Amino-4-[4-phenylamino-3-sulfophenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (39). According
to general procedure A, bromaminic acid (0.2 mmol) and 4-anilino-
3-sulfoaniline (2 equiv, 0.4 mmol) were heated in the microwave
oven at 120 °C for 5 min. Analytical data: yield 46%, mp >300
2H, 4-NHCH2), 7.29 (br, 1H, 4′-H), 7.38 (m, 4H, 2′-H, 3′-H, 5′-H,
6′-H), 7.77 (s, 1H, 3-H), 7.80 (m, 2H, 6-H, 7-H), 8.24 (m, 2H,
5-H, 8-H), 10.07 (br, 2H, 1-NH2), 10.96 (s, 1H, 4-NH). 13C NMR:
δ 46.05 (4-NHCH2), 109.28 (C-9a), 109.42 (C-4a), 121.21 (C-3),
125.90 (C-5), 126.04 (C-8), 127.33 (C-2′, C-4′, C-6′), 128.83 (C-
3′, C-5′), 132.69 (C-6, C-7), 134.02 (C-10a), 134.15 (C-8a), 138.92
(C-1′), 143.35 (C-4), 143.66 (C-2), 145.13 (C-1), 181.39 (C-9),
181.80 (C-10). LC-MS (m/z): 426 [M - Na + NH4+]+, 409 [M -
Na]+, 407 [M - Na]-. Purity by HPLC-UV (254 nm) ESI-MS:
97%.
1
°C, blue powder. H NMR: δ 6.91 (dd, 1H, 4′′-H), 7.12 (m, 3H,
2′′-H, 6′′-H, 5′-H), 7.30 (m, 3H, 3′′-H, 5′′-H, 6′-H), 7.50 (b, 1H,
2′-H), 7.83 (s, 1H, 3-H), 7.83 (m, 2H, 5-H, 8-H), 8.28 (m, 2H,
6-H, 7-H), 12.12 (br, 1H, 4-NH). 13C NMR: δ 109.11 (C-9a), 110.42
(C-4a), 115.79 (C-4′′), 118.28 (C-2′′, C-6′′), 120.98 (C-6′), 122.68
(C-3), 124.10 (C-5′), 126.07 (C-2′), 126.13 (C-1′′), 129.51 (C-3′′,
C-5′′), 132.78 (C-6), 133.01 (C-7), 133.89 (C-10a), 134.28 (C-8a),
135.31 (C-4′), 137.88 (C-3′), 142.38 (C-1′), 142.76 (C-4), 143.25
(C-2), 144.25 (C-1), 181.75 (C-9), 181.88 (C-10). LC-MS (m/z):
564 [M - 2Na]-, 281[M - 2Na]2-, 583 [M - 2Na + NH4+]+.
Purity by HPLC-UV (254 nm) ESI-MS: 100%.
Sodium 1-Amino-4-[3-(4,6-dichloro-[1,3,5]triazine-2-ylamino)-
5-carboxyphenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sul-
fonate (31). According to general procedure B. Analytical data: yield
1
95%, blue powder, mp >300 °C. H NMR: δ 7.56 (td, 1H, H-6′),
7.69 (td, 1H, H-3), 7.85 (m, 2H, H-6, H-7), 7.97 (s, 1H, H-3), 7.95
(d, 1H, H-2′), 8.26 (m, 2H, H-5, H-8), 10.80 (br, 1H, NH-4′), 11.39
(br, 2H, NH2-1), 11.88 (br, 1H, NH-4). 13C NMR: δ 109.57 (C-
9a), 112.68 (C-4a), 117.58 (C-2′), 118.54 (C-5′), 119.11 (C-3),
122.96 (C-6′), 126.21 (C-5, C-8), 132.88 (C-4′), 133.03 (C-6),
133.55 (C-1), 133.62 (C-10a), 134.29 (C-8a), 139.33 (C-1′), 139.77
(C-3′), 140.55 (C-4), 142.77 (C-2), 144.67 (C1), 154.11 (C-1′′),
1-Amino-2-methyl-4-[4-phenylamino-3-sulfophenylamino]-
9,10-dioxo-9,10-dihydroanthracene Sodium Salt (40). According
to general procedure A, 1-amino-4-bromo-2-methylanthraquinone
(0.2 mmol) and 4-anilino-3-sulfoaniline (2 equiv, 0.4 mmol) were
heated in the microwave oven at 120 °C for 5 min. Analytical data:
1
yield 30%, mp >300 °C, blue powder. H NMR: δ 2.25 (s, 3H,

Antagonists of Platelet P2Y12 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3791
CH3), 6.91 (dd, 1H, 4′′-H), 7.11 (d, 2H, 2′′-H, 6′′-H), 7.18 (dd,
1H, 6′-H), 7.28 (m, 2H, 3′′-H, 5′′-H), 7.33 (d, 1H, 5′-H), 7.51 (d,
1H, 2′-H), 7.81 (m, 2H, 5-H, 8-H), 8.27 (m, 2H, 6-H, 7-H), 8.55
(s, 1H, 3-H), 12.27 (br, 1H, 4-NH). 13C NMR: δ 18.77, 107.9, 108.6,
115.9, 118.2, 120.9, 123.9, 124.3, 125.9, 126.1, 126.2, 129.4, 129.5,
132.7, 134.1, 135.2, 137.6, 137.7, 142.4, 143.7, 147.0, 181.2, 181.8.
LC-MS (m/z): 498 [M - Na]-, 500 [M - Na]+. Purity by HPLC-
UV (254 nm) ESI-MS: 96%.
Cells were cultured in 175 cm2 cell culture flasks and maintained
in an incubator at 37 °C, 5% CO2, and 95% humidity. Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% fetal
calf serum, penicillin (100 units/mL), and streptomycin (100 µg/
mL) was used as a growth medium.
MTT Assays. Cells were detached from the 175 cm2 culture
flask and counted using a Neubauer hemocytometer. Then they were
resuspended in the growth medium to give a total of 2 × 105 cells
in 10 mL of the medium. Cell suspension (100 µL) was added into
each well of a 96-well plate to obtain a final concentration of 2000
cells per well and incubated for 24 h at 37 °C, 5% CO2, and 95%
humidity. The outer wells of the 96-well plate were filled with 200
µL of medium without cells to prevent evaporation. After 24 h,
the medium was removed and 180 µL of fresh growth medium per
well was added. Stock solutions (10 mM) of test compounds (5-
fluorouracil, 12, and 16) were prepared in DMSO and diluted with
medium to give 10× of final concentrations. Then test compound
solution (20 µL) was added to each well. The final DMSO
concentration was 1%. Compounds 12 and 16 were tested in
concentrations of 0.1, 1, and 100 µM. For 5-fluorouracil, full
dose-inhibition curves were determined. The cells were incubated
in the presence of the appropriate drug for 72-144 h. Then 40 µL
from a freshly made stock solution of MTT in phosphate-buffered
saline (5 mg/mL) was added to each well and the cells were
incubated for 1 h. After the incubation time, the medium containing
MTT was removed and 100 µL of DMSO was added to each well
in order to dissolve crystals that were formed. The spectrophoto-
metric absorbance was subsequently measured at 570 nm using a
UV-Multiwellreader Multiscan spectrophotometer (Thermo Elec-
tron, Dreieich) with a reference filter of 690 nm. The data were
analyzed using Microsoft Excel and Graphpad Prism 4. Results
were evaluated by comparing the absorbance of the wells containing
compound-treated cells with the absorbance of wells containing
1% DMSO without any drug () 100% viability). All experiments
were performed in triplicates in at least 3-8 separate experiments.
Sodium 1-Amino-4-[4-phenylamino-3-carboxyphenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (41). According
to general procedure A, 1-amino-4-bromo-2-carboxyanthraquinone
(0.2 mmol) and 4-anilino-3-sulfoaniline (2 equiv, 0.4 mmol) were
heated in the microwave oven at 120 °C for 5 min. Analytical data:
1
yield 55%, mp >300 °C, blue powder. H NMR: δ 6.93 (dd, 1H,
4′′-H), 7.13 (d, 2H, 2′′-H, 6′′-H), 7.21 (d, 1H, 6′-H), 7.31 (dd, 2H,
3′′-H, 5′′-H), 7.35 (d, 1H, 5′-H), 7.56 (d, 1H, 2′-H), 7.88 (m, 2H,
6-H, 7-H), 8.19 (s, 1H, 3-H), 8.29 (m, 2H, 5-H, 8-H), 11.71 (br,
1H, 4-NH). 13C NMR: δ 110.45 (C-9a), 113.86 (C-4a), 115.91 (C-
4′′), 118.25 (C-2′′, C-6′′), 121.00 (C-6′), 122.97 (C-3), 123.69 (C-
5′), 125.92 (C-2′), 126.18 (C-1′′), 126.29 (C-6), 129.17 (C-7),
129.56 (C-3′′, C-5′′), 133.19 (C-10a), 133.63 (C-8a), 134.13 (C-
4′), 135.25 (C-3′), 137.72 (C-1′), 140.82 (C-4), 142.37 (C-2), 147.24
(C-1), 167.68 (COOH), 182.29 (C-9), 183.13 (C-10). LC-MS (m/
z): 263 [M - Na]2-, 527 [M - Na]-, 529 [M - Na]+, 547 [M -
Na + NH4+]+. Purity by HPLC-UV (254 nm) ESI-MS: 98%.
Sodium 1-Amino-4-[4-([1,3]diazine-2-ylamino)phenylamino]-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (43). According
to the general procedure C, a solution of 160 mg (1 mmol) of
2-bromopyrimidine and 255 mg (0.5 mmol) of 1-amino-4-(4-
aminophenylamino)-9,10-dioxo-9,10-dihydroanthracene 2-sulfonic
acid sodium salt (14) was stirred at 0-5 °C and then the temperature
was increased to refluxing at 120 °C for one day. RP-FCC was
performed with methanol:water (2:3) as an eluent. Analytical data:
1
yield (295 mg) 90%, blue powder, mp >300 °C. H NMR: δ 6.84
(dd, 1H, 4′′-H), 7.21 (d, 2H, 6′-H, 2′-H), 7.84 (m, 2H, 6-H, 7-H),
7.96 (s, 1H, 3-H), 8.28 (m, 4H, 5-H, 8-H, 3′-H, 5′-H), 8.48 (d, 2H,
3′′-H, 5′′-H), 9.72 (br, 1H, 4′-NH), 10.12 (br, 2H, 1-NH2), 12.13
(br, 1H, 4-NH). 13C NMR: δ 109.1 (C-9a), 110,5 (C-4a), 112.5
(C-4′′) 120.0 (C-2′, C-6′), 122.8 (C-3), 124.4 (C-3′, C-5′), 126.0
(C-5), 126.2 (C-8), 132.4 (C-1′), 132.8 (C-6), 133.1 (C-7), 133.8
(C-10a), 134.3 (C-8a), 137.9 (C-4′), 142.2 (C-4), 143.1 (C-2), 144.3
(C-1), 158.2 (C-3′′, C-5′′), 160.1 (C-1′′), 181.7 (C-9), 181.9 (C-
10). LC-MS (m/z): 505 [M - 2Na + NH4+]+. Purity by HPLC-
UV (254 nm) ESI-MS: 95%.
Acknowledgment. Y.B. thanks the Deutscher Akademischer
Austauschdienst (DAAD) for a Ph.D. Scholarship. Support by
the Deutsche Forschungsgemeinschaft (DFG, GRK804 and
GRK677) for Y.B. and K.A. is also gratefully acknowledged.
We thank Jonas Esche (student of molecular biomedicine) and
Beate Sepanski (student of pharmacy) for performing the
proliferation experiments, and Dr. Ali El-Tayeb for synthesizing
the precursor for the P2Y12 radioligand.
Pharmacology: Human Platelet Membrane Preparation.
Membranes were prepared from human outdated platelets from the
blood bank. Platelets were obtained from the University of Bonn
blood bank 4-5 days after donation. Platelet rich plasma was
washed by centrifugation (10 min at 1000g) in a buffer consisting
of 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 150 mM NaCl.
The supernatant was centrifuged twice (48400g for 60 min), and
the resulting platelet pellets were resuspended in 5 mM Tris-HCl
(pH 7.4) containing 5 mM EDTA. The platelets were homogenized,
and the final suspension was stored frozen as multiple aliquots at
-80 °C until needed. Protein concentrations were determined by
the method of Lowry using a Sigma Chemie protein assay kit.
P2Y12 Radioligand Binding Assay. The precursor of [3H]PSB-
0413 was synthesized as described6 and custom-labeled by GE
Healthcare UK Ltd., Whitchurch, Cardiff, UK,6 with a specific
activity of 74 Ci/mmol, or 94 Ci/mmol, respectively. Radioligand
binding assays were performed using 5 nM [3H]PSB-0413 and 100
µg of protein in Tris-HCl buffer 50 mM, pH 7.4, as previously
described in a final volume of 400 µL.6 Competition by 10 µM of
test compound was initially determined. The mixture was incubated
for 1 h at rt, followed by filtration through GF/B filters. Nonspecific
binding was determined with 1 mM ADP. For potent compounds,
concentration-inhibition curves were determined using at least 6-7
different concentrations spanning 3 orders of magnitude. At least
three independent experiments were performed each in triplicate.
Cell Culture. Cell lines (human 1539 melanoma cells and human
1321N1 astrocytoma cells) were grown as previously described.38
Supporting Information Available: Mechanism of clopidogrel
activation, results from cell proliferation studies, results from P2Y12
receptor radioligand binding studies of therapeutically used an-
thraquinone derivatives and related drugs, additional experimental
details on the synthesis of anthraquinone derivatives, 1H NMR and
13C NMR spectroscopic data, HPLC-UV analyses and purification
procedure by reversed phase flash chromatography. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Guile, S. D.; Ince, F.; Ingall, A. H.; Kindon, N. D.; Meghani, P.;
Mortimore, M. P. The medicinal chemistry of the P2 receptor family.
Prog. Med. Chem. 2001, 38, 115–187. (b) Lambrecht, G.; Braun, K.;
Damer, M.; Ganso, M.; Hildebrandt, C.; Ullmann, H.; Kassack, M. U.;
Nickel, P. Structure-activity relationships of suramin and pyridoxal-
5′-phosphate derivatives as P2 receptor antagonists. Curr. Pharm. Des.
2002, 8, 2371–2399. (c) Gao, Z. G.; Jacobson, K. A. Partial agonists
for A3 adenosine receptors. Curr. Top. Med. Chem. 2004, 4, 855–
862.
(2) (a) Khakh, B. S.; Burnstock, G.; Kennedy, C.; King, B. F.; North,
R. A.; Se´gue´la, 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

3792 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Baqi et al.
to therapy. Pharmacol. ReV. 2006, 58, 281–341. (c) Brunschweiger,
A.; Mu¨ller, C. E. P2 receptors activated by uracil nucleotidessan
update. Curr. Med. Chem. 2006, 13, 289–312. (d) Mu¨ller, C. E. P2-
pyrimidinergic receptors and their ligands. Curr. Pharm. Des. 2002,
8, 2353–2369.
Humphries, R. G.; Hunt, S. F.; Ince, F.; Ingall, A. H.; Kirk, I. P.;
Leeson, P. D.; Leff, P.; Lewis, R. J.; Martin, B. P.; McGinnity, D. F.;
Mortimore, M. P.; Paine, S. W.; Pairaudeau, G.; Patel, A.; Rigby,
A. J.; Riley, R. J.; Teobald, B. J.; Tomlinson, W.; Webborn, P. J. H.;
Willis, P. A. From ATP to AZD6140: The discovery of an orally active
reversible P2Y12 receptor antagonist for the prevention of thrombosis.
Bioorg. Med. Chem. Lett. 2007, 17, 6013–6018.
(3) Gachet, C. Regulation of platelet functions by P2 receptors. Annu.
ReV. Pharmacol. Toxicol. 2006, 46, 277–300.
(4) (a) Le´on, C.; Vial, C.; Gachet, C.; Ohlmann, P.; Hechler, B.; Cazenave,
J. P.; Lecchi, A.; Cattaneo, M. The P2Y1 receptor is normal in a patient
presenting a severe deficiency of ADP-induced platelet aggregation.
Thromb. Haemostasis 1999, 81, 775–781. (b) Cattaneo, M.; Gachet,
C. ADP receptors and clinical bleeding disorders. Arterioscler.,
Thromb., Vasc. Biol. 1999, 19, 2281–2285.
(5) Ten Boekel, E.; Bartels, P. Abnormally short activated partial
thromboplastin times are related to elevated plasma levels of TAT,
F1 + 2, D-Dimer and FVIII:C. Pathophysiol. Haemostasis Thromb.
2002, 32, 137–142.
(6) El-Tayeb, A.; Griessmeier, K. J.; Mu¨ller, C. E. Synthesis and
preliminary evaluation of [3H]PSB-0413, a selective antagonist ra-
dioligand for platelet P2Y12 receptors. Bioorg. Med. Chem. Lett. 2005,
15, 5450–5452.
(7) Barnard, E. A.; Simon, J. An elusive receptor is finally caught:
P2Y(12′), an important drug target in platelets. Trends Pharmacol.
Sci. 2001, 22, 388–391.
(8) (a) Hollopeter, G.; Jantzen, H. M.; Vincent, D.; Li, G.; England, L.;
Ramakrishnan, V.; Yang, R. B. P.; Nurden, A.; Julius, D.; Conley,
P. B. Identification of the platelet ADP receptor targeted by anti-
thrombotic drugs. Nature 2001, 409, 202–207. (b) Zhang, F. L.; Luo,
L.; Gustafson, E.; Lachowicz, J.; Smith, M.; Qiao, X.; Liu, Y.-H.;
Chen, G.; Pramanik, B.; Laz, T. M.; Palmer, K.; Bayne, M.; Monsma,
F. J., Jr. ADP is the cognate ligand for the orphan G protein-coupled
receptor SP1999. J. Biol. Chem. 2001, 276, 8608–8615. (c) Takasaki,
J.; Kamohara, M.; Saito, T.; Matsumoto, M.; Matsumoto, S.; Ohishi,
T.; Soga, T.; Matsushime, H.; Furuichi, K. Molecular cloning of the
platelet P2T(AC) ADP receptor: pharmacological comparison with
another ADP receptor, the P2Y1 receptor. Mol. Pharmacol. 2001, 60,
432–439.
(9) (a) Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.; Picard,
C.; Maffrand, J. P.; Pascal, M.; Herbert, J. M.; Identification and
biological activity of the active metabolite of clopidogrel. Thromb.
Haemostasis 2000, 84, 891–896. (b) Sugidachi, A.; Asai, F.; Ogawa,
T.; Inoue, T.; Koike, H. The in vivo pharmacological profile of CS-
747, a novel antiplatelet agent with platelet ADP receptor antagonist
properties. Br. J. Pharmacol. 2000, 129, 1439–1446.
(10) (a) Ingall, A. H.; Dixon, J.; Bailey, A.; Coombs, M. E.; Cox, D.;
McInally, J. I.; Hunt, S. F.; Kindon, N. D.; Teobald, B. J.; Willis,
P. A.; Humphries, R. G.; Leff, P.; Clegg, J. A.; Smith, J. A.;
Tomlinson, W. Antagonists of the platelet P2T receptor: a novel
approach to antithrombotic therapy. J. Med. Chem. 1999, 42, 213–
220. (b) Boeynaems, J. M.; van Giezen, H.; Savi, P.; Herbert, J. M.
P2Y receptor antagonists in thrombosis. Curr. Opin. InVestig. Drugs.
2005, 6, 275–282.
(17) (a) Serebruany, V. L.; Stebbing, J.; Atar, D. Dyspnoea after antiplatelet
agents: the AZD6140 controversy. Int. J. Clin. Pract. 2007, 61, 529–
533. (b) Weitz, J. I.; Hirsh, J.; Samama, M. M. New antithrombotic
drugs. Chest 2008, 133, 234S–256S.
(18) Post, J. M.; Alexander, S.; Wang, Y. X.; Vincelette, J.; Vergona,
R.; Kent, L.; Bryant, J.; Sullivan, M. E.; Dole, W. P.; Morser, J.;
Subramanyam, B. Novel P2Y12 adenosine diphosphate receptor
antagonists for inhibition of platelet aggregation (II): pharmaco-
dynamic and pharmacokinetic characterization. Thromb Res. 2008,
4, 533–540. (b) Bryant, J.; Post, J. M.; Alexander, S.; Wang, Y. X.;
Kent, L.; Schirm, S.; Tseng, J. L.; Subramanyam, B.; Buckman,
B.; Islam, I.; Yuan, S.; Sullivan, M. E.; Snider, M.; Morser, J. Novel
P2Y12 adenosine diphosphate receptor antagonists for inhibition of
platelet aggregation (I): in vitro effects on platelets. Thromb Res.
2008, 4, 523–532. (c) Wang, Y. X.; Vincelette, J.; da Cunha, V.;
Martin-McNulty, B.; Mallari, C.; Fitch, R. M.; Alexander, S.; Islam,
I.; Buckman, B. O.; Yuan, S.; Post, J. M.; Subramanyam, B.;
Vergona, R.; Sullivan, M. E.; Dole, W. P.; Morser, J.; Bryant, J.
A novel P2Y12 adenosine diphosphate receptor antagonist that
inhibits platelet aggregation and thrombus formation in rat and dog
models. Thromb. Haemostasis. 2007, 5, 847–855.
(19) Burnstock, G. Introduction: P2 receptors. Curr. Top. Med. Chem. 2004,
4, 793–803.
(20) Tuluc, F.; Bu¨ltmann, R.; Gla¨nzel, 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.
(21) Kulick, M. B.; von Ku¨gelgen, I. P2Y-receptors mediating an
inhibition of the evoked entry of calcium through N-type calcium
channels at neuronal processes. J. Pharmacol. Exp. Ther. 2002, 303,
520–526.
(22) (a) Gla¨nzel, M.; Bu¨ltmann, R.; Starke, K.; Frahm, A. W. Consti-
tutional isomers of reactive blue 2sselective P2Y-receptor antago-
nists. Eur. J. Med. Chem. 2003, 38, 303–312. (b) Gla¨nzel, M.;
Bu¨ltmann, 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.
(23) Atzler, K. J. G-Protein-gekoppelte Rezeptoren als Arzneistoff-Targets:
In-Vitro-Charakterisierung neuer Liganden und Untersuchung der
Wirkstoff-Rezeptor-Interaktion (G protein-coupled receptors as drug
targets: in vitro characterization of new ligands and investigation of
drug-receptor interaction). Ph.D. Thesis. University of Bonn: Bonn,
Germany, 2006.
(24) Haynes, S. E.; Hollopeter, G.; Yang, G.; Kurpius, D.; Dailey, M. E.;
Gan, W.-B.; Julius, D. The P2Y12 receptor regulates microglial
activation by extracellular nucleotides. Nat. Neurosci. 2006, 9, 1512–
1519.
(25) (a) Baqi, Y.; Weyler, S.; Iqbal, J.; Zimmermann, H.; Mu¨ller, 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. (b) Weyler, S.; Baqi, Y.; Hillmann, P.; Kaulich, M.; Hunder,
A. M.; Mu¨ller, I. A.; Mu¨ller, C. E. Combinatorial synthesis of
anilinoanthraquinone derivatives and evaluation as non-nucleotide-
derived P2Y2 receptor antagonists. Bioorg. Med. Chem. Lett. 2008,
18, 223–227.
(26) Baqi, Y.; Mu¨ller, C. E. Rapid and efficient microwave-assisted
copper(0)-catalyzed Ullmann coupling reaction: general access to
anilinoanthraquinone derivatives. Org. Lett. 2007, 9, 1271–1274.
(27) Schumacher, B.; Scholle, S.; Ho¨lzl, J.; Khudeir, N.; Hess, S.; Mu¨ller,
C. E. Lignans isolated from valerian: identification and characterization
of a new olivil derivative with partial agonistic activity at A1 adenosine
receptors. J. Nat. Prod. 2002, 65, 1479–1485.
(28) Mosmann, T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
(11) van Giezen, J. J.; Humphries, R. G. Preclinical and clinical studies
with selective reversible direct P2Y12 antagonists. Semin. Thromb.
Hemostasis 2005, 31, 195–204.
(12) (a) Douglass, J. G.; Patel, R. I.; Yerxa, B. R.; Shaver, S. R.; Watson,
P. S.; Bednarski, K.; Plourde, R.; Redick, C. C.; Brubaker, K.; Jones,
A. C.; Boyer, J. L. Lipophilic modifications to dinucleoside poly-
phosphates and nucleotides that confer antagonist properties at the
platelet P2Y12 receptor. J. Med. Chem. 2008, 51, 1007–1025. (b)
Johnson, F. L.; Boyer, J. L.; Leese, P. T.; Crean, C.; Krishnamoorthy,
R.; Durham, T.; Fox, A. W.; Kellerman, D. J. Rapid and reversible
modulation of platelet function in man by a novel P2Y12 ADP-receptor
antagonist, INS50589. Platelets 2007, 18, 346–356.
(13) Johnson, F. L.; Boyer, J.; Crean, C. S.; Durham, T.; Krishnamoorthy,
R.; Kellerman, D. J. Phase 1 pharmacokinetic, pharmacodynamic, and
safety evaluation of a novel rapidly reversible intravenous platelet
P2Y12 ADP receptor antagonist, INS50589. J. Thromb. Haemostasis
2005, 3 (1), P2206. http://www.inspirepharm.com/pubs05.html.
(14) Inspire Pharmaceuticals, Inc. (ISPH) Terminates Mid-Stage Trial; Cites
Bleeding Complications; Aug 8, 2006; http://www.biospace.com/
news_story.aspx?NewsEntityId)26661.
(15) Cristalli, G.; Podda, G. M.; Costanz, S.; Lambertucci, C.; Lecchi, A.;
Vittori, S.; Volpini, R.; Zighetti, M. L.; Cattaneo, M. Effects of 5′-
phosphate derivatives of 2-hexynyl adenosine and 2-phenylethynyl
adenosine on responses of human platelets mediated by P2Y receptors.
J. Med. Chem. 2005, 48, 2763–2766.
(16) (a) Tantry, U. S.; Bliden, K. P.; Gurberl, P. A. AZD6140. Expert Opin.
InVest. Drugs. 2007, 16, 225–229. (b) van Giezen, J. J.; Humphries,
R. G. Preclinical and clinical studies with selective reversible direct
P2Y12 antagonists. Semin. Thromb. Hemostasis. 2005, 31, 195–204.
(c) Springthorpe, B.; Bailey, A.; Barton, P.; Birkinshaw, T. N.;
Bonnert, R. V.; Brown, R. C.; Chapman, D.; Dixon, J.; Guile, S. D.;
(29) Mu¨ller, C. E.; Baqi, Y. Novel P2Y12 receptor antagonists, PCT Int.
Appl. WO 2008107211 A2, 2008, 111 pp.
(30) Hoffmann, K.; Baqi, Y.; Mu¨ller, C. E.; von Ku¨gelgen, I. Potent
antagonistic action of an analogue of reactive blue 2 at the human
platelet P2Y12-receptor. Purinergic Signalling 2008, 4, S20.
(31) Colourants for Food Contact Plastics: Aspects of Product Safety;
Eurocolour, Ecological and Toxicological Association of Dyes and
Organic Pigments Manufacturers (ETAD) Verband der Mineralfar-
benindustrie (VdMi), 2002; Chapter 4.21, Anthraquinone pigments,
p 49; available from www.cefic.be/files/Publications/FDL_en.pdf.

Antagonists of Platelet P2Y12 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3793
(32) (a) Ishimura, M.; Suda, M.; Morizumi, K.; Kataoka, S.; Maeda, T.;
Kurokawa, S.; Hiyama, Y. Effects of KP-496, a novel dual antagonist
at the cysteinyl leukotriene receptor 1 and the thromboxane A2 receptor,
on airway obstruction in guinea pigs. Br. J. Pharmacol. 2008, 153,
669–675.
(33) (a) Ernster, L.; Dallner, G. Biochemical, physiological and medical
aspects of ubiquinone function. Biochim. Biophys. Acta 1995, 1271,
195–204. (b) Dutton, P. L.; Ohnishi, T.; Darrouzet, E.; Leonard, M. A.;
Sharp, R. E.; Cibney, B. R.; Daldal, F.; Moser, C. C. Coenzyme Q
oxidation reduction reactions in mitochondrial electron transport. In
Coenzyme Q: Molecular Mechanisms in Health and Disease; Kagan,
V. E., Quinn , P. J., Eds.; CRC Press: Boca Raton, FL, 2000; Chapter
4, pp 65-82.
(34) (a) Shindo, Y.; Witt, E.; Han, D.; Epstein, W.; Packer, L. Enzymic
and non-enzymic antioxidants in epidermis and dermis of human skin.
J. InVest. Dermatol. 1994, 102, 122–124. (b) Aberg, F.; Appelkvist,
E. L.; Dallner, G.; Ernster, L. Distribution and redox state of
ubiquinones in rat and human tissues. Arch. Biochem. Biophys. 1992,
295, 230–234.
(35) Ebadi, M.; Govitrapong, P.; Sharma, S.; Muralikrishnan, D.; Shavali,
S.; Pellett, L.; Schafer, R.; Albano, C.; Eken, J. Ubiquinone (coenzyme
Q10) and mitochondria in oxidative stress of Parkinson’s disease. Biol.
Signals Recept. 2001, 10, 224–253.
(36) Teng, C. M.; Lin, C. H.; Lin, C. N.; Chung, M. I.; Huang, T. F.;
Frangulin, B. An antagonist of collagen-induced platelet aggregation
and adhesion, isolated from Rhamnus formosana. Thromb. Haemo-
stasis 1993, 70, 1014–1018.
(37) Pearson, J. C.; Burton, S. J.; Lowe, C. R. Affinity precipitation of
lactate dehydrogenase with a triazine dye derivative: selective
precipitation of rabbit muscle lactate dehydrogenase with a procion
blue H-B analog. Anal. Biochem. 1986, 158, 382–389.
(38) (a) Hillmann, P.; Ko, G.-Y.; Spinrath, A.; Raulf, A.; von Ku¨gelgen, I.;
Wolff, S. C.; Nicholas, R. A.; Kostenis, E.; Ho¨ltje, H.-D.; Mu¨ller, 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. (b) Iqbal,
J.; Jirovsky, D.; Lee, S. Y.; Zimmermann, H.; Mu¨ller, C. E. Capillary
electrophoresis-based nanoscale assays for monitoring ecto-5′-nucleotidase
activity and inhibition in preparations of recombinant enzyme and melanoma
cell membranes. Anal. Biochem. 2008, 373, 129–140. (c) Brunschweiger,
A.; Iqbal, J.; Umbach, F.; Scheiff, A. B.; Munkonda, M. N.; Se´vigny, J.;
Knowles, A. F.; Mu¨ller, C. E. Selective nucleoside triphosphate diphos-
phohydrolase-2 (NTPDase2) inhibitors: nucleotide mimetics derived from
uridine-5′-carboxamide. J. Med. Chem. 2008, 51, 4518–4528.
JM9003297