N-substituted phenoxazine and acridone derivatives: Structure-activity relationships of potent P2X4 receptor antagonists

Article abstract of DOI:10.1021/jm300845v

P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was N-(benzyloxycarbonyl)phenoxazine (26, PSB-12054) with an IC₅₀ of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. N-(p-Methylphenylsulfonyl)phenoxazine (21, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC₅₀: 0.928-1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure-activity relationship analysis of P2X4 antagonists.

Full text of DOI:10.1021/jm300845v

Article
pubs.acs.org/jmc
N‑Substituted Phenoxazine and Acridone Derivatives: Structure−
Activity Relationships of Potent P2X4 Receptor Antagonists
Victor Hernandez-Olmos, Aliaa Abdelrahman, Ali El-Tayeb,† Diana Freudendahl, Stephanie Weinhausen,
and Christa E. Muller*
̈
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121
Bonn, Germany
S
* Supporting Information
ABSTRACT: P2X4 receptor antagonists have potential as
drugs for the treatment of neuropathic pain and neuro-
degenerative diseases. In the present study the discovery of
phenoxazine derivatives as potent P2X4 antagonists is
described. N-Substituted phenoxazine and related acridone
and benzoxazine derivatives were synthesized and optimized
with regard to their potency to inhibit ATP-induced calcium
influx in 1321N1 astrocytoma cells stably transfected with the
human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7)
were investigated. The most potent P2X4 antagonist of the present series was N-(benzyloxycarbonyl)phenoxazine (26, PSB-
12054) with an IC50 of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. N-(p-
Methylphenylsulfonyl)phenoxazine (21, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in
all three species (IC50: 0.928−1.76 μM). The compounds showed an allosteric mechanism of action. The present study
represents the first structure−activity relationship analysis of P2X4 antagonists.
subtypes, have been advanced as targets for novel analgesics.11
INTRODUCTION
■
Antagonists for P2X1,12 P2X2,13,14 P2X3,15,16 the heteromeric
The purine nucleotide ATP (1) not only is an important
intracellular source of energy but also is released from the cells
P2X2/P2X3,16 and P2X717,18 receptors have been developed,
and structure−activity relationships have been studied for those
receptor subtypes.
and activates specific cell membrane receptors. These
nucleotide or P2 receptors are subdivided into two structurally
distinct families: the P2X receptors (P2XR), which are ligand-
gated ion channels (ionotropic receptors), and the P2Y
receptors, which are G-protein-coupled (metabotropic) re-
ceptors. P2X receptors are homomeric or heteromeric cation
channels permeable for Ca2+ and Na+ ions, each containing a
two-transmembrane (2TM) motif.1 Seven subunits exist,
P2X1−7, with a degree of sequence identity ranging from
26% for P2X2 and P2X7 to 47% for P2X1 and P2X4. All P2X
receptors are activated by ATP.2 In contrast, the eight P2Y
receptors are activated by different nucleotides depending on
the receptor subtype: P2Y1, P2Y12, and P2Y13 by ADP; P2Y2 by
UTP and ATP; P2Y4 by UTP; P2Y6 by UDP; P2Y11 by ATP;
P2Y14 by UDP-glucose.3 Enzymes, called ectonucleotidases,
break down ATP to ADP, AMP, and adenosine and thus limit
the activation of ATP receptors.4 While adenosine activating
purinergic P1 receptors (A1, A2A, A2B, and A3) show analgesic
properties,5 ATP is pronociceptive. ATP is involved in
neurotransmission as a co-transmitter in all nerve types of the
peripheral and central nervous system.6,7 A remarkable
functional aspect of ATP receptors, of particular therapeutic
importance, is that they play a crucial role in the interaction
between neurons and glia in the CNS.8,9 Purinergic signaling
has been shown to be involved in different disorders of the
CNS,10 and purinergic receptors, especially the P2X3 and P2X7
Recently, the first crystal structure of a P2X receptor, the
P2X4 receptor from zebra fish, has been published but without
a ligand.19 On the basis of mutagenesis and molecular modeling
studies, ATP is proposed to bind to an intersubunit site around
45° from the ion channel domain, inducing conformational
changes within and between subunits.20,21
The P2X4 receptor is highly expressed in various CNS areas,
on immune cells and peripheral macrophages.6 The location
and specific up-regulation of the P2X4 receptors in CNS spinal
and/or supraspinal, injury-induced, activated microglia link the
receptor to pathophysiologic processes underlying persistent
and neuropathic pain,22−24 traumatic brain injury,25 cerebral
ischemia,26 and spinal cord injury.27 The link between up-
regulation of P2X4 receptors and microglial activation points to
a neuroinflammatory mechanism, the pharmacological attenu-
ation of which may bear therapeutic potential in the treatment
of chronic neuropathic pain and in neuroprotection. P2X4
receptor knockout mice showed a strong reduction in
mechanical allodynia and hyperalgesia in the spinal nerve
ligation model of neuropathic pain.28
Received: June 15, 2012
Published: October 17, 2012
© 2012 American Chemical Society
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However, to date, there is a lack of sufficiently powerful
pharmacological tools to selectively activate or block P2X4
receptor signaling.29 This led us to pursue the development of
P2X4 antagonists in order to allow the characterization of the
receptor and its (patho)physiological roles. Figure 1 shows
identified N-substituted phenoxazine derivatives as novel P2X4
receptor antagonists (see Figure 2 and Table 1).
Figure 2. N-Substituted phenoxazine, acridone, and benzoxazine
derivatives.
Phenoxazine and its derivatives show antioxidant proper-
ties.39,40 N-Substituted phenoxazine derivatives have been
described to possess sedative, antiepileptic, tranquillizing,
spasmolytic, antitubercular, and anthelminthic activities.41
Certain derivatives were also found to inhibit the protein
kinase Akt, thereby inhibiting apoptosis of human cell lines.42
In the present study, we describe the synthesis of a series of
mostly novel N-substituted phenoxazine derivatives and
structurally related benzoxazines and acridones (for basic
structures see Figure 2) and their evaluation as novel P2X4
receptor antagonists. Starting from simple N-substituted
phenoxazine derivatives, we explored a broad variation of N-
substituents and additionally investigated a smaller series of
bioisosteric acridone derivatives as well as some smaller, related
bicyclic benzoxazine derivatives.
RESULTS AND DISCUSSION
■
Chemistry. Phenoxazine derivatives were obtained starting
from phenoxazine (7). A wide variety of different N-substituted
compounds was prepared including N-alkyl-, amido-, sulfona-
mido-, ureyl, carbamoyl-, and thiocarbamoyl-substituted de-
rivatives (Scheme 1). N-Benzyl substituted phenoxazine
derivative 10 was prepared by alkylation of phenoxazine using
benzyl chloride in the presence of sodium hydride in
tetrahydrofuran (THF), providing the desired product in 80%
yield. N-Acetylphenoxazine derivative 11 was obtained in 43%
yield by reaction of phenoxazine with sodium cyanate and
acetic acid. Aromatically substituted amides 12, 13, and 14 were
obtained by reaction of phenoxazine with the corresponding
benzoic acid chloride and triethylamine in dichloroethane
(DCE) (see Scheme 1), while 15 was obtained by catalytic
hydrogenation of 13 with Pd/C (10%) as a catalyst. Compound
16, with one extra carbon in the amide linker, was synthesized
by treatment of phenoxazine with 2-phenylacetyl chloride in
the presence of sodium hydride. The urea derivatives 17−20
were obtained in good yield via reaction of phenoxazine with
the corresponding carbamoyl chloride and lithium diisopropy-
lamide (LDA) in THF. The synthesis of sulfonamide derivative
21 was carried out by treatment of phenoxazine with tosyl
chloride and triethylamine in DCE. Compound 22, with one
extra carbon atom in the linker, was obtained by reaction of
phenoxazine with phenylmethanesulfonyl chloride in the
presence of sodium hydride in THF.
Figure 1. Structures of physiological and synthetic agonists (1−3) and
antagonists (4−6) at P2X4 receptors.
some of the currently available and most frequently used P2X4
agonists and antagonists and their potency at P2X4 receptors.
Besides the physiological agonist ATP (1),30−32 some closely
related derivatives like 2-methylthio-ATP (MeS-ATP, 2)30−32
and 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate
(BzATP, 3) activate the P2X4 receptor but are nonselective.33
The ATP derivative 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-
triphosphate (TNP-ATP, 4) is a competitive antagonist.34,35
The antidepressant drug paroxetine (5) was reported to be a
noncompetitive P2X4 antagonist with an IC50 in calcium flux
studies of around 2 μM at both human and rat P2X4
receptors;36 however, Khakh and co-workers suggested an
indirect effect by paroxetine, which reduced the expression of
P2X4 receptors in a microglial cell line.37 Paroxetine is much
more potent as a serotonin reuptake inhibitor than as a P2X4
antagonist. Another P2X4 antagonist that has been described is
5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diaze-
pin-2-one (5-BDBD, 6); it was reported to block currents
induced by human P2X4 receptors expressed in Chinese
hamster ovary cells with an IC50 of 0.50 μM.38
Phenoxazine carbamates and thiocarbamates were obtained
as shown in Scheme 1. Compound 23 was synthesized by
refluxing phenoxazine in a solution of methyl chloroformate at
80 °C, while 24 was obtained using isopropyl chloroformate
and performing the reaction at 110 °C. The intermediate
compound phenoxazine-N-carbonyl chloride (25) was obtained
from phenoxazine via reaction with triphosgene in the presence
of triethylamine in DCE. Reaction of 25 with benzyl alcohol at
So far, studies on structure−activity relationships (SARs) of
P2X4 receptor antagonists have not been reported. Because of
the lack of potent and selective P2X4 receptor antagonists and
because of their high potential as novel therapeutics, we
screened part of our in-house library (http://mueller-group.
pharma.uni-bonn.de/mueller-laboratory/compound-library) to
discover new scaffolds suitable for optimization. We thereby
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a
a
Scheme 1. Synthesis of Phenoxazine Derivatives
Scheme 2. Synthesis of Acridone Derivatives
a
Reagents and conditions: (a) ClSO2NCO, DCM, yield 38%; (b)
BzCl, DCE, pyridine, 80 °C, yield 67%; (c) (1) triphosgene, DCE,
Et3N, (2) NH3 25%, yield 22%; (d) RCH2Cl, NaH, THF, yield 14−
58%; (e) PhMgBr, THF, yield 64%.
synthesis attempts to obtain the acridone-N-carbonyl chloride
derivative (the analogue of phenoxazine derivative 25; see
Scheme 1), but both were unsuccessful in our hands. In the first
trial, acridone (8) was treated with chlorosulfonyl isocyanate in
dichloromethane (DCM), but the isolated compound was
identified as derivative 4643 (Scheme 2). In the second trial,
acridone (8) was reacted with triphosgene in DCM in the
presence of triethylamine. However, the isolated products in
this case were 46 and also 48. Benzoylacridone 47 was obtained
by treatment of acridone with benzoyl chloride and pyridine in
DCE at 80 °C. The alkylated derivatives 49−52 were obtained
in low to moderate yield according to literature proce-
dures.44−46 Finally the tertiary alcohol 53 was prepared by a
Grignard reaction from 49 in good yield (Scheme 2).44
a
Reagents and conditions: (a) BnCl, NaH, DMF, yield 80%; (b)
NaOCN, AcOH for 11, yield 43%; R1COCl, Et3N, DCE for 12−14,
yield 44−82%; BnCOCl, NaH, THF for 16, yield 37%; (c) LDA,
THF, R1NCOCl, yield 60−85%; (d) TsCl, Et3N, ClCH2CH2Cl for 21,
yield 41%; BnSO2Cl, NaH, THF for 22, yield 20%; (e) CH3OCOCl,
70 °C for 23, yield 81%, i-PrOCOCl, 110 °C for 24, yield 50%; (f)
triphosgene, ClCH2CH2Cl, Et3N, yield 95%; (g) BnOH, 110 °C for
26, yield 48%; NaH, THF, R2(CH2)nOH, n = 1, 2 for 27−31, yield
13−71%; NaH, THF, BnCH2SH for 32, yield 70%; (h) 25% aqueous
NH3 for 33, yield 96%; R1R2NH, DCE for 34−45, yield 50−91%.
As a third scaffold, bicyclic benzoxazine derivatives were
prepared as phenoxazine analogues lacking the second benzene
ring (Scheme 3). 2-Aminophenol (54) was alkylated with the
respective carbonyl chlorides in the presence of calcium
carbonate in dioxane. The obtained compounds 55−58 were
further reacted with 1,2-dibromoethane in the presence of
potassium carbonate to afford the corresponding cyclic
analogues 59−61. The morpholinyl-substituted derivative 55
was obtained in 47% yield with only traces of the dimeric
byproduct. However, by use of phenyl substituted derivative 56,
benzoxazine 60 was formed in 58% yield, accompanied by 11%
of the dimer 63. The double O-alkylation rather than N-
alkylation was indicated by the typical NMR shifts of the
110 °C afforded 26. Products 27−32 were also synthesized
from intermediate 25 by treatment with the corresponding
alcohol or thiol, respectively, in the presence of sodium hydride
in THF (for details see Scheme 1).
1
methylene hydrogens (O-alkylation, 63, H NMR (500 MHz,
A series of urea derivatives was also prepared (compounds
33−45; see Scheme 1). The unsubstituted derivative 33 was
obtained by treatment of phenoxazine with triphosgene and
triethylamine followed by the addition of an aqueous ammonia
solution. Urea derivatives 34−45 were obtained via 25 by
treatment of 7 with triphosgene and triethylamine followed by
reaction with the corresponding amine in DCE, providing the
final products in moderate to good yields (Scheme 1).
A small series of structurally related acridone (8) derivatives
was prepared as depicted in Scheme 2. Initially, there were two
CDCl3) δ 4.49 (s, 4H) vs N-alkylation, 60, δ 4.40−4.33 (m,
2H), 4.05−3.97 (m, 2H)). Menthyl-substituted phenol 57
could not be converted to the benzoxazine derivative under the
applied reaction conditions but afforded almost exclusively
bromo derivative 62 (95% yield) after 90 h of refluxing.
Benzoxazine 62 was obtained without any side products in 62%
yield using carbamate 60 as a starting material.
Altogether, 52 final products were synthesized, analyzed, and
tested, 36 of which are new compounds not previously
described in the literature. Of the 16 previously reported
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a
Scheme 3. Synthesis of Benzoxazine Derivatives
a
Reagents and conditions: (a) carbonyl chloride, CaCO3, dioxane, 70−80 °C; (b) BrCH2CH2Br, K2CO3, acetone, reflux, 24−90 h.
compounds, complete analytical data (1H and 13C NMR) were
lacking and have now been provided. Pure compounds (≥95%
purity) were mostly obtained after purification by flash
chromatography. In some cases additional recrystallization
was required (see Experimental Section). The structures of the
the potent antagonists interacted with the ATP binding site,
radioligand binding studies were performed at membrane
preparations of 1321N1 astrocytoma cells transfected with the
human P2X4 receptor using [35S]ATPγS as a radioligand (see
Figure S1).
Structure−Activity Relationships. A series of 36 N-
substituted phenoxazine derivatives were investigated (com-
pounds 10−45; see Table 1). A substituent was connected to
the nitrogen atom of phenoxazine via one of the following
linkers: (i) one-atom linkers, including methylene (−CH2),
carbonyl (−CO) yielding amides, or sulfonyl (−SO2)
yielding sulfonamides and (ii) two-atom linkers, including
−CONH yielding ureas, −C(O)O yielding carbamates, or
−C(O)S yielding thiocarbamates.
A simple N-benzyl-substituted phenoxazine (10) was
virtually inactive (14% inhibition at 10 μM). Replacement of
the methylene linker in 10 by a carbonyl group (yielding
compound 12) strongly increased P2X4-antagonistic potency,
resulting in an IC50 of 4.11 μM. Therefore, amides were further
explored. Smaller residues instead of phenyl such as Cl (in 25)
or methyl (in 11) clearly reduced inhibitory potency. The same
was observed with the larger benzyl group (in 16), which was
not tolerated by the receptor, in contrast to a smaller phenyl
ring (compound 12). p-Substitution of the phenyl residue in 12
was explored, and p-nitrobenzoyl- (13), p-chlorobenzoyl- (14),
and p-aminobenzoyl- (15) substituted phenoxazine derivatives
were investigated. Compounds 13 and 14 bearing electron-
withdrawing groups showed about the same potency as the
unsubstitued benzoylphenoxazine (12). The aminobenzoyl-
substituted derivative 15 was slightly less potent exhibiting an
IC50 of 10.8 μM. Two sulfonamide derivatives were prepared
(21 and 22). The test results showed that the carbonyl linker
could be bioisosterically replaced by a sulfonyl function. While
benzyl derivative 22 showed moderate potency (46% inhibition
of ATP-induced calcium influx at 10 μM), similar or perhaps
slightly better than the corresponding benzyl derivative of the
carbonyl series (compound 16, 31% inhibition at 10 μM), the
p-methylphenyl-substituted sulfonamide derivative 21 was a
potent P2X4 antagonist exhibiting an IC50 of 1.38 μM (Table
1).
1
synthesized compounds were confirmed by H and 13C NMR
spectroscopy, in addition to HPLC analysis coupled to
electrospray ionization mass spectrometry (LC/ESI-MS)
which was also used to determine the purity.
Biological Activity. The compounds were initially tested at
10 μM for their potency to inhibit ATP-induced calcium influx
in 1321N1 astrocytoma cells stably transfected with the human
P2X4 receptor. For potent compounds that showed greater
than 50% inhibition full concentration−response curves were
determined and IC50 values were calculated. An ATP
concentration of 1 μM, which caused ∼80% of the maximal
effect, was used for receptor stimulation. Results are
summarized in Table 1, and concentration−response curves
for the most potent compounds are depicted in Figure 3.
Figure 3. Concentration−response curves of selected P2X4
antagonists at human P2X4 receptors expressed in 1321N1
astrocytoma cells. Cells were preincubated for ∼30 min with test
compound and subsequently stimulated with 1 μM ATP approx-
imately corresponding to the EC80 of ATP. For determined IC50 values
see Table 1.
Selected compounds were further tested at rat and mouse P2X4
receptors using the same assay system to investigate potential
species differences (Table 2). Furthermore, selectivity of the
most potent compounds was assessed versus the other
homomeric functional human P2X receptor subtypes, P2X1,
P2X2, P2X3, and P2X7, stably transfected in 1321N1
astrocytoma cells (Table 2). In order to investigate whether
Next we explored two-atom linkers (urea and (thio)-
carbaminic acid ester derivatives); most investigated examples
belonged to this second group of compounds. The unsub-
stituted phenoxazinecarboxamide, the urea derivative 33,
showed only low potency (40% inhibition at 10 μM).
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Table 1. Potency of Phenoxazine, Acridone, and Benzoxazine Derivatives at Human P2X4 Receptors
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Table 1. continued
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Table 1. continued
a
b
c
d
n = 3 unless otherwise noted. Percent inhibition at 10 μM test compound. Percent inhibition at 100 μM test compound. Percent inhibition at
IC50 was significantly lower than 50%: 12, 37%; 13, 27%; 14, 15%; 21, 39%; 27, 27%; 28, 39%; 30, 21%; 42, 35%; 44, 36%; 53, 35%.
Monosubstitution with alkyl residues including methyl (34),
isopropyl (35), cyclohexyl (36), (N,N-dimethylamino)ethyl
(37), or 4-(N-methylpiperidinyl) (38) did not improve the
potency of the urea derivatives.
In contrast, N,N-disubstitution with small alkyl residues
resulted in the following sequence of potency: dimethyl (17,
IC50 = 24.6 μM) ≤ diethyl (18, 17.4 μM) ≤ diisopropyl (19,
12.4 μM) < dipropyl (43, 2.69 μM). The piperidine derivative
44, which can be envisaged as a sterically fixed analogue of the
potent dipropyl derivative 43, was about 3-fold less potent
(IC50 = 10.7 μM). The analogous morpholine (20) and N-
methylpiperazine derivative (45) were completely inactive
indicating that a polar function is not tolerated.
comparison to the corresponding phenoxazine derivative 12
(IC50 = 4.11 μM) it was almost 7-fold less potent. Similarly, the
N,N-diethyl-substituted urea derivative 48 (IC50 = 39.1 μM)
was somewhat less potent than the corresponding phenoxazine
derivative 18. In contrast, N-benzylacridone (49) was relatively
potent (IC50 = 5.97 μM), while the corresponding N-
benzylphenoxazine (10) had been inactive. p-Substitution of
the benzyl residue did significantly alter the potency of the
acridones (compounds 50−52); p-nitro-substitution resulted in
the most potent compound of this series (52, IC50 = 2.49 μM).
Virtually the same potency was observed for the N1-benzyl-
substituted 6-hydroxy-6-phenyldihydroacridine (53, IC50 = 2.44
μM).
Benzyl substitution was not well tolerated in the urea series
(compound 39), but p-chloro substitution of the benzyl ring
increased potency (40, IC50 = 9.54 μM), while the 3,5-dichloro-
substituted derivative 41 was inactive. However the larger 2-
phenylethyl substitution (compound 42) resulted in a potent
P2X4 antagonist with an IC50 of 1.78 μM.
A third series of heterocyclic scaffolds that was investigated
was the bicyclic benzoxazine derivatives, which are related to
phenoxazines but are lacking one of the benzene rings. The
oxazine ring in the investigated compounds 59−61 was
partially hydrogenated (see Table 1). In fact, all three
investigated derivatives, including the benzylcarbamate 61, the
analogue of the corresponding phenoxazine derivative 26,
which had been the most potent compound in that series, were
inactive at P2X4 receptors. Several intermediate and side
products obtained during the efforts to synthesize benzoxazine
derivatives were also tested (55−58, 62, and 63; Table 1). All
of them were inactive except for one compound, the o-
hydroxyphenylcarbamate 57 (IC50 = 3.39 μM), which is
substituted with a bulky chiral (−)-menthyl residue. This result
indicates that voluminous residues are tolerated by the P2X4
receptor like it had previously been shown for the P2X7
receptor.47 Thus, compound 57 may serve as a new lead
structure for further optimization.
Some of the compounds did not lead to complete inhibition
of the ATP-induced calcium signal even at high concentrations,
and this did not correlate with their potency (see Figure 3 and
Table 1 where the percent inhibition at 100 μM is provided).
For example, the most potent antagonist 26 showed a maximal
inhibition of 77%, while the weaker compound 19 showed
complete inhibition at a high concentration of 100 μM.
Incomplete inhibition may indicate an allosteric mode of action.
When percent inhibition of the compounds at their IC50 was
considered, two-thirds of the compounds showed nearly 50%
The second series of compounds with a two-atom linker
were the carbamates 23, 24, and 26−31 and the thiocarbamate
32 (Table 1). They are analogues of the urea derivatives in
which the exocyclic nitrogen atom is replaced by an oxygen or a
sulfur atom. However, the structure−activity relationships were
quite different in the carbamate series compared to the urea
series of compounds. In contrast to the ureas, monosubstitution
with small alkyl residues was tolerated in the carbamate series:
the methyl ester (23) was weak (IC50 = 52.3 μM), but the
isopropyl ester (24) showed high potency exhibiting an IC50 of
2.35 μM. Also contrary to the results with the urea derivatives,
the unsubstituted benzylcarbamate (26; compare to urea
derivative 39) was a very potent antagonist; in fact 26 was
the most potent P2X4 antagonist of the present series with an
IC50 of 0.189 μM. p-Substitution did not further increase its
potency, but p-methyl- (27, IC50 = 2.28 μM), p-methoxy- (30,
IC50 = 2.64 μM), and p-chloro-substitution (28, IC50 = 1.02
μM) were well tolerated resulting in compounds with IC50
values in the low micromolar range, while a p-nitro group (in
29) abolished activity. The larger 2-phenylethyl residue (in 31,
IC50 = 3.93 μM) was also well tolerated but did not increase
potency in the carbamate series in comparison to the benzyl
residue, as it had in the urea series of phenoxazine derivatives.
The corresponding 2-phenylethyl-substituted thiocarbamate 32
was even less potent.
inhibition (49
7%, 95% confidence interval of 45−53%).
One-third of the compounds, 12, 13, 14, 21, 27, 28, 30, 42, 44,
and 53, showed significantly lower inhibition between 15% and
39% at their IC50 value. This was reflected by a maximal
inhibition of only around 50−70% at a high concentration of
100 μM. However, clear SARs for partial versus full inhibition
of P2X4-receptor induced calcium influx could not be deduced.
As a related heterotricyclic scaffold a small series of acridone
derivatives and closely related analogues was investigated (46−
53). 6-Chloroacridine (46) was inactive. N-Benzoylacridone
(47) showed moderate potency (IC50 = 26.9 μM). In
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Table 2. Potency of Selected Derivatives at P2X4 Orthologues and Different P2X Receptor Subtypes
IC50 SEM (μM)
compd
human P2X4
rat P2X4
mouse P2X4
human P2X1
human P2X2
human P2X3
human P2X7
b
b
b
b
TNP-ATP (4)
1.46 0.29
1.38 0.33
2.35 0.71
0.189 0.059
2.28 0.58
1.78 0.28
2.69 0.38
2.44 1.05
4.71 0.71
0.928 0.512
6.08 1.52
2.10 1.09
1.28 0.01
1.76 1.09
nd
nd
>10 (−5%)
nd
nd
a
a
a
a
21
24
26
27
42
43
53
≥10 (45%)
≥10 (38%)
6.52 1.68
>10 (32%)
>10 (6%)
a
a
a
a
a
>10 (1%)
>10 (13%)
>10 (11%)
>10 (10%)
a
a
a
1.77 0.34
2.81 0.51
>10 (13%)
∼10 (49%)
>10 (8%)
>10 (9%)
a
a
ac
,
a
a
>10 (24%)
>10 (24%)
>10 (−84%)
>10 (27%)
>10 (29%)
a
a
a
a
a
a
≥10 (42%)
≥10 (36%)
>10 (5%)
>10 (23%)
>10 (4%)
>10 (13%)
a
a
a
a
2.71 1.21
2.66 0.57
2.02 0.80
>10 (33%)
≥10 (44%)
0.429 0.141
>10 (2%)
a
4.04 1.22
0.602 0.169
≥10 (46%)
1.27 0.36
a
b
c
Inhibition of calcium flux at 10 μM. Not determined. Compound significantly increased ATP effect (positive modulator).
Species Selectivity. For some classes of P2X (e.g., P2X7)
receptor antagonists, large species differences between
potencies at human versus rodent P2X receptors have been
described.48 Therefore, we investigated selected compounds of
the new series of P2X4 antagonists at rat and mouse, in
addition to human P2X4 receptors in calcium influx assays
determining the antagonist-induced inhibition of ATP-medi-
ated receptor activation (Table 2). For comparison, the known
P2X4 antagonist TNP-ATP (4), an ATP derivative, was also
included. TNP-ATP did not show any significant species
differences, being similarly potent at human (IC50 = 1.46 μM)
and mouse P2X4 receptors (IC50 = 1.28 μM) and only slightly
(3-fold) less potent at rat P2X4 receptors (IC50 = 4.71 μM).
Six of the most potent phenoxazine derivatives were
investigated at P2X4 receptors of all three species: the
sulfonamide 21, the carbamates 24, 26, and 27, and the urea
derivatives 42 and 43. In addition, the acridine derivative 53
was investigated. The latter did not show any species
differences but inhibited P2X4 receptors across species with
nearly identical IC50 values. The same was true for N-(p-
methylphenylsulfonyl)phenoxazine (21). In contrast, the other
phenoxazine derivatives, carbamate as well as urea derivatives,
exhibited some species selectivity. Compounds 24 (isopropyl-
carbamate derivative) and 42 (phenylethylurea derivative) were
somewhat weaker at rat and nearly inactive at mouse P2X4
receptors than at the human orthologue. The p-methylbenzyl-
carbamate 27 and the N,N-dipropylurea derivative 53 were
equipotent at human and mouse receptors but much weaker at
the rat P2X4 receptor. These results showed that species
differences may be common for P2X4 receptor antagonists, and
new ligands have to be characterized at the species of interest
before they are being used as pharmacological tools.
Receptor Selectivity. In order to assess the selectivity of
the new P2X4 receptor antagonists versus closely related
targets, selected compounds were tested in calcium influx assays
at the other functional homomeric human P2X receptor
subtypes, namely, P2X1, P2X2, P2X3, and P2X7, at 10 μM (see
Table 2). Most of the compounds showed good selectivity for
the P2X4 receptor. For example, the most potent antagonist of
the present series, phenoxazine derivative 26, was at least 35-
fold selective versus all other human P2X receptor subtypes.
There were two exceptions: the phenoxazine derivative 43,
which was equipotent at P2X1 receptors, and the dihydroacri-
dine derivative 53, which was nonselective inhibiting all P2X
receptor subtypes at 0.429 μM (P2X3), 0.602 μM (P2X1), 1.27
μM (P2X7), 2.44 μM (P2X4), and ∼10 μM (P2X2) (see Table
2, Figure 4). These results indicate that starting points can be
found to develop phenoxazine and acridine derivatives as
antagonists for other P2X receptor subtypes as well.
Figure 4. Concentration-dependent inhibition of ATP-induced
calcium influx by compound 53 at different human P2X receptor
subtypes. For IC50 values see Table 2. An ATP concentration
corresponding to the EC80 was used.
Phenoxazine 27 (a p-methylbenzylcarbamate) showed a
peculiar effect at the P2X2 receptor: it did not inhibit ATP-
induced receptor activation but had the opposite effect − it
significantly enhanced the ATP effect (by 84% at 10 μM; ATP
concentration of 1 μM corresponding to the EC80). This effect
was highly reproducible. Compound 27 can thus be
characterized as a positive allosteric modulator (PAM) at
P2X2 receptors.
The two most potent P2X4 receptor subtype-selective
phenoxazine derivatives 21 and 26 were additionally inves-
tigated at a panel of 80 important drug targets comprising a
variety of receptors, ion channels, and transporters (http://
www.cerep.fr/cerep/users/pages/catalog/Assay/catalog.asp).
At 10 μM both compounds showed no significant interaction
with the majority of the investigated targets (see Supporting
Information). Compound 21 only showed significant affinity
for 2 of the 80 investigated targets, namely, adenosine A3 and
benzodiazepine receptors (>80% inhibition at 10 μM), while
compound 26 only showed significant binding to one target:
serotonin 5-HT2B receptors (for details see Supporting
Information). Thus, both compounds appear to be relatively
selective for P2X4 receptors.
Radioligand Binding Studies. The new P2X4 receptor
antagonists may exhibit a competitive mechanism of inhibition
interacting with the ATP binding site, or they may modulate
ATP binding by interacting with an allosteric site on the
receptor. In order to investigate their potential interaction with
the ATP binding site, radioligand binding studies (competition
assays) were performed using [35S]ATPγS as a radioligand.49
Results for all tested compounds (21, 24, 26, 27, 42, 43, 53)
are shown in Supporting Information (Figure S5). None of the
compounds competed with the binding of ATP in the
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concentration range where they inhibited functional receptor
response. Only at very high concentrations (>10 μM)
compounds 21 and 24 appeared to reduce radioligand binding.
In contrast, the ATP derived P2X4 antagonist ATP-TNP (4)
inhibited the binding of [35S]ATPγS in a concentration-
receptor antagonists possess higher affinity than the best
antagonists described so far in the literature (compounds 5 and
6). Furthermore, compounds 21 and 26 have also shown good
selectivity versus the other P2X receptor subtypes. The
phenoxazine and acridone derivatives can be obtained by
simple, straightforward syntheses, and these structural classes of
compounds still present room for further optimization with
regard to affinity and improvement of their physicochemical
properties. The new P2X4 receptor antagonists will be further
optimized, and their usefulness as pharmacological tools for in
vitro and in vivo studies will be investigated in due course.
dependent manner showing a low Ki of 48.9
11.1 nM.
These results clearly show that the ATP derivative 4 is a
competitive antagonist, while the new antagonists described in
the present study appear to act as negative allosteric modulators
showing a noncompetitive mechanism of action. This is in
agreement with the lipophilic nature of the developed P2X4
antagonists, while orthosteric ligands are expected to be polar,
negatively charged molecules. It will therefore in general be
easier to develop P2X4 antagonists with druglike properties
that interact with allosteric sites than with the positively
charged ATP-binding site. On the other hand, some of the
most potent compounds of the present series possess low water
solubility. The sulfonamide derivative 21 was less lipophilic and
more water-soluble than the carbamate 26. In the future we will
therefore introduce solubilizing function and extend the series
of sulfonamide derivatives.
Investigation of Hydrolytic Stability. Since carbamates
may be hydrolytically unstable, stability tests were performed
for the most active compound of the series, carbamate 26, at
three different pH values. In the first experiment, a 100 μM
solution of 26 in DMSO/HCl (aq, pH 1, 1:4) was stirred for 6
h at room temperature. In the second experiment, a 100 μM
solution of 26 in DMSO/HBSS buffer, pH 7.4 (1:4), was
stirred for 6 h at 80 °C. In the third experiment a 10 μM
solution of 26 in DMSO/NaHCO3 (aq, pH 8.4, 2.5:97.5) was
stirred for 6 h at 37 °C. In all three cases LC−MS analyses
showed no degradation of 26. These results confirm the
hydrolytic stability of the carbamate group of phenoxazine
derivative 26 under neutral conditions as they are found in
blood, under acidic conditions, as present in the stomach, and
under basic conditions, which are found in the intestine.
EXPERIMENTAL SECTION
■
General. All commercially available reagents were obtained from
various producers (Acros, Alfa Aesar, and Sigma-Aldrich) and used
without further purification. Solvents were used without additional
purification or drying unless otherwise noted. The reactions were
monitored by thin layer chromatography (TLC) using aluminum
sheets with silica gel 60 F254 (Merck). Column chromatography was
carried out with silica gel 0.060−0.200 mm, pore diameter of ∼6 nm.
1H and 13C NMR data were collected on a Bruker Avance 500 MHz
NMR spectrometer at 500 MHz (1H) and 126 MHz (13C),
respectively. CDCl3 was used as a solvent. Chemical shifts are
reported in parts per million (ppm) relative to the deuterated solvent,
that is, δ 1H, 7.26 ppm; 13C, 70.0 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). Melting points were
determined on a Buchi 530 melting point apparatus and are
̈
uncorrected. The purities of isolated products were determined by
ESI mass spectra obtained on an LC−MS instrument (Applied
Biosystems API 2000 LC−MS/MS instrument, HPLC Agilent 1100
instrument) using the following procedure. The compounds were
dissolved at 0.5 mg/mL in H2O/MeOH = 1:1, containing 2 mM
ammonium acetate. Then 10 μL of the sample was injected into an
HPLC column (Phenomenex Luna 3 μm C18, 50 mm × 2.00 mm).
Elution was performed with a gradient of water/methanol (containing
2 mM ammonium acetate) from 90:10 to 0:100 for 30 min at a flow
rate of 250 μL/min, the gradient starting 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. The purity of the
compounds was generally ≥95%.
CONCLUSIONS
■
N-Substituted phenoxazine and related acridone derivatives
have been discovered as potent and selective allosteric P2X4
receptor antagonists. The present study represents the first SAR
study of P2X4 antagonists. Structural optimization resulted in
the development of several compounds that will be useful as
pharmacological tools. The most potent P2X4 antagonist of the
present series was N-(benzyloxycarbonyl)phenoxazine (26,
PSB-12054) with an IC50 of 0.189 μM at human P2X4
receptors, maximal receptor inhibition of 77%, and good
selectivity versus the other human P2X receptor subtypes. The
compound was shown to possess hydrolytic stability at neutral
and acidic pH values. N-(p-Methylphenyl)sulfonylphenoxazine
(21, PSB-12062) was identified as a potent and selective P2X4
antagonist that was about equally active at P2X receptors of
different species (IC50: rat, 0.928 μM; mouse, 1.76 μM; human,
1.38 μM, maximal inhibition of 68%). N10-Benzyl-9-phenyl-
9,10-dihydroacridine-9-ol (53) was found to be a potent P2X4
antagonist (IC50 = 2.44 μM) but showed even higher potency
at human P2X3 (IC50 = 0.429 μM) and P2X1 (IC50 = 0.602
μM) receptors and thus may be a new starting point for the
development of potent and selective P2X1 and/or P2X3
receptor antagonists. N-(p-Methylbenzyloxycarbonyl)-
phenoxazine (27), a potent and selective P2X4 receptor
antagonist, was found to be also a positive allosteric modulator
of P2X2 receptor function. Some of the newly identified P2X4
Compounds 49,44 50,45 52,46 and 5344 were synthesized as
described in the literature. Compounds 10,50 11,51 13,52 21,53 23,54
25,55 44,54 46,43 47,56 55,57 56,58 58,59 and 6060 have previously been
described but were obtained by new methodologies, and additional
structural characterization data are provided in the present study (for
detailed synthesis and experimental data see Supporting Information).
General Procedure for the Synthesis of Compounds 12−14.
Benzoyl chloride (163 μL, 1.4 mmol) was added to a solution of
phenoxazine (187 mg, 1.0 mmol) and triethylamine (209 μL, 1.5
mmol) in dichloroethane (DCE, 3 mL) at 0 °C under argon. The
resulting mixture was stirred at room temperature for 2 h. Then the
reaction was quenched with aqueous HCl solution (20 mL, 0.5 M),
and the aqueous portion was extracted with dichloromethane (DCM)
(3 × 20 mL). The organic layers were combined, dried over MgSO4,
and subsequently evaporated. Pure compounds were isolated after
flash chromatography (for conditions see the individual compounds)
and crystallized (for crystallization solvents see the individual
compounds).
General Procedure for the Synthesis of Compounds 17−20.
To a solution of lithium diisopropylamide (LDA, 1 mL, 2 M in
tetrahydrofuran (THF), 2 mmol) was added a solution of phenoxazine
(187 mg, 1 mmol) and the corresponding carbamoyl chloride (2.5
mmol) in dry THF (3 mL) at 0 °C under an argon atmosphere. The
mixture was allowed to warm to room temperature and stirred for 2 h.
Then the reaction was quenched with aqueous HCl (30 mL, 0.4 M),
and the aqueous portion was extracted with DCM (3 × 20 mL). The
combined organic layers were washed with brine (50 mL) and dried
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over MgSO4. Pure compounds were isolated after flash chromatog-
raphy (for conditions see the individual compounds) and crystallized
(for crystallization solvents see the individual compounds).
7.43 (m, 2H), 7.38−7.35 (m, 3H), 7.27 (t, J = 8.0 Hz, 2H), 7.15−7.11
(m, 4H), 6.96−6.91 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 168.2,
150.3, 135.0, 130.6, 130.1, 128.9, 128.1, 126.5, 124.7, 123.3, 116.6.
LC−MS (m/z): positive mode 288 [M + H]+, 575 [2M + H]+. Purity
by HPLC−UV (254 nm)−ESI-MS: 100.0%; mp 157−158 °C.
Isobutyl 10H-Phenoxazine-10-carboxylate (24). 24 was
synthesized using isobutyl chloroformate (3 mL) at 110 °C for 3 h.
After crystallization (hexane/ethyl acetate, 3:1) the product was
obtained as a white powder (141 mg, 50% yield). 1H NMR (500 MHz,
CDCl3) δ 7.55 (dd, J = 7.5, 1.5 Hz, 2H), 7.14 (td, J = 7.5, 1.5 Hz, 2H),
7.09 (td, J = 8.0, 1.5 Hz, 2H), 7.07 (dd, J = 8.0, 1.5 Hz, 2H), 4.04 (d, J
= 6.5 Hz, 2H), 2.00 (m, 1H, CH), 0.95 (d, J = 6.5 Hz, 6H). 13C NMR
(126 MHz, CDCl3) δ 153.5, 150.4, 128.7, 126.2, 125.0, 123.1, 116.6,
72.9, 27.8, 19.2. LC−MS (m/z): positive mode 284 [M + H]+. Purity
by HPLC−UV (254 nm)−ESI-MS: 100.0%; mp 82−83 °C.
General Procedure for the Synthesis of Compounds 23 and
24. A solution of phenoxazine (187 mg, 1 mmol) in the corresponding
chloroformate (3 mL) was heated at 75 °C for compound 23 and at
110 °C for compound 24 for 3.5 h. Then the mixture was diluted with
DCM (60 mL) and washed with saturated aqueous NaHCO3 solution
(20 mL) and brine (20 mL). The organic phase was dried over MgSO4
and evaporated. Pure compounds were isolated after crystallization
(for crystallization solvents see the individual compounds).
General Procedure for the Synthesis of Compounds 26−31.
To a suspension of NaH (60% in mineral oil, 1 mmol, 40 mg) in dry
THF (1 mL) was added the corresponding alcohol (0.87 mmol),
dissolved in dry THF (1 mL), at 0 °C. After 30 min 10H-phenoxazine-
10-carbonyl chloride (25, 246 mg, 1 mmol) dissolved in dry THF (1
mL) was added dropwise, and the resulting mixture was allowed to stir
at room temperature overnight. The mixture was cooled to 0 °C and
quenched with water (20 mL), and aqueous HCl solution (5 mL, 2 M)
was added. The resulting solution was extracted with DCM (3 × 20
mL), and the combined organic layers were washed with saturated
aqueous NaHCO3 solution (50 mL) and brine (60 mL) and
subsequently dried with MgSO4 and evaporated to dryness.
General Procedure for the Synthesis of Compounds 34−45.
A solution of the appropiate amine (1.62 mmol) was added to a
solution of 10H-phenoxazine-10-carbonyl chloride (25, 200 mg, 0.81
mmol) in DCE (2 mL) at 0 °C under an argon atmosphere. The
suspension was allowed to warm to room temperature and stirred for 2
h. Then the reaction was quenched with water (30 mL), and the
aqueous portion was extracted with DCM (3 × 20 mL). The
combined organic layers were washed with brine (50 mL), dried with
MgSO4, and evaporated. Pure compounds were isolated after
crystallization (for crystallization solvents see the individual com-
pounds).
General Procedure for the Synthesis of Compounds 49−52.
Acridone (8, 394 mg, 2 mmol) was added portionwise to a stirred
suspension of NaH (60% in mineral oil, 2.4 mmol, 96 mg) in dry
dimethylformamide (DMF) (3.5 mL) at 0 °C under argon
atmosphere. The resulting mixture was allowed to warm to room
temperature and stirred for 1 h. Then a solution of benzyl chloride (3
mmol) in DMF (1.5 mL) was added and the mixture was stirred
overnight. Water (30 mL) was added, and the resulting solution was
extracted with ethyl acetate (3 × 20 mL). The combined organic
extracts were washed with brine (60 mL) and dried with MgSO4. Pure
compounds were isolated after flash chromatography (for conditions
see the individual compounds) and crystallized (for crystallization
solvents see the individual compounds).
General Procedure for the Synthesis of Compounds 56 and
57. A suspension of 2-aminophenol (54, 218 mg, 2 mmol) and
calcium carbonate (130 mg, 1.3 mmol) in dioxane (12 mL) was stirred
at 70 °C for 80 min. Then carbonyl chloride (3.3 mmol) was added
and the temperature was increased to 80 °C. After 90 min the mixture
was filtered while hot and ice-cold water was added to the filtrate. The
precipitate was filtered off, washed with 10 mL of ice cold water, and
dried under high vacuum.
General Procedure for the Synthesis of Compounds 59−63.
A solution of the carbamate (80.0 mg, 0.33 mmol), 1,2-dibromoethane
(228 μL, 495.9 mg, 2.64 mmol), and potassium carbonate (912 mg,
6.60 mmol) in acetone (7 mL) was heated to reflux for 36 h. The
reaction mixture was cooled to room temperature and filtered through
Celite. Water (25 mL) and ethyl acetate (25 mL) were added, and the
layers were separated. The aqueous layer was washed with ethyl
acetate (2 × 25 mL), dried with MgSO4, and evaporated. Pure
compounds were isolated after flash chromatography (for conditions
see the individual compounds).
Benzyl 10H-Phenoxazine-10-carboxylate (26). A solution of
10H-phenoxazine-10-carbonyl chloride (246 mg, 1 mmol) in benzyl
alcohol (3 mL) was heated at 110 °C for 6 h. Then the reaction was
quenched with water (30 mL), and then the aqueous portion was
extracted with DCM (3 × 20 mL). The combined organic layers were
washed with brine (60 mL). The organic phase was dried with MgSO4
and evaporated. After flash chromatography (petroleum ether/ethyl
acetate, 9:1) and crystallization (hexane/ethyl acetate, 3:1) the
1
product was obtained as a white powder (151 mg, 48% yield). H
NMR (500 MHz, CDCl3) δ 7.56 (dd, J = 8.0, 1.5 Hz, 2H), 7.40−7.32
(m, 5H), 7.14 (td, J = 7.5, 1.5 Hz, 2H), 7.10−7.05 (m, 4H), 5.30 (s,
2H). 13C NMR (126 MHz, CDCl3) δ 153.1, 150.4, 135.6, 128.6, 128.5,
128.3, 128.1, 126.4, 125.0, 123.2, 116.6, 68.3. LC−MS (m/z): positive
mode 318 [M + H]+. Purity by HPLC−UV (254 nm)−ESI-MS:
100.0%; mp 121−122 °C.
4-Methylbenzyl 10H-Phenoxazine-10-carboxylate (27). 27
was synthesized using 4-methylbenzyl alcohol (108 mg, 0.87 mmol).
Crystallization from hexane/ethyl acetate, 3:1, afforded the product as
a white powder (150 mg, 52% yield). 1H NMR (500 MHz, CDCl3) δ
7.55 (d, J = 7.5 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.5 Hz,
2H), 7.13 (td, J = 8.0, 1.5 Hz, 2H), 7.09−7.05 (m, 4H), 5.26 (s, 2H),
2.37 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 153.2, 150.4, 138.2,
132.6, 129.2, 128.6, 128.3, 126.3, 125.0, 123.2, 116.6, 68.3, 21.2. LC−
MS (m/z): positive mode 332 [M + H]+. Purity by HPLC−UV (254
nm)−ESI-MS: 100.0%; mp 144−145 °C.
4-Chlorobenzyl 10H-Phenoxazine-10-carboxylate (28). 28
was synthesized using 4-chlorobenzyl alcohol (125 mg, 0.87 mmol).
Crystallization from hexane/ethyl acetate, 3:1, afforded the product as
1
a white powder (77 mg, 25% yield). H NMR (500 MHz, CDCl3) δ
7.52 (dd, J = 7.5, 1.3 Hz, 2H), 7.35−7.32 (m, 4H), 7.14 (td, J = 8.0,
1.5 Hz, 2H), 7.10−7.06 (m, 4H), 5.25 (s, 2H). 13C NMR (126 MHz,
CDCl3) δ 153.0, 150.4, 134.3, 134.1, 129.6, 128.8, 128.4, 126.5, 124.9,
123.3, 116.7, 67.5. LC−MS (m/z): positive mode 352 [M + H]+.
Purity by HPLC−UV (254 nm)−ESI-MS: 100.0%; mp 182−183 °C.
4-Methoxybenzyl 10H-Phenoxazine-10-carboxylate (30). 30
was synthesized using 4-methoxybenzyl alcohol (110 mg, 0.87 mmol).
Flash chromatography (petroleum ether/ethyl acetate, 93:7) and
crystallization from hexane/ethyl acetate, 3:1, afforded the product as
colorless needles (128 mg, 42% yield). 1H NMR (500 MHz, CDCl3) δ
7.53 (dd, J = 8.0, 1.5 Hz, 2H), 7.34 (d, J = 9.0 Hz, 2H), 7.13 (td, J =
7.5, 1.5 Hz, 2H), 7.08−7.04 (m, 4H), 6.90 (d, J = 8.5 Hz, 2H), 5.23 (s,
2H), 3.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 159.7, 153.2,
150.4, 130.1, 128.6, 127.7, 126.3, 125.0, 123.2, 116.6, 113.9, 68.1, 55.3.
LC−MS (m/z): negative mode 316 [M − OCH3]−. Purity by HPLC−
UV (254 nm)−ESI-MS: 100.0%; mp 120−121 °C.
Phenethyl 10H-Phenoxazine-10-carboxylate (31). Phenylethyl
alcohol (121 μL, 1 mmol) was added to a solution of 10H-
phenoxazine-10-carbonyl chloride (246 mg, 1 mmol) dissolved in
pyridine (2 mL), and the resulting solution was stirred at 90 °C for 4.5
h. Water (30 mL) was added, and the resulting solution was extracted
with DCM (3 × 20 mL). The combined organic extracts were washed
with brine (60 mL) and dried with MgSO4. After crystallization from
hexane/ethyl acetate, 3:1, the product was isolated as pale orange
(10H-Phenoxazin-10-yl)(phenyl)methanone (12). 12 was
synthesized using benzoyl chloride (163 μL, 1.4 mmol). After flash
chromatography (petroleum ether/ethyl acetate, 9:1) and crystal-
lization (diethyl ether/DCM, 4:1) the product was obtained as a white
1
crystals (234 mg, 71% yield). H NMR (500 MHz, CDCl3) δ 7.41−
1
powder (236 mg, 82% yield). H NMR (500 MHz, CDCl3) δ 7.44−
7.39 (m, 2H), 7.31 (tt, J = 7.0, 1.5 Hz, 2H), 7.25 (tt, J = 7.5, 1.5 Hz,
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1H), 7.22−7.20 (m, 2H), 7.15−7.11 (m, 2H), 7.06−7.02 (m, 4H),
4.48 (t, J = 6.5 Hz, 2H), 3.01 (t, J = 7.0 Hz, 2H). 13C NMR (126 MHz,
CDCl3) δ 153.2, 150.4, 137.7, 129.0, 128.5, 128.5, 126.6, 126.3, 125.0,
123.2, 116.6, 67.3, 35.1. LC−MS (m/z): positive mode 332 [M + H]+.
Purity by HPLC−UV (254 nm)−ESI-MS: 100.0%; mp 78−80 °C.
N-Phenethyl-10H-phenoxazine-10-carboxamide (42). 42 was
synthesized using 2-phenylethylamine (254 μL, 2 mmol). After
crystallization (hexane/ethyl acetate, 3:1) the product was obtained as
a white powder (282 mg, 85% yield). 1H NMR (500 MHz, CDCl3) δ
7.36 (dd, J = 8.0, 1.5 Hz, 2H), 7.31−7.28 (m, 2H), 7.23 (td, J = 7.0,
1.5 Hz, 1H), 7.18−7.16 (m, 2H), 7.14−7.10 (m, 2H), 7.06−7.01 (m,
4H), 5.35 (t, J = 5.0 Hz, 1H, NH), 3.54 (q, J = 6.5 Hz, 2H), 2.85 (t, J =
7.0 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 154.5, 151.2, 138.9,
129.3, 128.8, 128.7, 126.5, 126.3, 124.5, 123.6, 117.0, 42.0, 35.7. LC−
MS (m/z): positive mode 331 [M + H]+, 661 [2M + H]+. Purity by
HPLC−UV (254 nm)−ESI-MS: 100.0%; mp 103−104 °C.
The assay was performed in a final volume of 200 μL. Compounds
were tested at seven to eight different concentrations.63
Radioligand Binding Studies. Membrane preparations of
astrocytoma cells expressing hP2X4 receptors were obtained similarly
as described before for Chinese hamster ovary cells expressing P2X4
receptors.49 The competition binding studies were performed in assay
buffer (50 mM Tris-HCl, pH 7.4) containing 1 mM EDTA and 0.2
nM [35S]ATPγS. The incubations were started by the addition of
membranes (10−15 μg) and were performed in a 250 μL final assay
volume. The reactions were terminated by vacuum filtration over GF/
B glass-fiber filters using a Brandell 48-well harvester. The filters were
rinsed three times with ice-cold Tris-HCl buffer (50 mM, pH 7.4).
The filters were punched out and transferred to 4 mL scintillation
vials. Then 2.5 mL of Ultima Gold scintillation cocktail was added, and
samples were counted after 6 h for 1 min each, using a liquid
scintillation counter (LSC). Nonspecific binding of [35S]ATPγS was
determined using 100 μM ATP. All data were analyzed with GraphPad
Prism, version 4.1 (GraphPad Inc., La Jolla, CA).
N,N-Dipropyl-10H-phenoxazine-10-carboxamide (43). 43
was synthesized using N,N-dipropylamine (277 μL, 2 mmol). After
crystallization (hexane/ethyl acetate, 3:1) the product was obtained as
1
ASSOCIATED CONTENT
a pale yellow powder (197 mg, 64% yield). H NMR (500 MHz,
■
S
CDCl3) δ 6.85−6.78 (m, 6H), 6.69−6.67 (m, 2H), 3.36 (t, J = 7.5 Hz,
4H), 1.65 (m, J = 7.5 Hz, 4H), 0.90 (t, J = 7.5 Hz, 6H). 13C NMR
(126 MHz, CDCl3) δ 154.9, 144.4, 130.4, 123.7, 122.9, 116.1, 114.2,
50.0, 21.5, 11.5. LC−MS (m/z): positive mode 311 [M + H]+, 621
[2M + H]+. Purity by HPLC−UV (254 nm)−ESI-MS: 100.0%; mp
71−72 °C.
* Supporting Information
1
Synthesis procedures and H and 13C NMR spectral data for
compounds 10, 11, 13−23, 25, 29, 32−40, 43−53, 55, 56 and
58−63; experimental procedures for investigating the hydro-
lytic stability of 26; and results from additional biological
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
N-(2-Hydroxyphenyl)-2-(((1R,2S,5R)-2-isopropyl-5-
methylcyclohexyl)oxy)acetamide (57). 57 was synthesized using
(−)-menthoxyacetyl chloride (2-(((1R,2S,5R)-2-isopropyl-5-
methylcyclohexyl)oxy)acetyl chloride) (383 μL, 3.3 mmol). The
AUTHOR INFORMATION
Corresponding Author
*Phone: +49-228-73-2301. Fax: +49-228-73-2567. E-mail:
christa.mueller@uni-bonn.de.
■
1
product was isolated as a yellow solid (367 mg, 40% yield). H NMR
(500 MHz, CDCl3) δ 9.11 (s, 1H), 8.60 (s, 1H), 7.13 (t, J = 8.4 Hz,
1H), 7.04−6.99 (m, 2H), 6.87 (t, J = 7.6 Hz, 1H), 4.25 (d, J = 15.6 Hz,
1H), 4.05 (d, J = 15.6 Hz, 1H), 3.28 (t, J = 10.6 Hz, 1H), 2.25−2.00
(m, 2H), 1.75−1.62 (m, 2H), 1.37 (dd, J = 12.3, 10.5 Hz, 2H), 0.96 (t,
J = 7.0 Hz, 10H), 0.84 (d, J = 7.0 Hz, 3H). 13C NMR (126 MHz,
CDCl3) δ 170.2, 148.7, 127.3, 124.8, 122.0, 120.3, 119.9, 81.1, 67.3,
48.0, 40.1, 34.3, 31.4, 26.3, 23.3, 22.2, 20.9, 16.3. LC−MS (m/z):
positive mode 306 [M + H]+. Purity by HPLC−UV (254 nm)−ESI-
MS: 100.0%; mp 171−172 °C.
Biological Assays. 1321 N1 astrocytoma cell lines stably
expressing the respective P2X receptor subtype were used for the
assays. Chimeric P2X1 and P2X3 receptors were used in which the N-
terminal domain and transmembrane domain I of the receptors were
replaced by the analogous region of the P2X2 receptor, which
abolished the fast desensitization kinetics typical for P2X1 and P2X3
receptors.61,62
Notes
The authors declare no competing financial interest.
†On leave from the University of Al-Azhar, Assiut, Egypt.
ACKNOWLEDGMENTS
■
We thank Stefanie Weyler and Scarlett Weigel for expert
technical assistance, Marion Schneider for LC−MS analyses,
Sabine Terhart-Krabbe and Annette Reiner for NMR spectra,
and Ralf Hausmann and Gunter Schmalzing (University of
̈
Aachen, Germany) for the gift of some P2X receptor clones.
This study was supported by the BMBF (German Federal
Ministry for Education and Research) within the BioPharma
initiative “Neuroallianz”.
Measurement of Intracellular Ca2+ in Transfected 1321N1
Astrocytoma Cells. P2X receptor function was determined by
measuring ATP-mediated increases in cytosolic Ca2+ concentrations.
The fluorescent Ca2+-chelating dye Fluo-4 was used as an indicator of
the relative levels of intracellular Ca2+ concentrations in a 96-well
format using a fluorescence imaging plate reader (Novostar, BMG).
Cells were grown to confluence in 96-well black-walled tissue culture
plates and loaded with Fluo-4 AM (2.4 μM) in Hank’s balanced salt
solution (containing 10 mM HEPES, pH 7.3, and 1% Pluronic F127)
for 1 h at 23 °C. After incubation the loaded cells were washed with
the same buffer to remove extracellular Fluo-4 AM. Compound
solutions were prepared in DMSO. Then an amount of 2 μL of
compound dilutions was transferred into the measurement plate.
Because of the low solubility of some of the compounds, the DMSO
solution should be directly pipetted into the wells without prior
dilution in buffer. The final DMSO concentration did not exceed 1%.
Fluorescence intensity was measured at 520 nm for 30 s at 0.4 s
intervals. Buffer or ATP solution was injected sequentially into
separate wells using the automatic pipetting device. At least three
independent experiments were performed, each in duplicate.
Antagonists were added 30 min before the addition of the agonist
ATP, applied at a concentration that corresponded to its EC80 value.
ABBREVIATIONSUSED
■
[35S]ATPγS, [35S]adenosine 5′-(γ-thio)triphosphate; 5-BDBD,
5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diaze-
pin-2-one; BzATP, 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-
triphosphate; HBSS, Hank's buffered salt solution; LLE, ligand-
lipophilicity efficiency; 2-MeSATP, 2-methylthioadenosine 5-
triphosphate; TNP-ATP, 2′,3′-O-(2,4,6-trinitrophenyl)-
adenosine 5′-triphosphate
REFERENCES
■
(1) King, B. F. Molecular Biology of P2X Purinoceptors. In
Cardiovascular Biology of Purines; Burnstock, G., Dobson, J. G.,
Laing, B. T., Linden, J., Eds.; Kluwer Academic Publications: Norwell,
MA, 1998; pp 159−186.
(2) Ralevic, V.; Burnstock, G. Receptors for purines and pyrimidines.
Pharmacol. Rev. 1998, 50, 413−492.
(3) Burnstock, G.; Williams, M. P2 Purinergic receptors: modulation
of cell function and therapeutic potential. J. Pharmacol. Exp. Ther.
2000, 295, 862−869.
9586
dx.doi.org/10.1021/jm300845v | J. Med. Chem. 2012, 55, 9576−9588

Journal of Medicinal Chemistry
Article
(4) Robson, S. C.; Sevigny, J.; Zimmermann, H. The E-NTPDase
family of ectonucleotidases: structure function relationships and
pathophysiological significance. Purinergic Signalling 2006, 2, 409−430.
(5) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.;
(24) Ulmann, L.; Hatcher, J. P.; Hughes, J. P.; Chaumont, S.; Green,
P. J.; Conquet, F.; Buell, G. N.; Reeve, A. J.; Chessell, I. P.;
Rassendren, F. Up-regulation of P2X4 receptors in spinal microglia
after peripheral nerve injury mediates BDNF release and neuropathic
pain. J. Neurosci. 2008, 28, 11263−11268.
(25) Zhang, Z.; Artelt, M.; Burnet, M.; Trautmann, K.; Schluesener,
H. J. Lesional accumulation of P2X4 receptor monocytes following
experimental traumatic brain injury. Exp. Neurol. 2006, 197, 252−257.
(26) Cavalieri, F.; Florenzano, F.; Amadio, S.; Fusco, F. R.; Viscomi,
M. T.; D’Ambrosi, N.; Vacca, F.; Sancesario, G.; Bernardi, G.;
Muller, C. E. International Union of Basic and Clinical Pharmacology.
̈
LXXXI. Nomenclature and classification of adenosine receptorsan
update. Pharmacol. Rev. 2011, 63, 1−34.
(6) Coddou, C. Activation and regulation of purinergic P2X receptor
channels. Pharmacol. Rev. 2011, 63, 641−683.
(7) Burnstock, G. Physiology and pathophysiology of purinergic
neurotransmission. Physiol. Rev. 2007, 87, 659−797.
̀
Molinari, M.; Volonte, C. Up-regulation of P2X2, P2X4 receptor and
(8) Fields, R. D.; Burnstock, G. Purinergic signalling in neuron-glia
interactions. Nat. Rev. Neurosci. 2006, 7, 423−436.
ischemic cell death: prevention by P2 antagonists. Neuroscience 2003,
120, 85−98.
(9) Burnstock, G.; De Ryck, M. UCB Pharma research day25
October 2007 “Glia-neuron interactions and purinergic receptors in
neurological disorders”. Purinergic Signalling 2008, 4, 79−84.
(10) Burnstock, G. Purinergic signalling and disorders of the central
nervous system. Nat. Rev. Drug Discovery 2008, 7, 575−590.
(11) Burnstock, G. Purinergic P2 receptors as targets for novel
analgesics. Pharmacol. Ther. 2006, 110, 433−454.
(27) Schwab, J. M.; Guo, L.; Schluesener, H. J. Spinal cord injury
induces early and persistent lesional P2X(4) receptor expression. J.
Neuroimmunol. 2005, 163, 185−189.
(28) Sandkuhler, J. Models and mechanisms of hyperalgesia and
̈
allodynia. Physiol. Rev. 2009, 89, 707−758.
(29) Tsuda, M.; Kuboyama, K.; Inoue, T.; Nagata, K.; Tokazi-Saitoh,
H.; Inoue, K. Behavioral phenotypes of mice lacking purinergic P2X4
receptors in acute and chronic pain assays. Mol. Pain 2009, 5, 28.
(30) Buell, G.; Lewis, C.; Collo, G.; North, R. A.; Surprenant, A. An
antagonist-insensitive P2X receptor expressed in epithelia and brain.
EMBO J. 1996, 15, 55−62.
(12) Kim, Y. C.; Brown, S. G.; Harden, T. K.; Boyer, J. L.; Dubyak,
G.; King, B. F.; Burnstock, G.; Jacobson, K. A. Structure−activity
relationships of pyridoxal phosphate derivatives as potent and selective
antagonists of P2X1 receptors. J. Med. Chem. 2001, 44, 340−349.
(13) Baqi, Y.; Hausmann, R.; Rosefort, C.; Rettinger, J.; Schmalzing,
(31) Soto, F.; Garcia-Guzman, M.; Gomez-Hernandez, J. M.;
G.; Muller, C. E. Discovery of potent competitive antagonists and
positive modulators of the P2X2 Receptor. J. Med. Chem. 2011, 54,
817−830.
(14) Wolf, C.; Rosefort, C.; Fallah, G.; Kassack, M. U.; Hamacher, A.;
Bodnar, M.; Wang, H.; Illes, P.; Kless, A.; Bahrenberg, G.; Schmalzing,
G.; Hausmann, R. Molecular determinants of potent P2X2 antagonism
identified by functional analysis, mutagenesis, and homology docking.
Mol. Pharmacol. 2011, 79, 649−661.
(15) Volpini, R.; Mishra, R. C.; Kachare, D. D.; Dal Ben, 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.
(16) Jahangir, A.; Alam, M.; Carter, D. S.; Dillon, M. P.; Du Bois, D.
J.; Ford, A. P. D. W.; Gever, J. R.; Lin, C.; Wagner, P. J.; Zhai, Y.; Zira,
J. Identification and SAR of novel diaminopyrimidines. Part 2: The
discovery of RO-51, a potent and selective, dual P2X3/P2X2/3
antagonist for the treatment of pain. Bioorg. Med. Chem. Lett. 2009, 19,
1632−1635.
̈
Hollmann, M.; Karschin, C.; Stuhmer, W. P2X4: an ATP-activated
̈
ionotropic receptor cloned from rat brain. Proc. Natl. Acad. Sci. U.S.A.
1996, 93, 3684−3688.
(32) Jones, C. A.; Chessell, I. P.; Simon, J.; Barnard, E. A.; Miller, K.
J.; Michel, A. D.; Humphrey, P. P. Br. J. Pharmacol. 2000, 129, 388−
394.
(33) He, M. L.; Gonzalez-Iglesias, A. E.; Stojilkovic, S. S. Functional
characterization of the P2X4 receptor orthologues. J. Biol. Chem. 2003,
278, 46270−46277.
(34) Bo, X.; Liu, M.; Schoepfer, R.; Burnstock., G. Characterization
and expression of ATP P2X4 receptor from embryonic chick skeletal
muscle. Drug Dev. Res. 2001, 53, 22−28.
(35) 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.
(36) Nagata, K.; Imai, T.; Yamashita, T.; Tsuda, M.; Tozaki-Saitoh,
H.; Inoue, K. Antidepressants inhibit P2X4 receptor function: a
possible involvement in neuropathic pain relief. Mol. Pain 2009, 5, 20.
(37) Toulme, E.; Garcia, A.; Samways, D.; Egan, T. M.; Carson, M. J.;
Khakh, B. S. P2X4 receptors in activated C8-B4 cells of cerebellar
microglial origin. J. Gen. Physiol. 2010, 135, 333−353.
(38) Donnelly-Roberts, D.; McGaraughty, S.; Shieh, C. C.; Honore,
P.; Jarvis, M. F. Painful purinergic receptors. J. Pharmacol. Exp. Ther.
2008, 324, 409−415.
(39) Hajieva, P.; Mocko, J. B.; Moosmann, B.; Behl, C. Novel imine
antioxidants at low nanomolar concentrations protect dopaminergic
cells from oxidative neurotoxicity. J. Neurochem. 2009, 110, 118−132.
(40) Behl, C.; Moosmann, B. Oxidative nerve cell death in
Alzheimer’s disease and stroke: antioxidants as neuroprotective
compounds. Biol. Chem. 2002, 383, 521−536.
(41) Thimmaiah, K. N.; Horton, J. K.; Seshadri, R.; Israel, M.;
Houghton, J. A.; Harwood, F. C.; Houghton, P. J. Synthesis and
chemical characterization of N-substituted phenoxazines directed
toward reversing vinca alkaloid resistance in multidrug-resistant cancer
cells. J. Med. Chem. 1992, 35, 3358−3364 and references cited herein..
(42) Thimmaiah, K. N.; Easton, J. B.; Germain, G. S.; Morton, C. L.;
Kamath, S.; Buolamwini, J. K.; Houghton, P. J. Identification of N10-
substituted phenoxazines as potent and specific inhibitors of Akt
signaling. J. Biol. Chem. 2005, 280, 31924−31935.
(43) Kivlehan, F.; Lefoix, M.; Moynihan, H. A.; Thompson, D.;
Ogurtsov, V. I.; Herzog, G.; Arrigan, D. W. M. Interaction of acridine-
calix[4]arene with DNA at the electrified liquid|liquid interface.
Electrochim. Acta 2010, 55, 3348−3354.
(17) Lee, W. G.; Lee, S. D.; Cho, J. H.; Jung, Y.; Kim, Y. H.; Hien, T.
T.; Kang, K. W.; Ko, H.; Kim, Y. C. Structure−activity relationships
and optimization of 3,5-dichloropyridine derivatives as novel P2X7
receptor antagonists. J. Med. Chem. 2012, 55, 3687−3698.
(18) Baraldi, P. G.; Nunez, M. C.; Morelli, A.; Falzoni, S.; Di Virgilio,
̃
F.; Romagnoli, R. Synthesis and biological activity of N-arylpiperazine-
modified analogues of KN-62, a potent antagonist of the purinergic
P2X7 receptor. J. Med. Chem. 2003, 46, 1318−1329.
(19) 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.
(20) Samwaysa, D. S. K.; Khakhb, B. S.; Dutertrec, S.; Egan, E. M.
Preferential use of unobstructed lateral portals as the access route to
the pore of human ATP-gated ion channels (P2X receptors). Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 13800−13805.
(21) Du, J.; Dong, H.; Zhou, H. X. Gating mechanism of a P2X4
receptor developed from normal mode analysis and molecular
dynamics simulations. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 4140−
4145.
(22) Tsuda, M.; Shigemoto-Mogami, Y.; Koizumi, S.; Mizokoshi, A.;
Kohsaka, S.; Salter, M. W.; Inoue, K. P2X4 receptors induced in spinal
microglia gate tactile allodynia after nerve injury. Nature 2003, 424,
778−783.
(23) Guo, L.; Trautmann, K.; Schluesener, H. J. Expression of P2X4
receptor by lesional activated microglia during formalin-induced
inflammatory pain. J. Neuroimmunol. 2005, 163, 120−127.
9587
dx.doi.org/10.1021/jm300845v | J. Med. Chem. 2012, 55, 9576−9588

Journal of Medicinal Chemistry
Article
(44) Nishimura, T.; Katoh, T.; Hayashi, T. Rhodium-catalyzed aryl
transfer from trisubstituted aryl methanols to alpha,beta-unsaturated
carbonyl compounds. Angew. Chem., Int. Ed. 2007, 46, 4937−4939.
(45) Gao, C.; Jiang, Y.; Tan, C.; Zu, X.; Liu, H.; Cao, D. Synthesis
and potent antileukemic activities of 10-benzyl-9(10H)-acridinones.
Bioorg. Med. Chem. 2008, 16, 8670−8675.
(46) Wang, C.; Hang, T.; Zhang, H. Microwave-promoted N-
alkylation of acridones without solvent. Synth. Commun. 2003, 33,
451−456.
(47) Lee, G. E.; Joshi, B. V.; Chen, W.; Jeong, L. S.; Moon, H. R.;
Jacobson, K. A.; Kim, Y. C. Synthesis and structure−activity
relationship studies of tyrosine-based antagonists at the human
P2X7 receptor. Bioorg. Med. Chem. Lett. 2008, 18, 571−575.
(48) Michel, A. D.; Ng, S. W.; Roman, S.; Clay, W. C.; Dean, D. K.;
Walter, D. S. Mechanism of action of species-selective P2X7 receptor
antagonists. Br. J. Pharmacol. 2009, 156, 1312−1325.
(49) Michel, A. D.; Miller, K. J.; Lundstrom, K.; Buell, G. N.;
̈
Humphrey, P. P. Radiolabeling of the rat P2X4 purinoceptor: evidence
for allosteric interactions of purinoceptor antagonists and monovalent
cations with P2X purinoceptors. Mol. Pharmacol. 1997, 51, 524−532.
(50) Bordell, F.; Hughes, D. L. SN2 reactions of nitranions with
benzyl chlorides. J. Am. Chem. Soc. 1984, 106, 3234−3240.
(51) Muller, P; Buu-Hoi, N. P.; Rips, R. Preparation and reactions of
̈
phenoxazine and phenoselenazine. J. Org. Chem. 1959, 24, 37−39.
(52) Fischer, H.; Buergin, W. Microbicidal Dibenzazoles. CH 653675
A5 19860115, 1986, .
(53) Burmistrov, S. I.; Karpishenko, L. S. Synthesis of benzene-
sulfonyl derivatives of phenoxazine. Khim. Geterotsikl. 1974, 11, 1576−
1577.
(54) Samolovova, V. G.; Gortinskaya, T. V.; Shchukina, M. N.
Phenoxazine series. VI. Synthesis of some 10-substituted phenoxazines.
Zh. Obshch. Khim. 1962, 32, 1085−1088.
(55) Gal, A. E.; Avakian, S. Nitrogen substituted phenoxazines. J.
Med. Chem. 1963, 6, 809−811.
(56) Chen, G.-S. J.; Gibson, M. S. Synthesis of 10-benzoyl-9-acridone
and 9-acridone from 2,2′- and 2,3′-disubstituted benzophenones. J.
Chem. Soc., Perkin Trans. 1 1975, 12, 1138−1139.
(57) Wagner, G.; Leistner, S. Effects of amines and hydrazines on
benzoxazolinone and benzoxalinethione. Pharmazie 1976, 31, 345−
347.
(58) Faler, C. A.; Joullie, M. M. Aminolysis of allyl esters with
bislithium aryl amides. Tetrahedron Lett. 2006, 47, 7229−7231.
(59) Chabrier, P.; Najer, H.; Giudicelli, R. New carbamates with local
anesthetic activity. II. Derivatives of p-aminophenol and o-amino-
phenol. Bull. Soc. Chim. Fr. 1955, 1353−1362.
(60) Coudert, G.; Guillaumet, G.; Loubinoux, B. A new synthesis of
3,4-dihydro-2H-1,4-benzoxazines using solid-liquid phase-transfer
catalysis. Synthesis 1979, 7, 541−543.
(61) Neelands, T. R.; Burgard, E. C.; Uchic, M. E.; McDonald, H. A.;
Niforatos, W.; Faltynek, C. R.; Lynch, K. J.; Jarvis, M. F. 2′,3′-O-
(2,4,6,Trinitrophenyl)-ATP and A-317491 are competitive antagonists
at a slowly desensitizing chimeric human P2X3 receptor. Br. J.
Pharmacol. 2003, 140, 202−210.
(62) Rettinger, J.; Schmalzing, G. Desensitization masks nanomolar
potency of ATP for the P2X1 receptor. J. Biol. Chem. 2004, 279,
6426−6433.
(63) Gee, K. R.; Brown, K. A.; Chen, W. N.; Bishop-Stewart, J.; Gray,
D.; Johnson, I. Chemical and physiological characterization of Fluo-4
Ca2+ indicator dyes. Cell Calcium 2000, 27, 97−106.
9588
dx.doi.org/10.1021/jm300845v | J. Med. Chem. 2012, 55, 9576−9588