Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.0c00435

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 μM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 μM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

Full text of DOI:10.1021/acs.jmedchem.0c00435

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Article
Discovery and structure-relationships of salicylanilide
derivatives as potent, non-acidic P2X1 receptor antagonists
Maoqun Tian, Aliaa Abdelrahman, Younis Baqi, Eduardo Fuentes, Djamil Azazna,
Claudia Spanier, Sabrina Densborn, Sonja Hinz, Ralf Schmid, and Christa E Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00435 • Publication Date (Web): 29 Apr 2020
Downloaded from pubs.acs.org on April 30, 2020
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Discovery and structure-relationships of
salicylanilide derivatives as potent, non-acidic
P2X1 receptor antagonists
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†
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‡
#
†
Maoqun Tian, Aliaa Abdelrahman, Younis Baqi, Eduardo Fuentes, Djamil Azazna,
Claudia Spanier, Sabrina Densborn, Sonja Hinz, Ralf Schmid, and Christa E. Müller†,*
†
†
†
§,║
†PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of
Bonn, An der Immenburg 4, D-53121 Bonn, Germany
‡Department of Chemistry, Faculty of Science, Sultan Qaboos University, PO Box 36, Postal
Code 123, Muscat, Oman
§Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 7RH,
United Kingdom
║Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester LE1
7
RH, UK
#Thrombosis Research Center, Medical Technology School, Department of Clinical
Biochemistry and Immunohaematology, Faculty of Health Sciences, Interdisciplinary Center
on Aging, Universidad de Talca, 3460000 Talca, Chile
KEYWORDS
Allosteric modulator; ATP; IMD-0345; multi-target drugs; P2X1 receptor; P2X4 receptor;
P2X7 receptor; anti-thrombotic; synthesis; platelets
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ABSTRACT
Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics,
and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives
were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1
astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of
ATP-induced calcium influx. Structure-activity relationships were analyzed and selectivity
versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-
bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC 0.0192 µM) and N-[3,5-
0
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bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC 0.0231 µM), displayed
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>500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7
receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative
allosteric modulators, and molecular modeling studies suggested an extracellular binding site.
Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor
inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric
P2X1 receptor antagonists reported to date.
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INTRODUCTION
Purinergic receptors are cell-surface proteins comprising P1 receptors activated by the
nucleoside adenosine, and P2 receptors activated by nucleotides, such as ATP, ADP, UTP, or
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UDP. P2 receptors are further subdivided into ATP-gated cation channels termed P2X, and G
protein-coupled P2Y receptors.2–4 Eight P2Y receptor subtypes (P2Y1,2,4,6,11,12,13,14) have been
identified.1–4 Seven P2X receptor subunits, P2X1-7, exist, that form homo- or hetero-trimeric
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ion channels. Several crystal structures of P2X3, P2X4 and P2X7 receptors have been
published in last years.5–7 The first one was the zebrafish P2X4 receptor in two states, a resting,
closed state determined at 3.1ꢀÅ resolution, and an ATP-bound, open-channel state at 2.9 Å.6,8
The ATP-bound crystal structure revealed that the ATP binding site is located ∼40 Å distant
from the membrane-spanning segments, which constitute the ionic pore of the P2X receptors.
Thus, ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the
opening of the ion channel.8,9
P2X receptors play important roles in a variety of physiological processes, such as central
transmission, contraction of smooth muscles, inflammation, thrombosis,10 and pain
sensation.2,11–15 Extracellular ATP acts as a danger signal,16,17 and therefore, P2X receptor
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antagonists have potential as therapeutic drugs for a range of diseases. Several clinical trials
evaluating P2X3, P2X4 or P2X7 receptor antagonists have been initiated (see
https://clinicaltrials.gov). Recently, the development of P2X3 receptor antagonists has attracted
much attention from pharmaceutical companies. For instance, the P2X3-selective antagonist
gefapixant (MK-7264/AF-219) was successfully tested in a phase II clinical study for the
1
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treatment of chronic cough, which can be envisaged as an inflammatory disease, and has been
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progressed to a phase III clinical study. P2X4 receptor antagonists have potential for the
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treatment of inflammatory and neuropathic pain. Several different classes of P2X4 receptor
antagonists have been identified, optimized and evaluated.22–25 A large number of P2X7
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receptor antagonists has been developed due to their potential for the treatment of
inflammation, chronic pain and depression.26–28
P2X1 receptors were shown to be highly expressed in smooth muscle cells of various organs
such as urinary bladder, arteries, and vas deferens.29–32 P2X1 antagonists have been suggested
for the treatment of overactive bladder.33 Interestingly, P2X1 knockout mice exhibited a
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resistance to acute thromboembolism in vivo, and a prothrombotic phenotype was observed
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upon overexpression of the platelet P2X1 receptor in transgenic mice. This suggests that
P2X1 receptors expressed on blood platelets may be promising drug targets for thrombosis and
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stroke prevention. Moreover, evidence for neuronal and glial P2X1 receptor expression was
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obtained. A strong upregulation of P2X1 receptors was observed following mechanical CNS
injury, and inhibition of P2X1 receptors showed neuroprotection after ischemic and toxic CNS
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injury. Therefore, the P2X1 receptor represents an attractive therapeutic drug target with
neuroprotective potential. P2X1 receptor knockout mice were found to show reduced LPS-
induced lethal endotoxemia indicating that P2X1 antagonists may also be useful for the
treatment of sepsis.38,39
Only few P2X1 antagonists have been described to date (Figure 1), including suramin40,41 and
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related polysulfonates, such as NF449, pyridoxal phosphate derivatives, e.g. PPADS; and
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ATP derivatives, such as TNP-ATP. However, all of these antagonists are negatively charged
(poly-anionic) compounds under physiological conditions and are therefore not suitable as
perorally bioavailable drugs, and are also not capable of penetrating into the brain. Moreover,
suramin and PPADS are only weakly potent and/or non-selective showing additional inhibition
of other P2 receptor subtypes. The peptidomimetic benzimidazole-2-carboxamide derivative
Ro 0437626 introduced by Roche represents the only, non-acidic P2X1 antagonist, albeit with
only moderate potency at micromolar concentrations.44
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O
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O
SO Na
SO Na
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3
H C
N
N
CH3
3
H
H
NH
HN
NaO S
SO Na
3
3
HN
O
O
NH
O
NH SO H
HO S HN
O
3
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SO H SO H
H
H
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SO H
SO H
NaO S
SO Na
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Suramin, IC50 1000-1700 nM
NF449, IC50 0.280 nM
NH2
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O
N
N
N
O
P
OH
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P
O
P
O
P
S
N
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OH
O
H
N
HO
H C
HO
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O
OH OH OH
N
H
N
O
O
N
HN
N
N
3
O N
2
NO2
HO
SO H
SO H
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3
OH
NO2
TNP-ATP, IC50 6 nM
PPADS, IC50 1000 nM
Ro 0437626 IC50 3000 nM
Figure 1. P2X1 receptor antagonists40–44
The aim of the present study was to identify potent, P2X1 antagonists suitable for in vitro
and in vivo studies. Besides selective P2X1 antagonists, compounds that block several P2X
receptor subtypes might also be of interest, e.g., as multi-target drugs for the treatment of
inflammatory diseases.
RESULTS AND DISCUSSION
Compound library screening
With the goal to develop potent, non-acidic and drug-like P2X1 antagonists, we initiated the
screening of part of our compound library. Initially, we screened 3000 proprietary and
commercial compounds including a commercial compound library (TOCRIS Bioscience, UK)
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containing 1140 diverse, drug-like molecules. The compounds were initially screened at a
concentration of 10 µM in a fluorimetric calcium influx assay at 1321N1 astrocytoma cells
recombinantly expressing the human P2X1 receptor. Blockade of ATP- (300 nM,
corresponding to its EC ) -induced calcium influx by the test compounds was measured. Initial
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hits were rescreened at 10 and 1 µM. Seven compounds, all from the TOCRIS compound
collection (0.6% hit rate for this library), were identified that showed >90% inhibition at 10
µM and retained >40% inhibition at 1 µM. Among the hit compounds were the known P2X1
antagonist suramin and N-(3,5-bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide (1,
also known as IMD-0354, Figure 2), a compound previously described as an inhibitor of
nuclear factor kappa-B kinase (IKK) with a potency in the high nano- to low micromolar
range at that target.45–48
CF3
OH
Cl
O
N
CF3
H
1
Figure 2. Structure of salicylanilide 1 (IMD-0354)
Preliminary studies confirmed a concentration-dependent effect of salicylanilide 1 at P2X1
receptors with an IC value in the nanomolar range. Due to its high inhibitory potency at P2X1
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receptors, its drug-like properties, and its core structure allowing easy access to a wide range
of modifications, salicylanilide 1 was selected for extensive structure-activity relationship
(SAR) studies. We re-synthesized hit compound 1 and prepared related salicylanilide
derivatives with broad variations of substituents on both aromatic rings, as well as analogs with
linker modifications. The products were evaluated as novel, non-acidic P2X1 receptor
antagonists.
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Chemistry
The salicylanilide derivatives were synthesized according to reported procedures with some
modifications (see Schemes 1–3). Reactions of salicylic acid derivatives with aniline
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derivatives in refluxing xylene in the presence of phosphorus trichloride (PCl ) yielded
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products 1, 4–9, and 10–54 in moderate to good yields (Scheme 1). Palladium-mediated
hydrogenation of nitro-substituted 9 yielded the amino-substituted derivative 10 in good yield.
Compound 11 bearing a benzylic hydroxyl group was synthesized by the reaction of phthalide
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with aniline 2 in the presence of aluminium chloride (AlCl ). The dihydroxy-substituted
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product 42 was obtained by demethylation of 40 with boron tribromide. Attempts to synthesize
5
6–58 by reaction of salicylic acid with 3-aminopyridine, 6-aminoindole, or 6-aminoquinoline,
respectively, were unsuccessful. The formation of the desired products 56–58 required an
activated salicylic acid derivative, namely phenyl salicylate 55, as a starting material, as well
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as harsh reaction condition (175 °C). In order to explore the SARs of the linker region,
analogues with modified linkage between both aromatic groups were synthesized (Scheme 2).52
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Scheme 1. Synthesis of salicylanilide derivatives 1, 4–58.a
CF3
CF3
CF3
OH
N
O
OH
O
OH O
N
CF3
N
CF3
N
N
CF3
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a
a
a
CF3
CF3
CF3
R1
O
HO
a
O
b
R2
N
CF3
N
H
CF3
H
H N
2
CF3
R3
R5
R4
11
2
1 4 10 12 25
,
-
,
-
OH
O
OH
O
OH
O
R1
d
N
OH
O
N
H
3
55
56
R2
d
c
d
R4
R3
R5
N
OH
O
OH
O
OH O
R1
R6
N
N
N
H
N
H
H
H
R2
29-54
58
57
R1
R2
R3
R4
R5
R1
R2
R3
R4
R5
R6
Compd.
Compd.
29
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OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
CF
H
H
H
H
H
H
H
H
H
CF
CF
H
3
H
H
H
H
H
H
H
H
H
CF
30
3
H
H
Cl
H
31
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3
H
OH
OMe
Cl
32
H
CF
Cl
F
3
Cl
H
33
H
34
H
NO
NH
H
2
2
H
35
H
NO
CN
H
2
0
2
3
4
5
H
36
H
OH
H
37
NO
Me
Cl
2
3
OH
OH
H
Cl
H
H
38
H
Me
Cl
H
39
H
H
CF
3
40
OMe
H
OMe
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50
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
F
H
OMe
OH
NO
NO
NO
CN
Cl
OH
NO
H
18
H
NO
F
2
2
2
2
2
1
9
H
20
H
OH
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
2
1
2
3
4
5
H
H
F
H
H
Me
NO
H
SO
2
CF
3
H
2
H
t-Bu
H
H
CCH
H
5
1
OCF
3
H
52
SO
Br
2
NH
2
H
5
3
H
H
54
H
CF
3
NO
2
a
Reagents and conditions: (a) salicylic acid derivative, xylene, PCl , reflux, 5h, yields: 32–
2%; compound 42: 40, BBr , dichloromethane (DCM), 8 h, yield 80%; compound 10: 9, H ,
Pd/C, MeOH, 4 h, yield 93%; (b) phthalide, AlCl , dichloromethane, rt, 8 h, yield 35%; (c)
arylamine, xylene, PCl , reflux, 5h, yields: 32–91%; (d) arylamine, 175 °C, 3 h, yield, 26–85%.
3
9
3
2
3
3
Substitution of 2-hydroxyphenacyl bromide (59a) with 3,5-bis(trifluoromethyl)aniline gave
product 59 with an elongated linker consisting of a glycine moiety. Compound 60 with a
reversed amide linkage was synthesized by reaction of 3,5-bis(trifluoromethyl)benzoyl
chloride (60a) with 5-chloro-2-hydroxyaniline (60b). Reductive amination starting from 5-
chloro-2-hydroxybenzaldehyde (61a) and 3,5-bis(trifluoromethyl)aniline yielded compound
61 lacking the carbonyl oxygen atom. Amide formation of 5-chlorosalicylic acid with 3,5-
bis(trifluoromethyl)benzylamine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(
EDC) as a condensing agent gave 62 containing the amide functionality with one extra
methylene group. N-Methylated amide derivative 63 and sulfonamide 64 were obtained by
reaction of aniline derivatives with the appropriate acid chloride derivatives, followed by
demethylation of the resulting amides with boron tribromide.
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2. Synthesis of products 59–64 containing modified linkers.
CF3
R1
R1
O
CF3
O
R2
Reaction conditions
Linker
+
CF3
R3
CF3
59b-64b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Cl
5
9a-64a
59-64
CF3
H C
3
O
Reaction
conditions
Reaction
conditions
Linker
CF3
63c, 64c
Cl
Product
R1
R2
R3
Reaction conditions
Linker
O
O
H
N
5
6
6
6
9
0
1
2
H
50 °C, 2h
H N
Br
2
H
N
O
NH2
H
H
H
toluene, reflux
Cl
O
O
H
NaBH , ethanol, reflux
N
H
H N
2
4
O
CO H
a
b
2
H N
2
EDC, DMAP, DCM
N
H
O
N
O
1. toluene, reflux;
H C
3
63
Me
Me
N
H
Cl
2. BBr , DCM
3
O O
S
N
H
1
2
. pyridine, DCM;
. BBr , DCM
O O
S
6
4
H N
2
Cl
3
a
DMAP, 4-(dimethylamino)pyridine
DCM, dichloromethane
b
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8
9
Finally, two urea derivatives, 66 and 67, were synthesized by reaction of isocyanate 65 with
8
-methoxy-1,2,3,4-tetrahydroquinoline, or 1-methylimidazolidin-2-one, respectively (Scheme
3).53
Overall, 63 products, including 17 novel compounds, were obtained. The structures and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
purities of all synthesized compounds were confirmed by H- and C-NMR spectroscopy, and
LC-MS analysis, respectively, and purities were shown to be greater than 95% in all cases.
Scheme 3. Synthesis of urea derivatives 66 and 67a
CH3
CF3
CF3
O
O
OH
O
a
b
N
N
H
CF3
N
N
H
CF3
CF3
66a
66
N
CF3
C
O
CF3
65
O
O
c
N
N
CF3
H C
3
N
H
67
aReagents and conditions: (a) 8-methoxy-1,2,3,4-tetrahydroquinoline, toluene, reflux; (b)
BBr , DCM, 0 °C to rt; (c) 1-methylimidazolidin-2-one, toluene, reflux.
3
Biological evaluation
The synthesized compounds were initially tested at 10 μM for their potency to inhibit ATP-
induced calcium influx in 1321N1 astrocytoma cells stably transfected with a non-desensitizing
mutant of the human P2X1 receptor. We employed a non-desensitizing P2X2/P2X1 chimera
which contained the entire P2X1 ectodomain and therefore has the same ligand specificity as
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the wildtype P2X1 receptor, but a different desensitization behaviour like that of the P2X2
receptor.10
For active compounds showing greater than 50% inhibition, full concentration-inhibition
curves were recorded and IC values were calculated. For stimulation of P2X1 receptors a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
concentration of 300 nM ATP was used which produces 80% of the maximal effect (EC ).
8
0
Under the experimental conditions established in our laboratory, the commercially available
P2X1 antagonist Ro 0437626 exhibited P2X1-antagonistic potency with an IC of 0.496 µM,
5
0
4
4
which is consistent with the reported results (3 µM). Another P2X1 standard compound,
suramin derivative NF449, displayed a similar potency in our assay (IC 236 nM), which was
50
within the range of reported data for the inhibitor, for which a wide range of potencies had been
obtained in different test systems.54–56
Structure-activity relationships
Modification of the salicylate ring
The hit compound 1 and modified salicylanilide derivatives 4–28, which share the 3,5-
bis(trifluoromethyl)phenyl residue as a common structural feature, were explored to investigate
the SARs on the salicylate ring (Table 1). Firstly, it was shown that removal of both, the 2-
hydroxy- and the 5-chloro-substituent, resulted in a significant drop in P2X1-antagonistic
potency (compound 4, IC = 2.22 µM). Removal of only the 2-hydroxy-substituent resulted in
5
0
a compound with similarly low potency (compound 5, IC = 1.39 µM), whereas removal of
5
0
the 5-chloro-substituent led to only a slight drop in potency (compound 6, IC = 0.0769 µM)
50
compared to lead compound 1. On the other hand, methylation of the hydroxyl substituent
completely abolished inhibitory activity (compound 7). Moreover, substitution of the 2-
hydroxy group by a chloro- or nitro-substituent (compounds 8 and 9), or bioisosteric
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9
substitution with an amino group (compound 10) did not show any enhancing effects on
potency. Notably, the hydroxyl substituent connected to the 2-position via a methylene unit (in
benzyl derivative 11, IC >10 µM) led to a complete loss of inhibitory activity. In addition,
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
shifting of the 2-hydroxyl substituent to the 3-position resulted in a weakly potent compound
12, IC 4.91 µM). These investigations confirmed that the 5-chloro-substituent was not
(
5
0
essential for P2X1-antagonistic activity, whereas the 2-hydroxyl substituent was found to be
crucial. Comparison between urea derivatives 66 (IC 10.4 µM) and 67 (IC >>10 µM) further
5
0
50
confirmed the importance of the hydroxyl group.
As a next step, compounds with different substituents and substitution patterns on the
salicylate ring were explored. Shifting of the 5-Cl atom to the 3-position (compound 13, IC50
0
.0580 µM) or 4-position (compound 14, IC = 0.0231 µM) did not significantly alter the
50
compounds’ potency compared to 1. Various substituents at the 5-position were subsequently
investigated. The rank order of potency was as follows: Cl (1, IC 0.0192 µM) >> Me (16,
5
0
IC 0.236 µM) > OMe (17, IC 0.755 µM) > F (19, IC 1.66 µM) ≥ NO (18, IC 2.53 µM)
5
0
50
50
2
50
≥
OH (20, IC 3.41 µM). 2-Hydroxy-5-(trifluoromethyl)benzoic acid is not readily available
50
as a starting material. Therefore, 5-trifluoromethyl-substituted compound 14 without 2-OH was
synthesized, which exhibited 5-fold lower potency (IC 7.18 µM) compared with the 5-chloro-
5
0
substituted compound 5 (IC 1.39 µM), indicating that a 5-trifluoromethyl substituent was
5
0
somewhat tolerated. Introduction of another hydroxyl substituent at the 6-position gave the
weakly potent compound 21 with an IC50 value of 8.00 µM. Additionally, a series of
compounds bearing substituents at the 3-position were investigated. The rank order of potency
was as follows: Cl (13, IC 0.0580 µM) ≥ H (6, IC 0.0769 µM) ≈ F (22, IC 0.0706 µM) >>
5
0
50
50
Me (23, IC 0.655 µM) ≥ NO (24, IC 0.869 µM). Presumably both electronic and steric
5
0
2
50
effects at that position influence the P2X1-antagonistic potency of the ligands. It should be
noted that annelation of another phenyl ring at the 2,3-position resulted in the moderately potent
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 25 with an IC of 0.341 µM. Finally, the investigation of aza-analogs (26, IC >10
5
0
50
µM; 27, IC 5.25 µM; 28, IC >10 µM) confirmed that additional polar residues (also see
5
0
50
compounds 20 and 21) were not well tolerated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Inhibitory potency of salicylanilide derivatives and standard antagonists in calcium
influx assays at the human P2X1 receptors
CF3
CF3
CF3
R1
O
OH
N
O
OH O
R2
R3
3
1
5
N
CF3
N
CF3
N
CF3
H
H
H
N
R5
R4
1, 4-25
26
27
CF3
CF3
CF3
OH
OH
O
O
O
N
O
N
N
CF3
N
N
H
CF3
H C
3
N
N
CF3
H
H
28
66
67
Compd. R1
R2
R3
R4
R5
Human P2X1
a
IC ± SEM (µM)
5
0
(max. inhibition at 10 µM
unless otherwise noted)
0.496 ± 0.132 (100%)
0.236 ± 0.022 (100%)
0.0192 ± 0.0041 (75%)
2.22 ± 0.05 (79%)
Ro 0437626 (for structure see Fig. 1)
NF449 (for structure see Fig. 1)
1
4
5
6
7
8
9
OH
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
1.39 ± 0.49 (82%)
OH
OMe
Cl
0.0769 ± 0.0106 (79%)
>1.00 (17%)b
Cl
H
5.39 ± 1.24 (100%)
8.60 ± 0.51 (61%)
NO2
H
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
NH2
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
9.93 ± 0.86 (67%)
>10 (37%)
CH OH
H
H
2
H
OH
Cl
H
H
4.91 ± 1.52 (100%)
0.0580 ± 0.0219 (83%)
0.0231 ± 0.0036 (59%)
7.18 ± 0.78 (63%)
0.236 ± 0.041 (91%)
0.755 ± 0.122 (91%)
2.53 ± 0.54 (99%)
1.66 ± 0.49 (96%)
3.41 ± 0.41 (109%)
8.00 ± 2.01 (75%)
0.0706 ± 0.0280 (80%)
0.655 ± 0.148 (102%)
0.869 ± 0.149 (112%)
0.341 ± 0.061 (105%)
OH
OH
H
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
CF3
Me
OMe
NO2
F
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
OH
H
H
F
H
Me
NO2
H
H
25
H
2
2
2
6
6
7
8
6
7
see above
see above
see above
see above
see above
> 10 (31%)
5.25 ± 0.43 (60%)
> 10 (9%)
10.4 ± 0.94 (77%)c
6
> 10 (0%)
a
n = 3.
inhibition at 1 µM
b
c
inhibition at 100 µM
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Modification of the aniline ring
In order to investigate the SARs of the aniline ring, 29 compounds, 29–58, with various
substituents on the aniline ring were explored (Table 2). Mono(trifluoromethyl)-substituted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds 29 (IC 0.0910 µM) and 30 (IC 1.30 µM) displayed considerably weaker P2X1
50
50
antagonistic activity than bis(trifluoromethyl)-substituted compounds 1 and 6. Interestingly,
changing the position of the mono-trifluoromethyl substituent from meta to ortho completely
abolished potency (31, IC >10 µM), whereas a single trifluoromethyl group in the para-
5
0
position resulted in a slight increase in potency (32, IC = 0.0383 µM). The para-position was
5
0
somewhat tolerant with the simple halogens (Cl, 33; F, 34) retaining some activity (albeit
considerably less than p-trifluoromethyl-substituted compound 32), whereas the corresponding
nitro- (35) and cyano- (36) substituted compounds lost activity. Next, we explored a series of
compounds with a 3,5-disubstitution pattern that displayed the following sequence of potency:
Cl (39, 0.0293 µM) ≥ 3-NO ,5-CF (37, 0.0316 µM) ≥ F (41, 0.0505 µM) > Me (38, 0.323 µM)
2
3
>
OMe (40, 0.915 µM) >> OH (42, IC >10 µM). Furthermore, compounds with single meta-
50
substituents were investigated. The test results showed that nitro (44, 0.172 µM), cyano (46,
.522 µM), chloro (47, 0.0756 µM), trifluoromethanesulfonyl (SO CF , 48, 0.335 µM), and
0
2
3
t-butyl 49, 0.567 µM) were tolerated in the meta-postion. In contrast, ethynyl (50, 1.34 µM),
trifluoromethoxy (51, 7.62 µM), and bromo (53, 2.69 µM) resulted in weakly potent
compounds with IC values in the (low) micromolar range. Moreover, introduction of the polar
5
0
sulfonamide as a substituent (in 52) or heterocycles such as pyridine (56), indole (57) or
quinoline (58) led to a complete loss of activity. Overall, it may be concluded that the P2X1-
antagonistic potency at the aniline ring seems to be largely dependent on the presence of
lipophilic, electron-withdrawing groups in the meta-position.
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Table 2. Potency of Salicylanilide Derivatives 29–58 at Human P2X1 Receptors
R2
R2
NO2
R1
R3
R4
R1
R3
R4
OH
O
OH
O
OH
O
Cl
N
CF3
N
H
N
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
Cl 29, 31-51
30, 52, 53
54
N
N
OH
O
OH
O
OH
O
N
H
N
H
N
N
H
H
56
57
58
Compd. R1
R2
R3
R4
Human P2X1
a
IC ± SEM (µM)
5
0
(max. inhibition at 10 µM
unless otherwise noted)
1
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
for structure see Fig. 2
0.0192 ± 0.0041 (75%)
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
H
H
H
H
H
H
H
H
H
H
0.0910 ± 0.0288 (75%)
1.30 ± 0.09 (75%)
> 10 (28%)
H
H
H
CF3
Cl
F
0.0383 ± 0.0058 (53%)
0.199 ± 0.043 (55%)
0.907 ± 0.126 (93%)
≥ 10 (43%)
H
H
H
NO2
CN
H
H
> 10 (38%)
NO2
Me
Cl
CF3
0.0316 ± 0.0108 (81%)
0.323 ± 0.059 (64%)
0.0293 ± 0.0055 (56%)
0.915 ± 0.198 (80%)
0.0505 ± 0.0154 (62%)
> 10 (4%)
H
Me
Cl
H
OMe
F
H
OMe
F
H
OH
NO2
NO2
H
OH
NO2
H
H
4.35 ± 0.67 (62%)
0.172 ± 0.049 (65%)
H
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Journal of Medicinal Chemistry
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13.5 ± 1.3 (80%)b
4
4
4
4
4
5
5
5
5
5
5
5
5
5
6
7
8
9
0
1
2
3
4
6
7
8
H
H
H
H
H
H
H
H
H
NO2
CN
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.522 ± 0.080 (60%)c
0.0756 ± 0.0155 (64%)
0.335 ± 0.083 (83%)
0.567 ± 0.123 (100%)
SO CF
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
t-Bu
CCH
OCF3
1.34 ± 0.23 (60%)
7.62 ± 2.98 (100%)b
SO NH
> 10 (21%)
2
2
2.69 ± 0.17 (98%)b
Br
0.219 ± 0.051 (83%)b
see above
see above
see above
see above
> 10 (3%)
> 10 (11%)
> 10 (28%)
a
b
c
n = 3.
Percent inhibition at 100 μM of test compound.
Percent inhibition at 1 μM of test compound.
Modification of the linker
Modification of the amide linker was not well tolerated and yielded either inactive
compounds like 60 bearing a reversed amide linkage, or compounds with IC values in the
5
0
micromolar range, such as glycine derivative 59 (IC 1.79 µM), amine 61 (IC 3.82 µM),
50
50
benzylamide 62 (IC 1.53 µM), and N-methylamide 63 (IC 4.06 µM), see Table 3. Notably,
5
0
50
there was one exception: bioisosteric replacement of the amide by a sulfonamide in 64 resulted
in an inhibitor with submicromolar potency (IC 0.439 µM). Further modifications of 64 may
50
be warranted in future studies.
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Page 19 of 57
Journal of Medicinal Chemistry
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8
9
Table 3. Potency of Inhibitors 59–64 at human P2X1 Receptors
CF3
OH
Linker
CF3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
Compd.
Linker
Human P2X1
a
IC ± SEM (µM)
5
0
(max. inhibition at 10 µM)
O
H
5
9
1.79 ± 0.48 (104%)
N
H
N
60
≥ 10 (48%)
O
6
6
1
2
N
H
3.82 ± 0.44 (122%)
1.53 ± 0.12 (111%)
O
O
N
H
63
4.06 ± 0.20 (88%)
N
O O
S
6
4
0.439 ± 0.051 (80%)
N
H
an = 3.
Figure 3 summarizes typical features required for P2X1 antagonists. A phenolic group in the
-position of the benzamide moiety is essential, while the chlorine substituent may be shifted
2
to the 3- or 4- position. On the other hand, lipophilic and electron-withdrawing groups are
required on the aniline moiety. An amide linker was found to be crucial for high potency.
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Journal of Medicinal Chemistry
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Summary of structure-activity relationships of salicylanilide derivatives as P2X1
receptor antagonists.
Receptor subtype selectivity
In order to assess the selectivity of the new P2X1 receptor antagonists versus closely related
receptors, selected compounds were tested in calcium influx assays at the other functional
homomeric human P2X receptor subtypes, namely P2X2, P2X3, P2X4 and P2X7 (see Table 4
and Figure 4). All of these compounds exhibited the highest potency at the human P2X1
receptor with the exception of 13, which was slightly more potent at the P2X4 receptor, and
2
5, which was slightly more potent at P2X3. None of the compounds showed antagonistic
activity at the human P2X2 receptor subtype. Regarding activity at P2X4 and P2X7 receptors,
these compounds may be divided into three groups: (a) compounds 1, 6, 14, 41 and 47 were
nearly equipotent at human P2X4 and P2X7 receptors, and were still more potent (2–10 fold)
at the human P2X1 receptor; (b) compounds 13, 29 and 37 showed comparable potencies at
P2X1 and P2X4 receptors, and were less potent at P2X7 receptor; (c) compounds 22, 32 and
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Page 21 of 57
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
3
9 were slightly less potent at P2X7 receptor compared with the P2X1 receptor, and were
significantly less potent at P2X4 receptors. One of the compounds, 25, was similarly potent at
four P2X receptor subtypes, P2X1,2,4 and 7.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Potency of selected salicylanilide derivatives at human P2X receptor
subtypesa
Compd.
1
P2X1
P2X2
P2X3
P2X4
P2X7
0.0192 ± 0.0041
>> 10
> 10 (11%)
0.156 ± 0.014
0.175 ± 0.073
(−7%)
6
0.0769 ± 0.0106
0.0580 ± 0.0219
0.0231 ± 0.0036
0.0706 ± 0.0280
0.341 ± 0.061
> 10
> 10 (30%)
> 10 (28%)
>10 (2%)
0.155 ± 0.069
0.0488 ± 0.0110
0.209 ± 0.083
≥ 10 (49%)b
0.315 ± 0.035
0.177 ± 0.011
0.196 ± 0.102
0.857 ± 0.220
0.797 ± 0.135
6.32 ± 2.60
(
15%)
> 10
1
1
2
2
2
3
4
2
5
9
(
13%)
>> 10
(−5%)
>> 10
> 10 (38%)
0.195 ± 0.113
>> 10 (4%)
>> 10 (−6%)
>> 10 (16%)
>> 10 (5%)
>> 10 (2%)
>> 10 (3%)
(−20%)
>> 10
0.729 ± 0.346
0.537 ± 0.13
(
6%)
>> 10
5%)
>> 10
3%)
>> 10
5%)
>> 10
−5%)
0.0910 ± 0.0288
0.0383 ± 0.0058
0.0316 ± 0.0108
0.0293 ± 0.0055
0.0505 ± 0.0154
0.0756 ± 0.0115
(
32
37
1.50 ± 0.87
0.371 ± 0.193
0.143 ± 0.035
0.0704 ± 0.0278
0.160 ± 0.083
0.599 ± 0.245
(
0.0426 ± 0.0108
0.712 ± 0.188
0.155 ± 0.047
0.135 ± 0.033
(
3
4
4
9
1
7
(
>> 10
(4%)
>> 10
(0%)
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Journal of Medicinal Chemistry
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
IC ± SEM (µM), n=3. Percent inhibition at 10 µM is given in brackets for inactive
50
compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
P2X1
P2X2
P2X3
7
6
5
P2X4
P2X7
1
6
13
14
22
25
29
32
37
39
41
47
Compound
Figure 4. Selectivity of selected salicylanilides at human P2X receptor subtypes.
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Page 23 of 57
Journal of Medicinal Chemistry
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5
6
7
8
9
These results provide the starting points to further improve P2X1 selectivity, and to develop
salicylanilide derivatives as antagonists for other P2X receptor subtypes as well.
Recently, multi-target drug discovery as an emerging area has attracted much attention from
the drug discovery community.57,58 P2X1/4/7 triple target antagonists may be of great interest
for the treatment of chronic inflammatory diseases such as asthma, since recent investigations
confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in the LAD2 human
mast cell line and in human lung mast cells.59
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Full and partial inhibition
The investigated compounds displayed different percentages of P2X1 receptor inhibition, some
completely, others only partially blocking ATP-induced calcium influx. The hit compound 1
showed about 75% maximal P2X1 receptor inhibition. In contrast, some antagonists exhibited
>
90% or even full inhibition, (8, 12, 16–20, 23–25, 34, 49, 51, 53), but no clear SARs could
be deduced with regard to antagonistic efficacy. In a few cases, especially for the weaker
compounds, the reason for maximal inhibition not being reached may have been limited
solubility of the compound.
Mechanism of action
There are two possibilities how the new antagonists may inhibit P2X1 receptor activity:
either by a competitive mechanism (blockade of the ATP binding site), or by an allosteric
mechanism. In order to assess their potential interaction with the ATP binding site,
concentration–response curves for ATP were determined in the absence and in the presence of
different concentrations (100 and 500 nM) of the selected antagonists 1 and 22. In either case,
the concentration–response curves (Figure 5) showed the characteristic behavior of a negative
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
allosteric modulator (NAM), that is, depression of the maximal response with moderate
alteration of the EC values.
5
0
1
00
1
00
50
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ATP
ATP
ATP + 100 nM 22
ATP + 100 nM 1
ATP + 500 nM 1
5
0
ATP + 500 nM 22
0
0-9
10-8
10-7
10-6
10-5
1
1
0-9
10-8
10-7
ATP], M
10-6
10-5
[ATP], M
[
Figure 5. ATP-induced intracellular calcium levels in 1321N1 astrocytoma cells stably
transfected with the human P2X1 receptors, measured in the absence and presence of different
concentrations of compound 1 (left) and compound 22 (right). Data were normalized to ATP
without inhibitor (100%) and buffer (0%).
Both antagonists did not show full receptor inhibition. Compared with full inhibitors, partial
inhibitors may have some advantages, such as less side effects and less risk of overdosing.
Partial receptor antagonism is consistent with the behavior of negative allosteric modulators,
while competitive antagonists typically lead to complete blockade of the signal. An allosteric
binding site is plausible since it is unlikely that these uncharged compounds bind to the polar,
positively charged orthosteric ATP binding site. In summary, there is compelling evidence for
an allosteric inhibition mechanism due to (i) only partial inhibition of ATP-induced calcium
influx by many of the investigated antagonists, (ii) the neutral structure of the antagonists which
are unlikely to bind to the orthosteric binding site containing positively charged residues, and
(iii) the observed decrease in maximal ATP-induced receptor activation by increasing
concentrations of antagonists. There is a fourth argument, namely the rough similarity of the
identified structures with some of the antagonists for other P2XR subtypes, for which an
allosteric binding site has been experimentally confirmed by X-ray crystallography or
mutagenesis studies.7,60
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Journal of Medicinal Chemistry
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7
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9
Molecular modeling studies
To provide detailed structural hypotheses for the observed structure-activity relationships,
binding poses were predicted using Rosetta Ligand docking for the selected potent compounds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
, 22, 39 and 41 and the human P2X1, P2X3, P2X4 and P2X7 receptors. The mean interface
delta scores of all runs for P2X1 receptor allosteric sites were consistently lower compared to
P2X4 and P2X7, and scores at the P2X3 receptor were consistently the highest (Supplemental
Table S4). In this respect the docking scores were in agreement with the P2X subtype
specificity for the four compounds where IC s for P2X1 are in the low nM range, P2X4 and
5
0
P2X7 have IC s in the sub-μM range, and no inhibition of P2X3 is observed. Comparing
50
scores between putative allosteric and orthosteric poses supports the picture above with P2X1
scores clearly favouring allosteric binding, while for P2X3 allosteric scores were only
marginally better than the corresponding orthosteric scores (Supplemental Table S4). Two
main putative binding modes for 1, 22, 39 and 41 are present in the docking results (Figure 6),
7
both overlap with the allosteric binding mode found in X-ray structures of P2X7 receptor. In
one of the predicted binding modes compounds 1, 22, 39 and 41 are located relatively close to
the receptor surface, and sit perpendicular to the known P2X7R allosteric binding mode (Figure
6
C). The other mode is located deeper in the allosteric pocket with the aniline moiety of 1, 22,
9 and 41 in a hydrophobic cavity embedded by Phe92, Val101, Phe291, Phe293, Phe308 and
3
Val310 (human P2X1 numbering). The region around the salicylate moiety of the compounds
is defined by Trp85, Phe106, Val108 and Trp164. A remarkable feature of this binding mode
is that the salicyl-OH group and the amide-carbonyl are forming H-bonds with His296 and
Arg295. As both residues are unique to the human P2X1R and not found in P2X3, P2X4 and
P2X7, this provides an appealing hypothesis to explain subtype selectivity.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
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5
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0
1
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5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
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9
0
1
2
3
4
5
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7
8
9
0
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Journal of Medicinal Chemistry
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9
Figure 6. Ligand docking. A. Cartoon representation of P2X1 model with docked 1 shown as
spheres. B. as in A but rotated by 90°, view perpendicular to membrane. C. Relative orientation
of alternative docking poses of 1 (CPK colours, C-atoms in yellow) superimposed onto the
allosteric P2X7 inhibitor AZ10606120 (PDBid: 5UIW, CPK colours, C-atoms in white). D.
Overlay of docking poses for 1 (C-atoms in yellow), 22 (C-atoms in blue), 39 (C-atoms in
green) and 41 (C-atoms in orange). For details of binding site see panels E and F. Two views
of the predicted P2X1 binding pose for 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition of platelet aggregation
Three different purinergic P2 receptors are involved in platelet aggregation, two purinergic G
protein-coupled receptors, P2Y and P2Y , both activated by ADP, and the ATP-gated ion
1
12
6
1
2+
+
+
channel receptor P2X1. The P2X1 receptor, a cation channel permeable for Ca , Na and K ,
mediates changes in platelet shape and amplifies the G protein-coupled P2Y1 receptor-evoked
q
calcium ion responses in ADP-induced platelet aggregation. Thus, co-stimulation of P2X1 and
2
+
P2Y receptors induces a transient rise in intracellular Ca concentration, platelet shape
1
change, and the initial, easily reversible platelet aggregation, whereas the G protein coupled
i
62–64
P2Y receptor mediates progressive and persistent platelet aggregation.
Since ATP and
1
2
ADP are co-released from dense granules during platelet activation, the P2X1 receptor strongly
participates in synergism and amplifies the formation of platelet aggregates in response to
collagen.65
In order to investigate the effect of selected potent P2X1 receptor antagonists of the present
series, compounds 1, 4, 14, 32, 39, 41, and 64 were investigated for their antiplatelet activity
on platelet aggregation assay in response to various agonists, such as collagen, TRAP-6, and
ADP (see Table 5). By adding an agonist to platelets, the platelets aggregate in a glycoprotein
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Journal of Medicinal Chemistry
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(
GP) IIb/IIIa-dependent manner. Thus, various agonists induce platelet aggregation by
different signaling pathways. These agonists include collagen that binds to the GPVI receptor
6
5
and which is positively regulated by P2X1 receptors; ADP, which may be released from
0
1
2
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4
5
6
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8
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0
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platelet-dense granules and induces activation of the P2Y and P2Y receptors; and TRAP-6
1
12
(thrombin receptor activator peptide-6), which acts through proteolysis of protease-activated
receptors (PARs).66
Table 5. Inhibition of platelet aggregation induced by collagen, TRAP-6 or ADP by selected
potent P2X1 receptor antagonists.
Compd.
Human P2X1 IC ± SD (µM) (or % inhibition at indicated concentration)
50
Collagena,b
TRAP-6a,b
3.64 ± 0.49
ADPa
IC (µM)
5
0
1
4
0.0192
2.22
0.57 ± 0.12
2.10 ± 0.45
1.14 ± 0.06
> 10 (7 ± 2%)
3.03 ± 0.41
≥ 10 (47 ± 1%)
3.13 ± 1.13
4.73 ± 0.38
5.57 ± 0.55
≥ 20 (47 ± 1%)
5.47 ± 0.32
14
0.0231
0.0383
0.0293
0.0505
0.439
4.80 ± 0.26
3
2
> 10 (6 ± 2%)
> 10 (13 ± 6%)
> 10 (5 ± 3%)
> 10 (8 ± 3%)
> 20 (23 ± 1%)
> 20 (26 ± 2%)
> 20 (14 ± 2%)
> 20 (27 ± 3%)
39
4
6
1
4
a
b
Sample: washed platelets. Agonists: ADP 4 µM, TRAP-6 10 µM and Collagen 1,2 µg/mL
Pretreatment: Apyrase 0.5 U/mL.
The two most potent P2X1 receptor antagonists of the present series, 1 and 14, inhibited platelet
aggregation induced by all three pathways (collagen, TRAP-6 and ADP). These compounds
were most potent in collagen-induced platelet aggregation showing high nanomolar to low
micromolar IC values. This is explainable by the fact that during platelet aggregation initiated
5
0
by collagen, early secretion of ATP results in the activation of the P2X1 receptor, which plays
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a key role in contributing to platelet aggregation in response to collagen. Thus our data
confirm that two most potent P2X1 receptor antagonists (1 and 14) inhibited collagen-induced
platelet aggregation and that this process is to a large extent P2X1 receptor-dependent.67
Similarly, NF449, a competitive antagonist of the platelet P2X1 receptor, was previously
shown to reduce collagen-induced aggregation. This compound was also a very weak
1
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antagonist of the P2Y -mediated inhibition of adenylyl cyclase activity. The lower
12
antiplatelet activity observed for compounds 1 and 14 versus the other inducers of platelet
aggregation may be due to the fact that the studied activation pathways (activated by ADP and
TRAP-6, respectively) would have a weak effect to trigger the release of ATP and therefore a
lower participation of the P2X1 receptor can be expected.
Compound 32, which inhibited the P2X1 receptor only by 53%, was inactive in inhibition of
platelet aggregation, possibly indicating that this partial inhibition was not sufficient for
inducing a biological effect on platelets. This is consistent with the moderate platelet
aggregation inhibitory potency of 39 and 41, which had also shown only 55% and 62%
maximal inhibition of the P2X1 receptor. Besides, physicochemical properties of the
compounds may also contribute to their weaker potency in the platelet aggregation assays.
Assessment of properties associated with peroral bioavailability
Drugs for peroral application require appropriate physicochemical properties. Therefore, lead
compound (1) and selected potent analogs (6, 13, 14, 22, 25, 29, 32, 37, 39, 41, and 47) were
assessed by computational methods (see Table S3, Supporting Information). All of these
compounds have low molecular masses of less than 400 D, which has been recommended for
68
oral drug candidates. The calculated logP (clogP) values were below 5 except for the naphthyl
derivative 25, for which it was slightly higher (clog P 5.15). Compounds show two H-bond
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