New P2X3 receptor antagonists. Part 1: Discovery and optimization of tricyclic compounds

Article abstract of DOI:10.1016/j.bmcl.2016.07.009

Purinergic P2X3 receptors are trimeric ligand-gated ion channels whose antagonism is an appealing yet challenging and not fully validated drug development idea. With the aim of identification of an orally active, potent human P2X3 receptor antagonist compound that can penetrate the central nervous system, the compound collection of Gedeon Richter was screened. A hit series of tricyclic compounds was subjected to a rapid, two-step optimization process focusing on increasing potency, improving metabolic stability and CNS penetrability. Attempts resulted in compound 65, a potential tool compound for testing P2X3 inhibitory effects in vivo.

Full text of DOI:10.1016/j.bmcl.2016.07.009

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3896–3904
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New P2X3 receptor antagonists. Part 1: Discovery and optimization
of tricyclic compounds
⇑
Gábor Szántó , Attila Makó, Imre Bata, Bence Farkas, Sándor Kolok, Mónika Vastag, Attila Cselenyák
Gedeon Richter Plc, Budapest 10, PO Box 27, H-1475, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Purinergic P2X3 receptors are trimeric ligand-gated ion channels whose antagonism is an appealing yet
challenging and not fully validated drug development idea. With the aim of identification of an orally
active, potent human P2X3 receptor antagonist compound that can penetrate the central nervous system,
the compound collection of Gedeon Richter was screened. A hit series of tricyclic compounds was sub-
jected to a rapid, two-step optimization process focusing on increasing potency, improving metabolic sta-
bility and CNS penetrability. Attempts resulted in compound 65, a potential tool compound for testing
P2X3 inhibitory effects in vivo.
Received 2 June 2016
Revised 1 July 2016
Accepted 3 July 2016
Available online 5 July 2016
Keywords:
Purinergic receptor
ATP
Ó 2016 Elsevier Ltd. All rights reserved.
Ion channel
P2X3 antagonist
HTS campaign
Airway hyperractivity
New binding site
Chirality
The theory of extracellular signaling via receptors that bind
antagonism suggested by experimental studies ranging from pre-
clinical observations to proof of concept studies in humans. Signif-
icant attention has been paid to the rational of P2X3 receptor
inhibition in pain management due to the unmet medical need,
which is still definite on this therapeutic field, and to the support-
0
adenosine 5 -tri-phosphate (ATP) as a transmitter was proposed
almost half a century ago by Burnstock.1 Since that time not only
has the existence of a membrane receptor family activated by
ATP been certified, but the possible roles of the members of this
family in various disease conditions have also been recognized
3
ive findings of numerous preclinical studies. However, the rela-
2
and widely studied. Purinergic receptors comprise two subfami-
tively poor translatability of rodent studies to humans poses
unacceptably high risk to pursue compounds to clinic in indica-
tions associated with chronic pain. Fortunately, there are many
other possible indications associated with airways hyperreactivity,
overactive bladder or even cardiovascular problems in which
pathological cellular mechanisms are ignited by P2X3 receptor
activation, and the blockade of P2X3 receptors may be converted
lies, namely the P2Y subfamily belonging to the metabotropic G
protein-coupled receptors, and the P2X subfamily, which contains
receptors that are cation channels gated by extracellular ATP.
These latter ionotropic receptors are expressed on both neuronal
and non-neuronal cells, and mediate fast current responses upon
ligand binding by permitting the passage of cations along their
electrochemical gradients. In excitable cells this leads to a great
influx of Na+ and Ca ions and resulting in a depolarization of
4
,5
into efficacy and effectiveness in clinical settings.
2+
Channels containing the P2X3 subunits are either homo- (P2X3)
or heterotrimers (P2X2/3), which may complicate in vitro assaying
and, not negligibly, the evaluation of the role of P2X3 at the level of
individual organisms. It is suggested however, that there is a clear
difference in distribution of P2X2/3 receptors between rodents and
primates due to the absence of functional P2X2/3 receptors in pri-
2+
the cell and triggering downstream Ca signaling. Functional
P2X receptors are homo- or heterotrimers formed by subunits with
two transmembrane domains and intracellular N- and C-termini.
Until now seven P2X receptor genes have been identified and
cloned, encoding receptor subunits termed P2X1–7.
6
The idea of specific targeting of the P2X3 receptor has raised
much pharmaceutical interest due to the restricted tissue distribu-
tion of the protein and the promise of therapeutic potential of its
mate sensory neurons, which justifies the search for pure P2X3
antagonists.
To date, a number of patents comprising P2X3-related com-
7
pounds have been filed, some of them molecules from Abbot
(1), Roche (2, 3) and Astra Zeneca (4). Nowadays among them only
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.07.009
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0

G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
3897
O
N
COOH
COOH
O
N
S
N
H
HOOC
N
N
NH2
O
O
O
N
N
MeO
N
NH2
I
1
(A-317491)
2 (RO-4)
3 (RO-85)
O
N
O
NH2
N
N
O
N
N
N
N
O
S
MeO
N
NH2
O
S
O
NH
N
N
O
S
O
F
NH2
Cl
OMe
4
(AZ-2)
5 (AF-219)
6
Figure 1. Structures of P2X3 receptor antagonists (1–5) and HTS hit (6).
N
O
N
O
S
N
O
S
S
H
Cl
N
O
S
b
a
c
O
N
O
HN
1
1
N
N
R
N
R
NH
1
N
2
R
N
R
O
O
7
8
9
6
5
% at 10µM
IC50=1300 nM
6% at 10µM
IC50 = 840 nM
Scheme 1. Synthesis of 6 with a general synthetic method (R1 = 2-MeO-phenethyl, R2 = 4-F-phenylsulfonyl). Reagents and conditions: (a) R –X (X = Br, Cl, OMs), Cs
6
1
2
3
CO , DMF,
2
0 °C, 2–3 h; 30–60% yield (b) HCl/dioxane or TFA/DCM, rt, 2–24 h, 60–98% yield; (c) R –Y (Y = OH, Cl, Br), DIPEA, DCM, rt, 2–24 h, 33–90% yield.
AF-219 (5) is in Phase 2 clinical trial8 to demonstrate improve-
,9
different groups on the far-end of the linker. In case of unsubsti-
tuted phenyl (8c) the activity dropped, while having pyridyl, pyr-
role, piperidine or i-propyl (8d–8g) we observed the complete
loss of activity.
4
ments in refractory chronic cough (Fig. 1).
Our goal was the identification of an orally available, CNS-pen-
etrant, potent human P2X3 receptor antagonist compound. Gedeon
Richter compound collection served as a starting point of an HTS
2
Introducing o-F or o-NO substituents to the phenyl ring (8h, 8i)
campaign in which a fluorometric [Ca2
+
]
the activity remained in the same few micromolar range as in case
of m- and p-methoxy groups (8j, 8k). However, introducing a sec-
ond methoxy group had a great impact on the P2X3 antagonist
activity; in case of 3,4- and 2,3-dimethoxy substitution (8l, 8m)
there were no big changes in the activity, but in contrast, 2,4-
and 3,5-dimethoxy analogues (8n, 8o) had sub-micromolar IC50
values. Conversely, 2,4,6-trimethoxy substitution (8p) resulted in
drop of activity (Table 1).
i
-assay using a cell line
expressing the hP2X3 receptor in an inducible manner was
1
0
deployed.
On our ongoing studies, a HTS campaign11 was performed and
we identified 6 as the best hit (Fig. 1) with an inhibitory action
on hP2X3 with an IC50 value of hardly less than 1 micromole
(
IC50: 840 nM). The next step was to find and identify a more
potent P2X3 antagonist molecule having lower molecular weight
M). For the exploration
(
M.W. < 500) and better solubility (ꢀ1.7
l
Considering these results, it seems that this region of the recep-
tor has two independent hydrophobic pockets, and therefore, the
left side of this large tricycle can bind there only with a proper size
of the chemical space around 6, resynthesis was carried out in
the following route: we started from known-tricycle 7,1 and after
alkylation we obtained intermediate 8. Removal of the t-butoxy-
carbonyl (BOC) protecting group yielded 9, from which, by sulfona-
tion, we successfully prepared target compound 6. Surprisingly,
intermediate 8 also showed significant P2X3 antagonist activity
2
1
3
and substituent pattern.
Subsequently, we investigated the role of the substituents on
the right side. After preparing 9 from 8 by removing the protecting
group, it was reacted with various acylating agents. Surprisingly,
the alkylated derivatives were chemically unstable, which ham-
pered their further investigation (data not shown).
Replacing the t-butoxy group of 8 with t-butylamino group (10),
the molecule became fivefold less potent. In case of the i-propy-
lamino group (11), the P2X3 activity dropped even more, however,
the dimethylamino group (12) turned out to be equipotent
(IC50 = 1300 nM) with 8. When we increased the size of the cyclic
amine attached to the carbonyl-group, the P2X3 activity decreased
accordingly (13–15). The phenylsulfonyl (16) and piperidinosul-
(
IC50: 1300 nM), which allowed us to perform a two-step SAR
investigation. First, we examined the role of the left side of the
molecule having BOC-group, then we moved on to explore the
right side with various acylating agents (Scheme 1).
Starting with the left side of the molecule, we investigated the
optimal distance between the tricycle and the o-metoxyphenyl
group. In case of both longer (8a) and shorter (8b) chain, we
observed a dramatic drop of activity. So, keeping the most effective
two carbon atom length chain, we synthesized analogues having

3
898
G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
Table 1
Table 2
Exploration of the R1 position of the tricyclic
Exploration of the R2 position of the tricyclic
N
N
S
S
O
1
N
N
2
R
N
N
R
O
O
O
OMe
R1
hP2X3a (IC50, nM)
1300
R2
hP2X3a (IC50, nM)
M.W.
500
Kin. sol.b
1.7
(lM)
Ex
Ex
O
S
F
8
6
840
OMe
OMe
O
O
8
a
7500
8
1300
7500
442
441
13.9
56.9
O
O
8
8
b
c
6300
9900
1
1
0
1
OMe
N
H
O
O
O
10,700
1300
427
413
91.5
100
N
H
8
8
8
8
d
e
f
4% at 10
6% at 10
l
l
M
M
N
N
12
N
N
N
11% at 10
36% at 10
l
M
13
1600
2100
439
453
83.1
47.2
g
lM
O
O
8
8
8
h
5000
5700
3700
14
N
F
i
1
1
1
5
6
7
N
3800
810
455
482
489
100
1.2
7.7
NO2
O
MeO
O
j
S
O
O
S
8
8
k
l
7500
3700
N
850
MeO
MeO
O
O
S
18
N
720
440
475
449
19.6
40.4
MeO
O
O
S
8
8
8
m
n
2400
580
OMe
19
N
OMe
O
a
Ca2+ assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated
with ,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of
20%.
MeO
MeO
OMe
a
±
b
For determination of the kinetic solubility 10
in a pH 7.4 phosphate buffer.
lM DMSO stock solution was used
o
390
OMe
OMe
8
p
38% at 10
l
M
These data suggest that there must be a new, unknown pocket
in that direction of the receptor, where a powerful hydrogen-
bridge bond is combined with a hydrophobic pocket. Luckily,
kinetic solubility could be improved in parallel with lowering of
the IC50 value of inhibition of P2X3 receptors (6 < 18 < 19).
MeO
OMe
a
Ca2+ assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated
with ,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of
20%.
a
±
The supraadditivity worked excellently when we combined the
1
best
R
group (3,5-dimethoxyphenethyl: 8o) with the most
2
fonyl (17) analogues were equipotent with the fluorophenylsul-
fonyl 6. Attempts to reduce the size of the sulfonamides, resulted
in the same tendency with respect to inhibitory effect on P2X3
receptors, observed with amides; i.e. the substitution of the sul-
fonyl group with pyrrolidine 18 was better than piperidine 17,
while dimethylamino derivative 19 was the most potent analogue
which yielded an IC50 value of 440 nM for inhibiting the P2X3
receptors (Table 2).
promising
R
groups (piperidinosulfonyl 17 and dimethy-
laminosulfonyl 19): the IC50 was 150 nM for compound 20, and
50 nM for 21. When the left side of this latter compound was mod-
ified by introducing a 2,5-dimethoxyphenethyl side chain, which
had not been examined previously, the obtained molecule 22
yielded an IC50 of 20 nM for inhibiting P2X3 receptors and, not neg-
ligibly, possessed a kinetic solubility value, higher than fifty micro-
moles (Table 3).

G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
3899
Table 3
Combination of the selected R and R2 group
1
N
O
S
1
N
2
R
N
R
Ex
R1
R2
hP2X3a (IC50, nM)
M.W.
Kin. sol.b
(lM)
hCLintc
(lL/min/mg)
1
2
3
4
5
A-317491
RO-4
RO-85
AZ-2
220
20
2400
25
566
400
425
484
353
500
P100
407
79
P100
0.2
8
4
13
0.2
AF-219
45
O
S
F
6
840
150
500
1.7
3.8
315
OMe
O
MeO
MeO
MeO
O
S
N
2
0
O
519
287
OMe
OMe
O
S
N
2
2
1
2
50
20
479
479
10.6
59.3
166
167
O
O
S
N
OMe
O
a
b
c
Ca2+ assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated with
For determination of the kinetic solubility 10 M DMSO stock solution was used in a pH 7.4 phosphate buffer.
Metabolic stability performed in human liver microsomes in the presence of NADPH at 37 °C.
a,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of ±20%.
l
N
O
N
O
S
S
N
O
S
O
S
O
a, b MeO
N
c
MeO
N
O
HN
NH
N
N
N
O
OMe
OMe
7
(5% at 10µM)
22a (IC50=11,200 nM)
22 (IC50=20 nM)
a, b
N
S
N
S
O
S
N
N
NH
N
N
MeO
O
c
MeO
O
O
OMe
OMe
2
2b (12% at 10µM)
22c (IC50=7400 nM)
Scheme 2. Synthesis of 22 and its derivatives. Reagents and conditions: (a) 2,5-dimethoxyphenylethyl mesylate, Cs
Me NSO Cl, DIPEA, DCM, rt, 24 h.
2 3
CO , DMF, 60 °C, 2 h; (b) HCl/dioxane, rt, 2 h; (c)
2
2
Compound 22 was obtained by utilizing the route depicted on
Analysis of metabolic pathways of compounds 20–22 clarified
that oxidation and demethylation were the primary reason for
the poor metabolic stability. To avoid oxidation, efforts were made
to introduce a sterical hindrance, while to avoid the demethylation
of the methoxy group, replacement of the methoxys by halogens
was attempted. By introducing a methyl group between the two
nitrogens of the tricycle (in A position of the structure in Table 4)
the inhibition of P2X3 activity decreased dramatically (23), yield-
ing an IC50 value for inhibiting the receptor almost in the micromo-
lar range compared to the anticipated low nanomolar range. The
P2X3 activity turned out to be even lower, when methyl or oxo
group was introduced to the side chain (24, 25 and 26). Although
5-chloro-2-methoxy analogue 27 was very potent (IC50 = 35 nM),
halogenated molecules not having methoxy groups were five to
tenfold less active (28, 29 and 30) than 27. In spite of these efforts,
not only did the microsomal stability of compounds remain very
Scheme 1, but in this case we isolated both the N-alkyl and O-alkyl
intermediates (22a and 22b). The synthesis was continued by
using both analogues. After testing these molecules, it turned out
that the O-alkyl derivative is 370-fold less potent at the P2X3
receptor than the N-alkyl derivative, so, later on, we stopped isolat-
ing these analogues (Scheme 2).
As both water solubility and penetrability properties of com-
pound 22 were deemed acceptable, with values of 59
l
M and
ꢂ6
3
1.1 ꢁ 10 cm/s, respectively, together with lack of an excessive
1
4
outward penetration property (having a PDR value of 0.9),
compound 22 was selected to serve as a lead for further opti-
mization. Compounds 6, 20–22 were suffering from poor stabil-
ity in human microsomes15 (hCLint; Table 3). Consequently, the
course of later optimization work was focused on improving this
property.

3
900
G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
Table 4
Modification of the side-chain of 22
A
N
O
S
O
S
O
1
N
R
N
N
Ex
R1
A
hP2X3a (IC50, nM)
50
clogPb
hCLintc
166
(lL/min/mg)
MeO
2
1
H
1.8
OMe
MeO
MeO
2
2
2
3
H
20
1.8
1.8
167
144
OMe
CH
3
590
OMe
CH3
MeO
MeO
24
25
26
H
H
H
1400
3300
2.2
2.2
1.2
174
282
84
OMe
CH
3
OMe
O
MeO
Cl
13% at 10
lM
OMe
27
28
29
H
H
H
35
2.6
3.3
3.4
266
211
248
OMe
Cl
280
180
Cl
OCF3
F
Cl
3
3
0
1
H
H
380
3.5
1.4
267
51
Cl
HO
inactive
OH
3
2
3
H
H
38% at 10
19% at 10
l
M
M
1.2
0.6
8
4
NH2
H N
2
3
l
N
MeO
3
4
5
H
H
7300
1.1
1.2
55
39
NH2
3
10% at 10
lM
N
MeO
N
3
6
7
N
H
H
7500
1.8
238
1
OMe
O
N
3
N
inactive
ꢂ0.5
a
b
c
Ca2+ assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated with
Calculated logP was determined by MarwinSketch 16.2.1.0 (ChemAxon ) program.
Metabolic stability performed in human liver microsomes in the presence of NADPH at 37 °C.
a,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of ±20%.
Ò

G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
3901
N
O
S
N
O
S
H N
2
S
b
X
a
c
MeO
N
+
HN
X
COOEt
X
O
X
N
EtOOC
OMe
2 2
X: C(O-CH -CH -O), SO
2
Scheme 3. Synthesis of tricyclic analogues. Reagents and conditions: (a) S
8
, morpholine, EtOH, rt, 24 h; (b) Form-amide, ammonium formiate, 140 °C, 16 h; (c) 3,5-
2 3
dimethoxyphenylethyl mesylate, Cs CO , DMF, 60 °C, 2 h.
Table 6
Table 5
N-Acyl and N-alkylsulfonyl derivatives of 22a
Tricyclic analogues (38–43) with different polar moieties
N
S
N
S
MeO
N
2
MeO
N
N R
X
O
OMe
O
OMe
Ex
R2
hP2X3a (IC50, nM)
hCLintb
(lL/min/mg)
2
2
4
2a
2
4
H
SO
CO–CH
11,200
20
140
N.D.
167
14
Ex
X
hP2X3a (IC50, nM)
hCLintb
166
(l
L/min/mg)
2
3 2
–N(CH )
3
O
S
21
38
39
N
N
50
45
35
25
44
O
O
4
4
6
7
59
NH
N
20% at 10
650
lM
N.D.
19
O
O
O
S
15
79
NH
2
O
O
4
4
5
8
9
0
10
40
15
133
196
191
4
0
65
66
O
O
O
4
4
1
2
O
6800
140
N.D.
170
N
O
S
O
5
5
1
2
40
15
138
177
4
3
S
1300
45
O
F
O
F
N.D. means not determined.
O
a
2+
Ca assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated
,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of
F
with
20%.
a
F
F
5
5
5
3
4
5
15
139
9
±
b
O
Metabolic stability performed in human liver microsomes in the presence of
NADPH at 37 °C.
OH
180
85
O
OH
29
low, but also the inhibitory potential of compounds on the activity
of P2X3 receptors was dampened (Table 4).
O
Applying a different approach, we tried to improve microsomal
stability by increasing the polarity (and lowering the logP) of the
left side of 22. Indeed, by replacing the 3,5-dimethoxyphenyl
5
5
6
7
OH
55
16
O
NH2
1400
1000
2
5
(21) moiety with 3,5-dihydroxyphenyl group (31), metabolic sta-
O
O
bility could be improved, but, simultaneously, the P2X3 inhibitory
action of the compound was completely lost. In case of aminophe-
nyl and aminopiridyl analogues (32 and 33), the metabolic stability
on human microsomes was excellent, however, activities on the
P2X3 receptors were unacceptable. Although introduction of a
methoxy group to compound 32 improved the value of IC50 for
inhibiting the P2X3 receptors (34), but in return, it also lessened
the metabolic stability of the compound. The same result was
observed in case of trisubstituted pyrrolo (35) and pyrimidine
N
58
5
9
NH
2
640
3
O
60
61
62
SO
SO
SO
2
2
2
-Me
-i-Pr
-c-Pentyl
40
20
40
36
135
190
N.D. means not determined.
Ca assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated with
,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of ±20%.
a
2+
(36) analogues. Investigation of the pyrimidinone analogue with
a
highly polar properties (37, clogP: ꢂ0.5) revealed that though
b
Metabolic stability performed in human liver microsomes in the presence of
the metabolic stability of the compound was excellent, its P2X3
NADPH at 37 °C.

3
902
G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
N
O
N
S
S
N
O
S
MeO
N
b
MeO
N
HN
a
COOH
COOEt
COOEt
O
OMe
OMe
6
3a
63 (IC50: 48 nM)
64 (IC50: 1000 nM)
N
S
N
O
N
O
S
S
O
MeO
N
O
MeO
N
O
MeO
+
N
d
c
O
N
N
N
OMe
OMe
OMe
6
5 (IC50: 33 nM)
(S)-(-)-65a (IC50: 21 nM)
(R)-(+)-65b (IC50: 668 nM)
Scheme 4. Synthesis of 65. Reagents and conditions: (a) 2,5-dimethoxyphenylethyl mesylate, Cs
2
CO
3
, DMF, 60 °C, 3 h; 62% yield; (b) 30% aqueous NaOH, EtOH, 60 °C, 2 h, 91%
18
2
yield; (c) Me NHxHCl, HOBt, EDCxHCl, trimethyl amine, DMF, rt, 24 h, 70% yield; (e) Chiral chromatography.
Table 7
Properties of the best references and new molecules
2
(RO-4)
5 (AF-219)
22
20
56
65
hP2X3a (IC50, nM)
M.W.
20
45
55
33
400
3.5
P100
8
30.6
0.7
>500
353
1.1
P100
0.2
0.7
12.1
>600
479
1.8
59.3
167
31.1
0.9
458
2.8
P100
16
39.7
1
>500
442
3.7
49.6
11
46.8
0.78
>300
clogPb
c
Kin. sol.
(
l
M)
lL/min/mg)
e
hCLintd
(
-6
VB-Caco-2 (x10 cm/s)
PDRe (efflux/influx)
hERG selectivity (fold)
>1500
a
b
c
Ca2+ assay: mean IC50, hP2X3, HEK293 cells loaded with Fluo-4 and stimulated with
Calculated logP was determined by MarwinSketch 16.2.1.0 (ChemAxon ) program.
Kinetic solubility measured used in a pH 7.4 buffer using HPLC with UV detection for quantification.
a,b-me-ATP; IC50 values are the mean of at least three experiments, with SD of ±20%.
Ò
d
e
Metabolic stability performed in human liver microsomes in the presence of NADPH at 37 °C.
15
VB-Caco-2 measurements and PDR determination based on the literature.
antagonist activity was completely lost. These data support the
earlier published presumption, that there are hydrophobic pockets
in this part of the receptor,14 and that is the reason, why polar ana-
logues bind very weakly, if they bind at all (Table 4).
other than identified so far on the P2X3 receptor. The SAR informa-
tion we have reached suggests that in order to achieve a strong
P2X3 inhibitory activity, a group (CO , S, SO ) being able to form
2 2
hydrogen-bridge bond is essential, which needs to be ‘covered’
with a smaller lipophilic group that fills the hydrophobic pocket
of the receptor (Table 5).
Findings with the N-acyl analogues of intermediate 22a are also
in accordance with the proposed theory. The subsequent work was
directed toward acylation of 22a with various small acid chlorides
and sulfonyl chlorides. As a result, compound 44 possessed a suffi-
Considering these results, it seemed that it was not possible to
cope with the metabolic liability issue by changing the left side of
the molecule, so we carried on to explore the right side in more
details. Since we could identify demethylated sulfonamide group
among the major metabolites of 21–22, we investigated what
polarity increase the right side of the molecule could tolerate
where the activity remained, but the metabolism decreased. Utiliz-
ing literature examples,16 the NH group of the tricycle was
replaced with other polar groups (carbonyl, sulfoxide, Scheme 3).
First, we investigated the close analogues of 21 (IC50 = 50 nM).
Its intermediate 38, as it was expected, demonstrated only weak
activity on P2X3 receptors. No higher potency on P2X3 inhibition
with the di-desmethyl analogue 39 compared to compound 21
was seen either (IC50 value of 650 nM for 39). However, the meta-
cient stability on human microsomes (14 lL/min/mg), while the
P2X3 inhibitory activity (IC50: 140 nM) was worse than that of
compound 22. Although the P2X3 inhibitory action of compounds
could be improved (Me < Et < i-Pro < c-Pro < t-Bu) by increasing the
2
size of R , deterioration of the metabolic stability was found in that
group of compounds (45–48). The most potent P2X3 antagonist
molecule that was synthesized in this series was the tert-butoxy
48 with having an IC50 value of 10 nM, which seemed to have
the optimal size of side chains to fit in to the binding pocket of
the P2X3 receptor. In case of larger groups (49–53) the activity
began to decrease compared to 48. To improve the metabolic sta-
bility, we introduced polar OH and NH groups to the small alkyl
chain. Compounds having amino groups (57–59) had excellent sta-
bility, however, their inhibitory activity on human P2X3 receptors
was only weak with IC50 values of ranging between 640 and
1400 nM. Analogues having OH groups were more active in terms
of P2X3 inhibition, so 56 was the first compound, which yielded an
bolic stability turned out to be significantly better (hCLint: 19
lL/
min/mg) compared to that of 21, which was only 166 L/min/mg
l
indicating that the methylated sulfonamide was indeed vulnerable
by the aspect of metabolic stability. Intermediate acetal 40 pos-
sessed a favorable inhibitory activity on P2X3 receptors with an
IC50 value of 65 nM, while the ketone 41 obtained from it by
hydrolysis turned out to be 2 orders of magnitude less potent than
4
0. Reacting it with thiazole, the intermediate 42 was obtained to
be active on P2X3 receptors, but to be unstable metabolically
hCLint: 170 L/min/mg). Tricyclic sulfon analogue 43 was 10-fold
(
l
acceptable metabolic stability (16 lL/min/mg) and, in parallel, an
less potent than 42, but despite the presence of a polar group in the
molecule, the metabolic stability did not improve significantly.
These data may further indicate the presence of a binding pocket
IC50 on P2X3 receptors of 55 nM, which was below the desired
100 nM level. Although the potencies of these alkylsulfonyl deriva-
tives (60–62) were tended to be better (i.e. IC50 were lower than

G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
3903
4
0 nM) than that of compound 56, alkylsulfonyl derivatives were
Research Division for their support on analytical studies in confir-
mation of the structures.
found to suffer from poor metabolic stability, similarly to the acyl
analogues (Table 6).
As a last attempt, we changed the NH to a CH
2
group and intro-
References and notes
duced a carboxyl-group in the right side of the tricycle. It was
assumed that converting it to an amide with dimethylamine, a
molecule that is as shaped as compound 22 could be achieved.
1
2
.
.
Burnstock, G. Pharmacol. Rev. 1972, 3, 509.
Burnstock, G.; Krügel, U.; Abbracchio, M. P.; Illes, P. Prog. Neurobiol. 2011, 95,
229.
The synthesis was carried out by applying the route depicted in
3. Ford, A. P. Pain Manage. 2012, 2, 267.
Scheme 4 by utilizing the known intermediates 63a.17
4.
Abdulqawi, R.; Dockry, R.; Holt, K.; Layton, G.; McCarthy, B. G.; Ford, A. P.;
Smith, J. A. Lancet 2015, 385, 1198.
All results obtained fulfilled the minimum requirements of the
project: the P2X3 inhibitory action of the ester 63 was in accordance
with our expectations with having an IC50 value of 48 nM, while of
the same value for compound 65 turned out to be even better, i.e.
5.
6.
7.
Ford, A. P.; Undem, B. J.; Birder, L. A.; Grundy, D.; Pijacka, W.; Paton, J. F. R.
Auton. Neurosci. 2015, 191, 16.
Serrano, A.; Mo, G.; Grant, R.; Paré, M.; O’Donnell, D.; Yu, X. H.; Tomaszewski,
M. J.; Perkins, M. N.; Séguéla, P.; Cao, C. Q. J. Neurosci. 2012, 32, 11890.
Bölcskei, H.; Farkas, B. Pharm. Patent Anal. 2014, 3, 1.
3
3 nM. The metabolic stability of this compound succeeded the
8. Ford, A. P.; Smith, S. A.; Dillon, M. P. FASEB J. 2013, 27, 5.
9. The structure of AF-219 was published in the Afferent patent (Ford, A. P.;
McCarthy, B. G. WO 2015/027212). All data of compound 16 are equivalent
5
desired 11
l
L/min/mg value on human microsome.1 Compared to
2
2, the human microsomal stability is far better, albeit the higher
with data of AF-219 in Lancet article (see reference 4).
clogP value of 65. The kinetic solubility and the penetration proper-
ties of compound 65 are also favorable and, in contrast to AF-219,
which is suffering from properties making the molecule probable
to be a substrate of efflux transporters (PDR value of AF-219 is higher
than 10), it is expected that compound 65 may exert its pharmaco-
dynamic effects also in the central nervous system in addition to
its actions in the periphery (Table 7).
2+
1
0. hP2X3 Ca assay protocol: recombinant HEK293 Tet-On cells expressing human
P2X3 receptors were loaded with Fluo-4 and stimulated with a,b-methylene-
ATP; IC50 values are the mean of at least three experiments, with SD of ±20%. All
reference compounds 1–5 were synthesized and measured in our test.
1
1
1
1.
A
screening concentration of 10
l
M
with 0.5% DMSO was applied. The
0
campaign was run in singletons on 384-well plates and yielded an average Z
of 0.63 ± 0.08. The selection criteria were 45% median-polished activity (0.55%
active rate).
2. Wu, C.-H.; Coumar, M. S.; Chu, C.-Y.; Lin, W.-H.; Chen, Y.-R.; Chen, C.-T.; Shiao,
H.-Y.; Rafi, S.; Wang, S.-Y.; Hsu, H.; Chen, C.-H.; Chang, C.-Y.; Chang, T.-Y.; Lien,
T.-W.; Fang, M.-Y.; Yeh, K.-C.; Chen, C.-P.; Yeh, T.-K.; Hsieh, S.-H.; Hsu, J. T.-A.;
Liao, C.-C.; Chao, Y.-S.; Hsieh, H.-P. J. Med. Chem. 2010, 53, 7316.
3. Human P2X3 is known to have a high sequence identify at the ATP binding site
with zebrafish P2X4 (see: Kawate, T.; Michel, J.C.; Birdsong, W.T.; Gouaux, E.
Nature, 2009, 460, 592) but until know there are only speculations about the
structure of P2X3 receptor binding site: i.e. ‘‘suspect that 2 methoxy groups
occupy separate hydrophobic pocket” in the article about the discovery of RO-4
(Carter, D. S.; Alam, M.; Cai, H.; Dillon, M. P.; Ford, A. P. D. W.; Gever, J. R.;
Jahangir, A.; Lin, C.; Moore, A. G.; Wagner, P. J.; Zhai, Y. Bioorg. Med. Chem. Lett.
As compound 65 carries a chiral center, separation of the enan-
tiomers has further improved the inhibitory potency of 65 on P2X3
receptors (Scheme 4). The absolute configuration of compound (ꢂ)
6
5a was assigned as (S) by comparison of its specific rotation with
19
the literature data of a structure analogue. This enantiomer was
much more active than the (+) (R) 65b enantiomer (IC50: 21 nM vs
2
0
6
68 nM).
In summary, we have discovered compounds with P2X3 antag-
2009, 19, 1628).
onistic properties based on a tricyclic core. Starting from a mildly
active HTS hit with poor physicochemical properties compounds
with favorable P2X3 potency, solubility and metabolic stability
could be achieved by the end of the optimization process. On the
basis of the SAR described in the present Letter, proposal for a
new (allosteric) binding site on the P2X3 receptor has been made.
This series of compounds provided us with useful pharmacological
tools, such as 65.
1
4. VB-Caco-2 measurements and PDR determination based on the literature: (a)
Hellinger, É.; Veszelka, Sz.; Tóth, A. E.; Walter, F.; Kittel, Á.; Bakk, M. L.; Tihanyi,
K.; Háda, V.; Nakagawa, S.; Duy, T. D. H.; Niwa, M.; Deli, M. A.; Vastag, M. Eur. J.
Pharm. Biopharm. 2012, 82, 340; (b) Hellinger, É.; Bakk, M. L.; Pócza, P.; Tihanyi,
K.; Vastag, M. Eur. J. Pharm. Sci. 2010, 41, 96.
1
5. 5 mg/mL human liver microsomes (n = 3) were incubated with the test
compound (at 1–2.5
lM) in the presence of NADPH at 37 °C. Clearance
classification ( L/min/mg): low: <11, Medium: between 11 and 38; High: >38.
l
1
6. Andersen, H. S.; Olsen, O. H.; Iversen, A. L.; Sørensen, S. B.; Christensen, M. S.;
Branner, S.; Hansen, T. K.; Lau, J. F.; Jeppesen, L.; Moran, E. J.; Su, J.; Bakir, F.;
Judge, L.; Shahbaz, M.; Collins, T.; Vo, T.; Newman, M. J.; Ripka, W. C.; Møller, N.
P. H. J. Med. Chem. 2002, 45, 4443.
Acknowledgements
1
7. Zhang, C.; Ladouceur, G. H.; Brennan, C., Chandler, B.; Dixin, J.; Miranda, K.; Fan,
D.; Zhu, Q.; Verma, S. K.; Dumas, J., WO Patent 010008, 2005.
18. Analytical chiral separation was performed on Chromasil 5-Cellucoat RP
The authors greatly appreciate Gedeon Richter R&D manage-
ment for the strong support, P2X3 project team members for their
contributions, Compound Profiling Laboratory for analytics, kinetic
solubility and chiral separation of compounds and Spectroscopic
4
8
.5 ꢁ 150 mm, Eluents: water/80%ACN-20%IPA; Ret.time of enantiomers: (R):
.96 and (S): 9.70 min.
1
9. Tanyeli, C., Turkut, E. Tetrahedron Asymm. 2004, 15, 2057. ‘‘Enzyme catalyzed
reverse enantiomeric separation of methyl (±) 3-cyclohexene-1-carboxylate”:
N
S
O
O
O
N
O
O
O
N
(
S)-65a
2
5
25
(
(
S)-(-) [a]
D
= -80.3 (c 1.0, CHCl
3
)
[a]
D
= -45.8 (c 0.1, pyridine)
N
S
O
O
O
N
N
O
O
O
(
R)-65b
2
5
25
R)-(+) [a]
D
= +86.5 (c 1.0, CHCl
3
)
[a]
D
= +40.5 (c 0.1, pyridine)

3
904
G. Szántó et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3896–3904
2
0. Literature findings suggest that the human P2X3 receptor is very sensitive for
chirality. Reports claim that huge differences of the P2X3 antagonistic effect
between the enantiomers exist.
IC50 of Ex.58 (R-enantiomer) = 10 nM vs Ex.87 (S-enantiomer) = 481 nM.
c. in a hydroxy-pyrrolone family in EP 2 336 109, 2010 patent by Shionogi:
IC50 of Ex.I-206 (S-enantiomer) = 630 nM vs -Ex.I-207 (R-enantiomer): 8 nM.
d. in pyrrolo-pyrimidinones published by AstraZeneca in Bioorg. Med. Chem.
Lett. 2012, 22, 2565, see reference 15 (‘‘In all cases the S-enantiomer was the
only one active. The R-enantiomer was consistently inactive.”).
a. the first non-nucleotide antagonist, A-317491 (1, S-enantiomer) is more
potent than R-enantiomer, A-317344 (Ki: 9 nM vs 1,100 nM) in PNAS, 2002,
1
7179.
b. in a pyrido[3,4-d]pyrimidine family in WO 2008/130481 patent by Renovis: