Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.12.044

Despite the extensive literature describing the role of the ATP-gated P2X₃ receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In th

Full text of DOI:10.1016/j.bmcl.2009.12.044

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1031–1036
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of RO-85, a novel drug-like, potent, and
selective P2X3 receptor antagonist
a
b
b
a
Christine E. Brotherton-Pleiss a, , Michael P. Dillon , Anthony P. D. W. Ford , Joel R. Gever , David S. Carter ,
*
Shelley K. Gleason a, Clara J. Lin a, Amy G. Moore a, Anthony W. Thompson a, Marzia Villa a, Yansheng Zhai a
a Department of Medicinal Chemistry, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
b Department of Biochemical Pharmacology, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite the extensive literature describing the role of the ATP-gated P2X3 receptors in a variety of phys-
iological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-
like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-
like, potent and selective P2X3 antagonist. High-throughput screening of the Roche compound collection
identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimi-
zation, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-like-
ness resulted in the discovery of RO-85.
Received 24 September 2009
Revised 6 December 2009
Accepted 10 December 2009
Available online 16 December 2009
Keywords:
P2X3
Ion channel
Ó 2009 Elsevier Ltd. All rights reserved.
RO-85
Inflammatory pain
Neuropathic pain
Drug-like
P2X3 receptor antagonist
P2X receptors are ligand gated ion channels activated by aden-
osine 50-triphosphate (ATP) and related di- and tri-phosphate
nucleotides. Seven P2X receptor subunits have been identified
P2X1-7 and each channel shown to be assembled from three sub-
units.1 Understanding the pharmacology of the channels is com-
plex in that, with the exception of P2X6, in addition to
homomeric channels, each subunit also has the ability to form het-
eromeric channels and a total of seven heteromeric P2X family
members have been identified.2 Homomeric P2X3, and the closely
related heteromultimeric P2X2/3, receptors are predominantly co-
localized on small to medium diameter sensory afferent neurons
and have become increasingly recognized as playing a major role
in mediating the primary sensory effects of ATP. P2X3-KO mice
demonstrate a reduced sensitivity to thermal stimuli and de-
creased pain behaviors.3 Furthermore reduction of P2X3 expression
through intrathecal administration of P2X3-selective antisense,4
and more recently siRNA,5 also causes a significant decrease in pain
behaviors in mice. By eliminating the P2X3 subunit these gene
deletion and knockdown techniques also eliminate the P2X2/3
receptor so a clear understanding of the receptor responsible for
these effects is hard to ascertain.
Despite significant advances in elucidating the pharmacology of
P2X3 receptors, the field has suffered from a lack of drug-like, po-
tent and selective ligands. Many of the reported selective P2X3
antagonists (Fig. 1), including the most recently reported highly
potent peptide antagonist Spinorphin (1),6 either do not report
selectivity over P2X2/3 or are dual P2X3/P2X2/3 antagonists like
TNP-ATP (2)7 and A-317491 (3),8 as well as our recently published
diaminopyrimidines, (4a) and (4b).9,10
In addition to the unknown, or lack of selectivity over P2X2/3
making their pharmacology hard to understand, these ligands also
show poor drug-like properties. TNP-ATP and A-317491, similar to
many of the non-selective P2X3 receptor antagonists, contain poly-
acidic functional groups that limit their bioavailability and overall
potential as drug candidates. Despite this A-317491 has been
shown to be active in a number of in vivo models of chronic inflam-
matory pain, neuropathic pain8 and overactive bladder.11 No data
has been published on the drug-like properties of spinorphin
which has also been shown to be effective in the tail pinch assay
following intracerebroventricular (ICV) administration.12 In this
Letter we report the discovery and optimization of a series of
drug-like potent and selective P2X3 antagonists.
A high-throughput screening campaign of the Roche compound
collection using the rat recombinant P2X3 receptor was performed.
Disappointingly, in addition to a very overall low hit rate of 0.01%,
many of the more potent sub-micromolar hits contained similar
* Corresponding author. Tel.: +1 408 569 3422.
E-mail address: brotherton.pleiss@gmail.com (C.E. Brotherton-Pleiss).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.044

1032
C. E. Brotherton-Pleiss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1031–1036
NH2
N
HN
N
HO
O
P
O
P
O
P
O
N
N
O
O
HO
O
O
O
N
OH OH OH
O
N
H
N
H
H
N
N
O
H
O
O
O2N
O
NO2
O
OH
O
N
OH
H
H2N
NO2
Spinorphin (LVVVPWT) (1)
COOH
COOH
TNP-ATP (2)
NH2
O
HOOC
N
O
O
N
NHR
MeO
N
I
(4a) R = H
(4b) R = CH (CH2OH)2
A-317491 (3)
Figure 1. Structures of known P2X3 antagonists.
poly-acidic functionality to the known antagonists with their im-
plied poor drug-like properties. These were not deemed suitable
starting points for a medicinal chemistry program aimed at pro-
ducing potential therapeutic agents so a careful analysis of the less
potent hits was undertaken. Four compounds, initially synthesized
as part of a ꢀ4500 member parallel synthesis library, were identi-
fied as hits. The library was based on a 2,4-disubstituted-oxazole-
5-carboxylic acid amide core structure. The hits were characterized
by a 2-phenyl substituent on the oxazole core and, more specifi-
cally, the presence of a 1-methyl-2-(4-pyrimidin-2-yl-piperazin-
1-yl)-ethylamine amide (Fig. 2).
The role of the oxazole scaffold was investigated using both a
general collection of acids fitting a similar motif and by a tradi-
tional iterative SAR approach. A representative set of similar
amides is shown in Table 1. For five-membered scaffolds, pyrazole
amides (6a) and (6b) and furans (6c) and (6d) had similar activity
to one of the other initial hits (5b). Substitutions that altered the
geometry of the amide bond, (6g) and (6h) were not tolerated. Sub-
stitution of the butyl or phenyl group for a methyl gave a signifi-
cant drop in activity (6i) versus (5a) and (5b). Interestingly,
substitution of the phenyl groups in a 3,4 relationship as in (6j)
and (6f) led to no loss in activity.
A detailed pharmacological and drug-likeness characterization
of (5a) was undertaken. (5a) was shown to be a selective antago-
nist of rP2X3 (pIC50 6.6) with no antagonist activity at P2X2/3 or
other family members tested (pIC50 <5). In a standard CEREP pro-
file of 80 other enzymes, receptors and ion channels only five other
A six-membered ring scaffold was also accepted, as shown in
examples (8a–8f), even with only one aryl substituent. An interest-
ing 5-5-bicylic core, 1-methyl-3-phenyl-1H-thieno[2,3-c] pyra-
zole-5-carboxylic amide (7), discovered using parallel synthesis
was found to show promising opportunities by allowing truncation
of the propyl group to a methyl group.
activities were identified in the 5–10 l
M range.13 In an assessment
of drug-likeness; with positive attributes of moderate molecular
weight (434) and calculated log P (2.7), and high permeability
without efflux it was disappointing to find high in vitro clearance
in rat and human microsomes and hepatocytes, and >99% protein
binding in rat plasma. In addition (5a) was a moderate inhibitor
Exploration of the piperazinyl-ethylamine linker of the mole-
cule showed little opportunity for modification (see Fig. 3) with
the exception of a preference for the R-stereochemistry (Table 2).
The pyrimidine group could, however be modified with other het-
erocycles, without losing activity at the P2X3 receptor (9a–9f) as
long as a nitrogen was present in the 2-position to act as an elector
acceptor (see Table 2). The m-pyridine and p-pyridine were inac-
tive (9g and 9h). Interestingly, a simple carbonyl group was shown
to be sufficient for good affinity as long as the group remained pla-
nar. For example, acetyl amide (10a), thioacetyl amide (10c) and
urea (10d) were all active, whereas the cyclopropylcarbonylamide
(10f) and other amides not shown here, the substituted urea (10e),
sulfonamide (10g) and ethyl carbamate (10h) were inactive. Of
particular significance was the promising activity of (10a) with a
concurrent improvement in molecular weight and lipophilicity.
of the CYP450 isozymes 3A4 and 2D6 with IC50’s <10 lM. A parallel
optimization approach exploring replacement of the oxazole core
and its substituents, and the amide portion of the molecule was
undertaken.
N
N
N
N
N
N
N
N
O
O
O
O
These analogs were synthesized easily starting from Boc-
D-alaninal
N
N
as shown in Scheme 1.
Reaction of Boc-D-alaninal with N-acetylpiperazine in the pres-
N
N
ence of NaBH(OAc)3 afforded intermediate (15) in 85% yield. Re-
moval of the Boc group with TFA, followed by coupling with
commercially-available 1-methyl-3-phenyl-1H-thieno[2,3-c]pyra-
zole-5-carboxylic acid using a HBTU coupling afforded the amide,
which was converted the oxalate salt (16).14 Similar reductive ami-
nation conditions, followed by deprotection were used to prepare
N-arylpiperidines (14a–14h), which were coupled to 2-(4-fluoro-
phenyl)-4-propyl-oxazole-5-carboxylic acid15 using DCC and HOBT
(5b) rP2X3 pIC50 5.9
(5a) rP2X3 pIC50 6.6
hP2X2/3 pIC50 < 5
hP2X2/3 pIC50 < 5
Figure 2. Hits structures identified by high-throughput screening and their pIC50’s
at the rat P2X3 receptor.

C. E. Brotherton-Pleiss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1031–1036
1033
Table 1
Heterocyclic core SAR
Table 1 (continued)
a,b,c
No.
R
pIC50
5.9
N
O
O
N
N
N
N
N
R
6j
N
Ph
Ph
a,b,c
No.
R
pIC50
6.0
Ph
O
Ph
N
N
N
N
N
N
6a
8a
8b
8c
5.9
5.3
6.4
N
Ph
Ph
Ph
N
O
O
O
O
O
O
Ph
N
N
N
N
6b
6c
6d
5b
6.0
6.2
5.7
5.9
N
N
N
Ph
Ph
Ph
Ar
Ar
O
Ph
Ph
O
O
O
O
N
N
N
8d
6.3
Ph
Ph
Ar
Ar
Ar
N
Br
O
N
8e
8f
6.3
6.9
Ph
Ph
O
O
N
N
6e
6f
5.4
5.7
O
Ph
Ar = p-F-phenyl
N
Ph
a
FLIPR: mean pIC50, rP2X3 CHO cell.
FLIPR: mean pIC50 <5.0, hP2X2/3 1321n1c cells.
pIC50 values are the mean of at least three experiments performed in triplicates,
b
c
Ph
Ph
standard deviation +20%.
O
to afford the corresponding amides (9a–9h). Intermediate (17),
prepared by reductive amination of Boc-D-alaninal with Cbz-piper-
azine, was coupled the same acid to provide intermediate (18). Re-
moval of the Cbz group by catalytic hydrogenation afforded the
free piperazine, which was functionalized to afford (10a–10f) using
standard conditions.
N
6g
6h
7
<5
<5
6.7
N
S
Ph
Ph
O
O
N
N
N
The thienopyrazole template, discovered with analog (7) and
which had the advantage of having good drug-likeness and for
not requiring a longer alkyl chain with the potential metabolic lia-
bilities, was explored further. The results are shown in Table 3.
Substitution of the phenyl ring in the 3-position with an ortho fluo-
rine also led to active analog (11a), however an ortho-methyl sub-
stituent was less tolerated (11b). Increasing the size of the N-1
substituent to an ethyl slightly decreased activity (11c) and intro-
duction of a phenyl group led to an inactive analog (11d). Changing
the sulfur to an oxygen to give (12) led to an inactive molecule.
Combining the substitution of an acetyl for a pyrimidyl group
and the thienopyrazole core template led to potent molecules
(11f–11h, Table 3). Resolution of the amide side chain resulted in
the identification of (R) as the preferred stereochemistry; thus 1-
methyl-3-phenyl-1H-thieno[2,3-c]pyrazole-5-carboxylic acid [(R)-
N
Ph
S
N
N
Ph
O
N
6i
5.3
N
O
Ph

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C. E. Brotherton-Pleiss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1031–1036
N
N
N
N
N
N
N
O
N
N
O
O
N
N
N
N
N
H
N
N
N
O
N
H
H
O
O
rP2X3 pIC50 < 5
F
(5a) rP2X3 pIC50 6.6
rP2X3 pIC50 5.6
F
N
N
N
N
N
O
N
O
N
N
O
N
N
N
N
N
N
N
H
N
N
N
H
O
O
O
F
rP2X3 pIC50 < 5
F
F
rP2X3 pIC50 < 5
rP2X3 pIC50 < 5
N
N
N
N
N
O
N
N
N
N
N
N
N
N
N
H
N
N
O
N
N
O
O
rP2X3 pIC50 < 5
rP2X3 pIC50 < 5
rP2X3 pIC50 < 5
F
F
F
Figure 3. Linker modifications and their pIC50’s at the rat P2X3 receptor.
Table 2
Acceptor group SAR
Table 2 (continued)
a,b,c
No.
R
Stereo-chem.
R,S
pIC50
<5
R
N
N
O
9g
N
N
N
*
O
9h
N
R
R
<5
10a
Ac
6.4
F
a,b,c
No.
R
Stereo-chem.
pIC50
7.3
O
O
10b
R
5.6
N
N
N
N
N
9a
R
S
10c
10d
R,S
R
7.0
6.0
9b
9c
9d
9e
9f
S
5.7
5.8
6.3
5.9
6.2
O
NH2
R
O
O
10e
10f
R
R
<5
<5
NHMe
N
N
R
N
N
10g
10h
–SO2Me
–CO2Et
R
R
<5
<5
R
a
b
c
FLIPR: mean pIC50, rP2X3 CHO cell.
FLIPR: mean pIC50 <5.0, hP2X2/3 1321n1c cells.
pIC50 values are the mean of at least three experiments performed in triplicates,
N
S
R,S
standard deviation +20%.

C. E. Brotherton-Pleiss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1031–1036
1035
O
O
a
b, c, d
Me
N
N
Me
Me
O
S
OHC
e, b
NHBoc
N
N
N
NHBoc
N
N
15
13
HO2CCO2H
16 (RO-85)
Cbz
N
O
Cbz
Me
f
X
N
Me
N
N
Me
N
N
N
N
NH3
+
O
NHBoc
17
18
14a, X = pyrimidin-2-yl
14c, X = 2-pyridyl
14d, X = pyrazin-2-yl
14e, X = pyrimidin-4-yl
14f, X = thiazol-2-yl
14g, X = 3-pyridyl
F
f
g, h
14h, X = 4-pyridyl
R
Ar
N
O
Me
N
O
Me
N
N
N
N
N
N
O
O
10a, R = -Ac
10b, R = 2-furanoyl
10d, R = -C(=O)NH2
10e, R = -C(=O)NHMe
10f, R = -C(=O)c-propyl
10g, R = -SO2Me
9a, Ar = pyrimidin-2-yl
9c, Ar = 2-pyridyl
9d, Ar = pyrazin-2-yl
9e, Ar = pyrimidin-4-yl
9f, Ar = thiazol-2-yl
9g, Ar = 3-pyridyl
10
F
F
9h, Ar = 4-pyridyl
10h, R = -CO2Et
Scheme 1. Reagents and conditions: (a) N-acetylpiperidine, 1.2 equiv, DCE, NaHB(OAc)3, 2 equiv, 20 h ; (b) TFA, CH2Cl2 2 h; (c) HBTU, 1.5 equiv, excess DIEA, THF; (d) oxalic
acid, 1.1 equiv, ether; (e) CH2Cl2; N-arylpiperazines (2-piperazin-1-ylpyrimidine, 1-pyridin-2-ylpiperazine, pyrazin-2-ylpiperazine, 4-piperazin-1-ylpyrimidine, 1-thiazol-2-
yl-piperazine, 1-pyridin-3-yl-piperazine, or 1-pyridin-4-yl-piperazine), 1.2 equiv, DCE, NaBH(OAc)3, 2 equiv; (f) (i) 2-(4-fluorophenyl)-4-propyl-oxazole- 5-carboxylic acid,
DCC on resin, HOBT, CH2Cl2, 2 h, (ii) amine (14a–14h, 17), CH2Cl2, DIEA 20 h; (g) Pd/C (10% Degussa), H2, EtOH; (h) for 10b 2-furoylchloride, CH2Cl2, DIEA; for 10d KOCN,
AcOH, H2O; for 10e CH3NCO, CH2Cl2; for 10f c-C3H5COCl, CH2Cl2, DIEA, for 10g CH3SO2Cl, CH2Cl2, DIEA, for 10h ClCO2Et, CH2Cl2, DIEA.
Table 3
original hit structure were also retained (MW 425, c log P 2.8).
SAR of 1H-thieno[2,3-c]pyrazole-5-carboxamides
RO-85 showed significantly higher in vitro metabolic stability16
in rat and human liver microsomes with 89% protein binding in
R3
rat plasma. No inhibition of the CYP450 isozymes was seen at con-
centrations <15 M. When dosed orally to rats RO-85 was 89% or-
N
O
l
N
X
R1
N
H
N
N
ally bioavailable with a half-life of 1.6 h.
In summary we have identified a novel, potent and selective
P2X3 receptor antagonist RO-85. This was discovered through the
rapid optimization of a hit series identified by high-throughput
screening facilitated by parallel synthesis. RO-85 is exquisitely
selective for the P2X3 receptor subtype over other P2X family
members and other pharmacological targets and has suitable phar-
macokinetic properties to explore the therapeutic potential of P2X3
antagonists in vivo. Additional SAR studies will be published in the
future.
R2
No.
X
R1
R2
Ph
R3
pIC50
7
S
S
S
S
S
S
S
S
S
O
Me
Me
Me
Et
Ph
Ph
Me
Me
Me
Me
Pyrimidyl
Pyrimidyl
Pyrimidyl
Pyrimidyl
Pyrimidyl
Pyrimidyl
Acetyl
Acetyl
Acetyl
Pyrimidyl
6.7
7.2
5.7
6.3
<5
11a
11b
11c
11d
11e
11f
11g
11h
12
o-F-Ph
o-Me-Ph
Ph
Ph
Me
Ph
5
7.0
7.4
7.4
<5
p-Me-Ph
o-F-Ph
Ph
References and notes
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man receptors (rP2X3 pIC50 = 7.5; hP2X3 = 6.4) selective over
hP2X2/3 and the other P2X family members tested (pIC50 <5). No
other pharmacological activity was seen in a standard CEREP panel
of 80 other enzymes, receptors and ion channels (no activity >50%
at 10 l
M).13 Favorable molecular properties comparable to the

1036
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16. HLM = 1.76
lL/min/mg (high stability), RLM = 43.7 lL/min/mg (medium
stability), rat hepatocytes = 3.93 mL/min/million cells (high stability).