Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective, dual P2X3/P2X2/3 antagonist for the treatment of pain

Article abstract of DOI:10.1016/j.bmcl.2009.01.097

The purinoceptor subtypes P2X₃ and P2X_2/3 have been shown to play a pivotal role in models of various pain conditions. Identification of a potent and selective dual P2X₃/P2X_2/3 diaminopyrimidine antagonist RO-4

Full text of DOI:10.1016/j.bmcl.2009.01.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1632–1635
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and SAR of novel diaminopyrimidines. Part 2: The discovery
of RO-51, a potent and selective, dual P2X3/P2X2/3 antagonist
for the treatment of pain
a
a
a
a
Alam Jahangir a, , Muzaffar Alam , David S. Carter , Michael P. Dillon , Daisy Joe Du Bois ,
*
Anthony P. D. W. Ford b, Joel R. Gever b, Clara Lin a, Paul J. Wagner a, Yansheng Zhai a, Jeff Zira a
a Department of Medicinal Chemistry, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
b Department of Biochemical Pharmacology, Roche Palo Alto LLC, 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:
The purinoceptor subtypes P2X3 and P2X2/3 have been shown to play a pivotal role in models of various
pain conditions. Identification of a potent and selective dual P2X3/P2X2/3 diaminopyrimidine antagonist
RO-4 prompted subsequent optimization of the template. This paper describes the SAR and optimization
of the diaminopyrimidine ring and particularly the substitution of the 2-amino group. The discovery of
the highly potent and drug-like dual P2X3/P2X2/3 antagonist RO-51 is presented.
Received 17 December 2008
Revised 28 January 2009
Accepted 29 January 2009
Available online 4 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
RO-51
P2X3/P2X2/3
Purinoceptor
ATP
Diaminopyrimidine
Neuropathic pain
RO-4
P2X2/3
P2X purinoceptors are ligand gated ion channels with adeno-
sine-50-triphosphate (ATP) as endogenous ligand.1,2 In the P2X
family of receptors, the homomeric P2X3 and the heteromeric
P2X2/3 receptors are mainly localized on sensory afferent neurons
and mediate the primary sensory effects of ATP. Studies using
P2X3-KO mice and antagonists in the chronic constriction injury
(CCI) and other models have clearly implicated a role in regulating
pain behavior in rodents.3–6
The discovery of the first selective, non-nucleotide dual P2X3/
P2X2/3 antagonist A-317491 and its in vivo efficacy in multiple
chronic inflammatory and neuropathic pain models validated the
potential use of P2X3/P2X2/3 antagonists as a new class of thera-
peutic agents for the treatment of pain.7,8 In the preceding commu-
nication, we reported the discovery of RO-4 (Fig. 1), a highly potent
and selective, drug-like dual P2X3/P2X2/3 antagonist.
pound collection. As a continuation of our work, we extended the
SAR of this series to substituents on the diaminopyrimidine ring.
Among several heterocycles investigated, the diaminopyrimi-
dine was the only ring system that retained reasonable potency.
All the attempts to replace the diaminopyrimidine ring with other
heterocycles resulted in compounds with potencies > 10 lM. Also,
substitution at C-6 consistently lowered the antagonistic potencies
at both P2X3 and P2X2/3 receptors, limiting the exploration of this
position. Although substitution on the C-4 amino group was toler-
ated, it generally resulted in decreased antagonist potency. These
initial results led us to investigate substitution at the C-2 position.
NH2
4
O
N3
This compound was discovered by an extensive structure–activ-
ity relationship (SAR) investigation of the phenyl ring substituents
and linkage atom of a novel 5-benzyl diaminopyrimidine screening
hit identified by a high-throughput campaign of the Roche com-
5
2
6
N1 NH2
MeO
I
RO-4
rP2X3(IC50
)
= 13 nM
hP2X2/3(IC50) = 25 nM
* Corresponding author. Tel.: +1 650 855 5346; fax: +1 650 855 6080.
E-mail address: alam.jahangir@roche.com (A. Jahangir).
Figure 1. Dual acting P2X3, P2X2/3 receptor antagonist RO-4.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.097

A. Jahangir et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1632–1635
1633
Removal of the C-2 amino group, or replacement with an alkyl
group, also resulted in loss of activity (Table 1, Entries 1 and 2).
Next, we investigated substituted amino groups at this position.
The chemistry and SAR pertaining to C-2 amino-substitutions are
presented here.
Compounds 12–14, substituted at the C-2 position of the pyrim-
idine ring, were prepared following the synthetic route shown in
Scheme 1.
over Pd/C in the presence of HCl, without further purification to the
corresponding isopropyl group (3).9 Attempts to purify the tertiary
alcohol 2 consistently produced varying amounts of the methyl-
styrene, produced by elimination of the hydroxyl group, which
was resistant to hydrogenation under various conditions.
Regioselective iodination ortho to the methoxy group was
achieved following the protection of the phenolic OH as the tosyl-
ate (4) and subsequent treatment with I2 in the presence of m-
CPBA with acetic acid as solvent (5).10 Hydrolysis of the tosylate
using aqueous NaOH in tBuOH produced phenol 6 which was alkyl-
ated with TsOCH2CN in THF to give 7 in 90% yield. Using either bro-
mo or iodoacetonitrile instead of TsOCH2CN consistently gave
lower yields. Phenoxy acetonitrile 7 was heated with Bredereck’s
reagent (tert-butoxy-bis(dimethyl-amino)methane) in DMF at
100 °C for 18 h. This reaction generally produced a mixture of
two adducts (8 and 9), both of which were transformed to enamine
10 on heating at 100 °C with aniline HCl in DMF.10 Finally, heating
intermediate 10 with the appropriate guanidine/amidine (11) in
the presence of NaOMe in EtOH produced the desired 2-amino-
substituted-pyrimidines (12–14) in satisfactory yields. Compounds
12a and 12b (Entries 1 and 2, Table 1), in which the C-2 position of
the pyrimidine ring was substituted with H and Me, were prepared
by condensing the enamine 10 with the corresponding amidines
(11: R = H, Me).
Treatment of acetophenone 1 with 2.2 equiv of MeMgCl at 5 °C
gave 90% yield of the tertiary alcohol (2) which was hydrogenated
Table 1
Effect of C-2 substituents on P2X3/P2X2/3 receptor affinity
NH2
4
N 3
2
O
5
6
N
R
MeO
1
I
a,c
b,c
Entry
Compound
R
P2X3
P2X2/3
1
2
3
4
5
6
12a
12b
RO-4
13a
13b
14
H
Me
NH2
NHMe
NHCH2CF3
NMe2
6.4
5.6
8.0
7.3
7.1
5.8
5.6
7.4
6.7
6.7
For synthesis of diaminopyrimidines (13), in which R0 is a
hydroxyalkyl group, the alcohol(s) on the corresponding guanidine
precursors (19) had to be protected prior to condensation with the
enamine 10. The general route for preparation of the protected
guanidines (11, R = NHR0) and hydroxyalkyl-2-aminopyrimidine
analogs is outlined in Scheme 2 and is exemplified by the synthesis
of 13t (RO-51).
<5.0
<5.0
a
FLIPR: mean pIC50, rP2X3 CHO cell.
FLIPR: mean pIC50, hP2X2/3 1321n1c cell.
pIC50 values are the mean of at least three experiments performed in triplicates,
b
c
standard deviation 20%.
Stirring the commercially available N,N-bis(benzyloxycar-
bonyl)-1H-pyrazole-1-carboxamidine (15) with 2-amino-1,3-pro-
panediol (16) for 7 h at ambient temperature produced guanidine
(17) in excellent yield.11 It was essential to protect the hydroxyls
before condensing the substituted guanidine fragment with the
intermediate 11. In this case, TBS protection of both hydroxyl
groups of 17 was achieved using standard conditions (TBDM-
SOTf/lutidine) to give 18.12 The desired guanidine intermediate
OH
O
OH
OH
O
OH
OH
b
a
c
MeO
MeO
MeO
MeO
1
2
3
OTs
OTs
e
d
f
OH
OH
NCbz
OH
NCbz
a
N
MeO
MeO
+
OH
N
CbzHN
b
N
H
CbzHN
H2N
I
H2N
I
15
16
17
4
5
6
OTBS
OTBS
OTBS
c
NH
NCbz
OTBS
CN
CN
CN
O
O
N
H
N
H
CbzHN
g
18
19
+
MeO
NMe2
NMe2
MeO
MeO
Me2N
NH2
N
d
I
I
I
9
OTBS
OTBS
O
7
8
CN
NHPh
O
N
N
MeO
H
MeO
NH2
I
20
I
10
i
O
CN
NHPh
O
h
N
NH2
N
H2N
HN
OH
OH
R
O
N
R
MeO
MeO
e
I
I
10
12: R = H, Me
13: R = NHR'
14: R = NMe2
11
N
N
H
MeO
I
13t (RO-51)
Scheme 1. Preparation of C-2 substituted compounds. Reagents and conditions: (a)
MeMgCl, THF, 5 °C to rt, 90%; (b) H2, Pd/C, EtOH–HCl; (c) TsCl, NaOH, THF–H2O, 95%;
(d) I2, m-CPBA, AcOH, 90–95%; (e) NaOH, tBuOH, H2O, 85–90%; (f) TsOCH2CN, KOtBu,
THF, 85–90%; (g) tBuOCH(NMe)2, DMF 100 °C; (h) PhNH3Cl, DMF, 100 °C, 90–95%
from 7; (i) 11, NaOMe, EtOH, microwave 160 °C, 15–30 min.
Scheme 2. Preparation of C-2 hydroxyalkylamino compounds. Reagents and
conditions: (a) THF, 25 °C, 7 h, 95%; (b) TBDMSOTf (3.2 equiv), lutidine, CH2Cl2,
1.5 h, 70%; (c) Pd/C, H2 (3 atm), EtOH, 2 h, 80%; (d) 10, microwave, NaOMe, EtOH,
160 °C 15–30 min; 86% (e) TBAF, (3.2 equiv), THF, 30 min, 84%.

1634
A. Jahangir et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1632–1635
Table 2
19, ready for cyclization, was then generated by the removal of the
Cbz groups with H2 over Pd/C. The required diaminopyrimidine
ring was constructed by condensing 19 with 10 under microwave
heating at 160 °C to produce 20 in excellent yield. Finally, the
deprotection of TBS groups was accomplished with TBAF under
standard conditions producing 13t (Scheme 2).
Initial SAR results (Table 1) clearly indicated that the C-2 posi-
tion of the pyrimidine ring must retain a single N–H group.
Replacement with either a non-amino moiety, such as H (12a, En-
try 1) or Me (12b, Entry 2) or a tertiary amino group, such as NMe2
(14, Entry 6) was detrimental to antagonist activity at both P2X3,
and P2X2/3 receptors. On the contrary, both monoalkyl amino ana-
logs 13a and 13b (Entries 4 and 5, Table 1) retained moderate
inhibitory potencies for both P2X3 and P2X2/3 receptor compared
to RO-4.
The essential requirement of NH at C-2 for P2X3 and P2X2/3
receptors activity prompted us to investigate the SAR of mono
substituted amines (R = NHR0) (Table 2). All compounds (13a–
13w) in Table 2 were prepared by condensing enamine (10) with
the appropriately substituted guanidines 11 (R = NHR0, Scheme 1).
In general, compounds containing basic amines (13c, 13f–13j,
Entry 1, 4–8; Table 2) exhibited only moderate antagonistic poten-
cies at rP2X3 and hP2X2/3 receptors. Interestingly, acylation of the
terminal C-2 NH2 of 13c, compound 13d (Entry 2, Table 2), resulted
in a 14-fold increase in rP2X3 and a 15-fold increase in hP2X2/3
antagonist potency.
With the exception of 13v (Entry 20), all the analogs containing
one or more OH groups were consistently highly potent. It is pos-
sible that the steric hindrance due to the gem-dimethyl group in
13v created the unfavorable conformation of the R group and
therefore had a negative impact on potencies relative to 13p and
13q (Entry 14 and 15). Conversion of the hydroxyl group of 13m
to the corresponding methyl ether 13n (Entry 12, Table 2) resulted
in substantial loss in potency at both targeted receptors. SAR of the
C-2 hydroxyalkylamino analogs also revealed a modest preference
for the two carbon alkyl chain over the three carbon one. Pyrimi-
dines 13m and 13t (Entries 11 and 18, Table 2) containing
hydroxyethyl moieties were more potent than their homologated
analogs 13o and 13u (Entries 13 and 19, Table 2).
Structures and activities of mono substituted amines at C-2
NH2
O
N
2
R'
N
H
MeO
N
I
Entry
Compound
R0
rP2X3a,c
hP2X2/3b,c
1
2
3
4
13c
13d
13e
13f
CH2CH2NH2
6.6
8.0
7.8
6.8
5.9
7.4
7.4
6.6
CH2CH2NHCOMe
CH2CH2SO2Me
CH2CH2NMe2
5
13g
13h
13i
13j
13k
13l
6.6
6.4
7.2
6.8
6.9
7.0
6.2
6.0
6.6
6.4
6.4
6.5
CH2CH2N
CH2CH2N
6
OH
7
CH2CH2N
CH2CH2N
NH
8
O
9
HC
HC
O
O
N S
O
10
11
12
13
13m
13n
13o
CH2CH2OH
CH2CH2OMe
CH2CH2CH2OH
8.0
7.2
7.8
7.8
7.2
7.2
OH
HC
14
15
13p
13q
8.2
8.0
7.8
7.5
CH3
OH
HC
CH3
CH3
16
17
13r
13s
7.9
8.1
7.6
7.5
OH
H2C
As evident from the pIC50 data in Table 2, compounds with di-
hydroxyalkyl groups (13s, Entry 17) were somewhat more potent
than their mono-hydroxyalkyl analogs (13o, Entry 13). Compound
RO-51 (13t, Entry 18) was the most potent analog in the series.
Compared to RO-4 (Entry 3, Table 1), RO-51 is seven- and fivefold
more potent as an antagonist at rP2X3 (IC50 2 nM) and hP2X2/3 (IC50
5 nM), respectively.
Encouraged by its high potency, RO-51 was chosen for further
pharmacological evaluation. This ligand proved to be highly selec-
tive for P2X3 and P2X2/3 exhibiting no antagonistic activity at other
P2X receptor family members tested (P2X1, P2X2, P2X4, P2X5, and
OH
CH2
OH
OH
HC
18
19
20
13t (RO-51)
13u
8.7
8.2
7.0
8.0
8.3
7.5
6.1
7.3
OH
OH
CH2
OH
P2X7) at concentrations up to 10
standard panel of 50 receptors, ion channels, enzymes, and trans-
porters, showed no significant activity (IC50 >10
M).13 In vitro
lM. CEREP profiling against the
CH3
CH2OH
CH3
13v
l
safety assessment (CYP450 inhibition, hERG blockade, Ames test,
phototoxicity) of RO-51 also revealed an attractive profile for fur-
ther evaluation. In permeability measurements using Caco-2 cells,
RO-51 proved highly permeable with Papp (AB) of 9.43 ꢀ 10ꢁ6 cm/s
and a lack of any efflux tendencies.
Single dose rat pharmacokinetics (SDPK) of RO-51 were also
evaluated. The results are summarized in Table 3 and are compara-
ble to those of RO-4.
OH
CH3
OH
21
13w
H2C
a
FLIPR: mean pIC50, rP2X3 CHO cell.
FLIPR: mean pIC50, hP2X2/3 1321n1c cell.
pIC50 values are the mean of at least three experiments performed in triplicates,
b
c
standard deviation 20%.
In rat both compounds suffered rapid clearance, short half-lives,
and high protein binding. However, in a dog SDPK assessment, RO-
51 (CL: 4.65; T1/2: 3.88 h; %F: 79) was comparatively superior to
RO-4 (CL: 13; T1/2: 1.5 h; %F: 72).
In summary, further optimization of the first selective, drug-like
P2X3/P2X2/3 antagonist RO-4 resulted in the discovery of the more
potent RO-51. Selectivity testing, in vitro safety assessment and

A. Jahangir et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1632–1635
1635
Table 3
4. Cockayne, D. A.; Hamilton, S. G.; Zhu, Q.; Dunn, P. M.; Zhong, Y.; Novakovic,
S.; Malmberg, A. B.; Cain, G.; Berson, A.; Kassotakis, L.; Hedley, L.; Lachnit,
W. G.; Burnstock, G.; McMahon, S. B.; Ford, A. P. D. W. Nature 2000, 407,
1011.
Rat SDPK dataa of RO-4 and RO-51
Compound
Tmax
Cmax
AUC
T1/2
CL
%F
(h)
(ng/ml)
(ng/h/ml)
(h)
(mg/min/kg)
5. Barclay, J.; Patel, S.; Dorn, G.; Wotherspoon, G.; Moffatt, S.; Eunson, L.; Abdel’al,
S.; Natt, F.; Hall, J.; Winter, J.; Bevan, S.; Wishart, W.; Fox, A.; Ganju, P. J.
Neurosci. 2002, 22, 8139.
6. Dorn, G.; Patel, S.; Wotherspoon, G.; Hemmings-Mieszczak, M.; Barclay, J.; Natt,
F. J. C.; Martin, P.; Bevan, S.; Fox, A.; Ganju, P.; Wishart, W.; Hall, J. Nucleic Acids
Res. 2004, 32, e49/1.
RO-4 (iv)
0.083
0.167
1.33
589
554
64.3
104
628
495
240
237
0.94
1.07
2.09
1.52
52.8
71.9
—
RO-51 (iv)
RO-4 (po)
RO-51 (po)
39
48
0.833
—
7. Jarvis, M. F.; Burgard, E. C.; McGaraughty, S.; Honore, P.; Lynch, K.; Brennan, T.
J.; Subieta, A.; Van Biesen, T.; Cartmell, J.; Bianchi, B.; Niforatos, W.; Kage, K.;
Yu, H.; Mikusa, J.; Wismer, C. T.; Zhu, C. Z.; Chu, K.; Lee, C.; Stewart, A. O.;
Polakowski, J.; Cox, B. F.; Kowaluk, E.; Williams, M.; Sullivan, J.; Faltynek, C.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 17179.
8. Ito, K.; Iwami, A.; Katsura, H.; Ikeda, M. Naunyn-Schmiedeberg’s Arch. Pharmacol.
2008, 377, 483.
9. Dillon, M. P.; Jahangir, A. U.S. Patent 2008207619, 2008; Chem. Abstr. 2008, 149,
307873.
a
Calculated from 2 mg/kg iv and oral (po) dosing.
SDPK evaluation indicated that RO-51 can serve as a valuable tool
in determining the in vivo pharmacological effects of dual P2X3/
P2X2/3 antagonists.
References and notes
10. Dillon, M. P.; DuBois, D. J.; Jahangir, A. U.S. Patent 20082076551, 2008; Chem.
Abstr. 2008, 149, 307874.
11. Brands, M.; Endermann, R.; Kruger, J.; Raddatz, S.; Stoltefub, J.; Belov, V. N.;
Nitzmov, S.; Sokolov, V. V.; Meijere, A. J. Med. Chem. 2002, 45, 4246.
12. Corey, E. J.; Cho, H.; Rucker, C.; Hua, D. H. Tetrahedron Lett. 1981, 22, 3455.
13. CEREP 50 screen performed by Cerep Corp. 15318 NE 95th St. Redmond, WA
98052 USA. www.cerep.com.
1. Gever, J. R.; Cockayne, D. A.; Dillon, M. P.; Burnstock, G.; Ford, A. P. D. W.
Pfluegers Archiv. 2006, 452, 513.
2. Dubyak, G. R. Mol. Pharmacol. 2007, 72, 1402.
3. Novakovic, S. D.; Kassotakis, L. C.; Oglesby, I. B.; Smith, J. A. M.; Eglen, R. M.;
Ford, A. P. D. W.; Hunter, J. C. Pain 1999, 80, 273.