From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor

Article abstract of DOI:10.1016/j.bmcl.2017.09.043

The G protein-coupled P2Y₂ receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y₂R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y₂ receptor discovered using the endogenous P2Y₂R agonist UTP as the chemical starting point.

Full text of DOI:10.1016/j.bmcl.2017.09.043

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
From UTP to AR-C118925, the discovery of a potent non nucleotide
antagonist of the P2Y receptor
2
⇑
Nicholas Kindon , Andrew Davis, Iain Dougall, John Dixon, Timothy Johnson, Iain Walters, Steve Thom,
⇑
Kenneth McKechnie, Premji Meghani, Michael J. Stocks
AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE11 5RH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The G protein-coupled P2Y receptor, activated by ATP and UTP has been reported as a potential drug tar-
2
Received 23 August 2017
Revised 14 September 2017
Accepted 20 September 2017
Available online xxxx
get for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in
the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been
impeded by the limited reported availability of stable, potent and selective P2Y
2
R antagonists. This article
describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of
the P2Y
2
receptor discovered using the endogenous P2Y
2
R agonist UTP as the chemical starting point.
Keywords:
Ó 2017 Elsevier Ltd. All rights reserved.
P2Y
2
Purinergic
AR-C118925
P2
Antagonist
Purinergic receptors are divided into P1 (adenosine) and P2
been reported that ATP released from tumor-cell activated plate-
lets, acting through the P2Y R, induce opening of the endothelial
barrier, leading to migration of tumor cells and hence cancer pro-
liferation. P2Y R antagonists, therefore have therapeutic potential
1
(
ATP, ADP) receptors, with P2 subdivided into P2X (trimeric ion
2
2
channels) and P2Y (metabotropic G-coupled receptors). The eight
members of the P2Y family of receptors, so far characterized, have
been further subdivided based on their primary signaling through
specific coupled G-proteins. The first subgroup P2Y1,2,4,6,11 act
2
8
as anti-metastatic agents.
O
O
3
through Gq and the second group, P2Y12,13,14, through Gi. The
NH
HN
P2Y
sues and cell types. Cell types include: epithelial cells, endothelial
cells, smooth muscles cells and leukocytes. A study using P2Y
2 2
receptor (P2Y R) has been found in a variety of different tis-
N
O
O
N
2
HO
HO
O
O
P
O
O
OH
OH
O
P
O
O
P
O
O
P
O
knockout mice revealed that the receptor mediates 85–95% of
nucleotide-stimulated chloride secretion in the trachea. This sug-
O
O
O
-
O
O
-
-
-
gests that P2Y
2
R agonists have therapeutic potential as a treatment
4Na+
for cystic fibrosis (CF), as activation of this chloride secretion chan-
2
nel could compensate for the defective chloride secretion in the
Despite the appeal of the P2Y R as an important drug target,
2
respiratory epithelium of CF patients.4 Indeed, a P2Y
,5
agonist,
9–11
2
limited reports on P2Y R antagonists have appeared to date
2
diquafosol 2, mediating chloride secretion, has been approved in
and indeed the only reported discovery of drug-like P2Y R antago-
2
Japan for the topical treatment of dry eye disease.6
12
nists is from an industrial research group within AstraZeneca,
1
3,14
Agonism of the P2Y
and neutrophil migration, indicating that P2Y
therapeutic potential as a treatment for psoriasis.
2
R can also lead to keratinocyte proliferation
disclosed within a series of chemical patents.
One of the most
2
antagonists have
potent and selective of these antagonists, AR-C118925, has been
5,7
It has also
used as a tool for pharmacological studies on the P2Y recep-
2
1
5,16
tor.
More recently, its selectivity profile against a range of P2
receptors has been published. AR-C118925 was at least 50-fold
selective against P2Y
4
, P2Y
6
, P2Y11, P2Y12, P2Y14, P2X
2
, P2X
4
and
⇑
Corresponding authors at: School of Pharmacy, The University of Nottingham,
17
7
P2X , whilst ꢀ40-fold against P2X
1
and ꢀ15-fold against P2X
3
.
Centre for Biomolecular Sciences, University Park, Nottingham NG7 2RD, UK.
E-mail addresses: Nicholas.Kindon@nottingham.ac.uk (N. Kindon), michael.
stocks@nottingham.ac.uk (M.J. Stocks).
https://doi.org/10.1016/j.bmcl.2017.09.043
0
960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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2
N. Kindon et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
H N
with lipophilic substituents (Table 1). The introduction of a ben-
zhydryl group into this position (5) gave a compound with a pA
for the P2Y R of 6. P2Y R activity was substantially increased by
either symmetrically adding substituents to the benzhydryl group,
compounds 6, 7, 8, and 9, or by linking the two phenyl rings of the
benzhydryl to form a tricycle, compounds 10, 11, 12, and 13, with
the highest activity achieved with the dibenzosuberenyl group,
2
O
N
N
2
HN
N
O
N
2
2
4
Na+
N
4Na+
O
OH
OH
OH
OH
O
P
O
O
P
O
P
O
O
P
O
P
O
P
O
-O
-O
O
O
-
O
O
-
-O
O-
O
-O
-
O
O
2
compound 13 (pA 8.5).
3
4
The P2Y R antagonist program ultimately required the develop-
2
2
1
Herein we report the design and synthesis of P2Y
which led to the discovery of AR-C118925.
In the absence of a high throughput screening (HTS), the
endogenous agonists, adenosine triphosphate (ATP) 3 and uridine
triphosphate (UTP) 4 were considered as starting points for the
2
R antagonists,
ment of an oral drug-like compound. This necessarily meant
moving away from the high molecular weight, highly charged sub-
stituted nucleotides. Removing the triphosphate group gave the
nucleoside analogue 14 (Table 2). Whilst this compound was sub-
stantially less potent than its parent triphosphate 13, it did retain
research program. The greater selectivity of UTP for P2Y
the other P2 purinoceptors led to its selection as the chemical
starting point. Replacing the b -oxygen of the triphosphate moiety
2
R over
2
some antagonist activity (pA 4.7). Furthermore, it was discovered
that the ribose ring could be replaced with the structurally less
complex 3-methylenebenzoic acid to give 15, with a slight gain
in activity. The rationale for having the carboxylic acid was to
mimic any binding interactions the alpha phosphate of UTP might
c
of UTP with a dichloromethylene unit and substitution of the
oxygen in the 4-position of the uracil with sulfur, gave enhanced
18–20
metabolic stability, whilst maintaining agonist activity.
Inter-
2
have with the P2Y R.
estingly, it was discovered that that P2Y R antagonism could be
achieved through substitution of the 5-position of the uracil ring
2
Changing the linker between the carboxylic acid and phenyl
ring from a direct bond to a one or two atom linker, potentially
Table 1
Exploration of the 5-position of the uracil ring.
S
Cl
R
HN
Na+
O
N
4
R =
O
P
O
P
O
O
OH
OH
O
P
-O
O
-
O
-O
-
Cl
O
Cl Cl
5
6
7
OMe
Cl
Cl
O
OMe
Cl
Cl
8
9
10
11
12
13
a,b
Compound
2 2
P2Y pA
5
6
7
8
9
1
1
1
1
6.0± 0.2
7.9± 0.2
7.8± 0.2
7.1± 0.2
7.7± 0.2
7.2± 0.2
7.3± 0.2
8.0± 0.2
0
1
2
3
8.5 ± 0.2
a
The assay used a human P2Y
2
R clone which was isolated from HL60 cells cDNA and then stably transfected into a Jurkat
cell line. The cloned receptor mediates an increase in intracellular calcium in the cell line, which possesses no endogenous
nucleotide receptor of its own. Inhibition of UTP mediated calcium responses were measured using 17 ktM fluo-3AM dye on a
SPEX Fluomax using 508 nm excitation and 525 nm emission wavelengths at room temperature.
b
n = ꢁ2 replicates.
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N. Kindon et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 2
Replacement of the ribose-triphosphate group.
O
X
OH
OH
O
HO
R =
HN
HO
O
N
R
14
15
O
O
O
HO
O
O
N
HO
HO
HO
O
16
17
18
19
O
O
O
S
O
N
N
HO
HO
N
HO
HO
O
S
0, 21
S
2
22
24
23
Compound
X
P2Y
2
pA2a
1
1
1
1
1
1
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
S
S
S
S
S
S
S
O
S
S
S
4.7 ± 0.2
5.4 ± 0.2
5.7 ± 0.2
5.6 ± 0.2
5.4 ± 0.2
5.9 ± 0.2
5.7 ± 0.2
4.4 ± 0.2
5.8 ± 0.2
5.2 ± 0.2
5.6 ± 0.2
a
n = ꢁ2 replicates.
placing the carboxylic acid group further into the triphosphate
binding region, gave a small increase in activity (16 and 17). In
Scheme 1, which shows the synthesis of compound 13, illus-
trates the general procedure for synthesising the compounds in
Table 1. Selective lithiation of the 5-position of the uracil ring
(32) using the method of Miyasaka followed by addition of 5-
dibenzosuberenone gave the 5-dibenzosuberenol (33). The alcohol
was then reduced using triethylsilane and borontrifluoride ether-
ate. During this reaction, there was some loss of the silyl protecting
groups, giving a mixture of mono, di and tri silylated products.
Reprotection using standard procedure gave the trisilylated pro-
duct (33). Thiation of the 4-position of the uracil was achieved
addition, the P2Y
replacements for the phenyl ring. For example, the furanyl
analogue 19 had a further slight gain in activity (pA 5.9). The
2
R appeared tolerant of a range of hetero aromatic
2
2
2
continuing importance of the 4-thiouracil relative to uracil was
demonstrated with thiazole replacement, where the uracil
analogue was >10-fold less potent (compare compound 20 with
2
1).
Having been able to replace the ribose triphosphate with a
structurally less complicated and synthetically less challenging
group, P2Y R antagonism could be increased through symmetrical
4
using P S10 in pyridine under reflux and removal of the silyl pro-
2
tecting groups using tetrabutylammonium fluoride gave the 5-sub-
0
substitution on the 5-suberenyl substituent 25, 26 and 27 (Table 3).
This combines the two features of change to the 5-benzhydryl
group in the ribose triphosphate series, which independently gave
an increase in receptor antagonism (Table 1).
In a similar manner to compounds discussed in Table 2, moving
the acidic group further into the triphosphate binding region gave
an increase in receptor antagonism. Although the dicarboxylic
acids 28 and 29 had similar activity to the mono carboxylic acid
stituted 4-thiouridine (14). Phosphorylation of the 5 -position of
the ribose was achieved using phosphorus oxychloride followed
by treatment with clodronic acid. The crude product was purified
using DEAE SephadexÒ ion exchange chromatography eluting with
a 0–1 M gradient of triethylammonium bicarbonate followed by
elution through a Na+ form DowexÒ ion exchange column to give
the sodium salt of the product (13).
The synthesis of AR-C118925 is shown in Scheme 2. Bromo
lithium exchange of 5-bromo-2,4-di-tert-butoxypyrimidine23 fol-
lowed by treatment with 2,8-dimethyldibenzosuberanone gave
the corresponding dibenzosuberanol product (34). Treatment
with acetic acid under reflux gave the 5-substituted uracil (35)
3
0, indicating perhaps that only one of the carboxylic acids is
involved in binding to the receptor. Finally, bioisosteric replace-
ment of the carboxylic acid was employed giving the most potent
compound, AR-C118925 (1).
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4
N. Kindon et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 3
Further changes to the substituent on the 1-position of the uracil ring.
R'
O
O
O
N
R =
HO
HO
S
S
HN
O
N
R
25, 26
27
R'
O
O
O
O
O
N
O
O
S
O
HO
HO
HO
HO
N
N
N
H
S
H
H
HO
N
O
O
30
28
29
O
O
O
O
S
O
N
N
N
O
HO
HO
N
H
N
N
H
N
N
H
H
O
1
3
1
0
a
Compound
R
2 2
P2Y pA
2
2
2
2
2
3
3
1
5
6
7
8
9
0
1
Me
Cl
7.0 ± 0.2
6.7 ± 0.2
6.6 ± 0.2
7.4 ± 0.2
7.6 ± 0.2
7.5 ± 0.2
7.2 ± 0.2
7.8 ± 0.2
Me
Me
Me
Me
Me
Me
a
n = ꢁ2 replicates.
in very good yield over the two steps. Bis-silylation of uracil (35)
followed by treatment with 5-bromomethyl-2-furancarboxylic
acid methyl ester, selectively alkylated in the 1-position of the
uracil. Treatment with Lawesson’s reagent then effected the con-
version to the 4-thio uracil. Hydrolysis of the methyl ester gave
the carboxylic acid (25) in good yield. Activation of the car-
boxylic acid with bromo-tris(pyrrolidino)phosphonium hexafluo-
rophosphate and coupling with 1-aminotetrazole gave AR-
C118925 (1).
O
N
O
N
HN
OH
HN
O
(b),(c),(d)
(a)
O
OTBS
OTBS
O
OTBS
OTBS
O
TBSO
TBSO
32
33
2
In conclusion, a potent and selective non nucleotide P2Y R
antagonist, AR-C118925, was discovered using the endogenous
agonist UTP as the chemical starting point for the program. AR-
C118925 has reasonable physicochemical properties for a tool
compound (MWt 537, LogD7.4 of 2.26) and was shown to have a
reasonable rat in vivo pharmacokinetics [i.v. clearance (75 mL/
min/kg), Vss 4.34 L/kg, T1/2 2.12 h)] but was not bioavailable (F
0%). This approach of starting with the endogenous ligand has also
S
S
HN
HN
(e)
O
N
4Na+
O
N
OH
OH
O
OH
OH
O
P
O
P
O
P
O
-
O
-
O
O
O
O-
O-
HO
Cl Cl
2
4
14
13
been used in the P2Y12 program and complimented the alterna-
tive, HTS – Active to Hit, Hit to Lead approach to initiating receptor
Scheme 1. Representative synthesis of nucleotide analogues (5–13). (a) (i) TMEDA
25–27
agonist/antagonist projects at AstraZeneca.
Utilization of AR-
(
(
(
2.2 eq), secBuLi (2.1 eq), THF, ꢂ78 °C (ii) 5-dibenzosuberenone (1.5 eq), THF, 55%
t
C118925 in the chemical literature supporting future potential
clinical applications for P2Y R antagonists has necessitated this
b) (i) Et
3
SiH (2 eq), BF
3
.Et
2 2 2 2
O (2 eq), CH Cl , 0 °C (ii) BuMe SiCl (1.5 eq), imidazole
1.5 eq), DMF, 70% (c) P
4
S
10, pyridine, reflux, 68% (d) TBAF, THF, r.t., 85% (e) (i) 1.3 eq
2
n
POCl
3
, (1.3 eq) proton sponge, PO(OEt)
3
, 16%.
3
3
(ii) clodronic acid (3 eq), Bu N (9 eq), PO
present disclosure and we will be reporting further studies in
due course.
(
OEt)
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N. Kindon et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
O
N
O
N
O
OH
Br
(a)
(b)
N
N
HN
O
O
O
N
H
35
3
4
S
N
S
HN
(f)
(c),(d),(e)
HN
O
O
N
O
O
O
O
H
N
N
N
NH
HO
N
1
25
n
Scheme 2. Synthesis of AR-C118925. (a) (i) BuLi, THF. ꢂ78 °C (ii) 2,8-dimethyldibenzosuberanone, THF (b) AcOH, reflux, 96% over two steps (c) (i) BSTFA (2.2 eq), 1,2-
dichloroethane, reflux (ii) 5-bromomethyl-2-furancarboxylic acid methyl ester, MeCN, reflux, 53% (d) Lawesson’s reagent (1 eq.), 1,4-dioxane reflux, 65% (e) LiOH (5 eq.), 3:1
2 3
MeOH:H O, r.t., 95% (f) aminotetrazole, (1.5 eq) pybrop (1.5 eq), NEt (3 eq), DMF, 60%.
1
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