Novel nucleotide triphosphates as potent P2Y2 agonists with enhanced stability over UTP

Article abstract of DOI:10.1016/j.bmcl.2006.10.038

The synthesis of a series of novel C-linked nucleotide triphosphates is reported. These exhibit excellent agonist potency and selectivity for the P2Y₂ receptor with a number of examples having EC₅₀ values below 10 nM. Representative compounds from the N-linked and C-linked series showed enhanced metabolic stability compared with that of the natural ligand UTP.

Full text of DOI:10.1016/j.bmcl.2006.10.038

Bioorganic & Medicinal Chemistry Letters 17 (2007) 558–561
Novel nucleotide triphosphates as potent P2Y2 agonists
with enhanced stability over UTP
Richard J. Davenport,a,* Paloma Diaz,a Frances C. A. Galvin,a Steve Lloyd,a
Stephen R. Mack,a Ray Owens,b Verity Sabina and Joanne Wynna
aUCB-Group, Granta Park, Abington, Cambridge CB1 6GS, UK
bWellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK
Received 30 August 2006; revised 16 October 2006; accepted 16 October 2006
Available online 19 October 2006
Abstract—The synthesis of a series of novel C-linked nucleotide triphosphates is reported. These exhibit excellent agonist potency
and selectivity for the P2Y2 receptor with a number of examples having EC50 values below 10 nM. Representative compounds from
the N-linked and C-linked series showed enhanced metabolic stability compared with that of the natural ligand UTP.
Ó 2006 Elsevier Ltd. All rights reserved.
Activation of P2Y2 receptors has potential therapeutic
utility in aiding mucociliary clearance and stimulating
tear production.1,2 UTP is a potent natural agonist for
the P2Y2 receptor, but is chemically unstable, readily
metabolised and consequently has a very short duration
of action. Inspire Pharmaceuticals have developed the
P2Y2 agonists INS365 and INS37217 for dry eye disease
and cystic fibrosis, respectively.3 Their enhanced stabili-
ty compared to UTP is attributed to them being capped
tetraphosphates.4,5 It has recently been reported that the
diadenosine penta(borano)phosphate 1 is a potent P2Y1
agonist with greatly enhanced metabolic stability,
achieved by the presence of a borano group at the enzy-
matic cleavage site.6 We considered whether the use of
nucleotide triphosphates bearing unnatural bases could
maintain good agonist potency against the P2Y2 recep-
tor and also improve stability. In the preceding article7
we disclosed a series of N-linked compounds, such as
the 7-fluoroisocarbostyril nucleotide triphosphate 2,
which had P2Y2 agonist potency comparable with that
of the natural ligand UTP.
H2N
NH2
N
N
N
N
O
P
O
P
O
P
O
P
O
P
N
N
O
N
O
O
O
O
N
O
O
O
OH
HO HO
BH3
OH
1
OH
HO
HO
OH
O
O
P
O
P
O
P
N
HO
O
O
O
F
OH OH OH
O
2
HO
OH
Three different C-linked unnatural bases, benzothiaz-
ole, benzoxazole and benzimidazole, were selected for
this work. The benzothiazole and benzoxazole exam-
ples were all synthesised from the same key intermedi-
ate, the commercially available tribenzoyl-protected
cyanoribose 3. In the case of the benzothiazoles, 3
was reacted directly with the appropriate 2-aminothi-
ophenol8 under basic conditions to yield the desired
tribenzoyl-protected C-linked benzothiazole nucleoside
4. If the appropriate thiophenol starting material was
not available then 3 was hydrolysed to the amide, con-
verted to the thioamide using Lawesson’s Reagent9 and
then reacted with the appropriate 2-iodoaniline in the
presence of palladium10 to yield the protected C-linked
nucleoside 4 (Scheme 1).
This article describes the further development of that
work, investigating whether a nucleotide triphosphate
incorporating a C-linked unnatural base could offer
potency comparable to that of the N-linked series, and
comparing the stabilities of an N- and a C-linked tri-
phosphate to that of UTP.
Keywords: P2Y2; UTP; Enhanced stability.
*
Corresponding author. E-mail: richard.davenport@ucb-group.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.038

R. J. Davenport et al. / Bioorg. Med. Chem. Lett. 17 (2007) 558–561
559
NH
The benzimidazole 6 was synthesised from a tribenzoyl-
BzO
BzO
O
O
protected ribose carboxylic acid 5. This was converted
to the acid chloride, which was coupled with 1,2-diami-
nobenzene to yield an amide intermediate. Ring closure
was achieved using POCl3, and the free NH methylated
using sodium hydride and methyl iodide to yield the de-
sired product (Scheme 2).
CN
i
OMe
.HCl
BzO
OBz
BzO
OBz
ii
3
N
The tribenzoyl-protected C-linked benzoxazole nucleo-
side 7 was synthesised by reacting the key intermediate
3 with methanolic HCl11 to yield an amidate ester, which
was converted to the benzoxazole by reacting with
2-amino-4-methylphenol12 under basic conditions
(Scheme 3).
BzO
O
O
7
BzO
OBz
Scheme 3. Reagents and condition: (i) HCl, MeOH; (ii) 2-amino-4-
methylphenol, Et3N, EtOH, reflux.
The tribenzoyl-protected compounds (4a–g, 6 and 7)
were treated with sodium methoxide in methanol to re-
move the benzoyl esters and the resulting nucleosides
converted to triphosphates 8a–i using standard condi-
tions13 (Scheme 4). The desired triphosphates were
separated from the crude reaction mixtures by prepara-
tive HPLC and isolated as the ammonium salts. Incor-
poration of the triphosphate unit was confirmed by
31P NMR spectroscopy and LC-MS.
O
P
O
P
BzO
O
P
i, ii
O
O
R
R
HO
O
O
O
OH OH OH
iii, iv
BzO
OBz
HO
OH
8a-i
Scheme 4. Reagents: (i) NaOMe, MeOH; (ii) POCl3, P(OMe)3, proton
sponge; (iii) H4P2O7Á1.5(Bu3N), Bu3N, DMF; (iv) NH4Cl, H2O.
The P2Y2 agonist potencies for the C-linked com-
pounds 8a–i are shown in Table 1. In general, the ben-
zothiazoles were well tolerated, with several examples
giving very active compounds (8b, 8c and 8d) with
P2Y2 agonist potencies similar to that of UTP. Inter-
estingly a larger substituent in the 4-position in the
case of 8g (methoxy) dramatically reduced activity,
whereas when this was much smaller, as in the case
of 8d (fluoro), the impact is far less detrimental. The
benzoxazole 8h and the benzimidazole 8i, though toler-
ated, led to reduced P2Y2 activity compared with those
of benzthiazoles and N-linked isocarbostyrils.7 We be-
lieve this is the first time that C-linked nucleotide tri-
phosphates have been reported, that have levels of
P2Y2 potency comparable to that of the natural ligand
UTP.
R
BzO
N
BzO
O
R
CN
i
O
S
iv
3
4a-g
BzO
OBz
BzO
OBz
ii
S
O
BzO
BzO
Some key compounds were tested against three other
purinergic receptor subtypes, P2Y1, P2Y4, and P2Y6.
The results of these experiments are shown in Table 2.
O
iii
O
NH2
NH2
BzO
OBz
BzO
OBz
All compounds tested were inactive against P2Y1.
While UTP had significant agonist activity at the
P2Y4 and P2Y6 receptors our nucleotide triphosphates
incorporating C-linked unnatural bases had no activity
against P2Y6 and, at worst, only micromolar activity
against P2Y4. Like our N-linked unnatural nucleotide
triphosphates,7 they therefore appear to provide a
more selective template than the uracil-derived P2Y2
agonists.
Scheme 1. Reagents and conditions: (i) appropriate 2-aminothiophe-
nol, Et3N, EtOH, reflux; (ii) HCl, dioxane; (iii) Lawesson’s reagent,
dioxane, reflux; (iv) appropriate 2-iodoaniline, PdCl2, CaO, DMF
80 °C.
O
N
BzO
BzO
O
O
Having generated some potent and selective P2Y2 ago-
nists, we were interested to assess the stability of these
compounds in comparison to that of the natural ligand
UTP. Stability was examined using a Human Bronchial
Epithelial Cell assay.16 UTP was compared with our N-
and C-linked triphosphates 2 and 8a. The results of this
i, ii, iii
N
OH
6
5
BzO
OBz
BzO
OBz
Scheme 2. Reagents and conditions: (i) (COCl)2, Et3N, 1,2-diamino-
benzene; (ii) POCl3; (iii) NaH, MeI, DMF.

560
R. J. Davenport et al. / Bioorg. Med. Chem. Lett. 17 (2007) 558–561
Table 1. Agonist potencies of compounds 8a–8f compared with those
of UTP and INS365 2
O
O
O
O
P
P
P
R
HO
O
O
O
OH
OH
OH
O
O
O
O
P
P
P
R
HO
OH
F
HO
O
O
O
8a-i
OH
OH
OH
R =
HO
OH
S
N
O
N
O
N
R groups:
S
N
H
O
Cl
S
S
UTP
100
2
8a
N
N
N
F
8a
8b
8c
8f
8i
80
60
40
20
0
Cl
OMe
S
N
S
N
S
N
CF3
8e
8h
8d
F
0
10
20
40
60
120
180
N
S
O
Time (minutes)
N
N
N
Me
Figure 1. Stability comparison of N- and C-linked nucleotide triphos-
phates with UTP.
8g
OMe
14
P2Y2EC50 (nM)
Compound
UTP
2
7
5
8a
8b
8c
8d
8e
8f
22
8
In conclusion, a number of novel UTP analogues have
been prepared and their agonist potencies against some
purinergic receptors determined. A number of these
were well tolerated, having agonist potencies compara-
ble to that of UTP. The metabolic stabilities of examples
from our N- and C-linked series were compared to that
of UTP, and both of our compounds were found to be
significantly more stable than UTP. We believe that this
enhanced stability might lead to an enhanced duration
of action should these compounds be used in a therapeu-
tic setting.
3
7
42
10
700
390
200
8g
8h
8i
Table 2. Agonist potencies against some other P2Y receptor subtypes
15
15
15
Compound
P2Y1 EC50
(nM)
P2Y4 EC50
(nM)
P2Y6 EC50
(nM)
Acknowledgments
UTP
8a
>2000
>20,000
>20,000
>20,000
39
4121
424
>20,000
>20,000
>20,000
The authors thank Slough and Cambridge NCE Project
Biology groups.
8d
8f
4233
>20,000
References and notes
study are shown in Figure 1. In our hands UTP was
essentially all gone after 120 min. However, both the
N- and C-linked unnatural UTP analogues offered en-
hanced stability. In the case of the C-linked compound
8a, after 120 min there was approximately 50% unme-
tabolised parent remaining, with at least 20% remaining
after 180 min. The result for the N-linked compound 2
was even better, with approximately 70% parent remain-
ing after 120 min and 50% remaining at 180 min. This
increased stability should lead to an increased duration
of pharmacological action. One could also envisage that
preparation of the dinucleotide tetraphosphate analogue
of either 2 or 8a would maintain potency and further in-
crease stability, as was the case with INS365 compared
with UTP.
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