Synthesis and SAR of pyrimidine-based, non-nucleotide P2Y14 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.084

A weak antagonist of the pyrimidinergic receptor P2Y₁₄ containing a dihydropyridopyrimidine core was identified through high-throughput screening. Subsequent optimization led to potent, non-UTP competitive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound 18q was identified as a 10 nM P2Y₁₄ antagonist with good oral bioavailability and provided sufficient exposure in mice to be used as a tool for future in vivo studies.

Full text of DOI:10.1016/j.bmcl.2011.03.084

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2832–2835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of pyrimidine-based, non-nucleotide P2Y14
receptor antagonists
Daniel Guay, Christian Beaulieu, Michel Belley, Sheldon N. Crane, Jeancarlo DeLuca, Yves Gareau,
Martine Hamel, Martin Henault, Huda Hyjazie, Stacia Kargman, Chi Chung Chan, Lijing Xu, Robert Gordon,
Lianhai Li, Yael Mamane, Nicolas Morin, Joseph Mancini, Michel Thérien, Geoffrey Tranmer,
⇑
Vouy Linh Truong, Zhaoyin Wang, W. Cameron Black
Merck Frosst Center for Therapeutic Research, PO Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8
a r t i c l e i n f o
a b s t r a c t
Article history:
A weak antagonist of the pyrimidinergic receptor P2Y14 containing a dihydropyridopyrimidine core was
identified through high-throughput screening. Subsequent optimization led to potent, non-UTP compet-
itive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound
18q was identified as a 10 nM P2Y14 antagonist with good oral bioavailability and provided sufficient
exposure in mice to be used as a tool for future in vivo studies.
Received 22 February 2011
Revised 22 March 2011
Accepted 23 March 2011
Available online 30 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
G protein-coupled receptors (GPCRs) are a large family of recep-
tors characterized by their ability to respond to diverse extracellu-
lar stimuli such as photons, odorants, chemokines, nucleotides,
lipids, amino acids and peptides. Importantly, modulation of GPCRs
serves as the basis of many disease treatments.1–3 Growing evi-
dence shows that nucleotides not only play key intracellular func-
tions in signaling and genetics, they also behave as potent
extracellular signaling molecules.4 They are released from most tis-
sues and exert their effects by interacting with two classes of
membrane receptors, P2X (ligand gated ion channels) and P2Y
(GPCRs) which are expressed on virtually all cell types.5 P2Y recep-
tors are activated specifically by adenine and uridine nucleotides
and nucleotide sugars. Specifically, P2Y1,11,12,13, respond to ADP
and ATP; in contrast, P2Y2,4,6 respond to UDP or UTP and P2Y14
to UDP-sugars such as UDP-glucose.6 We as well as others have re-
cently shown that UDP is also a potent agonist of P2Y14.7 P2Y14 sig-
nals via Gi and the consequent inhibition of cAMP8 and via Gq and
the consequent increase in calcium flux.9 It is expressed in a broad
range of tissues such as in the stomach, intestine, placenta, spleen,
adipose tissue, brain, lung, heart and several types of immune
cells.10 Several studies have demonstrated UDP-glucose-mediated
signaling in neutrophils,8 dendritic cells9 and airway epithelium11
thus suggesting a potential role of P2Y14 in immune response mod-
ulation. The precise physiological role of P2Y14 remains unknown.
To date, the only known agonists or antagonists of P2Y14 had
been nucleotide derivatives.12,13 The identification of selective,
non-nucleotide small molecule tools would prove invaluable in
deciphering the cellular function of P2Y14. Herein, we report the
first series of non-nucleotide small molecule antagonists of
P2Y14. Using a P2Y14 FLIPR (fluorometric imaging plate reader) cal-
cium mobilization assay in HEK cells, a high-throughput screening
(HTS) of the Merck sample collection identified compound 1 as a
weak antagonist of P2Y14 (Fig. 1). Although not very potent
(IC50 = 4.9 lM) this non-nucleotide lead was attractive based on
its modular structure that seemed amenable to SAR studies.
In order to explore the SAR of this series of compounds, a flex-
ible synthesis of the core tetrahydropyrido pyrimidine was re-
quired. Initial efforts involved exploration of the SAR of the
phenyl urea moiety and with this in mind, we developed a concise
synthesis of the late stage intermediate 7 (Scheme 1). Condensa-
tion of Boc-protected ethyl 4-oxopiperidine-3-carboxylate 2 with
pyridine-3-carboximidamide 3 gave intermediate 4 in excellent
yield. Introduction of the 2-tolyl group was accomplished by Suzu-
ki coupling of tosylate 5 with 2-methylphenyl boronic acid to give
6 in 89% yield. Removal of the Boc group gave the key intermediate
7 which was coupled with various isocyanates to afford analogs 8.
In order to explore replacements of the tolyl group, the steps of
the synthesis were rearranged. 4-Oxopiperidine 9 was reacted with
N
N
H
N
N
N
mouse P2Y14
O
FLIPR IC50 = 4.9 µM
1
⇑
Corresponding author. Tel.: +1 514 358 9294.
E-mail address: cameron.black@kaneq.ca (W.C. Black).
Figure 1. Hit from high-throughput screening.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.084

D. Guay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2832–2835
2833
N
N
H2N
N
COOEt
N
H2N
N
COOEt
N
NH
NH2
N
N
N
O
O
a
b, c
a
Boc
N
N
+
Boc
N
N
N
N
Boc
Boc
Boc
OH
Boc
N
HCl
OH
2
13
2
3
4
14
N
Br
N
Cl
N
N
N
f
e
d
c
b
N
N
N
NH
Boc
HCl
N
N
Boc
OTs
5
15
16
6
Ar
Cl
N
N
N
N
H
N
H
N
N
N
N
N
N
N
g
R
H
N
d
e
O
O
N
NH
N
N
O
17
18
Scheme 3. Reagents and conditions: (a) guanidine HCl, aq K2CO3, MeOH, reflux,
16 h, 92%; (b) TsCl, Et3N, DMAP cat., CH2Cl2, rt, 16 h, 85%; (c) o-tolyl boronic acid,
K3PO4, Pd(OAc)2, 2-(dicyclohexyl-phosphino)biphenyl, DMF, H2O, 80 °C, 16 h, 62%;
(d) t-BuONO, SbBr3, CH2Cl2, 0 °C, 16 h, 23%; (e) 4 M HCl/dioxane, rt, 3 h, 100%; (f)
3-EtC6H4NCO, Et3N, CH2Cl2, rt, 2 h, 92%; (g) ArB(OH)2, PdCl2(dppf), Na2CO3 aq, DMF,
90 °C, 16 h, 45–85%.
7
8
Scheme 1. Reagents and conditions: (a) aq K2CO3, MeOH, reflux, 16 h, 94%; (b) TsCl,
Et3N, DMAP cat., CH2Cl2, rt, 4 h, 92%; (c) o-tolyl boronic acid, K3PO4, Pd(OAc)2,
2-(dicyclohexylphosphino)biphenyl, DMF, H2O, 90 °C, 2 h, 89%; (d) HCl/dioxane or
TFA, CH2Cl2, rt, 4 h, 97%; (e) RC6H4NCO, Et3N, CH2Cl2, rt, 2 h, or RC6H4NH2,
(Cl3CO)2CO, CH2Cl2, aq NaHCO3, 0 °C to rt, 16 h, 77–94%.
of 2 with guanidine hydrochloride under basic conditions gave 13 in
very good yield. Selective formation of the O-tosylate followed by
the usual Suzuki coupling afforded intermediate 14. Conversion of
this 2-aminopyrimidine into the 2-bromopyrimidine 15 was accom-
plished using modified Sandmeyer conditions.15 Removal of the Boc
group with dry hydrochloric acid also effected a halogen exchange of
the heterocyclic template to give 16 quantitatively. Reaction of 16
with3-ethylphenyl isocyanate secured intermediate 17 whichcould
be coupled with a variety of phenyl, aryl and heteroaryl boronic
acids to give the desired analogs 18.
O
H
N
O
b
a
N
HCl
NH
EtOOC
O
EtOOC
10
9
N
N
N
N
c, d
H
N
H
N
A whole cell Ca2+ mobilization assay was used to assess com-
pound potencies. Specifically, we tested the ability of compounds
to inhibit UDP-glucose-stimulated Ca2+ release in human embry-
N
N
N
N
OH
O
O
R
onic kidney (HEK) cells co-expressing P2Y14 and Gaqi5. All com-
pounds were tested against both the mouse and chimpanzee
receptors. The selection of chimpanzee P2Y14 (99% identical to hu-
man) was driven by the larger window (signal/background) with
this receptor over the human P2Y14, while the mouse receptor
(83% identical to human) was used to enable future evaluation of
compounds in mouse models of disease.
11
12
Scheme 2. Reagents and conditions: (a) 3-EtC6H4NCO, Et3N, CH2Cl2, rt, 2 h, 99%; (b)
3, aq K2CO3, MeOH, reflux, 7 h, 94%; (c) TsCl, Et3N, DMAP, CH2Cl2, rt, 16 h, 82%; (d)
RC6H4B(OH)2, K3PO4, Pd(OAc)2, 2-(dicyclohexylphosphino)biphenyl or DavePhos,
DMF, H2O, 90 °C, 2–12 h, 50–92%
The effect of having different substituents on the phenyl urea
was studied first and results are summarized in Table 1. Addition
of a chloride at the ortho-, meta- or para-position revealed that sub-
stitution at the ortho-position was detrimental to potency whereas
substitution at the meta-position was beneficial (8a–c). Replacing
the 3-chloro for a 3-methoxy group (8d) did not affect the potency
on the chimpanzee receptor but reduced 10-fold its activity on the
mouse receptor. On the other hand, opting for a more lipophilic 3-
methylthio substituent had no effect on the potency for the mouse
receptor but increased the potency on the chimpanzee receptor by
25-fold (8f vs 8b). Switching to alkyl groups, a similar trend was
found where small non-polar substituents in the 3-position pro-
vided the most potent P2Y14 receptor antagonists. A simple methyl
group as in 8g only marginally improved the potency compared to
the parent 1 but one carbon homologation to the ethyl analog 8h
led to a 60-fold potency increase on the mouse receptor and over
3-ethylphenyl isocyanate to give urea 10 (Scheme 2). Condensation
of 10 with the carboximidamide 3 gave intermediate 11 in high
yield. Formation of the corresponding tosylate provided the key
intermediate for SAR of the bottom phenyl moiety. Suzuki coupling
with various phenyl boronic acids was typically carried out using
palladium acetate, potassium carbonate and 2-(dicyclohexylphos-
phino)biphenyl to afford the desired compounds 12. However, for
sterically hindered examples such as 2,6-disubstituted phenyl
boronic acids, this coupling required DavephosÒ as the ligand.14
Finally, replacement of the 3-pyridyl group with other aromatic
moieties could be carried out according to chemistry described
above and replacing 3 with different aryl amidines. However, in
order to be more convergent, we decided to slightly modify the ap-
proach so we could access a key intermediate such as 17 and intro-
duce diverse aryl moieties at the final step (Scheme 3). Condensation

2834
D. Guay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2832–2835
Table 1
Table 2
Potency of antagonists 8 on the mouse and the chimpanzee P2Y14 receptors
Potency of antagonists 12 on the mouse and the chimpanzee P2Y14 receptors
N
N
N
N
R
H
N
H
N
N
N
N
N
O
O
R
12
8
a
Compd
R
Mouse P2Y14 IC50
M)a
Chimpanzee P2Y14 IC50
(lM)
a
a
Compd
R
Mouse P2Y14 IC50
M)
Chimpanzee P2Y14 IC50
(lM)
(
l
(
l
8h
2-Me
2-Cl
2-F
3-Cl
2-Me, 4-Cl
2-Et
2-OMe
2-OH
2-CH2OH
2-Me, 6-
Me
2-Me, 6-Cl
2-Cl, 6-Cl
0.081
0.44
3.7
12
7.0
0.39
3.3
6.5
1.1
0.009
0.025
0.014
0.93
>20b
6.3
2.5
18
0.56
3.2
0.032
1
H
2-Cl
3-Cl
4-Cl
3-MeO
3-EtO
3-MeS
3-Me
3-Et
3-Pr
4.9
10
12a
12b
12c
12d
12e
12f
12g
12h
12i
8a
8b
8c
8d
8e
8f
8g
8h
8i
>20b
0.58
5.8
>20b
2.1
6.7
2.3
5.1
0.42
0.63
1.9
0.081
0.15
1.2
1.2
6.5
8.2
0.86
0.081
1.0
0.025
0.024
0.098
0.040
6.9
8j
3-(CH@CH2)
3-CH(CH2)2
3-COCH3
3-
12j
12k
0.050
0.051
0.016
0.034
8k
8m
8n
a
6.0
Values are means of at least two experiments where values were typically
CH(OH)CH3
3-CHFCH3
3-CF2CH3
3-Et, 5-Cl
within 3-fold of each other.
b
8p
8q
8r
0.029
0.62
0.16
0.033
0.062
0.017
Less than 50% inhibition at a maximum concentration of 20 lM.
a
Values are means of at least two experiments where values were typically
both ortho-positions could have a methyl or a chloro substituent
to provide P2Y14 antagonists with very good potency on both
receptors (12i–k).
within 3-fold of each other.
b
Less than 50% inhibition at a maximum concentration of 20 lM.
The third moiety we wanted to explore in terms of SAR modifi-
cation was the 3-pyridyl appended at the 2-position of the pyrim-
idine core template. For this, we elected to keep constant the 3-
ethylphenyl urea and the o-tolyl moieties and results are summa-
rized in Table 3. Oxidation of the pyridine nitrogen led to a large
loss of potency (18a). While the 2-pyridyl isomer 18b was totally
inactive, the 4-pyridyl analog 18c was found to be 17-fold less po-
tent on the mouse receptor but at least 3-fold more potent on the
chimpanzee ortholog. Changing the 3-pyridyl for a phenyl had no
effect on the potency on the mouse receptor and only reduced its
activity 3-fold on the chimpanzee receptor (18d). Addition of alkyl
substituents was not well tolerated (e.g., 18e–h) but small elec-
tron-withdrawing groups such as fluorine and nitrile provided very
potent antagonists on both receptors (e.g., 18i–m). Replacing the
phenyl with a 2-naphthyl (18n) was somewhat detrimental but a
3-quinolyl group gave a potent antagonist on the mouse receptor
(18p). The 3,4-methylenedioxyphenyl derivative 18q was found
to be potent with IC50’s of 10 nM on the mouse receptor and of
81 nM on the chimp receptor. Finally, we found that small nitrogen
containing heterocycles bearing electron-withdrawing groups pro-
vided the most active P2Y14 receptor antagonists (e.g., 18r–u).
Compounds 18r and t were eliminated from consideration due
to potent binding to the hERG channel (<200 nM) following screen-
ing in a binding assay with 35S-labelled MK-0499.16 Compounds
400-fold increase of potency on the chimpanzee ortholog. The pro-
pyl-substituted derivative 8i was similar in potency while the vinyl
and cyclopropyl derivatives (8j, k) were significantly less potent on
the mouse receptor but retained most of their activity on the chim-
panzee receptor. Polar substituents were not well tolerated (e.g.,
8m, n) and electron-withdrawing groups such as the 1,1-difluoro-
ethyl compound 8q maintained good potency on the chimpanzee
receptor but suffered 8-fold potency loss on the mouse receptor.
Although the monofluoroethyl analog 8p was found to be the most
potent in both assays, the electrophilic nature of this moiety pre-
cluded its further evaluation. Eventually, we found that 3,4- and
3,5-disubstituted phenyl ureas were also potent antagonists as
long as the 3-ethyl is present as in analog 8r. Overall, it appeared
that the 3-ethylphenyl urea was the best motif for these P2Y14
antagonists and that the chimpanzee receptor was more tolerant
of the nature of substituents on the phenyl ring.
Next, we examined the SAR of the o-tolyl group appended at the
4-position of the core heterocyclic template while keeping other
portions of the molecule constant. Changing the 2-methyl substitu-
ent of 8h for a 2-chloro led to a slight increase of potency on the
chimpanzee receptor but also resulted in a 5-fold loss of potency
on the mouse receptor (12a, Table 2). The smaller and more elec-
tronegative fluorine atom resulted in a very significant loss of po-
tency (12b). Moving the chloride atom to the meta- (12c) or to the
para-positions (12d) led to a dramatic loss in potency compared to
the starting compound. These results seemed to indicate little tol-
erance for substituents at the 3- and 4-positions of this phenyl ring.
Increasing the size of the 2-methyl to a 2-ethyl group (12e) proved
to be much more detrimental for potency on the chimpanzee
receptor with a 100-fold loss compared to the mouse receptor
(5-fold). Introduction of a polar group also led to significant reduc-
tion of activity in the assays (e.g., 12f– h). Finally we found that
18q, s and u all were >5 lM in this assay (data not shown). The
pharmacokinetic profiles of these promising antagonists were
determined in C57B6 mice (Table 4). The compounds were dosed
as oral suspensions in 0.5% methocel at 50 mg/kg. Intravenous dos-
ing was at 5 mg/kg in 80% aqueous PEG-200. Serial tail bleeds from
two animals for each dosing route were used to generate the re-
ported data. The compounds were characterized by high volumes
of distribution and long half-lives. The exposure achieved with
18q which gave exposures well above the cell-based IC50s through
24 h (C24 h = 0.4 lM) suggesting that this compound would be suit-

D. Guay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2832–2835
2835
Table 3
able for in vivo experiments to elucidate the pharmacological role
of P2Y14. Compound 18s, while being 10-fold more potent than
18q, gave exposures ꢀ10-fold lower at 8 h and 24 h, thus negating
much of its utility. Compound 18u was found to be poorly toler-
ated in mice following single dose administration.
Potency of antagonists 18 on the mouse and the chimpanzee P2Y14 receptors
Ar
N
H
N
N
N
Compound 18q was further characterized in a 3H-UDP filtration
binding assay in membranes prepared from HEK cells overexpress-
O
12
3
ing chimpanzee P2Y14. Compound 18q was unable to displace
H-UDP in the binding assay suggesting the compound is non-com-
petitive with UDP.
We have succeeded in identifying a series of non-nucleotide
P2Y14 antagonists. A 4.9 lM hit from high-throughput screening
was optimized to give non-UDP competitive antagonists with
IC50s of <10 nM. These compounds were orally bioavailable and
provide sufficient exposure in mice to serve as useful tools for
a
a
Compd Ar
Mouse P2Y14IC50
M)
Chimpanzee P2Y14 IC50
M)
(l
(l
8h
3-Pyr
3-Pyr N-oxide
2-Pyr
4-Pyr
Ph
4-(Me)Ph
3-(Me)Ph
4-(i-Pr)Ph
4-(CF3)Ph
3-(CN)Ph
4-(CN)Ph
4-(F)Ph
0.081
2.8
0.025
3.8
18a
18b
18c
18d
18e
18f
18g
18h
18i
>20b
1.4
>20b
0.007
0.093
0.86
0.087
0.062
1.3
the delineation of the biological role of P2Y14
.
0.73
>20b
7.5
>20b
12
References and notes
0.14
0.13
18j
0.016
0.025
0.030
0.88
0.013
0.010
0.003
0.012
0.001
0.031
0.082
0.089
7.1
1. Williams, C.; Hill, S. J. Methods Mol. Biol. 2009, 552, 39.
2. Thompson, M. D.; Percy, M. E.; McIntyre Burnham, W.; Cole, D. E. Methods Mol.
Biol. 2008, 448, 109.
3. Conn, P. M.; Ulloa-Aguirre, A.; Ito, J.; Janovick, J. A. Pharmacol. Rev. 2007, 59,
225.
4. Lecca, D.; Ceruti, S. Biochem. Pharmacol. 2008, 75, 1869.
5. Burnstock, G. Cell. Mol. Life Sci. 2007, 64, 1471.
6. Jacobson, K. A.; Ivanov, A. A.; de Castro, S.; Harden, T. K.; Ko, H. Purinergic
Signalling 2009, 5, 75.
7. Carter, R. L.; Fricks, I. P.; Barrett, M. O.; Burianek, L. E.; Zhou, Y.; Ko, H.; Das, A.;
Jacobson, K. A.; Lazarowski, E. R.; Harden, T. Mol. Pharmacol. 2009, 76, 1341.
8. Scrivens, M.; Dickenson, J. M. Eur. J. Pharmacol. 2006, 543, 166.
9. Skelton, L.; Cooper, M.; Murphy, M.; Platt, A. J. Immunol. 2003, 171, 1941.
10. Chambers, J. K.; Macdonald, L. E.; Sarau, H. M.; Ames, R. S.; Freeman, K.; Foley, J.
J.; Zhu, Y.; McLaughlin, M. M.; Murdock, P.; McMillan, L.; Trill, J.; Swift, A.;
Aiyar, N.; Taylor, P.; Vawter, L.; Naheed, S.; Szekeres, P.; Hervieu, G.; Scott, C.;
Watson, J. M.; Murphy, A. J.; Duzic, E.; Klein, C.; Bergsma, D. J.; Wilson, S.; Livi,
G. P. J. Biol. Chem. 2000, 175, 10767.
18k
18m
18n
18p
18q
18r
18s
18t
3-(F)Ph
2-Naphthyl
3-Quinolyl
3,4-(OCH2O)-Ph
4-(CN)-3-Pyr
5-Pyrimidyl
2-(CN)-5-
Pyrimidyl
Me2-4-
0.23
0.081
0.004
0.008
0.001
18u
0.004
0.002
isoxazolyl
a
Values are means of at least two experiments where values were typically
within 3-fold of each other.
b
Less than 50% inhibition at a maximum concentration of 20 lM.
11. Muller, T.; Bayer, H.; Myrtek, D.; Ferrari, D.; Sorichter, S.; Ziegenhagen, M. W.;
Zissel, G.; Virchow, J. C., Jr.; Luttmann, W.; Norgauer, J.; Di Virgilio, F.; Idzko, M.
Am. J. Respir. Cell. Mol. Biol. 2005, 33, 601.
12. Fricks, I. P.; Maddileti, S.; Carter, R. L.; Lazarowski, E. R.; Nicholas, R. A.;
Jacobson, K. A.; Harden, T. K. J. Pharmacol. Exp. Ther. 2008, 325, 588.
13. Das, A.; Ko, H.; Burianek, L. E.; Barrett, M. O.; Harden, T. K.; Jacobson, K. A. J.
Med. Chem. 2010, 53, 471.
14. Wolfe, J. P.; Singer, R. A.; Yang, B. H.; Buchwald, S. L. J. Am. Chem. Soc. 1999, 121,
9550.
15. Robins, M. J.; Uznanski, B. Can. J. Chem. 1981, 59, 2608.
16. Ok, D.; Li, C.; Abbadie, C.; Felix, J. P.; Fisher, M. H.; Garcia, M. L.; Kaczorowski, G.
J.; Kathryn, A.; Lyons, K. A.; Martin, W. J.; Priest, B. T.; Smith, M. M.; Williams, B.
S.; Wyvratt, M. J.; Parsons, W. H. Bioorg. Med. Chem. Lett. 2006, 16, 1358.
Table 4
Pharmacokinetic parameters for select compounds in mouse following dosing at
50 mpk po and 5 mpk IV
Compd
F (%)
Cmax
(lM)
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
18q
18s
18u
134a
40
31
2.4
6.4
2.0
48
38
43
19
11
9.3
11
12
5.6
a
This calculated value may be attributable to non-linear absorption at the high
oral dose used.