The identification of 4,7-disubstituted naphthoic acid derivatives as UDP-competitive antagonists of P2Y14

Article abstract of DOI:10.1016/j.bmcl.2011.03.081

A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY ₁₄ with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y₁₄ with a good pharmacokinetic profile.

Full text of DOI:10.1016/j.bmcl.2011.03.081

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2836–2839
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification of 4,7-disubstituted naphthoic acid derivatives
as UDP-competitive antagonists of P2Y14
Jacques Yves Gauthier, Michel Belley, Denis Deschênes, Jean-François Fournier, Sébastien Gagné,
Yves Gareau, Martine Hamel, Martin Hénault, Huda Hyjazie, Stacia Kargman, Geneviève Lavallée,
Jean-François Levesque, Lianhai Li, Yaël Mamane, Joseph Mancini, Nicolas Morin, Erin Mulrooney,
⇑
Joël Robichaud, Michel Thérien, Geoffrey Tranmer, Zhaoyin Wang, Jin Wu, W. Cameron Black
Merck Frosst Centre for Therapeutic Research, 16711 TransCanada Hwy, Kirkland, Quebec, Canada H9H 3L1
a r t i c l e i n f o
a b s t r a c t
Article history:
A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY14 with a naphthoic acid core
was identified through high-throughput screening. Optimization provided compounds with improved
potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of
metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuron-
idation and improved the pharmacokinetic profile. Additional optimization led to the identification of
compound 38 which is an 8 nM UDP-competitive antagonist of P2Y14 with a good pharmacokinetic
profile.
Received 22 February 2011
Revised 22 March 2011
Accepted 23 March 2011
Available online 31 March 2011
Keywords:
Pyrimidinergic receptor
P2Y14
Ó 2011 Elsevier Ltd. All rights reserved.
GPR105
Competitive antagonist
SAR
Glucuronidation
P2Y14 is a G-protein-coupled receptor that binds UDP-glucose
and is widely expressed in a broad range of tissues.1,2 The physio-
logical role of P2Y14 is currently unknown and mechanistic studies
would benefit from the identification of small-molecule tools. In
O
O
OH
the previous Letter in this journal, we have described the identifi-
3
cation of the first non-nucleotide antagonist of P2Y14
.
This non-
competitive antagonist was derived from a hit in an HTS campaign
using the FLIPR-based calcium flux assay in HEK cells over-
1
Figure 1. A UDP competitive naphthoic acid antagonist of P2Y14
.
expressing either mouse or chimpanzee P2Y14
.
Using the same assay, additional hits were identified, including
the 4,7-disubstituted naphthoic acid derivative 1 (Fig. 1). This com-
pound had FLIPR IC50’s of 3.5 lM on both the mouse and chimpan-
To explore the SAR of the naphthoic acid lead 1 we utilized both
single compound and library syntheses. For both, ethyl 4-hydroxy-
7-bromo-2-naphthoate 2 was the ideal building block (Scheme 1).
It was prepared from 3-bromobenzaldehyde and 4-tert-butyl-1-
ethyl-2-(diethoxyphosphoryl) succinate via Stobbe condensation
and Friedel–Crafts cyclization as described by Boger et al.4 Alterna-
tively, derivatives bearing an aryl group at C-4 and an oxygen-linked
substituent at C-7 were prepared from ethyl 4-hydroxy-7-benzyl-
oxy-2-naphthoate 3 (obtained using the same method). Derivatiza-
tion of the substituents at the 4- or the 7- positions was
accomplished by appropriate sequencing of Pd-catalyzed cross-cou-
plings of activated derivatives (the bromide 2, the triflate 6 or the
pinacolato arylboronic ester 9). Mitsunobu and/or base-catalysed
zee receptors, was fully reversible and inhibited UDP-glucose
mediated signaling. In addition, 1 was competitive with 3H-UDP
in
a
recombinant simian P2Y14 binding assay with
a Ki of
0.16
l
M, indicating that it was an active site antagonist. Since we
felt that the successful identification of a potent, competitive
antagonist would be advantageous to the elucidation of the phar-
macological role of P2Y14, we decided to pursue the optimization
of 1.
⇑
Corresponding author. Tel.: +1 514 358 9294.
E-mail address: cameron.black@kaneq.ca (W.C. Black).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.081

J. Y. Gauthier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2836–2839
2837
Ar-CH2-O
COOH
Ar
COOH
HO
COOEt
b, c
5
c
Ar
O-CH2-Ar
7
8
Ar
a, g
BnO
COOEt
Ar-CH2-O
COOH
Ar
COOEt
O-SO2-CF3
6
O-CH2-Ar
10
O-CH2-Ar
4
a, b
R= Bn
d
R= Br
f, b, c
R
COOEt
O
B
Br
COOEt
F
COOEt
O
i
e, b
R= Br
R= Br
OH
2, R= Br
3, R= OBn
OH
O-CH2-Ar
15
9
11
14
a, d, a, c
R= Br
a, d
f, d, a, c
Ar
COOH
Ar
COOH
F
Ar
COOEt
O-CH2-Ar
Ar
16
O-SO2-CF3
12
e
j
Ar
COOEt
Ar
COOH
Ar
COOH
OH
c, h
B
Ar
O
O
13
Ar
17
Scheme 1. Reagents and conditions: (a) Ar-B(OH)2, PdCl2dppf, 2 M Na2CO3, DMF, 85 °C; (b) ArCH2X (X = Cl, Br, I), K2CO3, DMF; (c) NaOH, EtOH, THF; (d) (CF3SO2)2O, pyridine,
CH2Cl2; (e) bis(pinacolato)diboron, KOAc, PdCl2dppf, dioxane, 85 °C; (f) H2O2 MeOH; (g) H2, Pd/C, EtOAc; (h) Ar-Br, PdCl2dppf, 2 M Na2CO3, DMF, 85 °C; (i) N-fluoropyridinium
triflate, 1,2-dichloroethane; (j) LiOH, DMSO, 150 °C.
alkylations, followed by ester hydrolysis generated the carboxylic
acids as shown in Scheme 1.
was evidenced by the loss of activity observed upon reversal of the
benzyloxy to a phenoxymethyl as in 23.
The 3-fluoro derivative was prepared from 2 using N-fluoropy-
ridinium triflate5 to produce ethyl 3-fluoro-4-hydroxy-7-bromo-2-
naphthoate 15 and then elaborated as described above to furnish
16. The fluorine atom could then be displaced with hydroxide to
produce the 3-hydroxy derivative 17.
Our initial evaluation of 1 focused on replacement of the car-
boxylic acid by a methyl ester, acylsulfonamide, acetic acid, or
phosphate, but all of these derivatives were found to be markedly
less active than 1 (data not shown).
Replacement of the C-7 benzyloxy moiety by either a methoxy
group (18) or a phenoxy group (19) led to a significant loss of po-
tency (Table 1). An acidic substituent on the benzyl ring (20) was
not tolerated.
Conversely, the addition of lipophilic substituents to the benzyl-
oxy moiety increased functional activity and improved binding.
The addition of chlorine atoms or methyl groups as shown in 21
and 22 led to potent antagonists. The importance of the linker itself
We then explored the substituent at C-4 of the naphthalene
ring. Removing the phenyl group entirely (24) resulted in complete
loss of activity; however the benzyloxy replacement 25 showed
comparable potency to 22. The C-4 phenyl group could also be re-
placed by a thienyl group such as in 26, which provided a 10-fold
increase in potency in the chimpanzee functional assay. Combining
these observations and adding a fluorine atom to the C-7 substitu-
ent to enhance metabolic stability led to compound 27 with a 2,6-
dimethyl-4-fluoro-benzyloxy group at C-7. 27 is 250-fold and
nearly 1000-fold more potent than 1 in the mouse and chimpanzee
functional assays respectively with a Ki of 14 nM in the chimpan-
zee binding assay.
To evaluate its pharmacokinetic properties, 27 was dosed in
C57BL/6 mice at 50 mg/kg P.O. and 2 mg/kg I.V. An oral bioavail-
ability of 12% was observed with a half-life of 2.7 h. To further
understand the metabolic fate of this compound, we carried out
a bile duct cannulation study using five wild-type and five Mrp2

2838
J. Y. Gauthier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2836–2839
Table 1
In vitro activity of selected analogs of the lead naphthoic acid antagonist 1
O
R7
OH
R3
R4
Compound R7
R4
R3
Mouse P2Y14 IC50
M)a
Chimpanzee P2Y14 IC50
M)a
Chimpanzee P2Y14 binding Ki
(l
M)a
(
l
(l
1
18
19
20
21
22
23
24
25
26
27
–OCH2Ph
–OCH3
–OPh
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
3.5
3.5
0.16
4.0
1.5
4.9
4.1
>40
0.091
0.041
1.5
4-Carboxy-benzyloxy–
2,6-Cl2-benzyloxy–
2,6-Dimethyl-benzyloxy–
2,6-Dimethylphenoxymethyl–
2,6-Dimethyl-benzyloxy–
2,6-Dimethyl-benzyloxy–
2,6-Dimethyl-benzyloxy–
2,6-Dimethyl-4-fluoro-
benzyloxy–
>13
0.19
0.11
2.1
0.029
0.042
0.34
13
6.5
–OCH2-Ph
3-Thienyl
3-Thienyl
0.11
0.026
0.014
0.089
0.006
0.004
0.021
0.016
0.014
28
29
30
31
4-CF3O-phenyl–
4-CF3O-phenyl–
4-CF3O-phenyl–
4-CF3O-phenyl–
3-Thienyl
3-Thienyl
3-Thienyl
4-(3-((CF3)2COH)-
thienyl-
H
F
0.054
0.38
0.014
0.11
0.49
0.029
0.11
0.17
OH 1.3
H
0.35
0.091
0.054
32
33
34
35
36
4-CF3O-phenyl–
4-CF3-phenyl–
4-CF3-phenyl–
4-CF3-phenyl–
4-CF3-phenyl–
4-(3-CH3SO2)-thienyl-
4-(3-CH3SO2)-thienyl-
4-(4-CH3SO2)-phenyl-
4-(4-CF3CHOH)-phenyl-
4-(R-4-CF3CHOH)-
phenyl-
H
H
H
H
H
0.33
0.19
0.020
0.013
0.004
0.086
0.048
0.007
0.005
0.001
0.032
0.013
0.007
0.014
0.010
37
38
4-CF3-phenyl–
4-CF3-phenyl–
4-(S-4-CF3CHOH)-
phenyl
4-(R-4-
H
H
0.051
0.008
0.011
0.001
0.021
0.004
CHF2CHOH)phenyl-
aIC50 and Ki values represent an average of at least three independent titrations where values were typically within 3-fold of each other. See Ref. 1 for assay conditions.
in particular by introducing electron-withdrawing substituents.6
F
OH
O
To monitor the effect of such modifications on the extent of metab-
olism, we used an in vitro mouse hepatocyte assay7 and tried to
establish a correlation with pharmacokinetic properties (Table 2).
We first replaced the electron-donating benzyloxy group of 27
with the electron-withdrawing 4-trifluoromethoxyphenyl group
obtaining 28. This modification led to a dramatic increase in met-
abolic stability in vitro, as measured by the amount of parent drug
remaining following a 2 h incubation with mouse hepatocytes (62%
vs 8%). Moreover, the modification did not significantly affect func-
tional activity or binding to the receptor. In an attempt to maxi-
HO
OH
O
In vivo
O
O
CO2H
27
39
S
Figure 2. In vivo studies indicated that 27 is excreted in the bile as the
acylglucuronide metabolite 39 (>99%).
mize electronic effects we introduced
a fluorine (29) or a
hydroxyl group (30) at C-3 of the naphthyl ring. Although these
modifications improved both hepatocyte stability and pharmacoki-
netic parameters, the resulting compounds were less potent than
27. An alternative way of improving the stability of these com-
pounds was found by introducing a polar group on the thienyl ring.
The bis-(trifluoromethyl)carbinol 31 showed good hepatocyte sta-
bility (91% parent remaining) and had improved bioavailability,
clearance and half-life relative to 27. The methylsulfone-substi-
tuted thiophene 32 had similar potency, but was not as effective
at reducing metabolism.
Additional work in the C-7 position revealed that the trifluor-
ophenyl substituent of 33 was somewhat superior to the trifluoro-
methoxyphenyl of 32. Holding this group constant and optimizing
the C-4 position led to the observation that the phenyl sulfone of
34 was markedly superior to the thienylsulfone, with a Ki of
7 nM. As observed previously, introducing fluorinated alcohol moi-
eties on the C-4 aryl group provided compounds with improved
(-/-) mice dosed with unlabeled 27. Bile, feces and urine were col-
lected over 24 h in the presence of 1% formic acid (to stabilize any
acyl glucuronide that may be present) and analyzed by LC-MS/MS.
Little parent or glucuronide were detected in the urine or feces,
suggesting that 27 is well-absorbed. More than 99% of the admin-
istered dose was recovered in the bile and identified as the acyl
glucuronide 39 (Fig. 2). Only traces of oxidative metabolites were
observed in the bile and were not characterized. A total recovery
of nearly 100% was determined based on the initial dose and
authentic standards for both 27 and 39. In addition, there were
no significant differences in drug excretion between the wild-type
and Mrp2 (-/-) mice suggesting that this transporter is not a major
contributor to the excretion of 27.
Based on these data, we attempted to reduce the extent of glu-
curonidation. It has been reported that acyl glucuronidation can be
modulated by varying the electronic density of the carboxylic acid,

J. Y. Gauthier et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2836–2839
2839
Table 2
2-naphthoic acid core substituted by an electron-withdrawing
4-trifluoromethylphenyl group directly attached to C-7 and a chiral
4-difluoromethylphenethyl alcohol attached at C-4. This combina-
tion resulted in a metabolic profile where glucuronidation of the
acid group was minimized, providing a compound with high oral
exposure and low clearance. Compound 38 is an excellent candi-
date for delineating the biological role of P2Y14 in in vivo models.
In vitro–in vivo correlation of metabolism in mouse hepatocytes with PK profile in
mice
Compound
% Parent
%Fb
Cl
T1/2
(h)
Cmax
(l
M)b
remaininga
(mL/min/kg)c
27
28
29
30
31
32
38
8
62
85
93
91
16
39
12
92
129
240
45
4
7.1
18
2.7
1.1
4.3
13
4.1
3.2
3.0
23
18
89
34
37
16
3.3
7.6
1.9
3.7
1.6
References and notes
1. Jacobson, K. A.; Ivanov, A. A.; de Castro, S.; Harden, T. K.; Ko, H. Purinergic Signal
2009, 5, 75.
67
113
a
Following 2 h incubation with freshly isolated mouse hepatocytes (2 Â 106
2. 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.
cells/mL) with 20 lM of the test compound.
b
Following an oral dose of 50 mg/kg in 5% Tween-80 except for compound 28,
dosed at 25 mg/kg.
c
Following an IV dose of 2 mg/kg in 60% PEG-200.
3. Guay, D.; Beaulieu, C.; Belley, M.; Crane, S. N.; DeLuca, J. C.; Gareau, Y.; Hamel,
M.; Henault, M.; Hyjazie, H.; Kargman, S.; Chan, C. C.; Xu, L.; Gordon, R.; Li, L.;
Mamane, Y.; Morin, N.; Mancini, J.; Therien, M.; Truong, V. L.; Wang, Z.; Black, W.
C. Biorg. Med. Chem. Lett. 2011, this issue, doi:10.1016/j.bmcl.2011.03.084.
4. Boger, D. L.; Han, N.; Tarby, C. M.; Boyce, C. W.; Cai, H.; Jin, Q.; Kitos, P. A. J. Org.
Chem. 1996, 61, 4894.
5. Umemoto, T.; Fukami, S.; Tomizawa, G.; Harasawa, K.; Kawada, K.; Tomita, K. J.
Am. Chem. Soc. 1990, 112, 8563.
6. Wong, Y. C.; Zhang, L.; Lin, G.; Zuo, Z. Expert Opin. Drug Metab. Toxicol. 2009, 11,
1399.
potency in the functional assays and good metabolic stability
(compounds 35-38). The most promising derivative was 38
(Ki = 4 nM), containing a chiral difluoromethylcarbinol. This com-
pound was orally bioavailable in mice (67%) with low intrinsic
clearance (1.6 mL/min/kg).
7. Mouse hepatocytes were isolated by a two-step collagenase (50 U/mL) perfusion
and incubations performed as described previously: Houle, R.; Raoul, J.;
Levesque, J.-F.; Pang, S.; Nicoll-Griffith, D. A.; Silva, J. M. Drug Metab. Dispos.
2003, 31, 447.
In summary, a highly potent, competitive and bioavailable
antagonist of P2Y14 was identified starting from a 3.5
lM hit from
high throughput screening. The key structural features combine a