Fluorinated pyrazole acids are agonists of the high affinity niacin receptor GPR109a

Article abstract of DOI:10.1016/j.bmcl.2007.07.101

A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency. One example from the series was shown to have improved properties in vivo compared to niacin.

Full text of DOI:10.1016/j.bmcl.2007.07.101

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5620–5623
Fluorinated pyrazole acids are agonists of the high affinity
niacin receptor GPR109a
Philip J. Skinner,a,* Martin C. Cherrier,a Peter J. Webb,a Young-Jun Shin,a
Tawfik Gharbaoui,a Andrew Lindstrom,a Vu Hong,a Susan Y. Tamura,a
Huong T. Dang,b Cameron C. Pride,b Ruoping Chen,b
Jeremy G. Richman,b Daniel T. Connollyb and Graeme Semplea
aMedicinal Chemistry, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA, 92121, USA
bDiscovery Biology, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA, 92121, USA
Received 1 June 2007; revised 23 July 2007; accepted 24 July 2007
Available online 23 August 2007
Abstract—A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the
human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity
niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency.
One example from the series was shown to have improved properties in vivo compared to niacin.
Ó 2007 Elsevier Ltd. All rights reserved.
Niacin (1) (Fig. 1) has long been used for the treatment
of lipid disorders and for the prevention of cardiovascu-
lar disease, the leading cause of death in the U.S., as a
result of its ability to raise high-density lipoprotein
(HDL) levels.1 Recent mechanistic investigations have
shown that niacin may exert its beneficial action through
activation of a G-protein-coupled receptor (GPCR)
located on adipocytes.2 The consequent decrease in
intracellular cAMP is believed to result in inhibition of
lipolysis by negative modulation of lipase activity and
perilipin phosphorylation, thereby decreasing plasma
free fatty acid (FFA) levels which has been postulated
to result in increased HDL.3 Two closely related human
orphan Gi-protein coupled receptors, both of which are
expressed in human adipocytes (termed GPR109b, or
HM74 and GPR109a, or HM74A; 95% identity), have
recently been identified as possible molecular targets
for niacin.4,5
appears to have arisen from evolutionary late gene
duplication. It differs from GPR109a and PUMA-G
mainly in the C-terminal region and a search of avail-
able genomic databases reveals it is found only in chim-
panzees and humans. Niacin has been shown to activate
GPR109a with an EC50 of 250 nM in a GTPcS assay
3
and displaces H-niacin from GPR109a expressing Chi-
nese hamster ovary (CHO) cell membranes with an IC50
of 81 nM.4 It is a much weaker ligand for GPR109b
with an EC50 in the millimolar range. The lack of a
GPR109b ortholog in rodents suggests that GPR109a
is sufficient for the antilipolytic activity of niacin
in vivo.7 Recent evidence shows that the cutaneous
flushing side effect in mice requires the presence of
PUMA-G,8 and thus suggests that the human flushing
response, observed in the majority of patients, most
likely occurs via GPR109a.
GPR109a is the human ortholog of the previously de-
scribed rodent receptor (PUMA-G),6 whereas GPR109b
Keywords: Niacin; GPR109a; GPR109b; GPCRs; Lipolysis; Free fatty
acids; Selectfluor; Cutaneous flushing; Pyrazole carboxylic acids;
HM74; HM74a; PUMA-G.
*
Corresponding author. Tel.: +1 858 453 7200; fax: +1 858 4537210;
e-mail: pskinner@arenapharm.com
Figure 1. Known ligands for GPR109a.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.101

P. J. Skinner et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5620–5623
5621
In addition to Niacin (1) two other agents that interact
with GPR109a have been shown to elevate HDL in ro-
dents and humans. Acipimox (2, EC50 = 2.0 lM) was
launched in 1985 by Pharmacia (now Pfizer) for the
treatment of hyperlipidemia and cutaneous flushing is
reported as a side effect with this compound.9 Acifran10
(3) also raises HDL in humans; however, it lacks
selectivity for GPR109a (EC50 = 2.1 lM) over GPR109b
(EC50 = 20 lM)4 and also induces cutaneous flushing.11
Additional agents have been shown to mediate lipid
levels in rats and to bind or activate GPR109a
in vitro. 5-n-Propyl- (4d), 5-i-propyl- (4e) and 5-n-bu-
tyl-pyrazole-3-carboxylic acid (4g) have been shown to
induce hypolipidemia in rats.12 An extension of the alkyl
pyrazole series also recently incorporated functionalized
5-benzyl-pyrazole-3-carboxylic acids which were pro-
posed as partial agonists of GPR109a.13 Recent investi-
gations have also extended the scope of the acifran
series, however, selectivity for GPR109b remains an
issue.14,15 As a part of our ongoing studies to develop
selective agonists of GPR109a, we investigated a series
of 4-functionalized-5-alkyl-pyrazole-3-carboxylic acids
(4–8).
5-Alkyl-pyrazole-3-carboxylic acids (4) were readily syn-
thesized via Claisen condensation of diethyl oxalate with
alkyl methyl ketones to give the corresponding a,c-dike-
to esters (Scheme 1). Cyclization with hydrazine hydro-
chloride afforded the pyrazole-3-carboxylic acid ethyl
esters (9), which upon hydrolysis provided the desired
5-alkyl-pyrazole-3-carboxylic acids (4). 5-Ethyl-4-meth-
ylpyrazole-3-carboxylic acid (5) was prepared in a simi-
lar manner from 3-pentanone (Scheme 2). Synthesis of
5-alkyl-4-fluoro-pyrazole-3-carboxylic acids (6) was
achieved via SelectfluorTM fluorination of the pyrazole-
3-carboxylic acid ethyl esters (9) in acetonitrile to
give 5-alkyl-4-fluoro-pyrazole-3-carboxylic acid ethyl
esters (10) and subsequent hydrolysis. Chlorination of
4-methylpyrazole-3-carboxylic acid ethyl ester (9b) with
N-chlorosuccinimide in CCl4 smoothly gave 3-chloro-4-
methylpyrazole-3-carboxylic acid ethyl ester which was
hydrolyzed to give 3-chloro-4-methylpyrazole-3-carbox-
ylic acid (7). 4-Butylpyrazole-3-carboxylic acid (4g) was
also readily brominated with bromine in acetic acid to
provide 3-bromo-4-butylpyrazole-3-carboxylic acid (7).
Scheme 2. Reagents and conditions: (i) EtO2CCO2Et, KOt-Bu, EtOH,
25 °C, 3 h; (ii) N2H4ÆHCl, EtOH, H2O, 80°; (iii) LiOH (aq), MeOH,
THF, 25 °C, 3 h; (iv) SelectfluorTM, MeCN, 65 °C, 18 h; (v) NCS, CCl4;
(vi) NaOH (aq); (vii) Br2, AcOH.
was measured using a cAMP whole cell Dynamic2
Homogenous Time-Resolved Fluorescence (HTRF) as-
say (Table 1). Twelve compounds were found to exhibit
an EC50 below 1 lM, and of these seven exhibited EC50
values of 100 nM or lower. All of the compounds pre-
pared, which had measurable activity and complete
dose–response curves, displayed efficacy values of 95–
100% relative to niacin (1) suggesting that they are all
full agonists. Niacin (1) has previously been shown to
be equally efficacious with b-hydroxybutyrate, which
has been proposed as a physiologically relevant ligand
for the receptor.17 Optimum activity was found for small
alkyl substituents at C(5)-R1, namely methyl, ethyl,
cyclopropyl, and butyl. Fluorination at C(4)-R2 was well
tolerated, resulting in compounds that were equipotent
with the non-functionalized analogs. Bromination, chlo-
rination, and insertion of a methyl group at C(4)-R2
however, resulted in significant loss of activity in the
HTRF assay. None of the compounds displayed any
activity on GPR109b at concentrations up to 50 lM
with the exception of 4e, 4g, and 4i, which displayed
activities two to three orders of magnitude less potent
than at GPR109a.
The biological activity at GPR109a of each of the 4-
functionalized-5-alkyl-pyrazole-3-carboxylic acids (4–8)
The effect of replacing the carboxylic acid moiety with
the commonly used tetrazole isostere was also investi-
gated. Formation of 5-functionalized-1H-pyrazol-3-yl-
1H-tetrazoles (11a, b, e) was achieved in three steps from
the related ethyl carboxylic ester (9) (Scheme 3). Ami-
Scheme 1. Reagents and conditions: (i) EtO2CCO2Et, KOt-Bu, EtOH,
25 °C, 3 h; (ii) N2H4ÆHCl, EtOH, H2O, 80°; (iii) LiOH (aq), MeOH,
THF, 25 °C, 3 h.

5622
P. J. Skinner et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5620–5623
Table 1. GPR109a and GPR109b agonist activity of selected 4,5-
disubstituted pyrazole-3-carboxylic acids and 4,5-disubstituted pyra-
zole-3-tetrazolesa
Compound C(5)-R1 C(4)-R2 GPR109a
pEC50 (n)
GPR109b
pEC50 (n)
1
—
H
—
H
H
H
H
H
H
H
H
H
Me
F
8.06 0.19 (17) 4.12 0.35 (8)
6.11 0.10 (2) NA (2)
7.61 0.26 (5) NA (2)
7.10 0.17 (5) NA (2)
6.74 0.15 (5) NA (2)
6.96 0.19 (5) 4.38 0.33 (2)
7.00 0.18 (5) NA (2)
7.07 0.18 (5) 4.93 0.19 (4)
5.65 0.14 (4) NA (2)
6.96 0.22 (5) 4.73 0.08 (4)
5.53 0.19 (3) NA (2)
7.38 0.27 (5) NA (2)
7.24 0.28 (5) NA (2)
6.57 0.30 (5) NA (2)
7.10 0.25 (5) NA (2)
5.60 0.22 (5) NA (2)
5.36 0.17 (3) NA (2)
4.77 0.07 (2) NT
4ab
4bc,d
4cd
4db,d
4eb,c,d
4fd
Me
Et
Pr
i-Pr
c-Pr
Bu
Scheme 3. Reagents and conditions: (i) NH3, MeOH, 50 °C, 18 h;
(ii) POCl3, MeCN, NaCl, 80 °C, 18 h; (iii) NaN3, ZnBr, DMF, 175 °C,
20 mins; (iv) BnBr, K2CO3, DMF, 55 °C, 18 h; (v) SOCl2, DMF, rt,
18 h; (vi) N3SiMe3, DMF, 175 °C, 20 mins, lW; (vii) DMSO, THF,
O2, 2 h.
4gb,c,d
4hd
4ic,d
5
c-Bu
Pentyl
Et
6B
6C
Me
Et
F
6F
c-Pr
Bu
F
receptor is sterically constrained and thus highly selec-
tive in this region. Weak activity at GPR109b was ob-
served for 12c and 11g, and moderate activity for 11d
(EC50 = 860 nM) and 11f (EC50 = 3.6 lM) with an
accompanying reversal of selectivity over GPR109a.
Increasing activity at GPR109b with larger substituents
is consistent with a predicted increase in the size of the
binding region of GPR109b over GPR109a.18 The
remaining tetrazoles (11 and 12) displayed no activity
at GPR109b.
6G
7
F
Me
Bu
Cl
Br
H
H
H
H
H
H
F
8
11a
11b
11d
11e
11f
11g
12b
12c
12f
12g
H
Me
Pr
5.82 0.11 (2) NT
5.11 0.31 (5) 6.14 0.18 (5)
4.39 0.56 (2) NT
i-Pr
c-Pr
Bu
4.74 0.49 (5) 5.49 0.08 (5)
4.73 0.27 (6) 5.12 0.21 (5)
5.82 0.07 (7) NA (3)
6.12 0.19 (7) 4.91 0.18 (2)
5.23 0.78 (4) NA (2)
5.46 0.18 (5) NA (3)
Me
Et
F
c-Pr
Bu
F
The reduction of free fatty acid (FFA) levels induced by
oral administration of 4-fluoro-5-methyl-pyrazole-3-car-
boxylic acid (6b) was measured in fasted male Sprague–
Dawley rats (Fig. 2). Significant reductions in plasma
FFA levels were observed upon administration of both
1 and 10 mg/kg 6b that were essentially equivalent in
magnitude to the response elicited by a 10 mg/kg dose
of niacin, but the effects were significantly longer lasting.
Only a very modest reduction of FFA levels was ob-
served following oral administration of 0.1 mg/kg of
6b which was not statistically significant. Cutaneous
vasodilation was also measured in male C57 Bl/6 mice
as a surrogate for the flushing side effect (Fig. 3). 6b,
administered at 100 mg/kg, elicited a cutaneous vasodi-
lation response that appeared notably less than that
F
a Activities were measured from 30 pM to 100 lM and are provided as
the negative log of the molar value of EC50. Errors are log SD.
Compounds that showed no response are designated NA (not active).
Compounds that were untested are designated NT. Efficacies were
observed as 95–100% relative to niacin where accurately measurable.
b Previously described by van Herk et al.13
c Previously described by Seki et al.12
d Previously described by Gharbaoui et al.16
nolysis provided the primary amide which was reduced
to the nitrile using phosphorus oxychloride allowing for-
mation of the desired tetrazole by cyclization with
sodium azide. Yields for the reduction step however,
were low, hence preparation of further tetrazoles (11d,
11f, 11g, 12b, 12c, 12f, 12g) was achieved in five steps
via a benzyl protected pyrazole. Benzyl protection of
the primary amide, reduction and cyclization with azi-
dotrimethylsilane or sodium azide gave the intermediate
benzyl protected tetrazole. Debenzylation in aerated
DMSO gave the desired pyrazole-tetrazoles (17 and
18). However, only moderate activity on GPR109a
was observed; all of the examples, with the exception
of 12c, having EC50 values of greater than 1 lM. In
addition, all the tetrazoles were significantly less potent
than the analogous carboxylic acids. As the pKa values
of the tetrazole and carboxylic acids are similar, this is
highly suggestive that the acid binding region of the
Figure 2. Free fatty acid reduction in male Sprague–Dawley rats with
niacin (1) and (6b).

P. J. Skinner et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5620–5623
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In summary, a series of 5-alkyl-pyrazole-3-carboxylic
acids were prepared and assessed for their activity at
the human GPCR GPR109a. Functionalization at the
4-position with either chlorine, bromine, or methyl sub-
stituents led to significant loss in potency relative to the
unsubstituted analogs. Compounds functionalized with
4-fluoro substituents retained activity, however. The
most potent compound from this series, 4-fluoro-5-
methyl-pyrazole-3-carboxylic acid (6b, EC50 = 42 nM),
was shown to effectively decrease plasma free fatty acid
levels in male Sprague–Dawley rats, an effect that was
much longer lasting than acute treatment with niacin
at 10 mg/kg. Although some cutaneous flushing was also
observed, flushing was notably lower than for a high
(100 mg/kg) dose of niacin, suggesting that there may
be a window of separation between the flushing and
antilipolytic effects of compounds of this type in rodents.
15. Jung, J.-K.; Johnson, B. R.; Duong, T.; Decaire, M.; Uy,
J.; Gharbaoui, T.; Boatman, P. D.; Sage, C. R.; Chen, R.;
Richman, J. G.; Connolly, D. T.; Semple, G. J. Med.
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Supplementary data
16. Gharbaoui, T.; Skinner, P. J.; Shin, Y.-J.; Averbuj, C.;
Jung, J.-K.; Johnson, B. R.; Duong, T.; Decaire, M.; Uy,
J.; Cherrier, M. C.; Webb, P. W.; Tamura, S. Y.; Zou,
N.; Rodriguez, N.; Boatman, P. D.; Sage, C. R.;
Lindstrom, A.; Xu, J.; Schrader, T. O.; Smith, B. M.;
Chen, R.; Richman, J. G.; Connolly, D. T.; Colletti, S.
L.; Tata, J. R.; Semple, G. Bioorg. Med. Chem. Lett.
2007, 17, 4914.
Synthetic methods, spectroscopic data and assay condi-
tions. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2007.07.101.
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