Novel 3,6,7-substituted pyrazolopyrimidines as positive allosteric modulators for the hydroxycarboxylic acid receptor 2 (GPR109A)

Article abstract of DOI:10.1021/jm300164q

A number of pyrazolopyrimidines were synthesized and tested for their positive allosteric modulation of the HCA₂ receptor (GPR109A). Compound 24, an efficacious and potent agonist and allosteric enhancer of nicotinic acid's action, was the basis for most other compounds. Interestingly, some of the compounds were found to increase the efficacy of the endogenous ligand 3-hydroxybutyrate and enhance its potency almost 10-fold. This suggests that the pyrazolopyrimidines may have therapeutic value when given alone.

Full text of DOI:10.1021/jm300164q

Brief Article
pubs.acs.org/jmc
Novel 3,6,7-Substituted Pyrazolopyrimidines as Positive Allosteric
Modulators for the Hydroxycarboxylic Acid Receptor 2 (GPR109A)
Clara C. Blad,† Jacobus P. D. van Veldhoven,† Corne Klopman, Dieter R. Wolfram, Johannes Brussee,
́
J. Robert Lane,‡ and Adriaan P. IJzerman*
Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden,
The Netherlands
S
* Supporting Information
ABSTRACT: A number of pyrazolopyrimidines were synthesized and tested for their positive allosteric modulation of the HCA2
receptor (GPR109A). Compound 24, an efficacious and potent agonist and allosteric enhancer of nicotinic acid’s action, was the
basis for most other compounds. Interestingly, some of the compounds were found to increase the efficacy of the endogenous
ligand 3-hydroxybutyrate and enhance its potency almost 10-fold. This suggests that the pyrazolopyrimidines may have
therapeutic value when given alone.
3-bromo building block 31 were synthesized from the
corresponding acids (15−19) and a range of amines by use
of coupling reagent EDC·HCl. Finally a Suzuki reaction with
building block 31 and various arylboronic acids, under
microwave conditions, yielded the 3-aryl compounds 32−43.16
INTRODUCTION
■
Hydroxycarboxylic acid receptor 2 (HCA2)1 or GPR109A is a
G-protein-coupled receptor (GPCR) that was first identified in
2001.2 Two years later its involvement in the antilipolytic
action of nicotinic acid (NA) was reported3−5 and 3-
hydroxybutyrate was identified as its endogenous ligand in
20056 (for reviews, see refs 1 and 7). A broad range of synthetic
ligands (agonists only) for this receptor has been developed.7−9
An intriguing class among those are substituted pyrazolopyr-
imidines, as recently reported by Shen and colleagues.10 These
compounds act as agonists, some with potencies comparable to
that of nicotinic acid, but were also suggested to bind
allosterically to HCA2. One compound in particular (25 in
the present study) was shown to behave as a positive allosteric
modulator (PAM) as well, by significantly enhancing the
potency of nicotinic acid at HCA2 (∼100-fold at 1 μM). We
decided to further investigate the pyrazolopyrimidines by
synthesizing a new series of derivatives and evaluating their
activity on HCA2 in several [3H]nicotinic acid and [35S]GTPγS
binding assays.
BIOLOGICAL EVALUATION AND DISCUSSION
■
To evaluate the compounds, we performed [3H]nicotinic acid
binding assays (see Supporting Information Table S1) and
[35S]GTPγS binding assays (Table 1) on HEK-HCA2
membranes. In Table 1 the following results are reported: (a)
the percentage of [35S]GTPγS binding in the presence of 10
μM test compound alone; (b) the shift in EC50 of nicotinic acid
in the presence of 10 μM test compound; (c) the Emax reached
at 100 μM nicotinic acid in the presence of 10 μM test
compound (100% is without test compound).
Structure−Activity Relationships. Compounds 20−23
show that linker lengths of two or three carbons between the
carboxamide and the phenyl group are preferred for agonism
and potentiation of nicotinic acid’s effects. However, linker
length does not appear to influence the Emax of nicotinic acid,
which was ∼130% for all four compounds. Introduction of an
ether function in the linker (24) resulted in a higher activity in
the GTPγS binding assay. So the compound acted as an agonist
in its own right, while the EC50 of nicotinic acid was increased
approximately 25-fold without a change in its Emax. This
compound also considerably slowed the dissociation of
[3H]nicotinic acid from the receptor and increased the
equilibrium specific binding of the radioligand to an extent
that was not surpassed by any of the other test compounds
(Supporting Information Table S1). Introduction of a methyl
substituent on the linker (25) was not beneficial in our hands,
as opposed to the findings of Shen et al.10 Removal of the
phenyl group yielded 26, which enhanced nicotinic acid’s
activity somewhat better. Replacing the ether of 24 by a
CHEMICAL SYNTHESIS
■
The synthetic route used to obtain the pyrazolo[1,5-a]-
pyrimidine-6-carboxamides 20−30 and 32−43 is depicted in
Scheme 1. The 1H-pyrazol-5-amine 110 or the commercially
available 2 was ring closed with the respective ethyl 2-
(ethoxymethylene)-3-oxoate11 (3, 5, or 6) or the available
diester 4 in EtOH at reflux temperature, resulting in the ethyl
pyrazolo[1,5-a]pyrimidine-6-carboxylates (7, 8, and 11−
13)10,12,13 in good yields. The 7-hydroxypyrazolopyrimidine 8
was converted into the 7-chloro analogue (9) in the presence of
13
POCl3 followed by palladium-catalyzed reductive dechlorina-
tion13 to give the pyrazolo[1,5-a]pyrimidine 10. The 3-position
of the pyrazolo[1,5-a]pyrimidine 1314 was functionalized using
NBS15 to the versatile 3-bromo analogue 14. Subsequently
hydrolysis of the ethyl esters 7, 10−12, and 14 with LiOH
smoothly resulted in the corresponding acids (15−19). The
final pyrazolo[1,5-a]pyrimidine carboxamides 20−30 and the
Received: October 24, 2011
Published: March 15, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
trifluoromethyl group (43) conferred good enhancement of
nicotinic acid potency and efficacy, paired with a moderate
agonistic activity. To investigate any correlation between the
agonistic and modulating effects of the pyrazolopyrimidines,
the [35S]GTPγS binding activation and the potency shift of
nicotinic acid, both at 10 μM, were plotted against each other
(Supporting Information Figure S2), yielding a nonlinear
correlation. The plot suggests that the ability of the
pyrazolopyrimidine agonists to stabilize active receptor
conformations contributes to, or even determines, the
modulator strength of the compounds.
Scheme 1. Synthetic Route to the Substituted Pyrazolo[1,5-
a]pyrimidinecarboxamides 20−43
a
Modulation of Nicotinic Acid Mediated Receptor
Activation. We next examined the effects at 1, 3, and 10
μM of five selected enhancers on the concentration−effect
curves for nicotinic acid in [35S]GTPγS binding assays. Figure 1
shows the results of a representative experiment, and the
average values obtained from three independent experiments
are discussed below. Compound 24 reached 71 17% receptor
activation at 10 μM without any nicotinic acid present, and
nicotinic acid further increased activation to 109 6%, with a
24-fold increased potency compared to control.
At concentrations of 3 and 1 μM, 24 increased [35S]GTPγS
binding to 51 14% and 30 5%, respectively, and caused
shifts in the EC50 of nicotinic acid of ∼5-fold for both
concentrations. Compound 27 caused modest increases in the
potency (2-fold at 1 μM, 5-fold at 3 μM, and 7-fold at 10 μM)
and the Emax (120 3% at 1 μM, 129 3% at 3 μM, and 139
10% at 10 μM) of nicotinic acid, which seemed to follow the
increase in [35S]GTPγS binding due to agonist activity alone
(13 2% at 1 μM, 31 2% at 3 μM, and 41 6% at 10 μM).
Compound 29 was highly efficacious in all aspects: an agonist
in its own right (28 3%, 53 5%, and 69 7% receptor
activation at 1, 3, and 10 μM, respectively) and a positive
allosteric modulator of nicotinic acid’s EC50 (5-, 9-, and 13-fold
a
Reagents and conditions: (a) EtOH, reflux, 3 h; (b) POCl3, N,N-
dimethylaniline, reflux, 3 h; (c) NaOAc, 5% Pd/C, H2 2.5 bar, rt, 1 h;
(d) NBS, DCM, 0 °C, 1.5 h, rt, 16 h; (e) LiOH, H2O/MeOH/THF,
rt, 16 h; (f) R3NH2, EDC·HCl, DCM, rt, 4 h; (g) R1B(OH)2, 31,
Pd(Ph3P)4, Na2CO3, toluene/H2O, microwave 150 °C, 2 h.
shifts) and Emax (122
7%, 129
6%, and 151
6% of
control). Both 38 and 42 were in every respect less potent and
efficacious than 29. As an agonist, 38 was more potent and
efficacious than 42, causing more receptor activation at 1 μM
(15 6% vs 5 3%), 3 μM (27 2% vs 16 3%), and 10 μM
secondary amine to obtain aniline 27 rendered the compound
more active than 25 in all respects.
Next, a small series was synthesized with varying substituents
on the 7-position (R2, Table 1). Absence of the methyl
substituent (28) severely reduced the activity compared to 24.
The ethyl and propyl substituted compounds (29 and 30)
behaved highly similar to each other and also to the parent
derivative 24 except for the enhancement of nicotinic acid’s
(50
3% vs 31
5%). The modulation of the EC50 of
nicotinic acid was similar at 1 μM (both 2-fold) and 3 μM (5-
fold vs 4-fold), but at 10 μM 38 was the more active compound
again (11-fold vs 5-fold shift). Both compounds increased the
Emax of nicotinic acid similarly (139 12%, 139 6% at 10
μM). However, 42 showed a relatively low potency compared
to 38, since no effect was seen at 1 μM (compared to 111 4%
E
max by 29 and 30 but not 24. Finally, a series of derivatives of
24 with varying substituents on the 3-phenyl ring was then
tested (R1, Table 1, 32−43). A methyl substituent on the para
position (32) resulted in more agonism and potentiation of
nicotinic acid compared to methyl substituents at the meta
(33) or ortho (34) positions. Compounds with ethyl (35) and
tert-butyl (36) substituents had activities highly similar to that
of the isopropyl-substituted 24 except that 35 and 36
significantly increased the Emax of nicotinic acid, which was
not affected by 24. Compounds substituted with methoxy (37)
or isopropoxy (38) were also highly active. These compounds,
like methyl derivatives 32 and 33, seemed to have a more
modest agonistic activity compared to their activity as
enhancers of nicotinic acid potency. A phenyl derivative (39)
retained activity in the [35S]GTPγS binding assay. Meta-
substitution (41), and to a lesser extent, para-substitution (40)
with chlorine diminished activity. The 3,4-dichloro-substituted
42 behaved very similarly to the 4-chloro derivative 40. A
for 38) and only a small effect at 3 μM (114
3% for 42
compared to 123 5% for 38). For EC50 values and Emax values
of 24, 29, 30, 32, 35, 36 and 38, tested again in the absence of
nicotinic acid, see Table S2 in Supporting Information. Some
further observations are discussed on page S7 of Supporting
Information.
Positive Allosteric Modulation of 3-Hydroxybutyrate
Potency and Efficacy. Allosteric modulators that enhance the
potency of nicotinic acid on the HCA2 receptor are of interest
clinically, since they could greatly reduce the daily dose of
nicotinic acid when used in combination therapy. Clearly the
pyrazolopyrimidines fit this picture, but many of them can also
activate HCA2 on their own. In vivo, the activity of the
endogenous ligand 3-hydroxybutyrate (3-OHB) may also be
enhanced by the pyrazolopyrimidines, but this cannot be
assumed a priori, since allosteric enhancement is probe
dependent.17 Therefore, 3-OHB dose−response curves were
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Journal of Medicinal Chemistry
Brief Article
e
Table 1. Different Measures for HCA2 Receptor Activation in [35S]GTPγS Binding Assays
a
b
Percentage of [35S]GTPγS binding in the presence of 10 μM test compound alone (100% is the Emax at 100 μM nicotinic acid). Shift in EC50 of
nicotinic acid (NA) in the presence of 10 μM test compound (1 is without test compound, and values less than unity indicate increased affinity of
c
nicotinic acid, e.g., where 0.1 means a 10-fold shift). Emax reached at 100 μM nicotinic acid (NA) in the presence of 10 μM test compound (100% is
without test compound). Also reported in ref 10 (26, racemate only). Values are the mean ( SEM) of three independent experiments performed
d
e
in duplicate.
recorded in the presence of five compounds, and four of these
increased the potency of 3-hydroxybutyrate approximately 8-
fold (Figure 2 and Supporting Information Table S3). The
modulators caused an increase in the intrinsic efficacy of the
endogenous ligand as well. In a [35S]GTPγS binding assay 3-
OHB was previously reported as a high-efficacy partial agonist
for HCA2,18 whereas it behaved as a low-efficacy partial agonist
with ∼30% intrinsic efficacy in our hands. This may have to do
with lower levels of receptor expression in our preparation, as
we explicitly selected clones for physiological rather than high
expression levels.
Future Perspectives and Conclusion. With the expanded
ligand repertoire reported in this study other functional assays
with the pyrazolopyrimidines can be performed, including ERK
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Journal of Medicinal Chemistry
Brief Article
Chemical shifts (δ) are reported in ppm relatively to Me4Si. Purity was
confirmed to be ≥95% by HPLC performed on a Gilson 306 system
(detection at 254 nm) equipped with an analytical C18 column in
combination with a gradient of mixture A (1 MeCN/9 H2O) and B (9
MeCN/1 H2O). High resolution mass spectra were recorded on a
mass spectrometer (Thermo Finnigan LTQ Orbitrap) with an
electrospray ion source in positive mode, resolution R = 60000 at
m/z 400 (mass range m/z = 150−2000). Reactions were routinely
monitored with TLC using Merck silica gel F254 plates. Microwave
reactions were performed in an Emrys optimizer (Biotage AB). Yields
were not optimized. The final products were purified by column
chromatography and/or by recrystallization.
General Procedure for the Synthesis of Pyrazolo[1,5-
a]pyrimidinecarboxamides (20−31). To a solution of the
appropriate pyrazolo[1,5-a]pyrimidine-6-carboxylic acid (15−19; see
Supporting Information) (1.0 equiv) and the substituted amine (1.2
equiv) in DCM (20 mL per mmol) was added EDC·HCl (1.2 equiv)
at room temperature. After 4 h the mixture was concentrated and
purified by column chromatography.
3-(4-Isopropylphenyl)7-methyl-N-(2-phenoxyethyl)-
pyrazolo[1,5-a]pyrimidine-6-carboxamide (24). The synthesis
started from acid 15 (0.33 mmol) and 2-phenoxyethylamine to give 90
1
mg (64%) as a yellow solid. H NMR (CDCl3): δ 8.48 (s, 1H), 8.14
(s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 11.2 Hz, 2H), 7.25 (d, J
= 8.0 Hz, 2H), 6.95 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 8.0 Hz, 2H), 6.76
(t, J = 5.6 Hz, 1H), 4.22 (t, J = 5.2 Hz, 2H), 3.82 (q, J = 5.2 Hz, 2H),
2.94 (m, 1H), 2.89 (s, 3H), 1.28 (d, J = 6.8 Hz, 6H). HRMS calcd for
[C25H26N4O2 + H]+ 415.212 85, found 415.212 74.
General Procedure for the Preparation of 3-(Aryl)-7-methyl-
N-(2-phenoxyethyl)pyrazolo[1,5-a]pyrimidine-6-carboxamides
via Suzuki Coupling (32−43). The synthesis was done according to
a modified procedure of Berger.15 A mixture of 31 (1.0 equiv), the
substituted phenylboronic acid (2.0 equiv), tetrakis-
(triphenylphosphine)palladium(0) (0.03 equiv), and sodium carbo-
nate (3.0 equiv) in toluene (3.0 mL) and H2O (0.5 mL) was heated in
the microwave for 2 h at 150 °C. Water was added, and the organics
were extracted with DCM. The organic layer was dried, concentrated,
and purified by column chromatography (1% MeOH/DCM). Final
products were obtained by recrystallization from MeOH.
Figure 1. Dose−response curves of nicotinic acid in the presence of 0,
1, 3, and 10 μM pyrazolopyrimidines. The data are from [35S]GTPγS
binding assays performed on HEK-HCA2 membranes. Shown are
representative graphs from one experiment performed in duplicate.
3-(4-Chlorophenyl)-7-methyl-N-(2-phenoxyethyl)pyrazolo-
[1,5-a]pyrimidine-6-carboxamide (40). The synthesis started from
31 (0.33 mmol) and (4-chlorophenyl)boronic acid (0.66 mmol).
Yield, 82 mg (62%). 1H NMR (CDCl3): δ 8.62 (s, 1H), 8.50 (s, 1H),
7.89 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 8.0 Hz,
2H), 7.00 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.42 (s, 1H,
NH), 4.21 (t, J = 5.2 Hz, 2H), 3.94 (q, J = 5.6 Hz, 2H), 3.03 (s, 3H).
HRMS calcd for [C22H19ClN4O2 + H]+ 407.126 93, found 407.126 79.
Biology. [35S]GTPγS Binding Assay. This assay was performed in
96-well format in 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2, 150
mM NaCl, pH 7.4 at 25 °C, with 1 mM DTT, 0.5% BSA, and 50 μg/
mL saponin freshly added. HEK-HCA2 membranes (5 μg of protein
per well in 25 μL) were preincubated with 25 μL of 40 μM GDP, in
absence or presence of test compound, and 25 μL of increasing
concentrations of the orthosteric ligand, for 30 min at room
temperature. Then 25 μL of [35S]GTPγS was added (final
concentration, 0.3 nM) and the mixture was incubated for 90 min at
25 °C with constant shaking. The incubation was terminated by
filtration over GF/B filter plates on a FilterMate harvester
(PerkinElmer). The filters were dried, and 25 μL of Microscint-20
(PerkinElmer) was added to each filter. After ≥3 h extraction the
bound radioactivity was determined in a Wallac MicroBeta TriLux
1450 counter.
Figure 2. Concentration−response curves of 3-hydroxybutyrate in the
presence and absence of 10 μM pyrazolopyrimidine. The data are from
[35S]GTPγS binding assays performed on HEK-HCA2 membranes.
Shown are representative graphs from one experiment performed in
duplicate (see also Table S3 in Supporting Information).
1/2 phosphorylation assays. Activation of ERK 1/2 induced by
HCA2 agonists in vitro has been suggested to be predictive of
the skin flushing side effect in vivo.19 Furthermore,
cooperativity with probes (orthosteric ligands) that are not
structurally related to nicotinic acid should be examined. In the
future, mutagenesis studies will hopefully shed light on the
binding mode of the allosteric modulators and on how these
ligands trigger the changes in receptor activation. In conclusion,
we presented several pyrazolopyrimidine derivatives that do not
displace [3H]nicotinic acid from HCA2 but are capable of
activating this receptor, which indicates an allosteric binding
mode. Next to their agonistic effects these compounds
potentiate the action of nicotinic acid and the endogenous
ligand 3-hydroxybutyrate. Therapeutically, such positive allos-
teric modulators may represent an interesting alternative in the
search for HCA2 receptor ligands.
Data Analysis. Analysis of the results was performed using Prism
5.0 software (GraphPad Software Inc., La Jolla, CA). Nonlinear
regression was used to determine IC50 values from competition
binding curves. The Cheng−Prusoff equation20 was then applied to
calculate Ki values. [35S]GTPγS curves were analyzed by nonlinear
regression to obtain EC50 values.
EXPERIMENTAL SECTION
■
1
Chemistry. H and 13C NMR spectra were recorded on a Bruker
AV 400 (1H NMR, 400 MHz; 13C NMR, 100 MHz) spectrometer.
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Journal of Medicinal Chemistry
Brief Article
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Hammond, M. L.; Tata, J. R.; Colletti, S. L. Discovery of
pyrazolopyrimidines as the first class of allosteric agonists for the
high affinity nicotinic acid receptor GPR109A. Bioorg. Med. Chem. Lett.
2008, 18, 4948−4951.
(11) Ife, R. J.; Brown, T. H.; Keeling, D. J.; Leach, C. A.; Meeson, M.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Tables S1−S3, Figures S1 and S2, additional notes, synthesis
and characterization of chemical compounds, and biological
methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +31715274651. E-mail: ijzerman@chem.leidenuniv.nl.
Present Address
‡Monash Institute of Pharmaceutical Sciences, Monash
University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
Author Contributions
†Both authors contributed equally.
(13) Bruni, F.; Selleri, S.; Costanzo, A.; Guerrini, G.; Casilli, M. L.;
Giusti, L. Reactivity of 7-(2-dimethylaminovinyl)pyrazolo[1,5-a]-
pyrimidines: synthesis of pyrazolo[1,5-a]pyrido[3,4-e]pyrimidine
derivatives as potential benzodiazepine receptor ligands. 2. J.
Heterocycl. Chem. 1995, 32, 291−298.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
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the HCA2 plasmid for cell line generation. This work was
performed within the framework of the Dutch Top Institute
Pharma (GPCR Forum, Project D1-105).
Miralles, A.; Merlos, M.; García-Rafanell, J.; Forn, J. Synthesis and SAR
of a new series of COX-2-selective inhibitors: pyrazolo[1,5-
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Gopalsamy, A.; Levin, J. I.; Kim, K.-H.; Xu, W.; Wilhelm, J.; Hu, Y.;
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ABBREVIATIONS USED
■
NA, nicotinic acid; 3-OHB, 3-hydroxybutyrate; [35S]GTPγS,
35S-labeled guanosine 5′-O-[γ-thio]triphosphate; BSA, bovine
serum albumin; DMEM, Dulbecco’s modified Eagle medium;
DTT, dithiothreitol; GDP, guanosine 5′-diphosphate; GPCR,
G-protein-coupled receptor; HCA, hydroxycarboxylic acid
receptor; HEK, human embryonic kidney cell; PAM, positive
allosteric modulator (allosteric enhancer)
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