Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A

Article abstract of DOI:10.1016/j.bmcl.2008.08.039

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby

Full text of DOI:10.1016/j.bmcl.2008.08.039

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4948–4951
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrazolopyrimidines as the first class of allosteric agonists
for the high affinity nicotinic acid receptor GPR109A
b
b
b
a
Hong C. Shen a, , Andrew K. P. Taggart , Larissa C. Wilsie , M. Gerard Waters , Milton L. Hammond ,
*
James R. Tata a, Steven L. Colletti a
a Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic
acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic
acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 30 July 2008
Revised 11 August 2008
Accepted 11 August 2008
Available online 14 August 2008
Keywords:
Pyrazolopyrimidines
Allosteric agonists
Nicotinic acid receptor
GPR109A
Nicotinic acid is a group B vitamin that in high doses reduces
morbidity and mortality from cardiovascular disease.1,2 These ben-
efits presumably arise via alterations in the serum lipid profile
including elevated high density lipoprotein cholesterol (HDL-C),
and reduced total plasma cholesterol, very low-density lipoprotein
cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C),
triglyceride (TG), and lipoprotein a (Lp(a)).3 However, the adverse
effect of cutaneous vasodilation (flushing) often limits patient
compliance to therapy.
domains of different orthosteric ligands may not exactly super-
impose, but do overlap to an extent that two orthosteric li-
gands cannot bind simultaneously. Therefore, orthosteric
ligands are competitive in equilibrium binding assays, and
the binding of one orthosteric ligand does not change the dis-
sociation kinetics of another. In contrast, with little or no over-
lap of their binding sites, an allosteric modulator and an
orthosteric ligand can bind to the same receptor simulta-
neously. As
a result, an allosteric modulator may modulate
GPR109A (also called PUMA-G in mice; protein-upregulated in
macrophages by interferon c) is a Gi-protein coupled receptor for
the affinity of an orthosteric ligand, either positively or
negatively.8
nicotinic acid that is highly expressed in adipocytes and myeloid-
lineage immune cells.4,5 Binding of agonists to GPR109A induces
reduction of intracellular cAMP levels which, in adipocytes, is
thought to impair triglyceride lipolysis and thus reduce levels of
circulating free fatty acid (FFA).6 Decreased plasma FFA is thought
to be responsible for the other observed changes in serum lipids
(e.g., reduction in LDL-C and increase of HDL-C).5 GPR109A ex-
pressed in epidermal Langerhans’ cells has also been shown to
mediate cutaneous flushing.6
G-protein-coupled receptor (GPCR) ligands can be classified
as either orthosteric or allosteric modulators.7 Orthosteric and
endogenous ligands bind to the same site, which is topologi-
cally distinct from the allosteric site. In general, the binding
Various orthosteric GPR109A agonists have been reported by
GlaxoSmithKline,9 Arena,10 Roche,11 Schering-Plough,12 Incyte,13
A.P. Ijzerman’s group at Leiden University,14 and ourselves,15
respectively. In contrast, to the best of our knowledge, allosteric
modulators of the nicotinic acid receptor have not been docu-
mented. We speculated that
a positive allosteric modulator
might improve the potency of b-hydroxybutyrate, the proposed
endogenous ligand for GPR109A,16 thereby mimicking, and pos-
sibly improving on, nicotinic acid’s therapeutic effects. It would
also be intriguing to examine whether allosteric modulation of
the receptor would induce the adverse effects of nicotinic acid
treatment, such as cutaneous flushing. Herein, we report our
discovery of pyrazolopyrimidines as the first class of allosteric
agonists for the nicotinic acid receptor GPR109A. In addition,
we present the in vitro characteristics of a representative ana-
log 9n.
* Corresponding author. Tel.: +1 732 594 1755; fax: +1 732 594 9473.
E-mail address: hong_shen@merck.com (H.C. Shen).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.039

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4948–4951
4949
a
H2N
OH
H2N
OPh
1
2
O
OH
HN
N
N
N
OEt
CN
CN
NH2
N
d
c
b
4
3
5
6
O
O
O
O
N
OPh
OH
N
N
N
N
f
e
OEt
OEt
H
N
N
8
9n
7
Scheme 1. Reagents and conditions: (a) PhI, CuI (0.05 equiv), Cs2CO3, pentanitrile, 125 °C, 16 h, 50%; (b) NaH, ethyl formate, tert-amyl alcohol, toluene, 75 °C, 12 h, 70%; (c)
hydrazine, acetic acid, EtOH, reflux, 16 h, 98%; (d) 8, ethanol, reflux, 1 h, 85%; (e) LiOH, THF/MeOH/water (3:1:1), rt, 2 h, 99%; (f) 2, HOBT, EDCI, CH2Cl2, rt, 12 h, 77%.
The synthetic route to pyrazolopyrimidine 9n as a represen-
tative example is summarized in Scheme 1. Starting with 4-iso-
phenoxy group in 9b, we were able to obtain compound
9m with improved activity as a racemate in the hGTP S assay
(EC50 = 0.7 (74%)). Further elaboration using enantiomeri-
cally pure 1-phenoxy-2-propylamine gave two enantiomers
with considerably different affinities for GPR109A. The R-enan-
tiomer 9n was 19-fold more active in the binding assay, and
c
propylphenylacetonitrile 3, an
condensation with hydrazine afforded aminopyrazole 5. The
ensuing reaction of 5 with -ethoxyvinylidene ethyl acetoace-
a-formylation followed by
lM
a
tate 8 generated pyrazolopyrimidine 6.17 The subsequent hydro-
lysis of 6 led to acid 7, which was then coupled with 1-
phenoxy-2-propylamine to give 9n. The requisite 1-phenoxy-2-
propylamine 2 was derived from a CuI-catalyzed chemoselective
O-arylation.18 This sequence was adopted to prepare most pyr-
azolopyrimidine analogs in this letter.
17-fold more active in the hGTPcS assay than its S enantio-
mer 9o (Table 1). As shown in the hGTPcS assay, 9n gave
approximately 75% of nicotinic acid’s maximum response
(Fig. 1).
In addition to their functional activation of GPR109A, com-
pounds of the pyrazolopyrimidine class also enhanced the bind-
ing of nicotinic acid to the receptor. For instance, 9n increased
the binding of 3H-nicotinic acid to the receptor (black curve)
in a concentration-dependent manner, whereas unlabeled nico-
tinic acid displaced 3H-nicotinic acid from the receptor (red
curve) (Fig. 2). We interpret enhanced 3H-nicotinic acid binding
as indicative of positive allosteric modulation of the receptor,
which taken together with the agonist properties of 9n desig-
nates this compound as an allosteric agonist of GPR109A. The
mechanism by which 9n enhances 3H-nicotinic acid binding will
be described elsewhere.
Nicotinic acid has been shown to inhibit forskolin-stimulated
cAMP accumulation in CHO-hGPR109A cells.19 A prediction of
enhanced nicotinic acid binding to GPR109A in the presence of
9n would be that the dose–response curve for nicotinic acid in
this assay might be left-shifted. Like nicotinic acid, 9n alone also
induced the reduction of cAMP levels but with less magnitude
(the bottom curve in Fig. 3). Furthermore, 9n potentiated the
nicotinic acid-induced cAMP reduction in a dose-dependent fash-
ion. As shown in Figure 3, the effect of nicotinic acid on the
cAMP accumulation was significantly left-shifted with increasing
concentration of 9n displaying a sensitization of nicotinic acid
activity.
The activity of compounds against GPR109A was measured
both by 3H-nicotinic acid competition binding assays and ago-
nist-induced exchange of GDP for 35S-GTP
cS on cell mem-
branes prepared from CHO cells overexpressing human
GPR109A.16 The maximum response to nicotinic acid in the
hGTPcS assay was defined as 100%, based on which we mea-
sured the maximum response level of our compounds. The
SAR of the pyrazolopyrimidine class regarding equilibrium
binding in the 3H-nicotinic acid and efficacy in the hGTP
cS as-
says is summarized in Table 1. The pyrazolopyrimidine core of
these analogs appeared to be crucial for activity against
GPR109A since modification of the core rendered compounds
inactive (data not shown). Extensive SAR studies of the pyraz-
olopyrimidine series established the importance of the 1,2-
aminoalkoxy functionality. For example, 9b was active, whereas
its carbon homolog 9c was not. The substitution pattern of the
phenoxy group in 9b clearly influenced activity. The introduc-
tion of para- or meta-methoxy group improved the EC50 values
in the hGTPcS assay, but with lower maximum response (41%
and 55%, respectively). Similar observations were also made
with chloro-, fluoro-, and methyl-substituted analogs (9h–l).
The substitution of methyl group of the 1-methoxy-2-propyla-
mino moiety with phenyl or benzyl groups (9p–q) improved
the EC50 values in the hGTP
similar maximum response level as 9a.
By combining the structural features of the
(with respect to the amide nitrogen atom) in 9a and the
c
S assay by twofold and gave a
In summary, we have discovered a pyrazolopyrimidine class
of allosteric agonists of the nicotinic acid receptor GPR109A.
To our knowledge, this is the first report of allosteric agonists
of this receptor. The observation of GPR109A allosteric agonism
a-methyl group

4950
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4948–4951
Table 1 (continued)
Table 1
In vitro SAR for pyrazolopyrimidine analogs
Compound
3H-nicotinic
acid binding
hGTP
c
l
S
(%) at
100 lM
3H-nicotinic
acid binding
IC50
Ma
hGTP
c
l
S
(%) at
100 lM
EC50
,
Ma
Compound
O
IC50, l
Ma
EC50
,
Ma
O
,
l
N
N
N
H
N
N
N
H
N
N
Nicotinic acid
O
0.15
3.4
1.0
3.2
100
43
O
1.3
0.7
0.12
3
74
75
42
9a
9m
Racemate
O
O
0.17
3.3
R
S
1.0
6.0
76
9b
9n
O
9c
na
—
na
0
9o
O
2.3
21
9d
—
—
1.9
1.4
37
31
9p
O
Racemic
na
na
0
9e
OMe
9q
O
O
—
—
—
1.3
41
55
40
Racemic
OMe
OMe
9f
a
Values were means of at least three experiments, average standard deviation is
O
about 20% (na, not active).
0.93
1.8
9g
F
O
O
9h
R2
4000
IC50
9i
—
—
—
—
0.46
2.3
24
32
35
22
Nicotinic acid
9n
2.618e-006
1.177e-007
0.9497
0.8531
F
Nicotinic acid
9n
3000
2000
1000
0
O
9j
Cl
Me
O
1.3
9k
-9
-8
-7
-6
-5
-4
-3
O
2.1
Compound (log[M])
Me
9l
Figure 1. GTPcS curve of 9n (n = 25).

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4948–4951
4951
6. (a) Offermanns, S. Trends Pharmacol. Sci. 2006, 27, 384; (b) Carballo-Jane, E.;
Ciecko, T.; Luell, S.; Woods, J. W.; Zycband, E. I.; Waters, M. G.; Forrest, M. J.
Inflamm. Res. 2007, 56, 254; (c) Benyo, Z.; Gille, A.; Bennett, C. L.; Clausen, B. E.;
Offermanns, S. Mol. Pharmacol. 2006, 70, 1844; (d) Maciejewski-Lenoir, D.;
Richman, J. G.; Hakak, Y.; Gaidarov, I.; Behan, D. P.; Connolly, D. T. J. Invest.
Dermatol. 2006, 126, 2637.
7. (a) Christopoulos, A. Nat. Rev. Durg. Disc. 2002, 1, 198; (b) Christopoulos, A.;
Kenakin, T. Pharmacol. Rev. 2002, 54, 323.
8. (a) Marino, M. J.; Conn, P. J. Curr. Opin. Pharmacol. 2006, 6, 98; (b) Gao, Z.-G.;
Jacobson, K. A. Drug Discov. Today: Disea. Mech. 2006, 11, 191.
9. (a) Rahman, S. S.; Smith, I. E. D. Patent WO 2005/016867 A2; (b) Campbell, M.;
Hatley, R. J. D.; Heer, J. P.; Mason, A. M.; Nicholson, N. H.; Pinto, I. L.; Rahman, S.
S.; Smith, I. E. D. Patent WO 2005/016870 A1.; (c) Pinto, I. L.; Rahman, S. S.;
Nicholson, N. H. Patent WO 2005/077950 A2.; (d) Campbell, M.; Mason, A. M.;
Pinto, I. L.; Pollard, D. R.; Smith, I. E. D. Patent WO 2006/085108 A1.; (e) Mason,
A. M.; Pinto, I. L.; Rahman, S. S. Patent WO 2006/085111 A1.; (f) Hatley, R. J. D.;
Mason, A. M.; Pinto, I. L.; Smith, I. E. D. Patent WO 2006/085112 A1.; (g) Pinto, I.
L.; Simpson, J. K. Patent WO 2006/085113 A2.; (h) Hatley, R. J. D.; Pinto, I. L.
Patent WO 2006/045564 A1.; (i) Hatley, R. J. D.; Pinto, I. L. Patent WO 2006/
045565 A1.; (j) Hatley, R. J. D.; Mason, A. M.; Pinto, I. L. Patent WO 2007/017261
A1.; (k) Hatley, R. J. D.; Heer, J. P.; Liddle, J.; Mason, A. M.; Pinto, I. L.; Rahman, S.
S.; Smith, I. E. D. Patent WO 2007/017262 A1.; (l) Heer, J. P.; Smith, I. E. D.
Patent WO 2007/017265 A1.
10. (a) Boatman, D. P.; Schrader, T. O.; Semple, G.; Skinner, P. J.; Jung, J.-K. Patent
WO 2006/069242 A2.; (b) Semple, G.; Schrader, T.; Skinner, P. J.; Colletti, S. L.;
Gharbaoui, T.; Imbriglio, J. E.; Jung, J.-K.; Liang, R.; Raghavan, S.; Schmidt, D.;
Tata, J. R. Patent WO 2005/044816 A1.; (c) Skinner, P. J.; Cherrier, M. C.; Webb,
P. J.; Shin, Y.-J.; Gharbaoui, T.; Lindstrom, A.; Hong, V.; Tamura, S. Y.; Dang, H.
T.; Chen, R.; Richman, J. G.; Connolly, D. T.; Semple, G. Bioorg. Med. Chem. Lett.
2007, 17, 5620.
R2
0.9839
0.9755
IC50
10000
7500
5000
2500
0
Nicotinic acid 1.505e-007
9n
1.728e-007
Nicotinic acid
9n
-11
-10
-9
-8
-7
-6
-5
-4
Compound (log[M])
Figure 2. Binding curve of 9n and nicotinic acid (n = 8).
Niicotinic acid
Nicotinic acid + 50 nM 9n
120
Nicotinic acid + 500 nM 9n
Nicotinic acid + 1uM 9n
9n
110
100
90
80
70
60
50
40
30
20
10
0
11. (a) Dehmlow, H.; Grether, U.; Kratochwil, N. A.; Narquizian, R.; Panousis, C.;
Peters, J.-U. Patent US 2006/0281810 A1.; (b) Dehmlow, H.; Grether, U.;
Kratochwil, N. A.; Narquizian, R.; Panousis, C. Patent WO 2007/0072873 A1.
12. Palani, A.; Su, J.; Xiao, D.; Huang, X.; Rao, A. U.; Chen, X.; Tang, H.; Qin, J.;
Huang, Y.; Aslanian, R. G.; Mckittrick, B. Patent WO 2006/124490 A2.
13. Cao, G.; Xue, C.-B.; Anand, R.; Huang, T.; Kong, L.; Glenn, J.; Feng, H. Patent WO
2007/015744 A1.
14. Herk, T.; Brussee, J.; Nieuwendijk, A. M. C. H.; Klein, P. A. M.; Ijzerman, A. P.;
Stannek, C.; Brumeister, A.; Lorenzen, A. J. Med. Chem. 2003, 46, 3945.
15. (a) Imbriglio, J. E.; Colletti, S. L.; Tata, J. R.; Liang, R.; Raghavan, S.; Schmidt, D.
R.; Smenton, A. R.; Chan, S. Y. Patent WO 2006/113150 A1.; (b) Colletti, S. L.;
Beresis, R. T.; Chen, W.; Tata, J. R.; Shen, H. C.; Marley, D. M.; Deng, Q.; Frie, J. L.;
Ding, F.-X. Patent WO 2006/052555 A2.; (c) Raghavan, S.; Colletti, S. L.; Ding, F.-
X.; Shen, H.; Tata, J. R.; Lins, A. R.; Smenton, A. L.; Chen, W.; Schmidt, D. Y.; Tria,
G. S. Patent WO 2007/002557 A1.; (d) Colletti, S. L.; Tata, J. R.; Shen, H. C.; Ding,
F.-X.; Frie, J. L.; Imbriglio, J. E.; Chen, W. Patent WO 2006/057922 A2.; (e)
Colletti, S. L.; Shen, H.; Tata, J. R.; Szymonifka, M. J. Patent WO 2007/027532
A2.; (f) Colletti, S. L.; Imbriglio, J. E.; Beresis, R. T.; Frie, J. L. Patent WO 2007/
035478 A2.; (g) Shen, H. C.; Szymonifka, M.; Deng, Q.; Carballo-Jane, E.; Cheng,
K.; Wu, K.; Wu, T.-J.; Wang, J.; Tong, X.; Ren, N.; Taggart, A.; Cai, T.; Waters, G.;
Hammond, M.; Tata, J. R.; Colletti, S. L. Bioorg. Med. Chem. Lett. 2007, 17, 6723;
(h) Shen, H. C.; Ding, F.-X.; Taggart, A.; Cheng, K.; Carballo-Jane, E.; Ren, N.;
Chen, Q.; Wang, J.; Wolff, M.; Waters, G.; Hammond, M.; Tata, J. R.; Colletti, S. L.
J. Med. Chem. 2007, 50, 6303; (i) Raghavan, S.; Tria, G. S.; Shen, H. C.; Ding, F.-X.;
Taggart, A. K. P.; Ren, N.; Wilsie, L. C.; Krsmanovic, M. L.; Holt, T. G.; Wolff, M.
S.; Waters, M. G.; Hammond, M. L.; Tata, J. R.; Colletti, S. L. Bioorg. Med. Chem.
Lett. 2008, 18, 3163.
-10
-12 -11 -10 -9
-8
-7
-6
-5
-4
-3
Compound (log[M])
Figure 3. Effects of 9n on cAMP accumulation in CHO-hGPR109A cells.
as reflected by these analogs may offer interesting implications,
worthy of further pharmacological investigation.
References and notes
1. (a) Mack, W. J.; Selzer, R. H.; Hodis, H. N.; Erickson, J. K.; Liu, C. R.; Liu, C. H.;
Crawford, D. W.; Blankenhorn, D. H. Stroke 1993, 24, 1779; (b) Nikkila, E. A.;
Viikinkoski, P.; Valle, M.; Frick, M. H. Br. Med. J. 1984, 289, 220.
2. (a) Canner, P. L.; Berge, K. G.; Wenger, N. K.; Stamler, J.; Friedman, L.; Prineas, R.
J.; Friedewald, W. J. Am. Coll. Cardiol. 1986, 8, 1245; (b) Pritzker, L. B. Crit. Rev.
Clin. Lab. Sci. 1998, 35, 603; (c) Taylor, A. J.; Sullenberger, L. E.; Lee, H. J.; Lee, J.
K.; Grace, K. A. Circulation 2004, 110, 3512.
16. Taggart, A. K. P.; Kero, J.; Gan, X.; Cai, T.-Q.; Cheng, K.; Ippolito, M.; Reng,
N.; Kaplan, R.; Wu, K.; Wu, T.-J.; Jin, L.; Liaw, C.; Chen, R.; Richman, J.;
Connolly, D.; Offermanns, S.; Wright, S. D.; Waters, M. G. J. Biol. Chem.
2005, 29, 26649.
3. Knopp, R. H. N. Engl. J. Med. 1999, 341, 498.
4. Schaub, A.; Futterer, A.; Pfeffer, K. Eur. J. Immunol. 2001, 31, 3714.
5. (a) Wise, A.; Foord, S. M.; Fraser, N. J.; Barnes, A. A.; Elshourbagy, N.; Eilert, M.;
Ignar, D. M.; Murdock, P. R.; Steplewski, K.; Green, A.; Brown, A. J.; Dowell, S. J.;
Szekeres, P. G.; Hassall, D. G.; Marshall, F. H.; Wilson, S.; Pike, N. B. J. Biol. Chem.
2003, 278, 9869; (b) Tunaru, S.; Kero, J.; Schaub, A.; Wufka, C.; Blaukat, A.;
Pfeffer, K.; Offermanns, S. Nat. Med. 2003, 9, 352.
17. Bruni, F.; Selleri, S.; Costanzo, A.; Curerrini, G.; Casilli, M. L. J. Heterocycl. Chem.
1995, 32, 291.
18. Job, G. E.; Buchwald, S. L. Org. Lett. 2002, 4, 3703.
19. Soga, T.; Kamohara, M.; Takasaki, J.; Matsumoto, S.; Saito, T.; Ohishi, T.;
Hiyama, H.; Matsuo, A.; Matsushime, H.; Furuichi, K. Biochem. Biophys. Res.
Commun. 2003, 303, 364.