Pyrazole acids as niacin receptor agonists for the treatment of dyslipidemia

Article abstract of DOI:10.1016/j.bmcl.2009.06.054

Niacin is an effective drug for raising HDL cholesterol and reducing coronary risks, but patients show low compliance with treatment due to severe facial flushing upon taking the drug. A series of bicyclic pyrazole carboxylic acids were synthesized and tested for their ability to activate the niacin receptor. One analog, 23, showed improved potency and lacked flushing at doses that effectively altered the lipid profile of rats.

Full text of DOI:10.1016/j.bmcl.2009.06.054

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4768–4772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazole acids as niacin receptor agonists for the treatment of dyslipidemia
a
a
b
b
Darby Schmidt a, , Abigail Smenton , Subharekha Raghavan , Ester Carballo-Jane , Silvi Lubell ,
Tanya Ciecko b, Tom G. Holt a, Michael Wolff a, Andrew Taggart b, Larissa Wilsie b, Mihajlo Krsmanovic b,
Ning Ren b, Daniel Blom b, Kang Cheng b, Peggy E. McCann b, M. Gerard Waters b, James Tata a,
Steven Colletti a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ 07065-0900, USA
b Department of Cardiovascular Disease, Merck Research Laboratories, Merck & Co., Inc., 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:
Received 24 March 2009
Revised 6 June 2009
Accepted 15 June 2009
Available online 17 June 2009
Niacin is an effective drug for raising HDL cholesterol and reducing coronary risks, but patients show low
compliance with treatment due to severe facial flushing upon taking the drug. A series of bicyclic pyra-
zole carboxylic acids were synthesized and tested for their ability to activate the niacin receptor. One
analog, 23, showed improved potency and lacked flushing at doses that effectively altered the lipid profile
of rats.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Atherosclerosis
Niacin
HDL
Elevated levels of LDL cholesterol and reduced HDL cholesterol
are both strong risk factors for the development of atherosclerotic
coronary heart disease. Statins can effectively lower LDL, but they
have shown only modest effects on HDL (5–15%).1 Even when LDL
levels are successfully lowered, patients with low HDL still face an
increased risk of mortality relative to patients with high HDL.
Therefore, the next generation of treatments for dyslipidemia must
address the issue of HDL cholesterol levels.
Niacin (1, Fig. 1) has been used for over 40 years for the treatment
of dyslipidemia. Niacin therapy leads to lowered free fatty acids (20–
40%), triglycerides (20–40%), lipoprotein A (30–40%), and LDL cho-
lesterol (20–40%), but also to a significant elevation of HDL choles-
terol (20–35%).2 In addition, long term studies have demonstrated
that niacin therapy reduces mortality from coronary heart disease.3
ThisprofilehasmadeniacinanattractivetreatmentforlowHDL cho-
lesterol, but side effects of the drug have limited its use. Most pa-
tients experience an intense and painful facial flushing after taking
the drug.3 In addition, niacin shows hepatotoxicity and reduces glu-
cose tolerance, which limits its use in prediabetic patients.4
the decrease in free fatty acids upon niacin treatment that is seen
in wild type mice, making this the likely target of niacin. Unfortu-
nately, niacin receptor knockout mice failed to show a niacin in-
duced flush, indicating that flushing was in fact mediated
through the niacin receptor. Thus, the development of a flush free
niacin receptor agonist represents a significant challenge.
The niacin receptor agonist 2 (Fig. 1) was developed twenty
years ago6 and was recently reinvestigated using niacin receptor
binding and functional assays. Further modifications of 2 led to
the discovery of compounds that were partial agonists7 with
equivalent or improved activity compared to niacin. Among them,
compound 3 was of interest to us because it was equipotent to
compound 2 and seemed amenable to rapid modifications. We
O
O
O
OH
OH
OH
O
N
N
N
N
H
N
H
Cl
Ar
Early efforts to improve upon niacin proved challenging be-
cause its mechanism of action was not known. Renewed interest
in HDL raising drugs spurred efforts to identify niacin’s cellular tar-
get, and a Gi coupled GPCR, GPR109A, in fat cells, also referred to as
HM74A, or 19CD, was identified. This receptor showed a high affin-
ity for niacin.5 In addition, knockout mice of GPR109A did not show
1
2
3
O
OH
OH
N
N
N
N
H
H
Cl
4
4a
* Corresponding author.
E-mail address: darbyrschmidt@verizon.net (D. Schmidt).
Figure 1. Structure of niacin and lead structures.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.054

D. Schmidt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4768–4772
4769
the functional assay, the same membrane preparations were incu-
bated with compound and then S. Retention of
32S]GTP
32S]GTP
S on the membrane preparation was measured to deter-
mine activation of the G proteins coupled to the niacin receptor.
We first synthesized the cyclohexyl compound 17, which
showed no activity at 25 lM (Table 1). Next, we examined the ef-
O
O
O
O
OEt
[
c
O
O
a
b
[
c
O
N
OEt
N
O
O
H
O
O
5
6
7
fect of an aryl substituent at the C5 and C6 position. Towards this
end, compounds 16 and 18 were synthesized. Compound 16 was
inactive. Interestingly, compound 18 showed weak activity in the
functional assay. Encouraged by this result, we decided to explore
the SAR of this series further. We first examined the effect of plac-
ing a fluorine atom in the ortho, meta, and para positions. The meta-
and para-fluorophenyl compounds had modest activity (data not
shown). However, the ortho-fluoro compound 19 showed a sixfold
improvement in activity compared to 18. Next the effect of meth-
oxy, methyl, and chloro groups on the phenyl ring was investi-
gated. Among them, only the ortho-chloro substituents, 20, had
activity similar to 19. Based on this data, we focused on fluoro sub-
stitution. A series of di- and trisubstituted fluorine compounds was
synthesized. The 2,6-difluoro compound 21 showed poor activity,
indicating that only one ortho position could be substituted. The
2,5-difluoro phenyl compound 22 had similar activity compared
to 19 in the binding assay, but showed a threefold improvement
in the functional assay. The 2,3,5-trifluoro compound 25 showed
further enhancement in in vitro activity compared to 19 (threefold
improvement in the binding assay; a fourfold improvement in the
functional assay). Furthermore, 22 and 25 showed a 25-fold in-
crease in functional activity compared to the unsubstituted phenyl
18. Compound 22 was selected for evaluation in pharmacokinetic
studies in mice. It showed a good half-life and low clearance with
oral bioavailability of 53% (Table 2).
The compounds described thus far were racemic mixtures.
Compound 22 was resolved to provide 23 and 24. Compound 23
showed a twofold improvement in the binding assay compared
to the racemic mixture, while the other enantiomer, 24, showed
weak activity. Compound 23 represented an improvement on com-
pound 18 of over 250-fold in the binding assay and 25-fold
improvement in the functional assay.
The fluoro compounds were active in the binding and functional
assays in the absence of serum. However, in the presence of serum
the activity decreased 5–6-fold. As this was a concern, we sought
to replace the phenyl ring with more polar groups with the goal
of reducing serum shift (Table 3). Towards this end, we examined
substituted pyridines. Two fluoropyridine compounds, 26 and 27,
were synthesized. Compound 27 had good binding activity compa-
rable to compound 19 and this activity was retained in the pres-
ence of serum (Table 3). Compound 27 when dosed in mice
showed threefold higher clearance than compound 22 with a sim-
ilar half-life (2.6 h) and oral bioavailability (47%) (Table 2).
All compounds synthesized thus far were full agonists. Previous
work had indicated that replacing the carboxylic acid with a tetra-
zole resulted in compounds that were partial agonists and did not
cause flushing in a mouse.9 Therefore, we synthesized the corre-
sponding tetrazole analogs of 22 and 27 (30 and 31). Compounds
30 and 31 were synthesized in a similar fashion to compound
O
O
OEt
OEt
O
O
c
e
d
N
N
N
N
H
Ts
8
9
O
O
O
OEt
OEt
g,h Ar
OH
TfO
Ar
f
N
N
N
N
Ts
N
N
H
Ts
10
11
12
Scheme 1. Synthesis of pyrazole acids. Reagents and conditions: (a) LDA, diethy-
loxalate, THF, À78 °C; (b) hydrazine hydrate, AcOH/EtOH, 65 °C, 68%; (c) 3 N HCl,
THF/EtOH, 62%; (d) NaH, TsCl, THF, 0 °C, 71%; (e) LDA, Comins’ reagent, THF, À78 °C,
77%; (f) Ar–B(OH)2, Na2CO3(aq), Pd(PPh3)4, THF, 63–100%; (g) Pd/C, formic acid,
ammonium formate, MeOH/THF; (h) LiOH, MeOH/THF/H2O, 10–30%.
sought to restrict the conformation of the aromatic ring in 3 by
appending a 6-membered ring onto the pyrazole. Thus, we under-
took the synthesis of a series of aryl cyclohexyl pyrazole acids (4).
Compounds described herein were synthesized using two different
routes (Schemes 1 and 2). Commercially available compound 1,4-
dioxaspiro[4.5]decan-8-one (5) was acylated with diethyloxalate
in the presence of LDA at À78 °C to afford 6. Cyclization with
hydrazine afforded the pyrazole 7. Removal of the ketal protecting
group, followed by treatment with sodium hydride and tosyl chlo-
ride afforded 9. Treatment of 9 with LDA at À78 °C followed by Co-
mins’ reagent gave vinyl triflate 10 as a single regioisomer.8 The
vinyl triflate 10 was coupled with a variety of boronic acids using
standard Suzuki reaction conditions. Finally, hydrogenation of the
double bond, followed by tosyl deprotection, and ester hydrolysis
gave the final pyrazole acids.
Compound 16 was synthesized using the route described in
Scheme 2. Commercially available 3-ethoxy-2-cyclohexen-1-one
(13) when treated with phenyl magnesium chloride followed by
an acidic workup afforded 14. Deprotonation of 14 with LDA at
À78 °C followed by treatment with diethyloxlate provided an
a
,b-unsaturated ketone that was hydrogenated to give 15. Com-
pound 15 was subsequently cyclized to the pyrazole with hydra-
zine hydrate under acidic conditions. Finally, hydrolysis of the
ester gave the final compound 16.
All compounds were evaluated in a binding assay and a func-
tional assay. For the binding assay, membrane preparations were
made from CHO–KI cells stably expressing the niacin receptor. Dis-
placement of radiolabelled niacin from the membrane preparation
was measured in the absence and presence of human serum. For
O
OH
O
O
O
O
a
b, c
d, e
CO2Et
N
N
H
Ph
EtO
Ph
Ph
13
14
15
16
Scheme 2. Synthesis and reagents. Reagents and conditions: (a) PhMgCl, THF, À78 °C to rt 70%; (b) Pd/C, H2, MeOH; (c) LDA, THF, À78 °C then diethyloxalate 90%; (d)
hydrazine hydrate AcOH:EtOH (1:10), 65 °C 72%; (e) LiOH, MeOH/THF/H2O 90%.

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D. Schmidt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4768–4772
Table 1
Binding and functional activity of compounds for niacin receptor
Compounds
h-3H-niacin IC50
0.14
(
lM)
h-3H-niacin with
hGTPcS
5% serum IC50
(l
M)
EC50
(lM)
NIACIN
0.14
1.0
O
OH
17
No activtiy at 25
lM
No activtiy at 100
No activtiy at 100
l
l
M
M
N
N
H
O
OH
16
63% at 25
19% at 25
0.27
l
M
N
N
H
O
OH
18
lM
35% at 25
18% at 25
20% at 25
>25.0
l
l
l
M
M
M
28
N
N
H
O
OH
19
4.4 (100%)
N
F
N
H
O
OH
20
0.46
2.5 (100%)
N
Cl
N
H
O
F
OH
OH
21
>25.0
30% at 100 lM
N
F
N
H
F
F
O
22 rac
23 end A
24 end B
0.22
0.09
1.4 (sixfold serum shift)
0.4
84% at 25 lM
1.3 (90%)
1.1 (90%)
N
N
H
56% at 25
lM
F
F
O
OH
25
0.067
0.37 (fivefold serum shift)
1.1 (100%)
F
N
N
H
Binding to niacin receptor determined by displacement of [3H]niacin in the presence or absence of 5% human serum. Functional assays were run using binding of hGTP
cS to
membrane preparations. Number in parentheses represents curve height as a percentage of the niacin curve height. Data are an average of two independent titrations having
calculated standard errors below 15%. The assay-to-assay variation was generally twofold. See Ref. 11 for assay protocols. All compounds containing a chiral center are
racemic unless otherwise indicated.
18.10 Tetrazole compounds 30 and 31 showed activity similar to
We next sought to test compound 23 in vivo. Free fatty acid lev-
the carboxylic acid analogs.
els in the blood decrease in rats upon treatment with niacin. It is

D. Schmidt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4768–4772
4771
Table 2
Pharmcokinetics of compounds in mouse
Compound
Clp (mL/min/kg)
8.3
Vdss (L/kg)
t1/2 (h)
AUC (
lM h kg/mg)
Cmax
(lM)
F (%)
F
O
OH
22
1.4
2.9
7.8
3.3
53
N
F
N
H
O
OH
N
27
21
2.4
2.6
2.9
2.3
47
N
F
N
H
For IV, dose =\ 1 mpk n = 2; For po, dose = 2 mpk, n = 3.
Table 3
Binding and functional activity of compounds for niacin receptor
Compounds
h-3H-niacin IC50
0.14
(l
M)
h-3H-niacin with 5% serum IC50
0.14
(
lM)
hGTPcS EC50 (lM)
NIACIN
1.0 (100%)
O
N
OH
26
60% at 25
l
M
69% at 25
lM
0% at 100 lM
F
N
N
H
O
OH
N
27 rac
28 end A
29 end B
0.60
0.20
90% at 25
0.85
0.37
88% at 25
4.5 (100%)
2.3 (100%)
67% at 25
N
F
N
H
l
M
l
M
M
lM
F
N
N
HN
N
30
0.15
17% at 25
l
1.3 (78%)
N
N
F
N
H
N
N
HN
31 rac
32 end A
33 end B
0.50
0.15
40% at 25
1.8 (fourfold serum shift)
0.15
3.8 (88%)
5.5 (92%)
28% at 100 lM
N
N
F
N
l
M
75% at 25
lM
H
See legend of Table 1.
currently postulated that a decrease in free fatty acids leads to the
improved lipid profiles observed in humans.6 Hence, a decrease in
free fatty acids was used to measure the effectiveness of our com-
pounds in vivo.
Rats were treated orally with 3, 10, 30, and 100 mpk of 23.
Blood samples were collected at 0 min, 20 min, 1, 2, and 4 h post
dose, and free fatty acid levels were measured. The data are sum-
marized in Figure 2. Compound 23 showed a maximal effect on
free fatty acid levels at 20 min post dose with effects comparable
to niacin at 100 mpk. A comparison of free fatty acid decrease
and drug levels led to a determination that the EC50 for 23 was
3 lM. Furthermore, free fatty acid decreases of 50% were seen at
doses as low as 3 mpk (Fig. 2).
For the side effect of facial flushing, a separate experiment in a
rat model was used. Laser Doppler effects were used to test
changes in blood perfusion of the rat’s ear after administration
of compound.11 Compound 23 was dosed in rats subcutaneously
at 3, 10, and 30 mpk. Higher doses could not be achieved in the
Figure 2. Free fatty acid decrease in rats treated with compound 23, niacin, or
vehicle po in 5% methylcellulose. Five rats were used for compound 23. Four
animals were used for niacin and vehicle experiments. Data are an average of two
independent titrations.

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D. Schmidt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4768–4772
has recently been reported that a related compound activates the
adenylate cyclase dependent pathway of GPR109A, but not the
MAPK kinase pathway.12 It will be useful to explore whether com-
pound 23 shows the same effects.
References and notes
1. Barter, P.; Kastelein, J.; Nunn, A.; Hobbs, R. Atherosclerosis 2003, 168, 195. and
references therein.
2. (a) Ashen, M.; Blumenthal, R. N. Eng. J. Med. 2005, 353, 1252; (b) Alderman, J. D.;
Pasternak, R. C.; Sacks, F. M.; Smith, H. S.; Monrad, E. S.; Grossman, W. Am. J.
Cardiol. 1989, 64, 725.
3. Knopp, R. Am. J. Cardiol. 2000, 86, 51L.
4. Etchason, J. A.; Miller, T. D.; Squires, R. W.; Allison, T. G. Mayo Clin. Proc. 1991,
66, 23.
Figure 3. Changes in rat ear blood perfusion when rats were treated with
compound 27, niacin, or vehicle subcuntaneous. in 5% BCD. Five rats were used
for each experiment.
5. (a) Tunaru, S.; Kero, J.; Schaub, A.; Wufka, C.; Blaukat, A.; Pfeffer, K.;
Offermanns, S. Nat. Med. 2003, 9, 352; (b) 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; (c) 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.
6. Seki, K.; Isegawa, J.; Fukuda, M.; Ohki, M. Chem. Pharm. Bull. 1984, 32, 1568.
7. van Herk, T.; Hartog, A. F.; Schoemaker, H. E.; Wever, R. J. Med. Chem. 2003, 46,
3945.
8. (a) Comins, D.; Dehghani, A. Tetrahedron Lett. 1992, 33, 6299; (b) Watanabe, N.;
Tanino, K.; Kuwajima, I. Tetrahedron Lett. 1999, 40, 8133.
9. Semple, G.; Fioravanti, B.; Pereira, G.; Calderon, I.; Uy, J.; Choi, K.; Xiong, Y.; Ren,
A.; Morgan, M.; Dave, V.; Thomsen, W.; Unett, D. J.; Xing, C.; Bossie, S.; Carroll,
C.; Chu, Z. L.; Grottick, A. J.; Hauser, E. K.; Leonard, J.; Jones, R. M. J. Med. Chem.
2008, 51, 5101.
Figure 4. Free fatty acid changes (solid lines) and drug levels (dashed lines) are
shown for rats treated with compound 23 subcutaneous. Drug and free fatty acid
levels were obtained from 3 animals for early time points (0–35 min) and from 2
different animals for later time points (36–60 min). Data are an average of two
independent titrations.
10. Commercial ketone
4
was deprotonated by LDA at À78 °C followed by
treatment with Comins’ reagent to give vinyl triflate 34. Compound 34 was
coupled with boronic acids using standard Suzuki reaction conditions to give
35. Hydrogenation and deprotection of the ketal protecting group gave ketone
36. Acylation with the tetrazole ester 37 and cyclization with hydrazine gave
the pyrazole tetrazoles.
O
OTf
Ar
vehicle used for subcutaneous administration. The flushing of the
rats’ ear was then monitored. No flushing was seen even at the
highest dose of compound 23 (Fig. 3). Drug levels could not be
monitored in the flushing experiment since removal of blood
samples would alter the blood perfusion of the ear, so drug levels
were measured in a separate, identical experiment (Fig. 4). At the
30 mpk dose, 135 lM drug levels were achieved. Thus, at drug
levels 45-fold over the EC50 of the compound, flushing is not
observed.
In summary, we synthesized a series of fused cyclohexylpyraz-
ole niacin receptor agonists and discovered that aryl and hetero-
aryl substitution on the cyclohexyl ring significantly improved
binding and functional activity. Among them, compound 23 was
evaluated in animal models and was shown to be a full agonist that
could separate the free fatty acid effects of the niacin receptor from
the flushing effect with a therapeutic index of greater than 45. This
result indicates that separation of the two effects can be achieved
and this series of compounds represents a promising new class of
niacin receptor agonists. Additionally, insight into the biological
basis for the observed separation of the flushing and free fatty acid
effects may be gained by further study of this class of molecule. It
a
b
c,d
O
O
O
O
O
O
O
4
34
35
N
N
Ar
N
NH
N
e,f
+
EtO
Ar
N
HN
N
N
N
O
36
H
37
38
(a) LDA, Comins’s reagent, THF, À78 °C to À20 °C, 100%;(b) Ar–B(OH)2,
Na2CO3(aq), Pd(PPh3)4; (c) Pd/C, H2, MeOH; (d) 3 N HCl, THF/EtOH (3:1), 92%;
(e) LDA, THF, À78 °C then 37; (f) hydrazine hydrate AcOH: EtOH (1:10), 65 °C
10–30%; (g) LiOH, MeOH/THF/H2O 90%.
11. All methods are described in Semple, G.; Schrader, T.; Skinner, P. J.; Colletti, S.
L.; Gharbaoui, T.; Imbriglio, J. E.; Jung, J.; Liang, R.; Subharekha, S.; Schmidt, D.;
Tata, J. R. WO 2005044816 A1, 2005.
12. Richman, J. G.; Kanemitsu-Parks, M.; Gaidarov, I.; Cameron, J. S.; Griffin, P.;
Zheng, H.; Guerra, N. C.; Cham, L.; Maciejewski-Lenoir, D.; Behan, D. P.;
Boatman, D.; Chen, R.; Skinner, P.; Ornelas, P.; Waters, M. G.; Wright, S. D.;
Semple, G.; Connolly, D. T. J. Biol. Chem. 2007, 282, 18028.