3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): A partial agonist of the nicotinic acid receptor, G-protein coupled receptor 109a, with antilipolytic but no vasodilatory activity in mice

Article abstract of DOI:10.1021/jm800258p

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro- cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism,

Full text of DOI:10.1021/jm800258p

J. Med. Chem. 2008, 51, 5101–5108
5101
3-(1H-Tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): A Partial Agonist of the
Nicotinic Acid Receptor, G-Protein Coupled Receptor 109a, with Antilipolytic but No
Vasodilatory Activity in Mice
Graeme Semple,*,† Philip J. Skinner,† Tawfik Gharbaoui,† Young-Jun Shin,† Jae-Kyu Jung,† Martin C. Cherrier,†
Peter J. Webb,† Susan Y. Tamura,† P. Douglas Boatman,† Carleton R. Sage,† Thomas O. Schrader,† Ruoping Chen,‡
Steven L. Colletti,§ James R. Tata,§ M. Gerard Waters,§ Kang Cheng,§ Andrew K. Taggart,§ Tian-Quan Cai,§
Ester Carballo-Jane,§ Dominic P. Behan,‡ Daniel T. Connolly,‡ and Jeremy G. Richman‡
Departments of Medicinal Chemistry and DiscoVery Biology, Arena Pharmaceuticals, 6166 Nancy Ridge DriVe, San Diego, California 92121,
Merck Research Laboratories, Rahway, New Jersey 07065
ReceiVed March 10, 2008
The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354),
a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects
in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose.
Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced
by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.
Introduction
Mechanistic investigations showed that nicotinic acid binds
to a G-protein coupled receptor (GPCR) expressed in rat spleen
and adipocytes,8 a finding that sparked a resurgence in the field.
Two Gi-coupled orphan GPCRs that share 95% identity and
that are both expressed in human adipocytes were subsequently
cloned and identified as putative molecular targets for nicotinic
acid.9 GPR109a (also called HM74a) is the human orthologue
of the previously described mouse receptor (PUMA-G, called
mGPR109a hereafter),10 whereas GPR109b (also called HM74)
differs from hGPR109a and mGPR109a mainly in the intracel-
lular C-terminal tail portion of the receptor and is not expressed
in rodents.9 Nicotinic acid was shown to activate hGPR109a in
a guanine nucleotide exchange assay and displace 3H-nicotinic
acid from hGPR109a expressing CHO cell membranes with
activity in the tens of nanomolar range, but is a much weaker
ligand for GPR109b.9 Further evidence in mGPR109a knockout
mice has demonstrated that the free fatty acid (FFA) and
triglyceride lowering effects of nicotinic acid are ablated in the
absence of this receptor.10 These data, coupled with the highly
restricted species expression of GPR109b, has brought hGPR109a
to the forefront as the more interesting potential drug target of
the two. The question still remains however, as to whether either
receptor is the molecular target responsible for the lipid
remodeling and antiatherogenic properties of nicotinic acid in
humans. The demonstration that other known compounds
previously shown to raise HDL in humans, acipimox11 and
acifran,12 are also agonists for hGPR109a13 is supportive of this
idea, but conclusive evidence is still lacking. A hypothesis has
been described whereby the initial activation of GPR109a
decreases intracellular cAMP levels in adipocytes, leading to
reduced protein kinase A (PKA) activity. This in turn results in
a decrease in hormone sensitive lipase activity, thereby reducing
intracellular triglyceride (TG) hydrolysis and FFA secretion. It
has been further postulated that this decrease in FFA levels
directly results in decreased production of TG and VLDL in
the liver. The use of knockout mice indicates that these
antilipolytic effects of nicotinic acid are receptor dependent.14
What is much less clear is whether the acute effects on plasma
FFA and TG can eventually lead to increases in HDL levels. It
has been hypothesized, though, that the reduction in the number
Nicotinic acid (sometimes called niacin) is a water-soluble
vitamin that at high doses in humans favorably modulates
essentially all serum lipid and lipoprotein parameters. As a
result, nicotinic acid has been used for the treatment of
cardiovascular disease for many years.1 Nicotinic acid can lower
very low-density lipoprotein cholesterol (VLDL-c),a low-density
lipoprotein cholesterol (LDL-c), and lipoprotein(a) (Lp(a)), but
the recent upsurge in interest in this area has focused on nicotinic
acid’s ability to increase high density lipoprotein-cholesterol
(HDL-c) to a greater extent than other currently marketed drugs,
as HDL-c levels are inversely correlated with the risk of
coronary heart disease.2 Indeed, in the Coronary Drug Project,
nicotinic acid was shown to reduce the number of cardiac events
over a six-year dosing period and to reduce all cause mortality
by 11% after 15 years.3,4 Subsequently, combinations of
nicotinic acid with the LDL-lowering statin class of drugs have
been shown to slow the progression of atherosclerosis, decrease
the number of cardiac events, and provide a therapeutic benefit
beyond that of statins alone.5,6
The use of nicotinic acid as a therapeutic, however, is limited
by a number of associated side-effects, most notably a highly
uncomfortable cutaneous flushing response that generally
manifests itself on the upper body and face which can limit
patient compliance.7 Hence the development of novel agents
with nicotinic acid-like effects on plasma lipid parameters and
atherosclerosis, but that do not induce flushing, has been
considered to be a high value goal for some time.
* To whom correspondence should be addressed. Phone: +1 858 453
7200. Fax: + 1 858 453 7210. E-mail: gsemple@arenapharm.com.
†
Medicinal Chemistry, Arena Pharmaceuticals.
Discovery Biology, Arena Pharmaceuticals.
Merck Research Laboratories.
‡
§
a Abbreviations: GPR109a, G-protein coupled receptor 109a (also known
as HM74a); GPR109b, G-protein coupled receptor 109b (also known as
HM74); PUMA-G, protein upregulated in macrophages by interferon gamma
(the mouse orthologue of GPR109a); VLDL-c, very low-density lipoprotein
cholesterol; LDL-c, low-density lipoprotein cholesterol; HDL-c, high density
lipoprotein-cholesterol; Lp(a), lipoprotein(a); CHO cells, Chinese hamster
ovary cells; TG, triglycerides; FFA, Free fatty acids; cAMP, 3′,5′-cyclic
adenosine monophosphate; MAPK, Mitogen activated protein kinase; ip,
intraperitoneal; PGD2, prostaglandin D2.
10.1021/jm800258p CCC: $40.75
2008 American Chemical Society
Published on Web 07/30/2008

5102 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Semple et al.
Scheme 1a
a Reagents and conditions: (i) (a) NaOEt, EtOH, (EtOCO)2, 75 °C; (b) H2NNH2 (aq.), 75 °C. (ii) Aqueous LiOH, THF/MeOH, 50-80 °C, 3 h. (iii) (a)
NH3, MeOH, reflux; (b) NaCl, POCl3, MeCN, r.t. 3 h. (iv) NaN3, DMF, 200 °C, microwave. (v) (a) NH3, MeOH, reflux or NH4OH, 1,4-dioxane, r.t.; (b)
BnBr, base, 1,4-dioxane, r.t. (vi) SOCl2, DMF, r.t. (vii) (a) NaN3, ZnBr2, DMF 120 °C or 200 °C, microwave; (b) DMSO, air, KOtBu, r.t. or HCO2H/MeOH,
Pd (black), r.t.
of VLDL particles may limit the cholesterol ester transfer protein
(CETP)-mediated exchange of cholesterol from HDL to VLDL,
and TG from VLDL to HDL, thereby leading to a net increase
in HDL levels.14 Clearly the identification of new agonists of
the receptor with plasma FFA lowering activity in vivo would
be of great interest to further explore this hypothesis. In addition,
the identification of compounds that lack the characteristic
flushing effect of nicotinic acid may lead to an improvement in
patient compliance.
At the outset of our program, it was unclear as to the
mechanism of the flushing effect and it appeared possible that
the two known pharmacological effects of niacin could be
separated with a molecule that selectively activated GPR109a
but did not interact with whichever protein was responsible for
the flushing. However, it has since been shown that both
reduction of plasma FFA and vasodilation by nicotinic acid in
mice requires the presence of mGPR109a.16 From these data,
it would appear unlikely that a separation of these two key
pharmacological effects can be achieved. Despite this supposi-
tion, we herein report the discovery of a new partial agonist of
GPR109a that retains the plasma FFA lowering effects in rodents
associated with receptor activation but that does not induce any
vasodilation in mice at the maximum feasible dose and that acts
as a competitive antagonist of nicotinic acid-induced vasodila-
tion in the same model.
with a tetrazole resulted in a loss in potency of 1-2 orders of
magnitude for GPR109a, although, on the whole, receptor
selectivity was maintained.18 Despite this unpromising prece-
dent, we sought to apply this isosteric replacement to the 5,5-
fused pyrazole analogues, and so a series of acids and tetrazoles
was prepared as outlined in Scheme 1.
Starting from the appropriate cyclic ketone (1a-d), acylation
with diethyl oxalate followed by cyclization of the resultant
diketo ester with hydrazine provided the bicyclic pyrazole esters
2a-d. Base catalyzed hydrolysis of the ester function provided
the acids 3a-c. Our original route to the tetrazole series
consisted of direct amidolysis of 2a-d to provide the primary
amides that were dehydrated by treatment with POCl3 to provide
the nitriles 4a-d (R ) H). Dipolar cycloaddition of 4a-d with
sodium azide under microwave heating provided the tetrazole
analogues 5a-d. All of the compounds containing alkyl groups
on the cyclopentyl ring were prepared only as racemates. As a
result of the very low yields observed in the latter two steps of
the tetrazole synthesis, we also carried out a similar reaction
sequence via the benzyl protected pyrazole amides 6a-d.
Dehydration to provide the nitriles 7a-d, followed by dipolar
cycloaddition with sodium azide and benzyl group deprotection,
again provided the tetrazole analogues 5a-d. The addition of
these two steps significantly improved the overall yield, but the
synthetic sequence was somewhat more cumbersome. For the
preparation of 5a on a larger scale, an alternative route was
developed whereby ketone 1a was acylated with the sodium
salt of 1H-tetrazole-5-carboxylic acid ethyl ester and the
resultant tetrazole diketone treated with hydrazine to form the
pyrazole tetrazole directly.19
Our lead identification strategy, which involved an extensive
SAR investigation starting from known small molecule com-
pounds that had been shown to activate the receptor, has been
described previously.17 This in vitro exploration of 5- and
6-membered heterocyclic acids led us to focus on a series of
pyrazole acid derivatives. Further screening and elaboration
provided a series of 5,5-fused pyrazole acids as suitable lead
compounds. We have also demonstrated for a series of 4,5- and
5- substituted pyrazoles, replacement of the acid functionality
A comparison of the in vitro agonist activity data of the
compounds synthesized is shown in Table 1. The 5,5-bicyclic
acid (3a)17 showed good potency and full efficacy at both the
cloned hGPR109a and mGPR109a receptors, whereas simple

MK-0354, Partial Nicotinic Acid Receptor Agonist
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5103
Table 1. Agonist activity of bicyclic pyrazoles in the whole cell cAMP assay at the homologous hGPR109a and mGPR109a receptors
efficacy
efficacy
hGPR109a
(% nicotinic
acid response)
mGPR109a
(% nicotinic
acid response)
compound
R1
R2
R3
EC50, µM (n)a
EC50, µM (n)a
3a
3b
3c
5a
5b
5c
5d
H
Me
H
H
Me
H
H
H
Me
H
H
H
H
H
H
H
H
Me
0.86 ( 0.05 (2)
106 ( 13
93 ( 14
n.e.
59 ( 15
n.e.
0.5 ( 0.14 (2)
97 ( 4
94 ( 5
n.e.
71 ( 15
n.e.
8.3 ( 0.2 (3)
12.2 ( 0.36 (2)
n.e.d
n.e.
1.65 ( 0.22 (23) b
1.08 ( 0.33 (16) c
n.e.
n.e.
n.e.
n.e.
n.e.
n.e.
Me
H
n.e.
n.e.
n.e.
n.e.
H
a EC50 from multiple determinations. Errors shown are ( SEM. b 95% confidence interval ) 1.3-2.0 µM. c 95% confidence interval ) 0.7-1.6 µM.
d n.e. ) no effect at maximum concentration tested (100 µM).
methyl substitution around the cyclopentyl ring (3b-c) gave a
significant reduction in potency or ablation of activity. In
contrast to our previous experience, when the carboxylic acid
moiety was replaced by a tetrazole, the unsubstituted analogue
5a retained comparable potency to the parent. None of the
methyl substituted bicyclic pyrazole tetrazole analogues, how-
ever, showed significant receptor activity in the cAMP assay.
Furthermore, 5a demonstrated clear and statistically significant
partial agonism in the cAMP assays for both the mouse and
human receptors with efficacy approximately 60-70% of that
of either nicotinic acid or ꢀ-hydroxy butyrate, a putative
physiologically relevant ligand for hGPR109a,20 in the same
assay platform. In addition, the compound showed no activation
of GPR109b in the cAMP assay at any concentration up to 100
µM. Following these interesting observations, we then prepared
a number of other 5,5-fused pyrazoles analogous to those that
showed receptor activity in our earlier studies. For example,
Figure 1. Effect of acute 5a and nicotinic acid on plasma FFA levels
insertion of either an oxygen or sulfur heteroatom into the
5-membered ring fused to the pyrazole, while maintaining
modest activity when the acid functionality was a carboxylate,17
showed barely measurable activity in the GPR109a cyclase assay
when this group was replaced by a tetrazole. Hence, compound
5a appeared to be somewhat unique among the members of
the pyrazole tetrazole series in having reasonable receptor
activity. As it was the first compound that we had discovered
that showed clear partial agonist character in our in vitro cAMP
assays for both hGPR109a and mGPR109a, we selected this
compound for more extensive profiling and closer comparison
with 3a.
Further characterization of 5a in a hGPR109a GTPγS assay
(EC50 ) 2.3 ( 0.4 µM, n ) 4; efficacy 72% of nicotinic acid
response) confirmed the partial agonist character observed in
the cAMP assay, whereas 3a was again a full agonist in this
assay (EC50 ) 10.4 ( 3.4 µM; n ) 4; efficacy 99% of nicotinic
acid response). Importantly for future in vivo studies, this partial
agonist activity was maintained on the rodent receptors (5a;
mGPR109a GTPγS assay, EC50 ) 0.4 ( 0.06 µM; n ) 3;
efficacy 68% of nicotinic acid response; rat GPR109a GTPγS
assay, EC50 ) 2.3 ( 0.6 µM, n ) 3; efficacy 38% of nicotinic
acid response). In binding studies, 5a was a competitive inhibitor
of 3H-nicotinic acid binding to hGPR109a (5a; Ki ) 505 ( 40
nM, n ) 6; nicotinic acid; Ki ) 50 ( 4 nM, n ) 6). In a further
demonstration of competition between 5a and nicotinic acid,
5a was shown to antagonize the effect of nicotinic acid in the
hGPR109a cAMP assay, with a maximum antagonist efficacy
consistent with its partial agonist character (see Supporting
Information). Despite being a partial agonist of the cAMP
pathway, 5a was able to fully inhibit isoproterenol stimulated
lipolysis in human adipocytes (5a; IC50 ) 3.1 ( 0.1 µM, n )
5) as, less surprisingly, was 3a, and so both of these compounds
were progressed into further studies. In line with the hypotheses
outlined above, we focused our in vivo screening first on testing
in fasted C57/BL6 mice. Compounds were administered ip in saline
20 min prior to sample collection.
for acute effects on plasma FFA as a potential marker for longer
term lipid profile modification and second on characterizing the
flushing side effect in a quantitative manner.
The time course of the nicotinic acid-induced plasma FFA
reduction was determined in mouse, and dose-response experi-
ments with 5a were performed at a single time point (20 min),
the time of maximum efficacy of nicotinic acid in this model
(Figure 1). Compound 5a was similar in efficacy and marginally
more potent than nicotinic acid in this acute test (ED50 for 5a
) 3.1 ( 0.9 mg/kg; ED50 for nicotinic acid ) 9.7 ( 3.6 mg/
kg), confirming what we had observed in vitro, that a partial
agonist was capable of fully suppressing lipolysis in these
systems. It has been previously shown that the antilipolytic effect
of nicotinic acid requires the presence of the receptor as the
compound has no effect on FFA in mGPR109a knockout mice.
Compound 5a was also without effect on FFA in mGPR109a
knockout mice (see Supporting Information). The measured
pharmacokinetic parameters in mice (Table 2) were consistent
with the observed in vivo pharmacodynamic effect and plasma
concentrations taken from the same samples from which plasma
FFA measurements were made and further confirmed the
pharmacokinetic-pharmacodynamic relationship.
Partial agonists of GPR109a have been previously described
along with a prediction that such compounds may have tissue
specific effects, but no in vivo data was included.21 More
recently, it has been reported that other agonists of the receptor,
both full and partial, including 5-isopropyl pyrazole-3-carboxy-
late, did not induce flushing in mice.22 In this paper, it was
concluded that some GPR109a agonists may differentially
activate parallel downstream receptor signaling pathways and
that differences in flushing in vivo were not necessarily a
function of efficacy at the receptor. Compounds that inhibit the

5104 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Semple et al.
Table 2. Pharmacokinetic Parameters in Mouse for 5a
(5a, MK-0354). This compound was found to be a partial agonist
of GPR109a in all species tested and was shown to compete
with nicotinic acid binding and activity in both a conventional
radioligand binding assays and in in vitro functional systems
measuring cAMP accumulation. In vivo, 5a possessed plasma
FFA lowering effects in mice comparable to those of nicotinic
acid. However, in contrast to nicotinic acid and the closely
related pyrazole acid analogue 3a, 5a did not induce vasodilation
in mice even at very high doses and was able to block the
vasodilation induced by nicotinic acid in the same model. In
addition, 5a showed no interaction with any other target tested
in a panel of over 120 other proteins, including the hERG
channel, was not an inhibitor of any of the major CYP isoforms
and had no effect in dog cardiovascular or mouse CNS safety
pharmacology models (data not shown). From these data, 5a
was identified as a compound of sufficient interest to progress
into further pharmacological studies in animals and eventually
into human trials, to test the hypothesis that lowering plasma
FFA by activation of GPR109a would result in similar HDL-c
elevating lowering effect observed with nicotinic acid. Data from
these advanced preclinical and clinical studies will be described
elsewhere in due course.
parametera
Clp (mL/min/kg)
Vd (L/kg)
T1/2 (h)
Cmax (µM)
Tmax (h)
52
13
10
16
0.083
93
Foral (%)
a Clp, plasma clearance (blood clearance for mice); Vd, volume of
distribution; T1/2, terminal half-life; Cmax, observed maximal plasma
concentration following oral dosing; Tmax, time to reach the Cmax; Foral, oral
bioavailability. IV doses were formulated in PBS and injected at 5.0 mg/
kg to male C57BL/6 mice. Peroral doses were formulated in PBS and given
by oral gavage at 10 mg/kg.
cAMP pathway were able to inhibit lipolysis. Compounds that
stimulated MAPK-induced phosphorylation of ERK1 and ERK2
were able to induce flushing, whereas those compounds that
did not signal through MAPK but were still able to inhibit
adenylate cyclase did not induce flushing. Consistent with these
observations is that the production of PGD2 requires the
generation of arachidonic acid and its subsequent metabolism
to prostaglandins, and this process is known to be regulated by
activation of the MAPK pathway.23 In our assays, 3a but not
5a was able to activate MAPK signaling in cells, overexpressing
either mGPR109a or hGPR109a. In addition, 5a was also a
competitive antagonist of nicotinic acid-induced MAPK signal-
ing in this model, showing that it can occupy the receptor but
still fail to initiate signaling through MAPK.24 Also consistent
with these observations is that 5a did not induce receptor
internalization, a process known to be ꢀ-arrestin and MAPK-
dependent and was able to block nicotinic acid-induced receptor
internalization (data not shown). Hence it would be predicted
that 3a but not 5a would be able to induce flushing in vivo in
mice. It is likely that this ability of 5a to distinguish between
receptor activation pathways is due, at least in part, to its partial
agonist character.
The vasodilation effect (a component of, and surrogate for,
flushing) may be quantified in anesthetized mice by use of a
laser-Doppler instrument to measure blood flow changes in the
exposed ear.15 Nicotinic acid induces a dose-dependent increase
in blood flow after ip injection in this model (Figure 2a), such
that a dose of 100 mg/kg ip results in a 100% increase over
baseline blood flow compared to vehicle treatment after 5 min.
Compound 3a also showed a similar dose-dependent effect in
mice in the same dose range (data not shown). In contrast, as
predicted from the in vitro data, there was no effect of increasing
ip doses of 5a, up to the maximal feasible dose (based on
solubility of 5a in the administration vehicle) of 400 mg/kg
(Figure 2). Subsequent analysis of plasma levels of 5a verified
that concentrations of at least 30-fold higher than those that
produced a maximal effect in the plasma FFA-lowering model
in mouse were achieved (510 ( 230 µM following a dose of
100 mg/kg ip; plasma levels not measured at 400 mg/kg). In
further experiments to characterize the effect of 5a in this model,
the compound was preadministered at 100 mg/kg ip to mice
that were challenged 5 min later with a dose of nicotinic acid
(30 mg/kg, ip) that normally produces robust vasodilation. A
complete inhibition of the expected nicotinic acid-induced
flushing effect was observed (Figure 3), consistent with the
competitive antagonist effect of 5a observed on the nicotinic
acid-induced activation of MAPK signaling in vitro. As would
also be expected, 5a failed to antagonize flushing induced by
PGD2, which acts downstream of GPR109a.15
Experimental Section
Proton and carbon nuclear magnetic resonance (1H and 13C
NMR) spectra were recorded on a Varian Mercury VX-400
equipped with a four-nucleus auto switchable probe and z-gradient
or a Bruker Avance-400 equipped with a Quad Nucleus Probe
(QNP) or a Broad Band Inverse (BBI) and z-gradient. Chemical
shifts are given in parts per million (ppm) with the residual solvent
signal used as reference. Coupling constants are reported in Hz.
NMR abbreviations are used as follows: s ) singlet, d ) doublet,
t ) triplet, q ) quartet, m ) multiplet, dd ) doublet of doublets,
dt ) doublet of triplets, br ) broad. Microwave irradiations were
carried out using the Emyrs synthesizer (Personal Chemistry). Thin-
layer chromatography (TLC) was performed on silica gel 60 F254
(Merck), and column chromatography was carried out on prepacked
silica gel columns using KP-Sil supplied by Biotage. Evaporation
was performed in vacuo on a Buchi rotary evaporator. Celite 545
was used for stated filtrations. Strong cation exchange (SCX)
columns were purchased from Phenomenex (Strata SCX 55 µm,
70 Å). All other reagents were purchased from Aldrich.
Analytical HPLC/MS was conducted on an AB/MDS Sciex API
150EX mass spectrometer with an electrospray source, using a
Shimadzu Inc. LC-10AD VP HPLC-pump, Shimadzu Inc. SCL-
10A VP HPLC system controller, Shimadzu Inc. SPD-10A VP UV
detector, monitoring at 214 nm, Leap Scientific CTC HTS, PAL
autosampler, Analyst 1.2 software and an (a) Alltech Prevail C18
column (5 µ, 50 mm × 4.6 mm), using a gradient of 5% v/v CH3CN
(containing 1% v/v TFA) in H2O (containing 1% v/v TFA) (t )
0.0 min) gradient to 100% v/v CH3CN in H2O (t ) 20.0 min), 3.5
mL/min, or (b) Waters YMC ODS-A C18 column (5 µ, 50 mm ×
4.6 mm), using a gradient of 5% v/v CH3CN (containing 1% v/v
TFA) in H2O (containing 1% v/v TFA) (t ) 0.0 min) gradient to
95% v/v CH3CN in H2O (t ) 4.0 min), 3.5 mL/min.
Preparative HPLC was conducted on a Varian Prostar reverse
phase HPLC using a Phenomenex Luna C18 column (10 µ, 250
mm × 50 mm), 5% (v/v) CH3CN (containing 0.1% v/v TFA) in
H2O (containing 0.1% v/v TFA) gradient to 95% CH3CN, 60 mL/
min, λ ) 220 nm, or using a Phenomenex Luna C18 column (10
µ, 250 mm × 21.20 mm), 5% (v/v) CH3CN (containing 0.1% v/v
TFA) in H2O (containing 0.1% v/v TFA) gradient to 95% CH3CN,
20 mL/min, λ ) 220 nm. Compound 3a was purchased from
Fluorochem but for larger amounts was also prepared using the
synthesis described below.
General Procedure for the Synthesis of 1H-Pyrazole-3-carboxy-
lic acid ethyl esters (2a-d). The appropriate ketone (1a-d) was
dissolved in ethanol (5 mL/mmol), and diethyl oxalate (1.2 eq.) and
In summary, we have described the discovery and profiling
of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole

MK-0354, Partial Nicotinic Acid Receptor Agonist
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5105
Figure 2. Quantification of the flushing response of nicotinic acid and 5a as measured by laser-Doppler recordings of blood flow in the ear of male
C57/BL6 mice.
4.35 (q, 2H, J ) 7.1, OCH2CH3), 3.16 (sextet, 1H, J ) 7.10, C(6)-
H), 2.87-2.77 (m, 1H), 2.77-2.62 (m, 2H), 1.36 (t, 3H, J ) 7.1,
OCH2CH3), 1.29 (d, 3H, J ) 6.9, CH3). HPLC/MS: (column b)
94%, tr ) 1.75 min.
4- and 5-Methyl-1,4,5,6-tetrahydro-cyclopentapyrazole-3-car-
boxylic Acid Ethyl Ester (2c and 2d). (0.570 g, 2.94 mmol, 59%,
ca. 1.3:1 mixture of 5-methyl (2d) and 4-methyl (2c) regioisomers).
m/z (ES+): 195 [M + H]+, 149 [M-OEt]+. Major regioisomer
5-methyl-1,4,5,6-tetrahydro-cyclopentapyrazole-3-carboxylic acid
ethyl ester (2d) 1H NMR (CDCl3): δ 10.9 (br s, 1H, NH), 4.42-4.30
(m, 2H, OCH2CH3), 3.02-2.90 (m, 2H), 2.43-2.32 (m, 2H),
2.08-1.98 (m, 1H), 1.40-1.34 (m, 3H, OCH2CH3), 1.21 (d, 3H, J
) 6.3, CH3); Minor regioisomer 4-methyl-1,4,5,6-tetrahydro-
cyclopentapyrazole-3-carboxylic acid ethyl ester (2c) m/z (ES+):
1
195 [M + H]+, 149 [M-OEt]+. H NMR (CDCl3): δ 10.9 (br s,
1H, NH), 4.42-4.30 (m, 2H, OCH2CH3), 3.24 (sextet, 1H, J )
Figure 3. 5a attenuates the vasodilation response induced by nicotinic
6.7, C(4)-H), 3.02-2.90 (m, 1H), 2.88-2.60 (m, 3H), 1.40-1.34
acid but not that induced by PGD2 as measured by laser-Doppler
(m, 3H, OCH2CH3), 1.29 (d, 3H, J ) 6.9, CH3).
recordings of blood flow in the ear of male C57/BL6 mice following
sequential treatment with compound (or vehicle), nicotinic acid, and
PGD2 at the time points shown. The first treatment in the sequence
(see legend) was injected at t ) 0, the second treatment at t ) 10 min,
and the third treatment at t ) 28 min. NA ) nicotinic acid.
General Procedure for the Synthesis of 1H-Pyrazole-3-carboxy-
lic acids (3a-c). The appropriate pyrazole-3-carboxylate ethyl ester
(2a-c) was dissolved in a solution of 1:5:1 MeOH:THF:1 M aq
LiOH (70 mL) or aq sodium hydroxide and heated to 50-80 °C
for 3 h or until hydrolysis was complete. Solvent was removed
under reduced pressure, and the resulting solid suspended in water
(50 mL). The mixture was acidified to pH 1 by the addition of 1
M HCl and then extracted with ethyl acetate (100 mL). The organic
portion was separated and solvent removed under reduced pressure
and the residue purified by preparative reverse-phase HPLC to give
the pyrazole-3-carboxylic acid (3a-c) as an off-white solid.
sodium ethoxide (1.1 eq.) were added at r.t. The mixture was heated
at 75 °C for 30 min and cooled to 4 °C in an ice bath. An aqueous
solution of hydrazine (2 equiv, 2 mL/mmol) was added, and the
resulting mixture was heated at 75 °C for 1 h. Solvent was removed
under reduced pressure, and the crude residue was partitioned between
DCM and water. The organic portion was separated and solvent was
removed under reduced pressure, and the residue was either purified
by column chromatography (n-hexane, to 50% EtOAc/n-hexane, silica)
or hydrolyzed directly to the corresponding 1H-pyrazole-3-carboxylic
acid (3) without further purification or analysis.
1,4,5,6-Tetrahydro-cyclopentapyrazole-3-carboxylic Acid Eth-
yl Ester (2a). (16.16 g, 90.0 mmol, 76%). m/z (ES+): 181 [M +
H]+. 1H NMR (CD3OD): δ 4.34 (q, 2H, J ) 7.1, OCH2CH3), 2.78
(t, 2H, J ) 7.0), 2.72 (br s, 2H), 2.49 (br s, 2H), 1.36 (t, 3H, J )
7.1, OCH2CH3).
1,4,5,6-Tetrahydrocyclopenta[c]pyrazole-3-carboxylic Acid (3a).
(5.10 g, 71%) HPLC/MS: (column a) 99%, tr ) 2.18 min, m/z
1
(ES+): 153 [M + H]+, 135 [M-OH]+. H NMR (DMSO-d6): δ
2.70-2.60 (m, 4H, C(4)-H and C(6)-H), 2.42-2.32 (m, 2H, C(5)-
H).
6-Methyl-1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carboxyl-
ic Acid (3b). HPLC/MS: (column a) 99%, tr ) 3.05 min, m/z (ES+):
167 [M + H]+, 149 [M-OH]+. 1H NMR (DMSO-d6): δ 2.98 (sextet,
1H, J ) 6.2, C(6)-H), 2.70-2.47 (m, 3H, C(4)-H and C(5)-H),
1.90-1.80 (m, 1H, C(5)-H), 1.11 (d, 3H, J ) 6.9, CH3).
6-Methyl-1,4,5,6-tetrahydro-cyclopentapyrazole-3-carboxylic Acid
Ethyl Ester (2b). (0.603 g, 3.11 mmol, 62%). m/z (ES+): 195 [M
+ H]+, 149 [M-OEt]+. 1H NMR (CDCl3): δ 10.7 (br s, 1H, NH),

5106 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Semple et al.
5-Methyl-1,4,5,6-tetrahydrocyclopenta[c]pyrazole-3-carboxyl-
ic acid (3c). HPLC/MS: (column a) 99%, tr ) 2.53 min, m/z (ES+):
167 [M + H]+, 149 [M-OH]+. 1H NMR (DMSO-d6): δ 2.90-2.45
(m, 3H), 2.22-2.12 (m, 2H), 1.07 (t, 3H, J ) 6.3, CH3).
Preparation of 1-Benzyl-1,4,5,6-tetrahydro-cyclopentapyrazole-
3-carboxylic acid amide (6a). 1,4,5,6-Tetrahydro-cyclopentapyra-
zole-3-carboxylic acid ethyl ester (2a) (0.808 g, 4.48 mmol) was
suspended in methanolic ammonia (ca. 7 M, 12 mL) and the mixture
heated under reflux overnight. The solution was cooled and the
resulting precipitate (1,4,5,6-tetrahydro-cyclopentapyrazole-3-car-
boxylic acid amide) collected by vacuum filtration as a white
crystalline solid (0.438 g, 2.90 mmol, 65%). m/z (ES+): 152 [M +
H]+. 1H NMR (CD3OD): δ 2.79 (t, 2H, J ) 6.9), 2.73 (t, 2H, J )
7.3), 2.55 (br s, 2H).
by column chromatography (30-75% EtOAc/hexanes, silica) to
give 1-benzyl-4-methyl-1,4,5,6-tetrahydro-cyclopentapyrazole-3-
carboxylic acid amide 6d (0.162 g, 0.64 mmol, 21% yield) as a
colorless oil. HPLC/MS: (column b) tr ) 2.39 min, m/z (ES+):
1
256 [M + H]+, 239 [M-NH2]+. H NMR (CDCl3): δ 7.37-7.26
(m, 3H), 7.17 (d, 2H, J ) 7.3), 6.73 (br s, 1H, CONHH), 5.71 (br
s, 1H, CONHH), 5.16 (s, 2H, CH2Ph), 2.51 (sextet, 1H, J ) 6.6,
C(4)-H), 2.75-2.62 (m, 1H), 2.58-2.48 (m, 1H), 2.46-2.35 (m,
1H), 2.10-2.00 (m, 1H), 1.30 (d, 3H, J ) 6.9, CH3). 13C NMR
(CDCl3): δ 164.7 (CONH2), 151.2, 139.0, 135.9, 134.1, 128.9
(C(2′)), 128.2 (C(4′)), 127.7 (C(3′)), 55.0 (CH2Ph), 39.9 (C(5)),
32.4 (C(4)), 23.3 (C(6)), 21.1 (CH3).
Also obtained was 1-benzyl-5-methyl-1,4,5,6-tetrahydro-cyclo-
pentapyrazole-3-carboxylic acid amide 6c (0.213 g, 0.84 mmol, 28%
1
yield) as a white solid. H NMR (CDCl3): δ 7.37-7.28 (m, 3H),
To a stirred solution of 1,4,5,6-tetrahydro-cyclopentapyrazole-
3-carboxylic acid amide (3.77 g, 25.0 mmol) in DMF (50 mL) at
25 °C was added K2CO3 (12.1 g, 87.4 mmol) followed by benzyl
bromide (11.7 g, 62.4 mmol). The reaction was heated to 55 °C
and stirred for 16 h. After cooling to ambient temperature, the
mixture was diluted with EtOAc (100 mL) and filtered. The filtrate
was washed with H2O (2 × 100 mL), the organic portion dried
over MgSO4, filtered, and the solvent removed under reduced
pressure. Purification by column chromatography (50-95% EtOAc/
hexanes, silica) provided the title compound (1.14 g, 4.73 mmol,
7.19-7.14 (m, 2H), 6.68 (br s, 1H, CONHH), 5.72 (br s, 1H,
CONHH), 5.17 (s, 2H, CH2Ph), 3.08-2.97 (m, 2H), 2.65 (dd, 1H,
J1 ) 15.3, J2 ) 7.5), 2.41 (dd, 1H, J1 ) 18.1, J2 ) 8.7), 2.09 (dd,
1H, J1 ) 15.51, J2 ) 6.2),1.150 (d, 3H, J ) 6.5, CH3). 13C NMR
(CDCl3): δ 164.9 (CONH2), 150.9, 139.2, 136.0, 129.0 (C(2′)),
128.23 (C(4′)), 128.18, 127.7 (C(3′)), 55.1 (CH2Ph), 41.2 (C(5)),
32.7, 32.5, 21.8 (CH3).
Preparation of 3-(2H-Tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclo-
pentapyrazole (5a). 1,4,5,6-Tetrahydro-cyclopentapyrazole-3-car-
boxylic acid amide (0.210 g, 1.39 mmol) was added to anhydrous
acetonitrile (12 mL), heated to 80 °C, and sodium chloride (2.0 g,
34 mmol) was added. After 15 min, phosphorus oxychloride (0.128
g, 0.83 mmol) was added and the solution was heated at 80 °C
overnight, cooled, filtered, and the collected solid washed with
acetonitrile. Solvent was removed from the combined solutions
under reduced pressure, and the resulting solid purified by prepara-
tive HPLC to provide 1,4,5,6-tetrahydro-cyclopentapyrazole-3-
carbonitrile as a deep-purple-colored solid (0.031 g, 0.23 mmol,
1
19% yield) as a white solid. m/z (ES+): 242 [M + H]+. H NMR
(CDCl3): δ 7.37-7.30 (m, 3H), 7.19 (m, 2H), 6.67 (br s, 1H), 5.34
(br s, 1H), 5.19 (s, 2H), 2.82 (m, 2H), 2.51 (m, 4H).
Preparation of 1-Benzyl-6-methyl-1,4,5,6-tetrahydro-cyclopen-
tapyrazole-3-carboxylic Acid Amide (6b). 6-Methyl-1,4,5,6-tet-
rahydro-cyclopentapyrazole-3-carboxylic acid ethyl ester (2b)
(0.603 g, 3.11 mmol) was dissolved in 1,4-dioxane (3.5 mL) and
ammonium hydroxide (25 mL) added. The resulting solution was
stirred overnight at room temperature. Solvent was removed under
reduced pressure and the residue dissolved in 1,4-dioxane (30 mL)
and 5 M aqueous sodium hydroxide (0.72 mL, 3.64 mmol) added,
followed by benzyl bromide (0.56 g, 3.30 mmol). The resulting
solution was stirred at 25 °C for 20 h. An additional 5 M aqueous
sodium hydroxide solution (0.30 mL, 1.5 mmol) and benzyl bromide
(0.25 g, 1.50 mmol) was added, and the solution was stirred at 25
°C for an additional 20 h. Solvent was removed under reduced
pressure and the residue partitioned between ethyl acetate and water.
The organic portion was separated, solvent removed under reduced
pressure, and the resulting residue purified by column chromatog-
raphy (30-60% EtOAc/hexanes, silica) to provide the title com-
pound (0.470 g, 1.84 mmol, 61% yield) as a colorless oil. HPLC/
MS: (column b) tr ) 2.35 min, m/z (ES+): 256 [M + H]+, 239
[M-NH2]+. 1H NMR (CDCl3): δ 7.35-7.25 (m, 3H), 7.13 (d, 2H,
J ) 7.2), 6.79 (br s, 1H, CONHH), 6.26 (br s, 1H, CONHH), 5.21
(q, 2H, J ) 15.7, CH2Ph), 3.00-2.90 (m, 1H), 2.90-2.78 (m, 1H),
2.78-2.65 (m, 2H), 2.10-2.00 (m, 1H), 1.10 (d, 3H, J ) 6.9, CH3).
13C NMR (CDCl3): δ 165.0 (CONH2), 155.4, 138.9, 136.3, 128.7
(C(2′)), 128.5, 127.9 (C(4′)), 126.9 (C(3′)), 54.4 (CH2Ph), 40.6
(C(5)), 32.0 (C(6)), 22.7 (C(4)), 19.3 (CH3).
Preparation of 1-Benzyl-5-methyl-1,4,5,6-tetrahydro-cyclopen-
tapyrazole-3-carboxylic Acid Amide (6c) and 1-Benzyl-4-methyl-
1,4,5,6-tetrahydro-cyclopentapyrazole-3-carboxylic Acid Amide
(6d). A mixture of 5- and 4-methyl-1,4,5,6-tetrahydro-cyclopen-
tapyrazole-3-carboxylic acid ethyl ester (2c and 2d) (0.570 g, 2.94
mmol) was dissolved in 1,4-dioxane (3.5 mL) and ammonium
hydroxide (25 mL) was added. The resulting solution was stirred
for 2 days at room temperature. Solvent was then removed under
reduced pressure and the residue dissolved in 1,4-dioxane (30 mL)
and 5 M aqueous sodium hydroxide (0.72 mL, 3.64 mmol) added,
followed by benzyl bromide (0.56 g, 3.30 mmol). The resulting
solution was stirred at room temperature for 20 h. Additional 5 M
aqueous sodium hydroxide solution (0.30 mL, 1.5 mmol) and benzyl
bromide (0.25 g, 1.50 mmol) was added, and the solution was stirred
at room temperature for an additional 20 h. Solvent was removed
under reduced pressure and the residue partitioned between ethyl
acetate and water. The organic portion was separated, solvent
removed under reduced pressure, and the resulting residue purified
1
17%). m/z (ES+): 134 [M + H]+. H NMR (CD3OD): δ 2.79 (t,
2H, J ) 7.3), 2.73 (t, 2H, J ) 7.1), 2.65-2.55 (m, 2H).
1,4,5,6-Tetrahydro-cyclopentapyrazole-3-carbonitrile (0.022 g,
0.165 mmol) and sodium azide (0.086 g, 1.30 mmol) were dissolved
in DMF (3 mL) and heated under microwave irradiation at 175 °C
for 20 min. The mixture was cooled to room temperature, filtered,
and the filtered solid washed with ethyl acetate. The combined
solutions was added to saturated aqueous sodium bicarbonate (20
mL) and washed with ethyl acetate (20 mL). The aqueous layer
was acidified to pH 1 with the addition of 1 M aqueous hydrochloric
acid and extracted with ethyl acetate (2 × 20 mL). The ethyl acetate
extracts were combined and solvent removed under reduced pressure
and the resulting solid purified by preparative HPLC to give 3-(2H-
tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a) as a white
solid (0.012 g, 0.068 mmol, 41%).
Alternative Preparation of 3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahy-
dro-cyclopentapyrazole (5a). Thionyl chloride (760 mg, 6.39 mmol)
was added dropwise to a solution of 1-benzyl-1,4,5,6-tetrahydro-
cyclopentapyrazole-3-carboxylic acid amide (6a, 1.03 g, 4.27 mmol)
in DMF (17 mL) at room temperature. The reaction mixture was
stirred for 17 h at which time NaHCO3 (saturated aq, 2 mL) was
added to quench excess thionyl chloride. The mixture was diluted
with EtOAc (100 mL) and washed sequentially with NaHCO3
(saturated aq, 75 mL) and H2O (2 × 100 mL). The organic portion
was separated and dried over MgSO4. The mixture was filtered and
solvent removed under reduced pressure to provide 1-benzyl-1,4,5,6-
tetrahydro-cyclopentapyrazole-3-carbonitrile (7a, 760 mg, 3.41
mmol, 80% yield) as a yellow oil, which was not purified further.
1H NMR (CDCl3): δ 7.40-7.34 (m, 3H), 7.23 (m, 2H), 5.22 (s,
2H), 2.70 (m, 2H), 2.52 (m, 4H).
To a solution of 7a (700 mg, 3.14 mmol) in DMF (6.8 mL) in
a heavy walled reaction vessel was added sequentially ZnBr2 (1.30
g, 4.98 mmol) and NaN3 (775 mg, 11.9 mmol). The vessel was
sealed and heated at 120 °C for 18 h. The resultant mixture was
cooled to room temperature and HCl (3 M aq, 2 mL) was added
whereupon stirring was continued for 5 min. The mixture was
diluted with EtOAc (50 mL) and washed with HCl (1M, aq, 50
mL). The organic portion was dried over MgSO4, the mixture

MK-0354, Partial Nicotinic Acid Receptor Agonist
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5107
filtered, and solvent removed under reduced pressure. Purification
by silica gel chromatography (50:50:0.2, hexanes:EtOAc:AcOH)
gave 1-benzyl-3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapy-
razole (450 mg, 1.69 mmol, 54% yield) as a white solid.
the cell lines and the radioligand binding protocol have been
described previously.20
[32S] GTPγS Binding Assay. [32S] GTPγS binding assays were
performed as previously described.20
Air was bubbled through a stirring solution of 1-benzyl-3-(2H-
tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (450 mg, 1.69
mmol) and KOt-Bu (11.8 mL of a 1 M solution in THF) in DMSO
(10 mL) for 1.5 h. The reaction mixture was acidified by the
addition of 3 M aq HCl (5 mL). The reaction mixture was then
adjusted to pH 3 by cautious addition of solid K2CO3. The mixture
was filtered and solvent removed under reduced pressure. The
residue was purified by reverse-phase HPLC to provide 3-(2H-
tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (207 mg, 1.18
mmol, 70% yield) (5a) as a white solid. HPLC/MS: (column a)
99%, tr ) 2.28 min, m/z (ES+): 177 [M + H]+, 149 [M-N2 +
H]+. 1H NMR (CD3OD): δ 2.88 (t, 2H, J ) 7.0), 2.82 (t, 2H, J )
7.3), 2.64 (quintet, 2H, J ) 7.1).
Preparation of 6-Methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahydro-
cyclopentapyrazole (5b). Thionyl chloride (5.5 mL, 0.5 M in DMF,
2.75 mmol) was added to a solution of 1-benzyl-6-methyl-1,4,5,6-
tetrahydro-cyclopentapyrazole-3-carboxylic acid amide (6b, 0.467
g, 1.83 mmol) in DMF (17 mL) and the resulting mixture stirred
at room temperature for 3 h. NaHCO3 was then added to quench
excess thionyl chloride and the solution stirred for an additional
30 min. The mixture was extracted with EtOAc (2 × 30 mL) and
the combined extracts washed with brine, dried over MgSO4,
filtered, and the solvent removed under reduced pressure to give
1-benzyl-6-methyl-1,4,5,6-tetrahydro-cyclopentapyrazole-3-carbo-
nitrile (7b), which was directly without further purification.
Zinc bromide (0.506 g, 2.25 mmol) and sodium azide (0.238 g,
3.66 mmol) were added to a solution of 7b in DMF (3 mL). The
resulting solution was heated under microwave irradiation to 200
°C for 10 min. Additional sodium azide (0.100 g, 1.54 mmol) was
added, and the solution was heated to 200 °C for a further 10 min.
Solvent was removed under reduced pressure, and the resulting solid
was suspended in aqueous 3 M hydrochloric acid and extracted
with ethyl acetate (2 × 20 mL). The organic solution was dried
over magnesium sulfate and solvent removed under reduced
pressure to give 1-benzyl-6-methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-
tetrahydro-cyclopentapyrazole, which was used directly without
further purification. HPLC/MS: (column b) 99%; tr ) 2.43 min;
m/z (ES+): 281 [M + H]+, 253 [M-N2 + H]+.
Measurement of Adenylyl Cyclase Inhibition. A 96-well ade-
nylyl cyclase activation flashplate assay kit (Perkin-Elmer) protocol
was developed and applied as described previously.22
Human Subcutaneous Fat Lipolysis Assay. Cultured human
subcutaneous adipocytes were received from Zen Bio., Inc. plated
in 96-well plates two weeks prior to performing the lipolysis assay.
Upon arrival, all media was removed and pooled (ZenBio Adipocyte
Maintenance Medium). Then 150 µL of this media was realiquoted
to each well. Cells were kept in a sterile, humidified incubator at
37 °C. On the day of the lipolysis assay, cells were washed twice
with 150 µL of Zen Bio Wash buffer. After the second wash and
removal of wash buffer, 75 µL of test compounds were added to
each well in triplicate. Compounds were prepared in Zen Bio Assay
buffer plus 1 µM theophylline. Cells were incubated for 5 h at 37
°C. Glycerol was determined using a free glycerol reagent from
Sigma (reagent A). Adipocyte media (50 µL) was removed and
transferred to a flat-bottom 96-well plate. Reagent A (50 µL) was
then added to each well. After 15 min, absorbance was read at
OD540 on a Spectramax 340PC microplate reader (Molecular
Devices). The amount of glycerol released was calculated based
on regression analysis of known glycerol concentrations using a
Glycerol Standard (Sigma).
In Vivo Assays. Animals. Animal studies were performed
according to the Guide for the Care and Use of Laboratory Animals
published by the National Academy of Sciences (1996) and
approved by the Arena Pharmaceuticals and Merck Research
Laboratories Animal Care and Use Committees. All mice used were
C57Bl/6 males, at least 8 weeks old (The Jackson Laboratories
West, Sacramento, CA). Animals were housed under standard
laboratory conditions with a 12 h dark, 12 h light cycle with constant
temperature and humidity. Mice were fed a standard rodent diet,
and had ad libitum access to food and water.
In Vivo Mouse Lipolysis. Prior to study, mice were fasted for
16 h. Compound or vehicle (0.5% methylcellulose) was adminis-
tered by oral gavage (po), and animals were euthanized 20 min
postdose by CO2 asphyxiation. Blood was collected via the inferior
vena cava, anticoagulated in EDTA, and plasma separated by
centrifugation on a tabletop microcentrifuge. Plasma was used for
the measurement of FFA, using an enzymatic method (NEFA C
Free Fatty-Acid Assay; WAKO Chemicals USA, Richmond, VA)
and for the measurement of compound levels, by API-4000 LC-
MS/MS after acetonitrile precipitation.
In Vivo Mouse Vasodilation. Mouse vasodilation was measured
by laser Doppler flowmetry as previously described.22 Briefly, male
C57/Bl6 mice (8-10 weeks old; ∼25 g) were anesthetized with
Nembutal via ip injection (80 mg/10 mL/kg). After 10 min, the
mouse was placed under an LDPI laser Doppler (PeriScan PIM II;
Perimed, Stockholm) and a needle and syringe containing vehicle
(PBS; 40% hydroxypropyl-ꢀ-cyclodextrin (HPBCD) or 0.5% me-
thylcellulose) or drug was placed in the intraperitoneal space and
a slight back pressure was applied to prevent premature delivery
of compound. The mouse’s right ear was turned inside-out to expose
the ventral side using forceps. The laser Doppler was focused in
the center of the ventral right ear and adjusted as follows: repeated
data collection; 15 × 15 image format, auto interval start, 20 s
delay, medium resolution, very fast scan speed, and 8-9 V intensity
(∼4.5 cm from ear). After a three minute baseline reading, vehicle
or compound was administered into the ip space (5 mL/kg through
the preinserted syringe) and readings continued for approximately
15 min. Vasodilation was expressed as “% change of perfusion
over baseline values”. At the end of the studies, mice were
euthanized and a blood sample was collected by cardiac puncture
and anticoagulated in EDTA. Plasma was obtained by centrifugation
and used for determination of compound concentration by LC-MS/
MS.
1-Benzyl-6-methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclo-
pentapyrazole was dissolved in formic acid (10% in methanol, 2.5
mL) and palladium black (350 mg) added. The resulting solution
was stirred at room temperature for 2 days, filtered, and the residue
applied to a 10 g SCX cartridge, which was then eluted with
methanol to remove no acidic species. Elution with ammonia (2
M in ethanol) provided 6-methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-
tetrahydro-cyclopentapyrazole ammonium salt (5b) as a white solid
(0.111 g, 0.53 mmol). HPLC/MS: (column a) 97%; tr ) 3.03 min;
m/z (ES+): 191 [M + H]+, 163 [M-N2 + H]+. 1H NMR (CD3OD):
δ 3.15 (sextet, 1H, J ) 7.1, C(6)-H), 2.95-2.85 (m, 1H), 2.82-2.68
(m, 2H), 2.12-2.00 (m, 1H), 1.29 (d, 3H, J ) 6.9, CH3).
Preparation of 5-Methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahydro-
cyclopentapyrazole (5c). 5c was prepared using the same method
described above for 5b and the title compound isolated as a white
solid (0.053 g, 0.25 mmol). HPLC/MS: (column a) 98%; tr ) 3.08
min; m/z (ES+): 191 [M + H]+, 163 [M-N2 + H]+. 1H NMR
(CD3OD): δ 3.65-3.55 (m, 1H), 3.12-2.85 (m, 2H), 2.41 (ddd,
1H, J1 ) 43.5, J2 ) 14.4, J3 ) 5.2), 1.28-1.13 (m, 3H, CH3).
Preparation of 4-Methyl-3-(2H-tetrazol-5-yl)-1,4,5,6-tetrahydro-
cyclopentapyrazole (5d). 5d was prepared using the same method
described above for 5b and the title compound isolated as a white
solid (0.032 g, 0.15 mmol). HPLC/MS: (column a) 99%; tr ) 3.30
min; m/z (ES+): 191 [M + H]+, 163 [M-N2 + H]+. 1H NMR
(CD3OD): δ 3.40 (sextet, 1H, J ) 6.51, C(4)-H), 2.85-2.60 (m,
3H), 1.26 (d, 3H, J ) 6.8, CH3).
In Vitro Assays. [3H] Nicotinic Acid Binding. Radioligand
binding assays were carried out on membranes derived from stably
transfected Chinese hamster ovary (CHO) cells. The derivation of

5108 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Semple et al.
(14) (a) Offermanns, S. Trends Pharmacol. Sci. 2006, 27, 384–390. (b)
Semple, G.; Boatman, P. D.; Richman, J. G. Recent progress in the
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DeV. 2007, 10, 452–459.
Supporting Information Available: HPLC-MS spectra, dose-
response curves for 5a, 5a antagonizes nicotinic acid, PK-PD
relationship for 5a and nicotinic acid in c57/bl6 mice, effect of 5a
and nicotinic acid in PUMA-G knockout mice. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Gottesdiener, K.; Wright, S. D.; Wang, Z.; O’Neill, G.; Lai, E.; Waters,
M. G. Antagonism of the prostaglandin D2 receptor 1 suppresses
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