Pyrazole derivatives as partial agonists for the nicotinic acid receptor

Article abstract of DOI:10.1021/jm030888c

Nicotinic acid as a hypolipidemic agent appears unique due to its potential to increase HDL cholesterol levels to a greater extent than other drugs. However, it has some side effects, among which severe skin flushing is the most frequent and often limits patients' compliance. In a search for novel agonists for the recently identified and cloned G protein-coupled nicotinic acid receptor, we synthesized a series of substituted pyrazole-3-carboxylic acids that proved to have substantial affinity for this receptor. The affinities were measured by inhibition of [³H] nicotinic acid binding to rat spleen membranes. Potencies and intrinsic activities relative to nicotinic acid were determined by their effects on [³⁵S]GTPγS binding to rat adipocyte and spleen membranes. Interestingly, most compounds were partial agonists. In particular, 2-diazabicyclo-[3,3,O ^4,8]octa-3,8-diene-3-carboxylic acid (4c) and 5-propylpyrazole-3-carboxylic acid (4f) proved active with K_i values of approximately 0.15 μM and EC₅₀ values of approximately 6 μM, while their intrinsic activity was only ～50% when compared to nicotinic acid. Even slightly more active was 5-butylpyrazole-3-carboxylic acid (4g) with a K_i value of 0.072 μM, an EC₅₀ value of 4.12 μM, and a relative intrinsic activity of 75%. Of the aralkyl derivatives, 4q (5-(3-chlorobenzyl)pyrazole-3-carboxylic acid) was the most active with a relatively low intrinsic activity of 39%. Partial agonism of the pyrazole derivatives was confirmed by inhibition of G protein activation in response to nicotinic acid by these compounds. The pyrazoles both inhibited the maximum effect elicited by 100 μM nicotinic acid and concentration dependently shifted nicotinic acid concentration-response curves to the right, pointing to a competive mechanism of action.

Full text of DOI:10.1021/jm030888c

J . Med. Chem. 2003, 46, 3945-3951
3945
P yr a zole Der iva tives a s P a r tia l Agon ists for th e Nicotin ic Acid Recep tor
T. van Herk,† J . Brussee,† A. M. C. H. van den Nieuwendijk,† P. A. M. van der Klein,† A. P. IJ zerman,*,†
C. Stannek,‡ A. Burmeister,‡ and A. Lorenzen‡
Leiden/ Amsterdam Center for Drug Research, Division of Medicinal Chemistry, P.O. Box 9502, 2300 RA Leiden,
The Netherlands, and Institute of Pharmacology, Im Neuenheimer Feld 366, University of Heidelberg,
D-69120 Heidelberg, Germany
Received May 5, 2003
Nicotinic acid as a hypolipidemic agent appears unique due to its potential to increase HDL
cholesterol levels to a greater extent than other drugs. However, it has some side effects, among
which severe skin flushing is the most frequent and often limits patients’ compliance. In a
search for novel agonists for the recently identified and cloned G protein-coupled nicotinic acid
receptor, we synthesized a series of substituted pyrazole-3-carboxylic acids that proved to have
substantial affinity for this receptor. The affinities were measured by inhibition of [3H]nicotinic
acid binding to rat spleen membranes. Potencies and intrinsic activities relative to nicotinic
acid were determined by their effects on [35S]GTPγS binding to rat adipocyte and spleen
membranes. Interestingly, most compounds were partial agonists. In particular, 2-diazabicyclo-
[3,3,04,8]octa-3,8-diene-3-carboxylic acid (4c) and 5-propylpyrazole-3-carboxylic acid (4f) proved
active with Ki values of approximately 0.15 µM and EC50 values of approximately 6 µM, while
their intrinsic activity was only ∼50% when compared to nicotinic acid. Even slightly more
active was 5-butylpyrazole-3-carboxylic acid (4g) with a Ki value of 0.072 µM, an EC50 value of
4.12 µM, and a relative intrinsic activity of 75%. Of the aralkyl derivatives, 4q (5-(3-
chlorobenzyl)pyrazole-3-carboxylic acid) was the most active with a relatively low intrinsic
activity of 39%. Partial agonism of the pyrazole derivatives was confirmed by inhibition of G
protein activation in response to nicotinic acid by these compounds. The pyrazoles both inhibited
the maximum effect elicited by 100 µM nicotinic acid and concentration dependently shifted
nicotinic acid concentration-response curves to the right, pointing to a competive mechanism
of action.
In tr od u ction
Nicotinic acid is one of the drugs used in hyperlipi-
demia. It inhibits the formation of nonesterified fatty
acids from adipose tissue. As a consequence the hepatic
synthesis of triglycerides, the secretion of VLDL, and
its conversion into LDL are reduced. Interestingly,
nicotinic acid increases serum HDL concentrations by
approximately 25% at a standard daily dose of 1-2 g.
The latter characteristic is very beneficial, and not seen
with any of the other standard regimens, including the
use of statins.1
F igu r e 1. Structures of nicotinic acid (I) and acipimox (II).
trinsic activity of such compounds. These methods
proved also instrumental in the cloning of the nicotinic
receptor that was reported very recently. It appears that
two receptor subtypes may exist with high and low
affinity for nicotinic acid, respectively.4
The target for nicotinic acid in the adipocyte is a G
protein-coupled receptor. It has been characterized
pharmacologically in rat adipocytes and spleen and
mouse macrophages.2,3 Nicotinic acid is a full agonist
and has submicromolar affinity for this receptor, as has
acipimox, a close analogue that is also used clinically
(see Figure 1 for both structures). The receptor is
coupled to the enzyme adenylate cyclase through Gi/o
proteins; upon activation of the receptor adipocyte cAMP
levels are reduced. These findings have yielded facile
screening methods in the form of receptor binding
assays with [3H]nicotinic acid as the radioligand to
determine the affinity of novel chemical entities, and
[35S]GTPγS binding assays to assess potency and in-
The favorable characteristics of nicotinic acid can be
accompanied by disturbing side effects, including flush-
ing of the skin, an effect that is judged unbearable by
10% of patients. Apparently, the actions of nicotinic acid
are not tissue selective. Tissue selectivity may poten-
tially be achieved with partial agonists. Partial agonists
can behave as full agonists in tissues with high receptor
density and/or efficient coupling to G proteins and
effector enzymes, whereas in other tissues such com-
pounds may have reduced efficacy or may even be
“silent”. We have adopted that reasoning for the design
of partial agonists for the adenosine A1 receptor, and
were successful in synthesizing compounds with full
antilipolytic activity on rat adipose tissue, whereas their
cardiovascular effects were largely reduced.5,6
* Corresponding author. Tel: (+31) 71 5274651. Fax: (+31) 71
5274565. E-mail: ijzerman@lacdr.leidenuniv.nl.
† Leiden/Amsterdam Center for Drug Research.
‡ University of Heidelberg.
In analogy, we established a program for the design
of partial agonists for the nicotinic receptor as well. We
10.1021/jm030888c CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/01/2003

3946 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
van Herk et al.
Ta ble 1. Inhibition of [3H]Nicotinic Acid Binding to Rat Spleen
Membranes
Sch em e 1. Synthetic Route for Substituted
Pyrazole-3-carboxylic Acidsa
compd
R1
R2
H
Ki (µM, 95% CI)
nicotinic
acid
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
0.033
(0.028-0.040)
H
0.594
(0.522-0.675)
-CH2CH2O-
-C3H6-
-C4H8-
>100
0.156
3.54
0.683
0.143
0.072
21.4
101
63.7
107
37.9
1.25
3.61
(0.133-0.184)
(2.21-5.67)
(0.500-0.931)
(0.121-0.169)
(0.060-0.085)
(15.6-29.2)
(81.7-125)
a
(a) Na/ETOH, 18 h, rt; (b) H2NNH2/HAc, 8 h, reflux; (c) NaOH/
i-C3H7
C3H7
C4H9
H
H
H
H
H
H
H
H
H
H
dioxane, 18 h, rt.
were intrigued by a series of substituted pyrazole
derivatives, reported by Seki et al. two decades ago.7
These authors noticed substantial in vivo hypolipidemic
activity for some of their compounds when administered
to rats. We resynthesized a number of their derivatives
(4e-h in the present study), synthesized other ana-
logues as well, demonstrated their interaction with the
nicotinic acid receptor, and developed structure-activity
relationships. Most compounds tested appeared to be
partial agonists for this receptor.
C
11H23
C6H5
3-Cl-C6H4
4-Cl-C6H4
4-CH3-C6H4
C6H5-CH2
4-Cl-C6H4
-CH2
4-CH3-C6H4
-CH2
4-OCH3-C6H4
-CH2
(39.7-102)
(91.8-126)
(13.2-109)
(1.15-1.37)
(2.48-5.27)
4o
4p
H
H
20.3
66.0
0.504
(13.9-29.6)
(25.2-173.0)
Resu lts a n d Discu ssion
4q
4r
4s
3-Cl-C6H4-CH2
C6H5-C2H4
C6H5-C3H6
H
H
H
(0.450-0.566)
(1.29-1.92)
(5.51-7.20)
1.57
6.30
Ch em istr y. The synthetic route for the preparation
of 5-substituted-1H-pyrazole-3-carboxylic acids involved
a three-step synthesis and is depicted in Scheme 1. The
first step for compounds 4e-s consisted of a Claisen
condensation of the appropiate alkyl-methyl ketone 1
(R2 ) H) with diethyl oxalate by the use of sodium
ethoxide in absolute ethanol. This afforded the corre-
sponding R,γ-diketo esters 2.7,8 Alkyl-methyl ketones
that were not commercially available (1n -p ) were
prepared by reaction of methyllithium with the corre-
sponding carboxylic acid.9,10 The cyclic compounds 2b-d
were prepared in the same way starting from butyro-
lactone,11 cyclopentanone, and cyclohexanone,12 respec-
tively. The second step involved the addition of hydra-
zine hydrate in acetic acid to the R,γ-diketoesters 2 to
form the pyrazole-3-carboxylic acid ethyl esters 3.8,13 In
most cases, yields were high, except for the benzyl-
substituted derivatives 3m -q. In the latter case, enol
formation (step b in Scheme 1) also occurred at the
benzylic carbon position of the corresponding ketones,
which rendered yields of the desired intermediates less
than optimal. Hydrolysis of the ethyl esters with 0.25
M NaOH in H2O/dioxane and acidification of the reac-
tion mixture to pH 2 afforded the substituted pyrazole
carboxylic acids 4b-s.8
Biologica l Eva lu a tion . Table 1 displays radioligand
binding data for all the synthesized 5-substituted-1H-
pyrazole-3-carboxylic acids. For reasons of comparison,
data for nicotinic acid and potassium pyrazole-3-car-
boxylate (4a ) were also included. [3H]nicotinic acid (20
nM) was used as the radioligand. This compound binds
with reasonably high affinity to rat spleen membranes
(KD ) 23 nM) with an acceptable level of nonspecific
binding (<30% of total binding at KD).2 It has similar
binding characteristics on epididymal adipocyte mem-
branes (the target tissue in hyperlipidemia), but due to
higher yields per animal we preferred the spleen
membrane preparation. All compounds with the excep-
tion of 4b fully displaced [3H]nicotinic acid from the
receptor. The unsubstituted pyrazole-3-carboxylate 4a
was 18-fold less active in this respect than nicotinic acid,
suggesting that the pyrazole pharmacophore is some-
what less well accommodated by the receptor than the
pyridine ring system in nicotinic acid. For substitution
on R1 alkyl, aryl and aralkyl groups were chosen. An
alkoxy group linking positions R1 and R2 (as in 4b) was
not tolerated at all. Even at a concentration of 100 µM,
this compound displayed less than 50% displacement
of the radioligand. Smaller alkyl groups were introduced
in 4c-g, i.e., propyl, isopropyl, and butyl, either free or
linked to R2. The (free) butyl substituent on R1 (4g) led
to the highest affinity of the whole series, being only
2-fold less active than nicotinic acid. An extended alkyl
chain as in 4h was quite unfavorable for receptor
affinity. Next (substituted) phenyl groups were intro-
duced on R1 (4i-l). This modification yielded com-
pounds with poor receptor affinity. Even the most active
with a 4-methyl group on the phenyl ring (4l) was more
than 500 times less active than 4g. Going from phenyl
(4i) to benzyl (4m ), phenylethyl (4r ) and phenylpropyl
(4s), affinities improved with an optimum for benzyl
(1.25 µM) and phenylethyl (1.57 µM). With 4m as a lead,
we synthesized a few more derivatives (4n -q) based on
a substituent decision tree suggested by Topliss.14 The
3-chlorobenzyl derivative (4q) was slightly more active
than the parent compound.
The preferential coupling of the nicotinic acid receptor
to Gi/o proteins rendered the setup feasible of a [35S]-
GTPγS binding assay.2 In this assay, the binding of the
stable and radiolabeled GTP analogue [35S]GTPγS is
increased by the concomitant presence of receptor
agonists. Minute quantities of protein usually suffice in
such assays, which, in this case, allowed the use of both

Pyrazole Derivatives as Partial Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3947
F igu r e 3. Comparison of intrinsic activities at rat adipocyte
and spleen membranes. Relative intrinsic activities (RIA) of
pyrazole compounds were determined in G protein activation
studies and are relative to the maximum effect induced by
nicotinic acid (100%). Dashed line indicates 1:1 stoichiometry.
F igu r e 2. G protein activation by nicotinic acid and compound
4f in rat adipocyte membranes. [35S]GTPγS binding was
performed as described in Experimental Section with 1 µg of
membrane protein for 90 min at 25°C. Data from one repre-
sentative experiment are shown. All experiments (n ) 3) were
performed in triplicate. (Ctr ) control)
Ta ble 2. Potencies (EC50 Values) and Relative Intrinsic
Activities (RIA) in Stimulation of [35S]GTPγS Binding to Rat
Adipocyte and Spleen Membranes
adipocytes
spleen
EC50
(µM)
EC50
(µM)
comp
RIA (%)
RIA (%)
NA
4a
4c
4d
4e
4f
1.70 (0.94-3.07) 100
0.74 (0.57-0.96) 100
16.0 (12.5-20.5) 78.5 ( 3.4 21.5 (15.6-29.8) 85.3 ( 5.5
6.44 (3.31-12.5) 51.7 ( 4.5 6.97 (4.31-11.3) 55.9 ( 6.2
85.1 (70.6-103) 29.2 ( 2.7 64.6 (25.4-165) 47.2 ( 4.7
13.7 (8.16-22.9) 49.2 ( 6.9 21.8 (13.6-34.8) 67.7 ( 10.0
6.10 (4.78-7.79) 53.5 ( 2.2 5.09 (3.93-6.58) 69.9 ( 5.7
4.12 (3.03-5.60) 75.7 ( 0.6 2.26 (1.00-5.13) 80.7 ( 5.6
4g
4m 70.7 (35.0-143) 52.4 ( 7.5 86.9 (68.2-111) 50.3 ( 4.3
4q 46.6 (24.6-88.3) 39.4 ( 2.3 n.d. n.d.
rat spleen and rat adipocyte membranes. We selected
the compounds with the highest affinities from Table 1
and tested them with respect to potency (EC50 values)
and intrinsic activity relative to nicotinic acid (100%).
Interestingly, all these compounds proved to be partial
agonists in these assays. In Figure 2 the concentration-
response curves for nicotinic acid and 4f are graphically
represented, showing the reduced maximal effect of the
latter compound. Intrinsic activities varied between 29
and 85% for the pyrazole derivatives (Table 2). The
intrinsic activities did not grossly differ between adi-
pocyte and spleen membranes, which is also evident
from Figure 3 showing a close correlation between the
intrinsic activities in these two preparations. Com-
pounds 4d -f were somewhat more efficacious in spleen.
EC50 values are reported in Table 2 and ranged from
1.7 to 85.1 and 0.74 to 86.9 µM in the epididymal
adipocyte and spleen preparation, respectively. A strong
correlation (r ) 0.97) was found between ligand poten-
cies determined in adipocyte and spleen membranes
(Figure 4, upper panel). In these activation assays EC50
values for nicotinic acid were approximately 1 µM,
substantially higher than the Ki/KD values (approxi-
mately 30 nM) observed in the binding assays. The
experimental conditions in both assays are very differ-
ent, however. The inclusion of sodium ions and GDP in
the GTPγS binding assay, essential for a sufficient and
robust “read-out”, is known to reduce agonist affinity.
F igu r e 4. Comparison of affinities and potencies of nicotinic
acid receptor ligands. Since receptor occupancy depends on the
log concentration of ligands, both axes are log scale. The upper
panel shows the correlation between potencies to activate G
proteins at adipocyte and spleen membranes (dashed line
indicates 1:1 stoichiometry). The lower panel shows the
correlation between potencies in G protein activation and
binding affinities in spleen membranes.
Correlations between Ki and EC50 values from the
spleen preparation were also highly significant (r ) 0.97;
Figure 4, lower panel). The slopes of the two lines were

3948 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
van Herk et al.
F igu r e 5. Stimulation of basal [35S]GTPγS binding and
inhibition of nicotinic acid-stimulated [35S]GTPγS binding by
pyrazole derivatives. [35S]GTPγS binding was determined with
1 µg of adipocyte membrane protein in absence or presence of
100 µM nicotinic acid. Pyrazole derivatives (1 mM) stimulate
basal [35S]GTPγS binding (open columns) and inhibit the
stimulatory effect of nicotinic acid (grey columns). Unstimu-
lated binding of [35S]GTPγS was 1191 ( 86 cpm/µg, binding
in the presence of 100 µM nicotinic acid was 2634 ( 178 cpm.
All experiments (n ) 3) were performed in triplicate.
F igu r e 6. Inhibition by pyrazole 4f of G protein activation
by nicotinic acid. Concentration-effect curves for nicotinic acid
in the absence or presence of increasing concentrations of 4f
are shown. [35S]GTPγS binding to 1 µg of adipocyte membrane
protein was performed as described in Experimental Section.
The inset shows the Schild plot of the data. One out of four
experiments, performed in triplicate, is shown.
by Schild plot analysis revealed competitive antagonism
by pyrazole 4f with a KB value of 6.33 (4.36-9.19,
n ) 4) µM for inhibition of nicotinic acid effects, which
is in good agreement with the EC50 value determined
for 4f (6.10 µM).
close to unity, 1.18 (upper panel) and 0.92 (lower panel),
respectively, suggestive of identical receptors and a
common mechanism of action in all assays. Compounds
4c and 4f combined relatively high potency with sub-
stantially reduced intrinsic activity. Such materials may
serve as lead structures for the development of antago-
nists of nicotinic acid receptors, which have not been
described to date. They are, however, urgently required
for the characterization of this receptor and may also
be of therapeutic relevance, e.g., in the treatment of
obesity.
To confirm that pyrazoles were indeed partial ago-
nists at the nicotinic acid receptor, we investigated their
effects on nicotinic acid-induced G protein activation in
adipocyte membranes. [35S]GTPγS binding was mea-
sured in the presence of 1 mM of the compounds under
study, with or without the addition of 100 µM nicotinic
acid (Figure 5). The pyrazoles increased [35S]GTPγS
binding in the absence of nicotinic acid to less than the
maximum stimulation observed in the presence of 100
µM of this full agonist. When [35S]GTPγS binding was
measured in the presence of 1 mM of the pyrazoles and
a maximally stimulating concentration (100 µM) of
nicotinic acid under control conditions, it was observed
that the pyrazoles inhibited guanine nucleotide binding.
This inhibition of the effects of nicotinic acid, although
indicative only, demonstrates that the pyrazoles inves-
tigated also possess antagonist activities, which is an
intrinsic characteristic of partial agonists.
Therefore, this antagonistic effect was further inves-
tigated in more detail for compound 4f. Concentration-
response curves of G protein activation in adipocyte
membranes for nicotinic acid in the absence or presence
of increasing concentrations (1 µM to 1 mM) of pyrazole
4f are shown in Figure 6. Nicotinic acid concentration-
dependently stimulated [35S]GTPγS binding. With the
addition of increasing concentrations of 4f, control
binding in the absence of nicotinic acid increased, and
the concentration-response curves for nicotinic acid
were shifted to the right, yielding higher EC50 values
for this full agonist. Evaluation of the experimental data
Little information seems to be available on the
medicinal chemistry of nicotinic acid and related het-
erocyclic compounds, even for the pyrazole compounds
in the present study. We have found previously that
imidazole derivatives were inactive, and furan-3-car-
boxylic acid has only low potency at nicotinic acid
receptors.2 5-Methyl- and 5-ethylpyrazole-3-carboxylic
acid have been described as both hypoglycemic and
hypocholesteremic compounds as assayed in in vivo
animal models.15,16 Another compound based on an
isoxazole core rather than a pyrazole template, 3-me-
thylisoxazole-5-carboxylic acid, also proved effective as
an antilipolytic agent.17 Substitution of the carboxylic
group for a tetrazole function in nicotinic acid and
derivatives appeared allowed, i.e., such compounds
retained substantial antilipolytic activity.18
Con clu sion
We synthesized a series of pyrazole derivatives with
substantial affinity for the nicotinic acid receptor as
present on rat epididymal adipocytes and spleen tissue.
Some of the compounds had affinities comparable to
nicotinic acid itself. Interestingly, the pyrazole deriva-
tives were all partial agonists compared to nicotinic acid
as identified in [35S]GTPγS binding assays. Partial
agonists generally induce tissue selectivity, which may
be relevant in this particular case, since nicotinic acid,
although very useful as a lipid-lowering agent, displays
serious side effects occurring distantly from the target
tissue. This series of compounds may provide useful
tools for a more detailed functional characterization of
the nicotinic acid receptor, which was recently cloned.
Future medicinal chemistry efforts aiming at both
partial agonists with even higher affinity and antago-
nists appear worthwhile.
Exp er im en ta l Section
Ch em ica ls a n d Solven t s. Guanosine 5′(-γ-[35S]thio)-tri-
phosphate ([35S]GTPγS); 1250 Ci/mmol was obtained from

Pyrazole Derivatives as Partial Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3949
NEN Life Science Products (Rodgau, Germany). [5,6-3H]-
nicotinic acid was purchased from Biotrend (Cologne, Ger-
many). 4-Methoxyphenylacetone, 3-chlorophenylacetic acid,
4-tolylacetic acid, 4-chloroacetophenone, 4-chlorophenylacetic
acid, p-methylacetophenone, and methyllithium were pur-
chased from Acros Organics (‘s Hertogenbosch, The Nether-
lands). Nicotinic acid, 2-hexanone, hydrazine monohydrate,
and bovine serum albumin (fraction V) were purchased from
Sigma-Aldrich (Zwijndrecht, The Netherlands). Isopropyl-
methyl ketone was purchased from Fluka Chemica (Zwijn-
drecht, The Netherlands). Diethyloxalate, cyclopentanone, and
Silica gel 60 (0.040-0.063 mm) were purchased from Merck-
Schuchardt (Hohenbrunn, Germany). Cyclohexanone was
purchased from Avocado (Karlsruhe, Germany). 3-[(3-Chola-
midopropyl)-dimethylammonio]propanesulfonate (CHAPS) and
adenosine deaminase were purchased from Roche Biochemi-
cals (Almere, The Netherlands). Acipimox was a generous gift
from Pharmacia-Upjohn (Kalamazoo, MI). All other materials
were from standard sources and of the highest purity com-
mercially available.
CH, cis/trans isomers), 7.38 (d, J ) 8.41, 2H, arom.), 7.78 (d,
J ) 8.40, 2H, arom.).
4-(4-Meth yl-p h en yl)-2,4-d ioxo-bu tyr ic Acid Eth ylester ,
1
Sod iu m Sa lt (2l). H NMR: (MeOD) δ 1.33 (t, J ) 7.31, 3H,
CH3 ester), 2.36 (s, 3H, CH3 arom.), 4.24 (t, J ) 7.31 and 6.58,
2H, CH2 ester), 6.50 (s, 1H, CH), 7.19 (d, J ) 8.04, 2H, arom.),
7.71 (d, J ) 8.04, 2H, arom.).
2,4-Dioxo-5-p h en yl-p en ta n oic Acid Eth yl Ester , En olic
F or m (2m ). 1H NMR (CDCl3) δ 1.36 (t, 3H, J ) 7.3 Hz, CH3),
3.79 (s, 2H, CH2), 4.35 (q, 2H, J ) 7.3, OCH2), 6.38 (s, 1H,
dCH), 7.29 (m, 5H, arom.), 10.60 (broad s, 1H, OH).
5-(3-Ch lor o-p h en yl)-2,4-d ioxo-p en ta n oic Acid Eth yl-
ester , En olic F or m (2n ). 1H NMR: (CDCl3) δ 1.36 (t, J )
7.31, 3H, CH3 ester), 3.75 (s, 2H, CH2 benzylic), 4.34 (q, J )
7.31, 2H, CH2 ester), 6.36 (s, 1H, CH), 7.13 (m, 1H, arom.),
7.30 (m, 3H, arom.).
5-(4-Ch lor o-p h en yl)-2,4-d ioxo-p en ta n oic Acid Eth yl-
ester (2o). 1H NMR: (CDCl3) δ 1.35 (t, J ) 7.3, 3H, CH3 ester),
3.72 (s, 2H, CH2 benzylic), 4.33 (q, J ) 7.31, 2H, CH2 ester),
6.35 (s, 1H, CH), 7.36 (d, 2H, arom.), 7.76 (d, 2H, arom.).
5-(4-Meth ylp h en yl)-2,4-d ioxo-p en ta n oic Acid Eth yl-
ester (2p ). 1H NMR: (CDCl3) δ 1.01 (t, J ) 7.31, 3H, CH3
ester), 2.34 (s, 3H, CH3 ring), 3.73 (s, 2H, CH2 benzylic), 4.32
(q, J ) 7.31 and 6.58, 2H, CH2 ester), 6.36 (s, 1H, CH), 7.17
(m, 4H, arom.).
In str u m en ts a n d An a lysis. 1H NMR spectra were mea-
sured at 200 MHz with a Bruker AC 200 spectrometer
equipped with a PG 200 computer operating in the Fourier
transform mode. 13C NMR spectra were measured at 50 MHz.
1
Chemical shifts for H and 13C are given in ppm (δ) relative to
tetramethylsilane (TMS) as internal standard, and coupling
constants are given in Hertz. Melting points were determined
with a Bu¨chi capillary melting point apparatus and are
uncorrected. Combustion analyses of new target compounds
were performed by the analytical department of the Gorlaeus
Laboratories, Leiden University (The Netherlands), and are
within (0.4% of theoretical values unless otherwise specified.
Syn th esis. Compounds 2b-s were prepared according to
literature procedures.8,11,12,19,20
5-(4-Meth oxy-p h en yl)-2,4-d ioxo-p en ta n oic Acid Eth yl-
ester (2q). 1H NMR: (CDCl3) δ 1.35 (t, J ) 6.57 and 7.31,
3H, CH3 ester), 3.71 (s, 2H, CH2 benzylic), 3.02 (s, 3H, OCH3),
4.32 (q, J ) 7.31 and 6.58, 2H, CH2 ester), 6.35 (s, 1H, CH),
6.88 (d, J ) 8.78, 2H, arom.), 7.11 (d, J ) 8.78, 2H, arom.).
2,4-Dioxo-6-p h en yl-h exa n oic Acid Eth yl Ester , Sod iu m
Sa lt (2r ). 1H NMR (MeOD) δ 1.08 (t, 3H, J ) 7.3 Hz, CH3),
2.86 (m, 4H, 2× CH2), 4.20 (q, 2H, J ) 7.3, OCH2), 7.16 (m,
5H, arom.).
2,4-Dioxo-7-p h en yl-h ep ta n oic Acid Eth yl Ester (2s). 1H
NMR (CDCl3) δ 1.38 (t, 3H, J ) 7.3 Hz, CH3), 2.03 (m, 2H,
CH2), 2.51 (t, 2H, J ) 7.3 Hz, CH2), 2.67 (t, 2H, J ) 7.3 Hz,
CH2), 4.35 (q, 2H, J ) 7.3, OCH2), 6.34 (s, 1H, dCH), 7.24 (m,
5H, arom.).
Oxo-(2-oxo-tetr a h yd r ofu r a n -3-yl)-a cetic Acid Eth yl-
1
ester (2b).11 Prepared from butyrolacton. H NMR (CDCl3) δ
1.39 (t, J ) 7.31 and 6.58, 3H, CH3 ester), 3.30 (t, J ) 8.04
and 7.31, 2H, CH2), 4.37 (q, J ) 7.31 and 6.57 CH2 ester), 4.51
(t, J ) 7.31 and 7.30, 2H, CH2O).
1,2-Dia za -7-oxa -bicyclo[3,3,04,8]octa -3,8-dien e-3-ca r box-
ylic Acid Eth ylester (3b). 5.0 g (26.85 mmol) 2b was
dissolved in 10 mL of acetic acid at 0 °C. A total of 1.48 g (29.56
mmol) of hydrazine hydrate was slowly added. The mixture
was heated to reflux for 8 h. After cooling of the sample, the
solid matter was filtered and dried in vacuo, resulting in 4.9
g (26.90 mmol, ∼100%) of white crystals.
(2-Oxo-cyclop en tyl)-glycoxylic Acid Eth yl Ester , So-
d iu m Sa lt (2c).12 Prepared from cyclopentanone. 1H NMR
(MeOD) δ 1.31 (t, J ) 7.31, 3H, CH3 ester), 1.82 (q, J ) 7.31
and 8.04, 2H, CH2), 2.19 (t, J ) 7.31 and 8.04, 2H, CH2), 2.05
(t, J ) 6.58 and 7.31, 2H, CH2), 4.23 (q, J ) 7.31, 6.58, 2H,
CH2 ester).
(2-Oxo-cycloh exyl)-glycoxylic Acid Eth yl Ester (2d).12,19
1
1H NMR (CDCl3) δ 1.42 (t, J ) 6.58 and 7.31, 3H, CH3 ester),
3.01 (t, J ) 7.31 and 6.58, 2H, CH2-O), 4.26 (t, J ) 7.31 and
6.58, 2H, CH2), 4.44 (q, J ) 7.31 and 6.58, 2H, CH2 ester).
1,2-Dia za -b icyclo[3,3,04,8]oct a -3,8-d ien e-3-ca r b oxylic
Acid Eth ylester (3c)19. 1.07 g (21.34 mmol) of hydrazine
hydrate was slowly added to a cooled suspension of 4.00 g
(19.40 mmol) 2c in 10 mL of acetic acid. The mixture was
heated to reflux for 8 h, poured into ice-H2O, neutralized with
NaHCO3, and extracted with dichloromethane (3 × 100 mL)
and ethyl acetate (1 × 100 mL). The combined organic layers
were dried (Na2SO4), filtered, and concentrated. Yield 62%.
1H NMR (CDCl3) δ 1.37 (t, J ) 7.31 and 6.58, 3H, CH3 ester),
2.47 (m, 2H, CH2), 2.77 (m, 4H, 2× CH2), 4.35 (q, J ) 7.31
and 6.58, 2H, CH2 ester).
Prepared from cyclohexanone. H NMR (CDCl3) δ 1.38 (t, J )
7.31, CH3 ester), 1.72 and 2.46 (2× m, combination of 4× CH2),
3.76 (t, J ) 7.31, 1H, CH, ketoform), 4.34 (q, J ) 7.31 and
6.58, 2H (CH2 ester).
5-Meth yl-2,4-d ioxo-h exa n oic Acid Eth ylester , Sod iu m
Sa lt (2e).8,20 1H NMR (MeOD) δ 1.04 (d, J ) 6.58, 6H, 2× CH3
isopropyl), 1.31 (t, J ) 7.31 and 6.58, 3H, CH3 ester), 3.06 (m,
J ) 7.31 and 6.58, 1H, CH isopropyl), 4.19 (q, J ) 7.31 and
6.58, 2H, CH2 ester), 5.14 and 5.73 (2× s, 1H (cis/trans)).
2,4-Dioxoh ep ta n oic Acid Eth yl Ester , En olic F or m (2f).
1H NMR (CDCl3) δ 0.96 (t, 3H, J ) 7.3 Hz, CH3), 1.38 (t, 3H,
J ) 7.3 Hz, CH3 ester), 1.70 (m, 2H, CH2), 2.47 (t, 2H, J ) 7.3
Hz, CH2CdO), 4.36 (q, 2H, J ) 7.3 Hz, OCH2), 6.37 (s, 1H,
dCH), 12-13 (broad s, 1H, dCOH).
2,4-Dioxo-octa n oic Acid Eth ylester , En olic F or m (2g).
1H NMR: (CDCl3) δ 0.94 (t, J ) 7.31, 3H, CH3 butyl), 1.25 (m,
2H, CH2), 1.38 (t, J ) 7.31 and 6.58, 3H, CH3 ester), 1.65
(quintet, J ) 6.58 and 8.04, 2H, CH2), 2.50 (t, J ) 7.31 and
8.04, 2H, CH2), 4.36 (q, J ) 7.31, 2H, CH2 ester), 6.37 (s, 1H,
CH).
4,5,6,7-Tetr a h yd r o-1H-in d a zole-3-ca r boxylic Acid Eth -
ylester (3d ). Prepared as described for 3c. Yield 93%. 1H NMR
(CDCl3) δ 1.38 (t, J ) 7.31, 3H CH3 ester), 1.81 (m, 4H, 2×
CH2), 2.73 (m, 4H, 2× CH2), 4.37 (q, J ) 7.31, 6.58, 2H, CH2
ester).
5-Isop r op yl-1H-p yr a zole-3-ca r boxylic Acid Eth yl Ester
(3e). Prepared as described for 3c. Yield 89%. 1H NMR (CDCl3)
δ 1.29 (d, J ) 6.58, 6H, 2x CH3 isopropyl), 1.36 (t, J ) 6.58
and 7.31, 3H, CH3 ester), 3.03 (septet, J ) 7.31 and 6.58, 1H,
CH isopropyl), 4.37 (q, J ) 7.31 and 6.58, 2H, CH2 ester), 6.62
(s, 1H, CH pyrazole).
2,4-Dioxop en ta d eca n oic Acid Eth yl Ester , Sod iu m
1
Sa lt (2h ). H NMR (MeOD) δ 0.90 (t, 3H, J ) 7.3 Hz, CH3),
1.29 (m, 21H, CH2 and CH3 ester), 1.57 (m, 2H, CH2), 2.58 (t,
2H, J ) 7.3 Hz, CH2CdO), 4.18 (q, 2H, J ) 7.3 Hz, OCH2),
5.71 (s, 1H, dCH).
4-(4-Ch lor o-p h en yl)-2,4-d ioxo-4-p h en yl-bu t yr ic Acid
5-P r op yl-1H -p yr a zole-3-ca r b oxylic Acid E t h yl E st er
(3f). Prepared as described for 3b. Yield 76%. 1H NMR (CDCl3)
δ 0.97 (t, 3H, J ) 7.3 Hz, CH3), 1.39 (t, 3H, J ) 7.3 Hz, CH3
1
Eth ylester , Sod iu m Sa lt (2k ). H NMR (MeOD) δ 1.28 (m,
3H, CH3 ester), 4.27 (m, 2H, CH2 ester), 5.74 and 6.43 (2× s,

3950 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
van Herk et al.
ester), 1.69 (m, 2H, CH2), 2.70 (t, 2H, J ) 7.3 Hz, CH2), 4.38
(q, 2H, J ) 7.3 Hz, OCH2), 6.62 (s, 1H, dCH).
mixture was allowed to stand overnight at room temperature.
The mixture was acidified with conc HCl and concentrated in
vacuo. The precipitate was collected. Yield 64%; mp 216-217
°C. 1H NMR (MeOD) δ 3.00 (t, J ) 5.85 and 6.57, 2H CH2),
3.79 (t, J ) 6.58 and 5.85, 2H, CH2-O). 13C NMR (MeOD) δ
25.6 (CH2), 62.0 (CH2-O), 107.2 (C4), 136.9 (C3), 157.9 (C5),
160.0 (carbonyl). Anal. (C6H6N2O3 H2O) C, H, N.
1,2-Dia za -b icyclo[3,3,04,8]oct a -3,8-d ien e-3-ca r b oxylic
Acid (4c)19. Prepared as described for 4b. Yield 42%; mp 270
°C (dec). 1H NMR (MeOD) δ 2.52 (q, J ) 6.58 and 8.04, 2H,
CH2), 2.75 (m, 4H, 2× CH2). 13C NMR (DMSO) δ 23.4, 23.9
and 30.0 (3× CH2), 128.4 and 129.7 (C3 and C4), 159.3 (C5),
161.7 (carbonyl). Anal. (C7H8N2O2 0.1 H2O) C, H, N.
5-Bu tyl-1H-p yr a zole-3-ca r boxylic Acid Eth ylester (3g).
1
Prepared as described for 3c. Yield 93%. H NMR: (CDCl3) δ
0.90 (t, J ) 7.31, 3H, CH3 butyl), 1.33 (m, 2H, CH2), 1.34 (t, J
) 6.58 and 7.31, CH3 ester), 1.62 (m, 2H, CH2), 2.69 (t, J )
7.31 and 8.04, 2H, CH2), 4.35 (q, J ) 7.31 and 6.58, 2H, CH2
ester), 6.59 (s, 1H, CH pyrazole).
5-Un d ecyl-1H-p yr a zole-3-ca r boxylic Acid Eth yl Ester
(3h ). Prepared as described for 3b. Yield 97%. 1H NMR
(CDCl3) δ 0.88 (t, 3H, J ) 7.3 Hz, CH3), 1.26 (m, 18H, CH2),
1.39 (t, 3H, J ) 7.3 Hz, CH3 ester), 1.65 (m, 2H, CH2), 2.67 (t,
2H, J ) 7.3 Hz, CH2), 4.38 (q, 2H, J ) 7.3 Hz, OCH2), 6.62 (s,
1H, dCH).
5-(4-Ch lor oph en yl)-1H-pyr azole-3-car boxylic Acid Eth -
yl Ester (3k ). Prepared as described for 3b. Yield 94%. 1H
NMR: (CDCl3) δ 1.42 (t, J ) 6.94 and 7.31, 3H, CH3 ester),
4.42 (q, J ) 7.31 and 6.94, 2H, CH2 ester), 7.11 (s, 1H,
pyrazole), 7.41 (d, J ) 8.77, 2H, arom.), 7.72 (d, J ) 8.78, 2H,
arom.).
5-(4-Meth ylph en yl)-1H-pyr azole-3-car boxylic Acid Eth -
ylester (3l). Prepared as described for 3b. Yield 90%. 1H
NMR: (CDCl3) δ 1.40 (t, J ) 6.58 and 7.31, 3H, CH3 ester),
2.39 (s, 3H, CH3), 4.40 (q, J ) 7.31, 2H, CH2 ester), 7.07 (s,
1H, CH pyrazole), 7.25 (d, J ) 8.77, 2H, arom.), 7.61 (d, J )
8.04, 2H, arom.).
4,5,6,7-Tetr a h yd r o-1H-in d a zole-3-ca r boxylic Acid (4d ).
1
Prepared as described for 4b. Yield 53%; mp 247-248 °C. H
NMR (CDCl3) δ 1.89 (m, 4H, 2× CH2), 2.81 (m, 4H, 2× CH2).
13C NMR (DMSO) δ 21.2, 21.5, 22.0, and 22.5 (4× CH2), 119.4
(C4), 135.2 (C3), 144.8 (C5), 161.9 (carbonyl). Anal. (C8H10N2O2
1.9 H2O) C, H, N.
5-Isop r op yl-1H-p yr a zole-3-ca r boxylic Acid (4e).8 Pre-
pared as described for 4b. Yield 91%; mp 150-152 °C (Lit.
153-154°C). 1H NMR (CDCl3) δ 1.44 (d, J ) 7.31, 6H, 2x CH3
isopropyl), 3.28 (septet, J ) 6.58 and 7.31, 1H, CH isopropyl),
6.88 (s, 1H, CH pyrazole). 13C NMR (CDCl3) δ 21.7 (2× CH3
isopropyl), 25.9 (CH isopropyl), 106.1 (CH pyrazole), 138,9 (C3),
155.4 (C5), 160.0 (carbonyl).
5-P r op yl-1H-p yr a zole-3-ca r boxylic Acid (4f).8 Prepared
as described for 4b. Yield 42%; mp 186-188 °C (Lit. 189-190
°C). 1H NMR (MeOD) δ 0.96 (t, 3H, J ) 7.3 Hz, CH3), 1.69 (m,
2H, CH2), 2.64 (t, 2H, J ) 7.3 Hz, CH2), 6.56 (s, 1H, CH
pyrazole). 13C NMR (MeOD) δ 13.9 (CH3 propyl), 23.6 (CH2),
28.7 (CH2), 107.2 (CH pyrazole), 143.3 (C3), 148.6 (C5), 165.0
(carbonyl).
5-Ben zyl-1H -p yr a zole-3-ca r b oxylic Acid E t h yl E st er
(3m ). Prepared as described for 3b. Yield 79%. 1H NMR
(CDCl3) δ 1.36 (t, 3H, J ) 7.3 Hz, CH3), 4.05 (s, 2H, CH2),
4.36 (q, 2H, J ) 7.3, OCH2), 6.59 (s, 1H, dCH), 7.28 (m, 5H,
arom.), 11.00 (broad s, 1H, NH).
5-(3-Ch lor oben zyl)-1H-p yr a zole-3-ca r boxylic Acid Eth -
ylest er (3n ). Prepared as described for 3c. Purification by
silica gel column chromatography, eluens: ethyl acetate/
petroleum ether ) 2:3. Yield 42%. 1H NMR: (CDCl3) δ 1.33
(t, J ) 7.31 and 6.58, 3H, CH3 ester), 4.04 (s, 2H, CH2 benzylic),
4.34 (q, J ) 7.31 and 6.57, 2H, CH2 ester), 6.56 (s, 1H, CH
pyrazole), 7.11 (m, 1H, arom.), 7.20 (m, 3H, arom.).
5-(4-Ch lor oben zyl)-1H-p yr a zole-3-ca r boxylic Acid Eth -
yl Ester (3o). Prepared as described for 3c. Purification by
silica gel column chromatography, eluens: ethyl acetate/
petroleum ether ) 2:1. Yield 31%. 1H NMR: (CDCl3) δ 1.36
(t, J ) 7.31, 3H, CH3 ester), 4.02 (s, 2H, CH2 benzyl), 4.35 (q,
J ) 7.31 and 6.58, 2H, CH2 ester), 6.56 (s, 1H, pyrazole), 7.15
(d, J ) 8.78, 2H, arom.), 7.27 (d, J ) 8.77, 2H, arom.).
5-(4-Meth ylben zyl)-1H-pyr a zole-3-ca r boxylic Acid Eth -
ylest er (3p ). Prepared as described for 3c. Purification by
silica gel column chromatography, eluens: ethyl acetate/
petroleum ether ) 2:3. Yield 35%. 1H NMR: (CDCl3) δ 1.36
(t, J ) 7.31, 3H, CH3 ester), 2.33 (s, 3H, CH3 ring), 4.00 (s,
2H, CH2 benzylic), 4.35 (q, J ) 7.31, 2H, CH2 ester), 6.59 (s,
1H, CH pyrazole), 7.12 (s, 4H arom.), 10.73 (s, broad, 1H, NH).
5-(4-Met h oxyb en zyl)-1H -p yr a zole-3-ca r b oxylic Acid
Eth ylester (3q). Prepared as described for 3c. Purification
by silica gel column chromatography, eluens: ethyl acetate/
petroleum ether ) 2:1, followed by crystallization from ethyl
5-Bu tyl-1H-p yr a zole-3-ca r boxylic Acid (4g).8 Prepared
as described for 4b. Recrystallization from methanol. Yield
1
20%; mp 165-167 °C (Lit. 167-169 °C). H NMR: (MeOD) δ
0.95 (t, J ) 7.31, 3H, CH3 butyl), 1.37 (sextet, J ) 7.31, 8.04,
and 6.58, 2H, CH2), 1.64 (quintet, J ) 6.57, 8.05 and 7.31, 2H,
CH2), 2.67 (t, J ) 7.31 and 8.05, 2H, CH2), 6.56 (s, 1H, CH
pyrazole). 13C NMR: (MeOD) δ 14.1 (CH3 butyl), 23.1, 26.3,
and 32.4 (3× CH2), 107.1 (CH pyrazole), 143.5 (C3), 148.6 (C5),
165.2 (carbonyl).
5-Un d ecyl-1H-p yr a zole-3-ca r boxylic Acid (4h ).8 Pre-
pared as described for 4b. Yield 87%; mp 152-154 °C (Lit.
151-153 °C). 1H NMR (MeOD) δ 0.89 (t, 3H, J ) 7.3 Hz, CH3),
1.29 (m, 18H, CH2), 1.66 (m, 2H, CH2), 2.67 (t, 2H, J ) 7.3
Hz, CH2), 6.66 (s, 1H, CH pyrazole). 13C NMR (MeOD) δ 14.5
(CH3), 23.7, 26.7, 30.2, 30.3, 30.6, 30.7, 33.0, 107.1 (CH
pyrazole), 143.3 (C3), 148.8 (C5), 164.9 (carbonyl).
5-P h en yl-1H-p yr a zole-3-ca r boxylic Acid (4i).21 Prepared
as described for 4b. Yield 81%; mp 230-232 °C (Lit. 232-234
1
°C). H NMR: (MeOD) δ 7.19 (s, 1H, CH pyrazole), 7.39 (m,
3H, arom.), 7.82 (m, 2H, arom.).
5-(3-Ch lor op h en yl)-1H-p yr a zole-3-ca r boxylic Acid (4j).
1
Prepared as described for 4b. Yield 64%; mp 238-240 °C. H
NMR: (MeOD) δ 7.29 (s, 1H, CH pyrazole), 7.45 (m, 2H,
arom.), 7.80 (m, 1H, arom.), 7.90 (s, 1H, arom.). Anal.
(C10H7N2O2Cl 0.7 H2O) C, H, N.
1
acetate/petroleum ether. Yield 12%. H NMR: (CDCl3) δ 1.35
(t, J ) 7.30 and 6.58, 3H, CH3 ester), 3.79 (s, 3H, O-CH3),
3.98 (s, 2H, CH2 benzylic), 4.35 (q, J ) 7.31, 2H, CH2 ester),
6.57 (s, 1H, CH pyrazole), 6.84 (d, J ) 8.77, 2H arom.), 7.14
(d, J ) 8.77, 2H, arom.), 10.90 (s, broad, 1H, NH).
5-(2-P h en yl)eth yl-1H-p yr a zole-3-ca r boxylic Acid Eth yl
Ester (3r ). Prepared as described for 3b. Yield 56%. 1H NMR
(CDCl3) δ 1.37 (t, 3H, J ) 7.3 Hz, CH3), 2.99 (m, 4H, 2× CH2),
4.37 (q, 2H, J ) 7.3, OCH2), 6.60 (s, 1H, dCH), 7.19 (m, 5H,
arom.).
5-(4-Ch lor oph en yl)-1H-pyr azole-3-car boxylic Acid (4k).
1
Prepared as described for 4b. Yield 89%; mp 250-251 °C. H
NMR: (MeOD) δ 7.16 (s, 1H, CH pyrazole), 7.44 (d, J ) 8.78,
2H, arom.), 7.77 (d, J ) 8.77, 2H, arom.). 13C NMR (DMSO) δ
105.7 (C4), 127.1 (2C arom.), 129.0 (2C, arom.), 130.4 (ipso),
132.7 (ipso, C-Cl), 139.4 (C3), 147.3 (C5), 161.6 (carbonyl).
Anal. (C10H7N2O2Cl H2O) C, H, N.
5-(4-Meth ylp h en yl)-1H-p yr a zole-3-ca r boxylic Acid (4l).
1
5-(3-P h en yl)pr opyl-1H-pyr azole-3-car boxylic Acid Eth -
yl Ester (3s). Prepared as described for 3b. Yield 73%. 1H
NMR (CDCl3) δ 1.41 (t, 3H, J ) 7.3 Hz, CH3), 1.99 (m, 2H,
CH2), 2.70 (m, 4H, 2× CH2), 4.37 (q, 2H, J ) 7.3, OCH2), 6.63
(s, 1H, dCH), 7.21 (m, 5H, arom.).
1,2-Dia za -7-oxa -bicyclo[3,3,04,8]octa -3,8-d ien e-3-ca r box-
ylic Acid (4b). 1 g (5.49 mmol) of 3b was dissolved in 22 mL
of 1,4-dioxane. 22 mL of 0.5 M NaOH was added, and the
Prepared as described for 4b. Yield 34%; mp 246-248 °C. H
NMR: (CDCl3) δ 2.37 (s, 3H, CH3), 7.06 (s, 1H, CH pyrazole),
7.26 (d, J ) 8.05, 2H, arom.), 7.63 (d, J ) 8.04, 2H, arom.).
13C NMR: (MeOD) δ 21.3 (CH3), 105.9 (C4), 126.6 (2C, arom.),
128.6 (ipso), 130.6 (2C, arom.), 139.8 (ipso, C-CH3), 142.8 (C3),
149.0 (C5), 164.3 (carbonyl). Anal. (C11H10N2O2 0.9H2O) C, H, N.
5-Ben zyl-1H-p yr a zole-3-ca r boxylic Acid (4m ). Prepared
as described for 4b. Yield 33%; mp 216-217 °C. 1H NMR

Pyrazole Derivatives as Partial Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3951
(MeOD) δ 4.02 (s, 2H, CH2), 6.55 (s, 1H, CH pyrazole), 7.23
(m, 5H, arom.). 13C NMR (MeOD) δ 33.1 (CH2), 108.2 (C4),
127.7, 129.6, 129.7, 139.7, 142.6 (C3), 148.6 (C5), 164.2
(carbonyl). Anal. (C11H10N2O2 0.95 HCl) C, H, N.
and EC50 values for stimulation of [35S]GTPγS binding were
calculated as described previously.2 EC50, Ki and KB values are
given as geometric means with 95% confidence limits from at
least 3 experiments. Relative intrinsic activities (RIAs) are
given as arithmetic means ( SEM with reference to the
maximum stimulation induced by nicotinic acid as a full
agonist with its intrinsic activity assumed as 100%.
5-(4-Ch lor oben zyl)-1H-pyr azole-3-car boxylic Acid (4n ).
1
Prepared as described for 4b. Yield 81%; mp 224-225 °C. H
NMR: (MeOD) δ 4.01 (s, 2H, CH2 benzylic), 6.56 (s, 1H, CH
pyrazole), 7.22 (d, J ) 8.78, 2H, arom.), 7.31 (d, J ) 8.04, 2H,
arom.).13C NMR (DMSO) δ 31.7 (CH2 benzylic), 107.3 (CH
pyrazole), 128.7 (2C, arom.), 130.7 (2C, arom.), 131.4 and 138.4
(2× ipso), 140.9 (C3), 146.8 (C5), 162.8 (carbonyl). Anal.
(C11H9N2O2Cl) C, H, N.
Refer en ces
(1) Knopp, R. H. Drug treatment of lipid disorders. N. Engl. J . Med.
1999, 341, 498-511.
(2) Lorenzen, A.; Stannek, C.; Lang, H.; Andrianov, V.; Kalvinsh,
I.; Schwabe, U. Characterization of a G protein-coupled receptor
for nicotinic acid. Mol. Pharmacol. 2001, 59, 349-357.
(3) Lorenzen, A.; Stannek, C.; Burmeister, A.; Kalvinsh, I.; Schwabe,
5-(4-Meth ylben zyl)-1H-pyr azole-3-car boxylic Acid (4o).
1
Prepared as described for 4b. Yield 67%; mp 227-228 °C. H
NMR: (MeOD) δ 2.29 (s, 3H, CH3 ring), 3.93 (s, 2H, CH2
benzylic), 6.50 (s, 1H, CH pyrazole), 7.10 (s, 4H, arom.). 13C
NMR: (DMSO) δ 20.6 (CH3 ring), 31.6 (CH2 benzylic), 106.8
(CH pyrazole), 128.4 (2C, arom.), 129.1 (2C, arom.), 135.3
(ipso), 136.1 (ipso, C-CH3), 140.6 (C3), 146.9 (C5), 162.5
(carbonyl). Anal. (C12H12N2O2) C, H, N.
U.
G protein-coupled receptor for nicotinic acid in mouse
macrophages. Biochem. Pharmacol. 2002, 64, 645-648.
(4) Wise, A.; Foord, S. M.; Fraser, N. J .; Barnes, A. A.; Eslhourbagy,
N.; Eilert, M.; Ignar, D. M.; Murdock, P. R.; Steplewski, K.;
Green, A.; Bronw, A. J .; Dowell, S. J .; Szekeres, P. G.; Hassall,
D. G.; Marshall, F. H.; Wilson, S.; Pike, N. B. Molecular
identification of high and low affinity receptors for nicotinic acid.
J . Biol. Chem. 2003, 278, 9869-9874; Tunaru, S.; Kero, K.;
Schaub, A.; Wufka, C.; Blaukat, A.; Pfeffer, K.; Offermanns, S.
PUMA-G and HM74 are receptors for nicotinic acid and mediate
its anti-lipolytic effect. Nat. Med. 2003, 9, 352-355; Soga, T.;
Kamohara, M.; Takasaki, J .; Matsumoto, S.-I.; Saito, T.; Ohishi,
T.; Hiyama, H.; Matsuo, A.; Matsushime, H.; Furuichi, K.
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J .; Mathot, R. A.; IJ zerman, A. P. N,6C8-Disubstituted adenosine
derivatives as partial agonists for adenosine A1 receptors, J .
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A. P.; Danhof, M. Selectivity of action of 8-alkylamino analogues
of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-
lipolytic responses in rats, Br. J . Pharmacol. 1998, 124, 607-
618.
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lipidemic agents. II. Synthesis and pharmacological properties
of alkylpyrazole derivatives. Chem. Pharm. Bull. 1984, 32,
1568-1577.
(8) Royals, E. E. The use of sodium methoxide in the Claisen
reaction. J . Am. Chem. Soc. 1945, 7, 1508-1509.
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lic acids. Acta Chem. Scan. 1952, 6, 782-790.
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effects in elimination reactions. VI. Bimolecular eliminations
from 1-aryl-2-propyl and 2-aryl-1-propyl tosylates and bromides.
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(11) Moloney, G. P.; Martin, G. R.; Mathews, N.; Milne, A.; Hobbs,
H.; Dodsworth, S.; Sang, P. Y.; Knight, C.; Williams, M.;
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substituted benzyl carboxyamido-5-(2-ethyl-1-dioxoimidacolidi-
nyl)-N,N-dimethyltryptamine derivatives. Novel antagonists for
the vascular 5HT1B-like receptor. J . Med. Chem. 1999, 42, 2504-
2526.
5-(4-Met h oxyb en zyl)-1H -p yr a zole-3-ca r b oxylic Acid
(4p ). Prepared as described for 4b. Yield 64%; mp 229-230
°C. 1H NMR: (MeOD) δ 3.76 (s, 3H, O-CH3), 3.94 (s, 2H, CH2
benzylic), 6.50 (s, 1H, CH pyrazole), 6.85 (d, J ) 8.77, 2H,
arom.), 7.14 (d, J ) 8.78, 2H, arom.). 13C NMR: (DMSO) δ
31.2 (CH2 benzylic), 55.1 (OCH3), 106.8 (CH pyrazole), 114.0
(2C, arom.), 129.6 (2C, arom.), 131.1 (ipso), 140.8 (C3), 147.2
(C5), 157.9 (ipso, C-OCH3), 162.6 (carbonyl). Anal. (C12H12N2O3)
C, H, N.
5-(3-Ch lor oben zyl)-1H-p yr a zole-3-ca r boxylic Acid (4q).
1
Prepared as described for 4b. Yield 86%; mp 221-222 °C. H
NMR: (MeOD) δ 4.02 (s, 2H, CH2 benzylic), 6.57 (s, 1H, CH
pyrazole), 7.24 (m, 4H, arom.). 13C NMR: (DMSO) δ 32.9 (CH2
benzylic), 108.3 (CH pyrazole), 127.7, 128.0, 129.6, and 131.1
(4× arom.), 135.4 (ipso, C-Cl), 141.2 (ipso), 142.3 (C3), 148.1
(C5), 164.3 (carbonyl). Anal. (C11H9N2O2Cl) C, H, N.
5-(2-P h en yl)eth yl-1H-p yr a zole-3-ca r boxylic Acid (4r ).
1
Prepared as described for 4b. Yield 33%; mp 210-212 °C. H
NMR (MeOD) δ 2.96 (s, 4H, 2 x CH2), 6.53 (s, 1H, CH pyrazole),
7.21 (m, 5H, arom.). 13C NMR (MeOD) δ 28.9 (CH2), 36.5 (CH2),
107.5 (CH pyrazole), 127.2, 129.4, 142.1, 142.9 (C3), 148.3 (C5),
164.8 (carbonyl). Anal. (C12H12N2O2) C, N; H: calcd, 5.59;
found, 6.04.
5-(3-P h en yl)p r op yl-1H-p yr a zole-3-ca r boxylic Acid (4s).
1
Prepared as described for 4b. Yield 30%; mp 165-166 °C. H
NMR (MeOD) δ 1.97 (m, 2H, CH2), 2.67 (m, 4H, 2× CH2), 6.59
(s, 1H, dCH), 7.17 (m, 5H, arom.). 13C NMR (MeOD) δ 26.3
(CH2), 32.1 (CH2), 36.1 (CH2), 107.3 (CH pyrazole), 126.9,
129.4, 142.9, 143.2, 148.7, 164.9 (carbonyl). Anal. (C13H14N2O2)
C, H, N.
Biologica l Assa ys. [3H]Nicotin ic Acid Bin d in g. Equi-
librium binding of [3H]nicotinic acid to rat spleen membranes
was performed with 75 µg of membrane protein per tube in a
total volume of 250 µL in 50 mM Tris-HCl, pH 7.4, containing
1 mM MgCl2, 200 U/mL penicillin G, 200 µg/mL streptomycin,
and 0.02% CHAPS. Binding experiments were conducted in
the presence of 20 nM radioligand for 3.5 h at 25 °C.
Nonspecific binding was determined by addition of 100 µM
acipimox. Separation of membrane-bound from free radio-
ligand was done by filtration of the samples through nitro-
cellulose filters washing twice with 4 mL of 50 mM Tris-HCl
(pH 7.4) containing 0.02% CHAPS.
(12) Emerson, D. W.; Titus, R. L.; J ones, M. D. Ring opening reactions
of 6-oxo-substituted spiro-pyrrolidinediones: synthesis of 4-sub-
stituted-1,5-dihydro-3-hydroxy-2-oxo-1,5-diphenyl-2H-pyr-
roles. J . Heterocycl. Chem. 1998, 35, 611-617.
(13) ,2-Azoles: pyrazoles, isothiazoles, isoxazoles: reactions and
synthesis. In Heterocyclic Chemistry, 4th ed.; J oule, J . A., Mills,
K., Eds. Blackwell Science, Oxford, 2000; pp 432-448.
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synthesis in drug design. J . Med. Chem. 1972, 15, 1006-1011.
(15) UK patent 1,048,104 (Upjohn Company). Pharmaceutical com-
positions containing 3,5-disubstituted pyrazoles, 1964/1966
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3-carboxylic acid on carbohydrate and free fatty acid metabolism.
J . Pharmacol. Exp. Ther. 1965, 50, 491-498.
[
35S]GTP γS Bin d in g. G protein activation in rat membrane
(17) Pereira, J . N.; Holland, G. F. The development of resistance to
a potent lipolysis inhibitor, 3-methylisoxazole-5-carboxylic acid.
J . Pharmacol. Exp. Ther. 1967, 57, 381-387.
preparations was assessed as stimulation of [35S]GTPγS bind-
ing as described previously.2 Briefly, samples (100 µL) con-
tained 50 000 cpm (0.2 nM) [35S]GTPγS, 50 mM Tris-HCl, pH
7.4, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 100 mM
NaCl, 10 µM GDP, 0.5 U/mL adenosine deaminase, 0.005%
CHAPS, and 0.5% bovine serum albumin. Incubations with 1
to 1.5 µg of membrane protein were done for 90 min at 25 °C
and were terminated by filtration over GF/B glass fiber filters
(Whatman) followed by two 4-mL washes with ice-cold buffer
(50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.02% CHAPS).
Da ta An a lysis. Ki values from [3H]nicotinic acid binding
(18) Holland, G. F.; Pereira, J . N. Heterocyclic tetrazoles, a new class
of lipolysis inhibitors. J . Med. Chem. 1967, 10, 149-154.
(19) Auwers, K.; Noll, W. U¨ ber Trimethylen-pyrazole und -oxy-
pyridazone. J ustus Liebigs Ann. Chem. 1938, 636, 97-116.
(20) Lapworth, A.; Hann, A. C. O. Derivatives of normal and iso-
butyrylpyruvic acids. J . Chem. Soc. 1902, 81, 1485-1491.
(21) Lempert-Stre´ter, M.; Lempert, K. Alkaline cleavage of some (1-
phenylsulfonyl-2-pyrazolin-5-yl)-methyl ketones and related com-
pounds. Tetrahedron 1975, 31, 1677-1682.
J M030888C