Design, synthesis, and pharmacological activity of nonallergenic pyrazolone-type antipyretic analgesics

Article abstract of DOI:10.1021/jm101208x

To develop novel nonallergenic pyrazolone analgesics, we synthesized a series of compounds in which position 1 of the pyrazolone ring was substituted in place of the original methyl group in order to block the formation of allergenic metabolites via N-dea

Full text of DOI:10.1021/jm101208x

J. Med. Chem. 2010, 53, 8727–8733 8727
DOI: 10.1021/jm101208x
Design, Synthesis, and Pharmacological Activity of Nonallergenic Pyrazolone-Type
Antipyretic Analgesics
Naoto Uramaru,†,‡ Hidenari Shigematsu,‡ Akihisa Toda,§ Reiko Eyanagi,§ Shigeyuki Kitamura,‡ and Shigeru Ohta*,†
†Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan,
‡Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan, and
§Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
Received September 18, 2010
To develop novel nonallergenic pyrazolone analgesics, we synthesized a series of compounds in which
position 1 of the pyrazolone ring was substituted in place of the original methyl group in order to block
the formation of allergenic metabolites via N-dealkylation. These pyrazolone analogues were found to
show as potent an antipyretic and analgesic effect as antipyrine (AT). In an examination of allergenicity,
AT induced a typical skin reaction in guinea pigs, whereas the pyrazolone analogues were inactive.
When AT was administered (po) to rats, norantipyrine (NORA) as an active metabolite was detected in
the urine, whereas similar administration of the pyrazolone analogues did not afford NORA. We con-
clude that these novel pyrazolone analogues were nonallergenic because they were not converted to
allergenic metabolites in vivo. Because these compounds retain the antipyretic and analgesic activities of
AT, they are considered to be promising candidates for nonallergenic antipyretic analgesics.
Introduction
The pyrazolones are the oldest synthetic pharmaceuticals,
and antipyrine (AT)a, which has a pyrazolone skeleton, was
first prepared by Knorr as an alternative to quinine in 1883.1
Their derivatives, such as AT, aminopyrine, isopropylantipy
rine, and sulpyrine (Figure 1), are widely used as nonsteroidal
anti-inflammatory drugs that exhibit potent antipyretic and
analgesic activities.2-4 Unfortunately, however, the pyrazolones
areallergenic, and various sideeffects, includinganaphylaxis,5
cutaneous reactions,5,6 and agranulocytosis,5,7 have been re-
Figure 1. Basic structures of pyrazolone derivatives.
ported. Consequently, the clinical use of pyrazolones is re-
that blocking of N-demethylation at position 1 of the pyrazo-
stricted in several countries.
lone ring would be an effective approach to prevent allergenicity.
The metabolic pathways of pyrazolones have been well
Therefore, we designed and synthesized several pyrazolone
clarified by many researchers (Scheme 1),8-12 and it is con-
compounds with substituents that were expected to block
sidered that reactive intermediates generated by metabolic
N-elimination reaction. The antipyretic and analgesic activ-
activation are converted to dimer, trimer, and conjugates.13,14
ities of these pyrazolone analogues were examined in animal
For example, AT was converted to conjugates, dimer, and
models of fever and inflammation, and skin allergenicity was
trimer derived via norantipyrine (NORA).14 NORA itself is
examined in guinea pigs. Furthermore, metabolism of these
an unstable and active metabolite. Also, we reported that
compounds by rats was examined in vivo and in vitro.
NORA has antigenicity in guinea pig skin reaction.14 These
results strongly suggested that pyrazolones become antigenic
after binding to biomacromolecules following metabolic acti-
Results
Chemistry. Pyrazolone derivatives 1, 2, and 3 were synthe-
sized from hydrazine derivatives and diketene in chloroform
with triethylamine (Scheme 2) based on the method of Kato
et al..15 Compound 1 or 2 was synthesized from 1-phenyl-2-
(2,2,2-trifloroethyl) hydrazine or 1,2-diphenyl hydrazine,
respectively, in satisfactory yield. Compound 3 was synthe-
sized from 1-(1,1-dimethylethyl)-2-phenyl hydrazine and
diketene; two pyrazolone derivatives of the same molecular
weight were obtained, and their structures were elucidated as
1-(1,1-dimethylethyl)-5-methyl-2-phenyl-1,2-dihydro-3H-pyr-
azol-3-one (3a) and 2-(1,1-dimethylethyl)-5-methyl-1-phenyl-
1,2-dihydro-3H-pyrazol-3-one (3b) using a combination of
vation. We hypothesized that N-demethylation is the key
reaction in the pathway leading to allergenic products and
*To whom correspondence should be addressed. Phone: þ81-82-257-
5325. Fax: þ81-82-257-5329. E-mail: sohta@hiroshima-u.ac.jp.
a Abbreviations: AT, antipyrine; NORA, norantipyrine; NOESY,
nuclear Overhauser effect spectroscopy; IL-1β, interleukin-1β; MF,
mefenamic acid; ipl, intraplantar; IM, indometacin; id, intradermal;
NADPH, β-nicotinamide-adenine dinucleotide phosphate reduced
form; HPLC, high-performance liquid chromatography; HMA, hydro-
xymethylantipyrine; OHA, hydroxyantipyrine; TLC, thin layer chro-
matography; IR, infrared spectrometry; 1H NMR, 1H nuclear magnetic
resonance; TMS, tetramethylsilane; HRMS, high-resolution mass spec-
tra; icv, intracerebroventricular.
r
2010 American Chemical Society
Published on Web 12/01/2010
pubs.acs.org/jmc

8728 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Scheme 1. Metabolic Pathways of AT
Uramaru et al.
Scheme 2. Synthesis of Pyrazolone Derivatives 1, 2, and 3a
Analgesic Activity of Antipyrine and the Analogues on In-
flamedTissue. Hyperalgesia was induced by intraplantar (ipl)
injection of 0.1 mL of carrageenin (10 mg/mL) into the right
hindpaw. The nociceptive threshold of the carrageenin-treated
hindpaw decreased to about 30% of the basal value at 1 h,
and this was maintained for at least 3 h, followed by a slow
recovery. The carrageenin-induced nociception in the hyper-
algesic rat was completely blocked by indometacin (IM)
(2 mg/kg) and AT (100 mg/kg). Compounds 1, 2, and 3a
(100 mg/kg) also significantly suppressed the nociceptive
threshold from 1 h after administration, being as potent as
IM and AT (Figure 3).
Guinea Pig Skin Reaction. The antigenicity of AT and com-
pounds 1, 2, and 3a was examined using the guinea pig skin
reaction test to evaluate the delayed skin reaction on day 21
after sensitization with NORA. AT (1 mg/body) elicited a sig-
nificant skin reaction at 24 h after intradermal (id) injection,
whereas compounds 1, 2, and 3a (1 mg/body) did not. Figure 4
shows photographs of the excised abdominal skin.
a Reagents and conditions: (a) diketene, triethylamine, CHCl3, reflux.
2D NMR techniques and nuclear Overhauser effect spectros-
copy (NOESY).
N-Demethylation of Antipyrine and the Analogues by Rat
Microsomes. AT was incubated with liver microsomes of
untreated rats in the presence of β-nicotinamide-adenine
dinucleotide phosphate reduced form (NADPH) to obtain
metabolites as described in the Experimental Section. Three
peaks, which were not detected in the control, were found
on the high-performance liquid chromatography (HPLC)
of the extract of incubation mixtures. The retention times
of 3.0, 5.4, and 9.4 min corresponded to those of hydro-
xymethylantipyrine (HMA), NORA, and hydroxyanti-
pyrine (OHA), respectively (Figure 5a). The amounts of
N-demethylation products increased linearly for 20 min (data
not shown). When AT was incubated, HMA and OHA were
produced at similar levels of about 40 nmol/20 min/mg
Effect on Body Temperature of Rats with Interleukin-1β
(IL-1β)-Induced Fever. The mean temperatures of the experi-
mental animals, measured rectally, were 38.13 ( 0.06 °C
at 30 min before treatment. The body temperatures of the
IL-1β-treated rats significantly increased (þ1.75 ( 0.18 °C)
within 2 h after injection of IL-1β. Administration of mefe-
namic acid (MF) (100 mg/kg) or AT (100 mg/kg) signifi-
cantly decreased the IL-1β-induced fever, restoring the body
temperature to the level prior to IL-1β administration from
2 h. Compounds 1, 2, and 3a showed dose-dependent anti-
pyretic effects, and at the dose of 50 or 100 mg/kg, they were
as potent as MF (100 mg/kg) or AT (100 mg/kg) after 2 h
(Figure 2).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8729
Figure 2. Changes in the rectal temperature of rats. Each pain threshold value represents the mean ( SEM of four rats. AT (100 mg/kg), MF
(100 mg/kg), and compounds 1, 2, and 3a (10-100 mg) were administered orally, immediately after the icv injection of IL-1β (200 units). Rectal
temperature was measured in the large intestine 7 cm above the anus at 2 h intervals. ΔTrec was calculated as described in the Experimental
Section. *p < 0.05, **p < 0.01 compared with control.
Figure 3. Analgesic effects in rats, evaluated by means of Randall-Selitto’s method. Each pain threshold value represents the mean ( SEM of
four rats. AT (100 mg/kg), IM (2 mg/kg), and compounds 1, 2, and 3a (10-100 mg/kg) were administered orally, immediately after ipl injection
of 0.1 mL of carrageenin (10 mg/mL) into the right hindpaw. Analgesic effect was calculated as described in the Experimental Section. *p <
0.05, **p < 0.01 compared with control.
protein and NORA was formed at a level of 27 nmol/
20 min/mg protein (Figure 5b). When compounds 1, 2, and
3a were incubated under the same conditions, the amount
of NORA formed was below the HPLC detection limit
(0.1 nmol/mL in this experiment equivalent to 0.1 nmol/
20 min/mg protein) (Figure 5c).
Metabolism of Antipyrine and the Analogues in Rats in
Vivo. When AT was administered to rats, four peaks, which
were not detected in the control, were detected in an HPLC
chromatogram of the extract of the urine. The retention times
of 3.0, 5.4, 6.0, and 9.4 min corresponded to these of HMA,
NORA, AT, and OHA, respectively (Figure 6a). NORA was
detected in AT (100 mg/kg) treated rats, at a level corre-
sponding to approximately 2% of the dose of AT, together
with AT, HMA, and OHA (Figure 6b). On the other hand,
NORA was not detected in the urine of rats administered
compounds 1, 2, and 3a (100 mg/kg), i.e., it was below the
HPLC detection limit (0.2 nmol/mL in this experiment)
(Figure 6c).
equivalent analgesic effect to AT in a rat model of carrageen-
in-induced paw inflammation after single oral administration
at the same dose. Therefore, the antipyretic and analgesic ef-
fects were retained irrespective of the substituent at position 1.
On the other hand, compounds 1, 2, and 3a were not metab-
olized to NORA in vivo or in vitro, as expected, and did not
elicit an allergic reaction in guinea pigs, unlike AT.
Drug allergy is rare, but seriously and potentially life-
threatening. There are many reports that T-lymphocytes react
specifically tolow-molecular-weight compounds, even though
peptide structures are required for antigen presentation.16,17
The reason for such hypersensitivity is that some low-molec-
ular-weight drugs are metabolically activated and form com-
plexes with proteins in vivo.18,19 Himly et al. reported that
biomacromolecules bearing norantipyrine as an antigen were
formed following isopropylantipyrine administration, and this
supports the idea that the allergenicity of antipyrine involves a
similar mechanism.20
We found that the hydroxylation reaction at position 4
of the pyrazolone ring is the main metabolic pathway of AT
in rats, together with oxidation at position 5 and demethyla-
tion at position 1, both in vitro and in vivo, in agreement
with the findings of Shimeno et al.21 It is thought that the
N-demethylation is due to deprotonation of the R-carbon, lead-
ing to the formation of NORA by N-elimination reaction.
Discussion and Conclusion
The newly synthesized pyrazolone analogues 1, 2, and 3a
(100 mg/kg) showed antipyretic activity equivalent to that of
AT (100 mg/kg) in a rat fever model. Further, they showed

8730 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Uramaru et al.
Figure 4. Elicitation of skin reaction in guinea pigs sensitized with AT. Sensitization and elicitation were performed as described in the
Experimental Section. The photograph shows erythema at 24 h after id injection of 0.1 mL of corn oil, or a corn oil solution of AT (1 mg/body)
or compounds 1, 2, and 3a (1 mg/body).
However NORA was not detected as a metabolite of the newly
synthesized pyrazolone analogues, suggesting that these com-
pounds are metabolically stable in this respect, as anticipated.
As NORA is considered to mediate the allergenicity of AT,
compounds 1, 2, and 3a should not show NORA-mediated
allergenicity, and indeed, they did not elicit any allergic reac-
tion in the guinea pig skin test. Thus, we have obtained pyr-
azolone derivatives which retain the antipyretic and analgesic
effects of AT but lack the allergenicity of AT, as summarized
in Figure 7.
In conclusion, our results confirm the validity of our strat-
egy of blocking metabolic activation of pyrazolones by means
of structural modification to obtain compounds that can not
be metabolically activated to bind to biomacromolecules and
so do not show allergenicity but retain potent antipyretic and
analgesic activities. These new pyrazolones are considered to
be candidate nonallergenic analgesic agents.
a PerkinElmer 2400II CHNS/O recorder, and results were
within (0.4% of the theoretical values. The analytical HPLC
system consisted of SPD-20A Shimadzu chromatograph and
equipped with a CAPCELLPAK C18 UG120 column (Shiseido
Co., Ltd., Japan), 5 μm (4.6 mm ꢀ 250 mm). Elution was per-
formed with 0.1 M sodium acetate aqueous solution, adjusted
with acetic acid to pH 6.6, and acetonitrile (85:15, v/v). The flow
rate was 2 mL/min, and the column temperature was set to
30 °C.22 Column effluents were monitored at a wavelength of
254 nm. The determination of the substrate and metabolites,
AT, HMA, NORA, and OHA were used authentic samples. The
limits of detection (S/N value of 3) of NORA in the in vivo and in
vitro experiments were 0.2 nmol/mL and 0.1 nmol/mL, respec-
tively.
General Procedure for Synthesis of Pyrazolone Derivatives.15
A mixture of hydrazine derivative (0.01 mol) and diketene
(0.01 mol) in chloroform (30 mL) was stirred at room tempera-
ture. Triethylamine (0.01 mol) was added, and the reaction
mixture was refluxed for 3 h. After evaporation of the solvent,
the residue was dissolved in ethyl acetate (100 mL), and the
resulting solution was extracted with 10% hydrochloric acid.
The extract was basified with 5 M sodium hydroxide in an ice
bath and extracted with ethyl acetate. The organic layer was
washed with H2O and brine, dried over MgSO4, and evaporated
under reduced pressure. The residue was purified by column
chromatography on silica gel (n-hexane/ethyl acetate) or recrys-
tallized from n-hexane/ethyl acetate to give the corresponding
pyrazolone derivative.
5-Methyl-2-phenyl-1-(2,2,2-trifluoroethyl)-1,2-dihydro-3H-
pyrazol-3-one (1). According to the general procedure, com-
pound 1 was obtained in 68% yield as white needles after recrys-
tallization from n-hexane/ethyl acetate. mp 91-92 °C. IR (KBr)
cm-1: 1676 (CO). 1H NMR (500 MHz, CDCl3-d) δ: 7.48 (t, 2H,
J=10.0 Hz, ArH), 7.36-7.32 (m, 3H, ArH), 5.53 (s, CH), 4.09
(q, J = 5.0 Hz, CF3CH2), 2.31(s, CH3). HRMS (EI) m/z calcd
for C12H11F3N2O 256.0823; found 256.0814 [Mþ]. Anal. Calcd
for C12H11F3N2O: C, 56.25; H, 4.33; N, 10.93. Found: C, 56.20;
H, 3.99; N, 10.92.
Experimental Section
General Methods. All commercial chemicals and solvents are
reagent grade and were used without further purification. The
residue was purified by silica gel column (63-200 μm) chroma-
tography or recrystallized from an appropriate solvent. The prog-
ress of reactions was monitored by thin layer chromatography
(TLC) using commercially prepared silica gel 60 F254 glass-backed
plates. Compounds were visualized under ultraviolet light. Male
Sprague-Dawley rats (weighing 210-240 g) and male Hertly
guinea pigs (weighing 350-400 g) were purchased from Japan
SLC Inc.. Urine was stored at -80 °C until analyzed by HPLC.
Melting points were determined with a Yanagimoto hot-stage
melting point apparatus and are uncorrected. Infrared spectrom-
etry (IR) spectra were recorded on a Shimadzu FTIR-8400S
(KBr). The 1H nuclear magnetic resonance (1H NMR) spectra
and NOESY data were obtained on a JEOL JMN-ECA500 spec-
trometer. Proton chemical shifts were referenced to the tetra-
methylsilane (TMS) internal standard. J values are given in Hz.
High-resolution mass spectra (HRMS) were obtained with a
JEOL JMS-AX 505 W double-focusing spectrometer interfaced
to DA win data system. Elemental analysis was carried out with
5-Methyl-1,2-diphenyl-1,2-dihydro-3H-pyrazol-3-one (2).
According to the general procedure, compound 2 was obtained in
80% yield as white needles after recrystallization from n-hexane/

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8731
Figure 5. In vitro metabolism of AT by rat liver microsomes.
(a) HPLC chromatogram of HMA, NORA, AT, and OHA after
incubation of AT with rat microsomes; for details, see the Experi-
mental Section. (b) Quantitative analysis of metabolites. Each bar
represents the mean ( SEM of four rats. (c) Comparison of for-
mation of NORA from AT and compounds 1, 2, and 3a. ND: not
detected.
Figure 6. In vivo metabolism of AT by rats. (a) HPLC chromato-
gram of extract of rat urine after administration of AT; for details,
see the Experimental Section. (b) Quantitative analysis of metabo-
lites. Each bar represents the mean ( SEM of four rats. (c) Com-
parison of formation of NORA from AT and compounds 1, 2, and
3a. ND: not detected.
ethyl acetate. mp 126-127 °C. IR (KBr) cm-1: 1664 (CO).
1H NMR (500 MHz, CDCl3-d) δ: 7.37-7.09 (m, 10H, ArH), 5.56
(s, CH), 2.08 (s, CH3). HRMS (EI) m/z calcd for C16H14N2O
column chromatography (eluent: n-hexane/ethyl acetate 6/1);
1
mp 127-128 °C. IR (KBr) cm-1: 1663 (CO). H NMR (500
MHz, CDCl3-d) δ: 7.38 (t, 2H, ArH, J = 10.0 Hz), 7.29 (t, 1H,
ArH, J = 5.0 Hz), 7.21 (d, 2H, ArH, J = 5.0 Hz), 5.41 (s, CH),
1.83 (s, CH3), 1.40 (s, C(CH3)3). HRMS (EI) m/z calcd for
C14H18N2O 230.1419; found 230.1416 [Mþ]. Anal. Calcd for
250.1106; found 250.1099 [Mþ]. Anal. Calcd for C16H14N2O
3
1/2H2O: C, 74.11; H, 5.83; N, 10.80. Found: C, 74.13; H, 5.76; N,
10.79.
C14H18N2O 1/4H2O: C, 71.61; H, 7.94; N, 11.93. Found: C,
1-(1,1-Dimethylethyl)-5-methyl-2-phenyl-1,2-dihydro-3H-
pyrazol-3-one (3a). According to the general procedure, com-
pound 3a was obtained in 36% yield as white needles after col-
umn chromatography (eluent: n-hexane/ethyl acetate 6/1); mp
129-130 °C. IR (KBr) cm-1: 1678 (CO). 1H NMR (500 MHz,
CDCl3-d) δ: 7.38-7.34 (m, 4H, ArH), 7.20 (t, 1H, J = 5.0 Hz,
ArH), 5.41 (s, CH), 2.40(s, CH3), 1.24 (s, C(CH3)3). HRMS
(EI) m/z calcd for C14H18N2O 230.1419; found 230.1418 [Mþ].
3
71.92; H, 8.06; N, 11.68. Rf (n-hexane/ethyl acetate 1/1) = 0.29.
Cannula Implantation.23 The animals were anesthetized with
diethylether and positioned in a stereotaxic apparatus (SR-5R,
Narishige Scientific Instrument Lab., Japan). The overlying
skin and connective tissue were cleared from the skull. A hole
was drilled in the skull and a 22-G stainless steel guide cannula
was inserted unilaterally into the lateral ventricle. The stereo-
taxic coordinates used for cannula placement were 0.8 mm pos-
terior to bregma, 1.5 mm lateral from midline, and 4.2 mm below
the surface of the skull. The cannula was secured using dental
cement (Quick resin, Shofu Inc., Japan) and anchored to the
skull with two stainless steel screws. A wire dummy cannula was
used to seal the guide cannula. The animals were provided with
Anal. Calcd for C14H18N2O 1/4H2O: C, 71.61; H, 7.94; N,
3
11.93. Found: C, 71.60; H, 8.14; N, 11.55. Rf (n-hexane/ethyl
acetate 1/1) = 0.17.
2-(1,1-Dimethylethyl)-5-methyl-1-phenyl-1,2-dihydro-3H-
pyrazol-3-one (3b). According to the general procedure, com-
pound 3b was obtained in 14% yield as yellow needles after

8732 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Uramaru et al.
Figure 7. Summary of the effect of various substituents at position 1 of pyrazolone analgesics on the allergenicity of the compounds.
food and water ad libitum and were allowed to recover in in-
dividual cages for 7 days. Proper positioning of the cannula was
verified at the end of experiment by intracerebroventricular (icv)
injection of methylene blue.
Skin Reaction in Guinea Pigs.26 Sensitization: a corn oil solu-
tion of NORA (10 mg/mL) was administered subcutaneously to
the back of three guinea pigs at a volume of 0.1 mL of 10 mg/mL
solution per site (total volume of 1 mL). Elicitation: 21 days after
sensitization, a corn oil solution of AT (1 mg/body) or com-
pound 1, 2, or 3a (1 mg/body), or corn oil alone as a control, at a
volume of 0.1 mL was intradermally injected into the side of the
abdomen of guinea pigs shaved with an electric clipper and hair
remover. Erythema at the injected site was observed at 24 h after
id injection.
Induction of Fever and Drug Administration.23 Fever was
induced by repeated injection of IL-1β (200 units/rat) dissolved
in sterile pyrogen-free 10 mM sodium phosphate buffer contain-
ing 0.15 M sodium chloride (PBS, pH 7.5) into the lateral ven-
tricle of the brain. The degree of fever was assessed by measuring
rectal temperature with a thermoelectric monitor (CTM-303,
TERUMO, Japan) via a thermal probe inserted to a depth of
7 cm. To adapt animals to the technique and to minimize stress-
induced hyperthermia, the rectal temperature was measured
twice a day for 3 days before the IL-1β injection. Changes in
the rectal temperature (ΔTrec) were calculated from the follow-
ing formula: ΔTrec = T - T0, where T0 and T represent the rectal
temperatures before and after IL-1β injection, respectively. All
experiments were started at 8:00 to minimize the influence of
possible circadian variations.
Preparation of Microsomal Fraction from Rat Liver.27 Livers
were excised and homogenized in four volumes of 1.15% KCl
with a Potter-Elvehjem homogenizer. The homogenate was
centrifuged for 20 min at 9000g, and for 60 min at 105000g
successively to prepare the microsomal and cytosolic fractions.
The microsomal fraction was washed by resuspension in the KCl
solution and resedimented. The microsomal pellet was resus-
pended in the solution to make 1 mL equivalent to 1 g of liver.
Protein content was determined by the method of Lowry et al.
with bovine serum albumin as a standard protein.28
Nociceptive Assay.24,25 The paw pressure test described by
Randall and Selitto was applied to rats.24 Carrageenin is a nat-
ural polysaccharide (carbohydrate) extracted from red seaweed.
It is commonly used to induce acute inflammation in experi-
mental animal models.25 An analgesy meter (37215, Ugo Basile
Srl, Italy) with a pencil-shaped wooden paw-presser with a dull
tip was used; pressure was gradually applied to the hind paw at
an increasing linear rate of 16 g/s. The pressure required to elicit
nociceptive responses, such as squeaking and struggling, was
determined as the mechanical nociceptive threshold. A cutoff
value of 250 g was used to prevent damage to the paw. The
nociceptive threshold of each rat was measured 6-8 times, and
only rats with stable thresholds were used in experiments. The
control threshold for each rat was defined as the mean of the
values of the last four stable thresholds because the initial 2-4
values were, in general, high and unstable. Results are expressed
as a percentage of the control threshold.
Assay of Liver Microsomal Oxidase Activity.29 The incuba-
tion mixture consisted of 2 μmol of AT or an analogue, 10 μmol
of NADPH, 4 mg of Na2S2O5, and 0.2 mL of liver microsomes
equivalent to 200 mg of liver wet weight (2-2.5 mg protein) in a
final volume of 2 mL of 0.1 M K/Na phosphate buffer (pH 7.4).
Incubation was performed at 37 °C for 20 min. After addition of
670 μL of 20% trichloroacetic acid, the samples were centri-
fuged for 10 min at 4000 rpm. After addition of 80 mg of
Na2S2O5 and 0.4 mL of 4 M NaOH to the supernatant, un-
changed AT was extracted 3 times with 5 mL of toluene. The pH
of the aqueous phase was then adjusted to 7.0 by addition of
4 mL of 1 M potassium phosphate buffer (pH 6.0). After addition
of 1.3 g of NaCl, the samples were vortexed for at least 30 s. Then
50 nmol of phenacetin was added as an internal standard, and
the metabolites were extracted with 5 mL of chloroform/metha-
nol 9:1 (v/v). This extraction step was repeated once more with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8733
5 mL of chloroform/methanol 9:1 (v/v). The organic phases from
the two extractions were combined and evaporated to dryness,
the residue was dissolved in 0.2 mL of equal parts of methanol
and distilled water, and an aliquot (20 μL) was analyzed by
HPLC.
(12) Yoshimura, H.; Shimeno, H.; Tsukamoto, H. Metabolism of
Drugs. LXX. Further study on antipyrine metabolism. Chem.
Pharm. Bull. 1971, 19, 41–45.
(13) Shigematsu, H.; Hisanari, Y.; Yoshida, K.; Shimeno, H. Delayed
allergic reactions with aminopyrine metabolites. Skin Res. 1988, 30,
95–100.
Assay of Urinary Metabolites.30 AT or an analogue was given
orally to rats at a single dose of 100 mg/kg, and urine was
collected for 24 h. Then, a mixture of 1 mL of urine, 4 mg of
Na2S2O5, and 40 μL of β-glucuronidase/sulphatase in a final
volume of 4 mL of 0.1 M acetate buffer (pH 4.5) was incubated
at 37 °C for 24 h. Then 50 nmol of phenacetin was added as an
internal standard. The incubation mixture was extracted with
4 mL of chloroform/methanol 9:1 (v/v). The extract was evapo-
rated to dryness, the residue was dissolved in 0.2 mL of equal
parts of methanol and distilled water, and an aliquot (20 μL) was
analyzed by HPLC.
Statistical Analysis. All data were expressed as the mean (
SEM. The statistical significance of differences was evaluated by
means of two-way repeated-measures analysis of variance
(ANOVA) followed by contrasts for mean values comparison.
A p value <0.05 was considered significant.
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Acknowledgment. We are grateful to the SC-NMR Labo-
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the NMR experiment.
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