Synthesis and structure - Activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00093-2

A series of 1-phenylpyrazoles was evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds prepared, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700) had the most potent enzyme inhibition and displayed longer-lasting hypouricemic action than did allopurinol in a rat model of hyperuricemia induced by the uricase inhibitor potassium oxonate.

Full text of DOI:10.1016/S0960-894X(01)00093-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 879–882
Synthesis and Structure–Activity Relationships of
1-Phenylpyrazoles as Xanthine Oxidase Inhibitors
Seigo Ishibuchi,a,* Hiroshi Morimoto,b Takanori Oe,b Tsuguo Ikebe,b Hiroyoshi Inoue,c
Atsushi Fukunari,b Miho Kamezawa,b Ichimaro Yamadab and Yoichi Nakaa
aDrug Discovery Research Laboratories, Welfide Corporation, Shodai-Ohtani 2-chome, Hirakata-shi, Osaka 573-1153, Japan
bDrug Development Laboratories, Welfide Corporation, 955 Koiwai, Yoshitomi-cho, Chikujo-gun, Fukuoka 871-8550, Japan
cRadioisotope Institute for Basic and Clinical Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume,
Fukuoka 830-0011, Japan
Received 9 November 2000; accepted 26 January 2001
Abstract—A series of 1-phenylpyrazoles was evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds
prepared, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700) had the most potent enzyme inhibition and dis-
played longer-lasting hypouricemic action than did allopurinol in a rat model of hyperuricemia induced by the uricase inhibitor
potassium oxonate. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Via chemical modification of allopurinol (1) as shown in
Figure 1, we attempted to design novel XO inhibitors
(2) possessing no purine nuclei. The rationale for the
synthesis of these compounds included the following
considerations: (1) The introduction of a phenyl group
at the N-1 position in pyrazolo[3,4-d]pyrimidines would
exclude the possibility of these compounds being con-
verted, like allopurinol, to unnatural nucleotides; (2) As
a model for the mechanism of XO inhibition by purine
analogues, it was hypothesized that the molybdenum
site of the enzyme becomes attached to the oxygen of
their pyrimidine rings;7 we therefore replaced the 4-
hydroxypyrimidine moiety with a carboxyl group, the
presence of which might enhance coordination with
molybdenum.
Since gout can be prevented by reducing plasma levels
of uric acid (UA), therapy is possible with uricosuric
drugs that increase the urinary excretion of UA, or with
xanthine oxidase (XO) inhibitors that block the term-
inal step in UA biosynthesis.1 The purine analogue,
allopurinol is the only XO inhibitor administered in
clinical situations, and has been used for the therapy of
both the primary hyperuricemia of gout and that sec-
ondary to haematological disorders or antineoplastic
therapy.2 Unfortunately, it has been reported to be
associated with an infrequent but severe hypersensitiv-
ity,3 in which rises in the blood concentration of allo-
purinol metabolites point to involvement of drug
toxicity, although the exact mechanism or causative
agent has not been identified.4 Allopurinol and its
actual metabolite, oxipurinol, are substrates for N-1
ribosyl derivative-forming phosphoribosyl transferases.5
Oxipurinol is also converted by uridine phosphorylase
to the N-7 ribosyl derivative.5 Because of the possible
deleterious effects of these metabolites in long-term
allopurinol administration, there have been efforts to
find novel potent XO inhibitors with non-purine iso-
steres,6 but so far none has been made clinically available.
Here, we describe the synthesis and structure–activity
relationships of 1-phenylpyrazole derivatives as novel
XO inhibitors.
*Corresponding author. Tel.: +81-72-856-9283; fax: +81-72-857-
5020; e-mail: ishibuchi@welfide.co.jp
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00093-2

880
S. Ishibuchi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 879–882
Synthesis
conducted with a 1.5 mL reaction mixture containing
Tris–HCl buffer (pH 8.1), 0.1 mol/L; XO from bovine
milk (Sigma Chemical Co.), 0.0014 units; and xanthine,
0.12 mol/L, at various concentrations, or absent for
the purpose of the control reaction. The assays were
carried out aerobically at 37 ꢀC. The reactions were
started by adding xanthine to the mixture of the enzyme
and the test drug, and terminated by addition of
trichloroacetic acid at a final concentration of 10%
(w/v). Duplicate assays were repeated three to four
times. The inhibitory activity of each test compound
against XO was indicated in terms of IC50 value (con-
centration of compound producing 50% inhibition
compared to control reaction). IC50 and 95% con-
fidence limit were calculated based on a non-linear
regression analysis.
A general scheme for the synthesis of 1-phenylpyrazole
derivatives is given in Scheme 1.
Condensation of a series of phenylhydrazines (3a–e, g–i)
with ethyl ethoxymethylenecyanoacetate in refluxing
ethanol followed by deamination with isoamyl nitrite
gave 4a–e, g–i. Demethylation of 4b with AlCl3 followed
by nitration with HNO3 and alkylation with isobutyl
bromide afforded 4f. Similarly, demethylation of 4c with
AlCl3 gave the phenol derivative 4j, which was treated
with the corresponding alkyl bromide to afford 4k–n.
The pyrazole-4-carboxylic acid derivatives 5a–n were
synthesized by hydrolysis of 4a–n.
Biological Assays
Hypouricemic effect in potassium oxonate-treated rats
Inhibitory activity against XO
Male Sprague–Dawley rats (6 weeks old, n=8) were
repeatedly treated with the uricase inhibitor potassium
oxonate (250 mg/kg, sc) 1 h before oral dosing (0.3–
10 mg/kg) with the test compounds, and 3, 7 and 23 h
afterward. One hour and immediately before adminis-
tration of the test compounds, and 2, 4, 6, 10 and 24 h
afterward, blood samples were taken individually under
light anaesthetization with halothane. UA plasma level
was determined with phosphotungstic acid colorimetry
using a commercial kit (Uric Acid Test Kit, Wako Pure
Chemical Industries Ltd). The area under the effect–
time curves from time 0 to time 6 h (AUEC0ꢁ6 h) or 24 h
(AUEC0ꢁ24 h) after test compound administration was
calculated from mean UA plasma level at each time
point. The efficacy of the hypouricemic action of each
test compound was presented in terms of ED20 value
(effective dose reducing AUEC0ꢁ6 h or AUEC0ꢁ24 h of
control group by 20%), which was calculated based on
a linear regression analysis.
Xanthine-oxygen reductase activity of XO was mea-
sured spectrophotometrically by following the absor-
bance of UA at 292 nm. The enzyme assay was
Table 1. Inhibitory activity of 1-phenylpyrazole derivatives against
XO in vitro
Compd
R1
R2
XO
inhibitory activitya
IC50 (nmol/L)
b
4d
5a
5b
5c
OCH2CH(CH3)2A
Cl
OCH3
CNN
H
H
CN460 (350–590)
CN7.1 (6.2–8.1)
CN5.8 (5.4–6.3)
NO2
Br
NA
N
A
OCH3
5d
5e (Y-700)
5f
5g
5h
5i
5j
5k
OCH2CH(CH3)2
OCH2C(CH3)3
OCH2CH(CH3)2
OCH2CH(CH3)2
OCH2CH(CH3)2
H
33 (22–49)
43 (37–50)
4700 (2400–11,000)
H
CN3300 (2500–4400)
CN3600 (3400–3800)
CN15 (12–19)
OH
OC4H9
5l
5m
OC8H17
OCH2CH2N(CH3)2
OCH2CO2H
CN21 (11–40)
CN700 (350–1400)
CN1500 (1400–1600)
5n
Allopurinol (1)
260 (220–310)
Scheme 1. (i) EtOCH¼C(CN)CO2Et EtOH; (ii) isoamyl nitrite, THF;
(iii) AlCl3, ClCH2CH2Cl; (iv) NHO3, AcOH; (v) alkyl bromide, K2CO3,
DMF; (vi) 1 NNaOH, EtOH.
a95% Confidence limits for IC50 values are shown in parentheses.
bNA=Not active <50% inhibition @10,000nmol/L.

S. Ishibuchi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 879–882
881
Pharmacokinetics in potassium oxonate-treated rats
Results and Discussion
The oxonate-treated rats described above were used.
The test compound was orally administered at doses of
0.3 and 3 mg/kg to animals. Blood samples were
collected at designated time intervals and were cen-
trifuged for the separation of plasma. Plasma con-
centration of test compound was analyzed by the HPLC
method with reversed-phased C-18 column (Shim-pack
CLC-ODS(M), 150Â4.6 mm i.d., Shimadzu). The
area under the plasma concentration–time curves
The SARs of the 1-phenylpyrazole derivatives newly
synthesized as XO inhibitors are summarized in Table 1.
The 4-monosubstituted analogues 5a,b had no inhibi-
tory activity against XO, but addition of a cyano group
as an electrophilic substituent at the 3-position of the 4-
methoxyphenyl unit produced apparent inhibitory
activity (5b vs 5c).
We therefore proceeded to examine the influence of
substitution at the 4-position of the 3-cyanophenyl
moiety with compounds 5c–e and 5i–n. It was found
that increasing the size of the 4-substituent from
hydrogen to butoxy and octyloxy groups improved XO
inhibitory activity (5i vs 5k, l), but that compounds with
polar groups (5m, n) had much less potent inhibitory
activity. Specifically, the introduction of bulky groups
such as isobutoxy and neopentyloxy at the 4-posi-
tion produced optimal results in vitro (5d, e). These
compounds showed much more potent inhibitory activ-
ity than allopurinol (IC50=7.1 and 5.8 nmol/L, respec-
tively). Other electrophilic substituents in the form of
bromo and nitro groups at the 3-position of the phenyl
ring were tolerated, but a cyano group containing a p
bond was best (5f,g vs 5d). A free carboxyl group at the
4-position of the pyrazole ring was found to be essential
for XO inhibition (4d vs 5d). Since the 4-isobutoxy
analogue 5h and 3-cyano analogue 5i were much less
potent than the 3-cyano-4-isobutoxy analogue 5d, the
combination of a cyano group and a bulky alkoxy
group was concluded to be of great importance for the
XO inhibitory activity of the 1-phenylpyazole-4-car-
boxylic acids. However, the exact mechanism of enzyme
inhibition by 1-phenylpyazole derivatives remains to be
clarified.
from time 0 to time 6 h (AUC0ꢁ6 h) or 24 h (AUC0ꢁ24 h
)
was calculated by the linear trapezoidal method. The
apparent oral clearance (CL/F) was calculated as dose/
AUC0ꢁ1 h
.
Table 2. Hypouricemic activity of 5d, Y-700 (5e) and allopurinol (1)
Compd
ED20 (mg/kg, po)a
0–6 h
0–24 h
5d
Y-700 (5e)
Allopurinol (1)
2.5 (1.9–3.3)
0.55 (0.33–0.79)
3.5 (2.0–8.8)
>10
1.4 (1.1–1.9)
>10
a95% Confidence limits for ED20 values are shown in parentheses.
Since the isobutoxy analogue 5d and the neopentyloxy
analogue 5e (Y-700) showed more potent inhibitory
activity than all other compounds tested in vitro, their
hypouricemic effects were investigated and compared in
vivo with that of allopurinol. Repeated subcutaneous
injection of potassium oxonate to rats caused a marked
UA plasma level increase which was maintained
throughout the experiment (data not shown). Com-
pound 5d showed hypouricemic effects in hyperuricemic
rats. However, the potency of the effects proved unsa-
tisfactory with both ED20 (0–6 h) and ED20 (0–24 h)
values of 5d nearly equal to those of allopurinol (Table 2).
Figure 2. Plasma concentration of 5d and Y-700 (5e) after oral
administration to potassium oxonate-treated rats (meanꢂSD, n=4).
Table 3. Pharmacokinetic parameter of 5d and Y-700 (5e) after oral administration to potassium oxonate-treated ratsa
Compd
Dose
(mg/kg)
Pharmacokinetic parameter
Cmax
(mg/mL)
tmax
(h)
t1/2
(h)
AUC0ꢁ6 h
AUC0ꢁ24 h
AUC0ꢁ1 h
.
.
.
(mg h/mL)
(mg h/mL)
(mg h/mL)
5d
0.3
3
0.3
3
0.09ꢂ0.03
1.16ꢂ0.57
0.14ꢂ0.01
2.29ꢂ0.68
1.1ꢂ0.6
1.3ꢂ1.8
3.5ꢂ1.9
3.5ꢂ1.0
3.3ꢂ1.0
3.5ꢂ2.0
6.0ꢂ0.8
4.5ꢂ0.7
0.41ꢂ0.09
3.56ꢂ0.26
0.66ꢂ0.04
11.44ꢂ2.87
0.65ꢂ0.11
5.38ꢂ0.23
1.64ꢂ0.16
25.63ꢂ5.97
0.62ꢂ0.10
5.16ꢂ0.55
1.77ꢂ0.22
26.40ꢂ6.17
Y-700 (5e)
aMeanꢂSD, n=4.

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S. Ishibuchi et al. / Bioorg. Med. Chem. Lett. 11 (2001) 879–882
Y-700, on the other hand, showed potent and long-
lasting hypouricemic effects, although the XO inhibitory
activity of Y-700 was almost equipotent to that of 5d in
vitro (Table 1 and 2). Plasma concentration–time curves
for Y-700 and 5d after oral administrations are shown
in Figure 2. The AUC0ꢁ6 h and AUC0ꢁ24 h for Y-700 at
potent and long-lasting hypouricemic effect in a rat
model of hyperuricemia. Y-700 thus appears to be a
promising candidate for the treatment of hyperuricemia,
gout and other diseases in which XO may be involved.
.
3 mg/kg were 11.44ꢂ2.87 and 25.63ꢂ5.97 mg h/mL,
References
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To conclude, we prepared a novel series of 1-phe-
nylpyrazole derivatives and discovered a number of
compounds among them with more potent inhibitory
activity than allopurinol. Specifically, the 1-(3-cyano-4-
neopentyloxyphenyl)pyrazole-4-carboxylic acid Y-700
was found to be an orally effective XO inhibitor with