Design, synthesis, and pharmacological and pharmacokinetic evaluation of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives as xanthine oxidoreductase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.122

In an effort to find a potent xanthine oxidoreductase (XO) inhibitor, we discovered the best compound 2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-pyridin-4-yl)-1H-[1,2,4]triazol-3-yl]-benzonitrile 28. Here, we describe the following: (1) the design, synthesis, and structure-activity relationship of a series of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives by in vitro studies of XO inhibitory activity in bovine milk and in vivo studies of serum uric acid (UA) reductive activity in rats, (2) a drug interaction study by a cytochrome P450 3A4 (CYP3A4) assay, and (3) a pharmacokinetic (PK) study. Compound 28 exhibits potent XO inhibitory activity, serum UA-lowering activity in rats, weak CYP3A4 inhibitory activity, and moderate PK profile.

Full text of DOI:10.1016/j.bmcl.2008.10.122

Bioorganic & Medicinal Chemistry Letters 19 (2009) 184–187
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and pharmacological and pharmacokinetic evaluation
of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives as xanthine
oxidoreductase inhibitors
b
b
a,
Takahiro Sato a, , Naoki Ashizawa , Takashi Iwanaga , Hiroshi Nakamura , Koji Matsumoto b,
*
Tsutomu Inoue a, Osamu Nagata b
a Research Laboratories 1, Fuji Yakuhin Co., Ltd, 3936-2 Sashiougi, Nishi-ku, Saitama-shi, Saitama 331-0047, Japan
b Research Laboratories 2, Medicinal Chemistry Research Department, Fuji Yakuhin Co., Ltd, 636-1 Iidashinden, Nishi-ku, Saitama-shi, Saitama 331-0068, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to find a potent xanthine oxidoreductase (XO) inhibitor, we discovered the best compound
2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-pyridin-4-yl)-1H-[1,2,4]triazol-3-yl]-benzonitrile 28.
Here, we describe the following: (1) the design, synthesis, and structure–activity relationship of a series
of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives by in vitro studies of XO inhibitory activity in bovine
milk and in vivo studies of serum uric acid (UA) reductive activity in rats, (2) a drug interaction study
by a cytochrome P450 3A4 (CYP3A4) assay, and (3) a pharmacokinetic (PK) study. Compound 28 exhibits
potent XO inhibitory activity, serum UA-lowering activity in rats, weak CYP3A4 inhibitory activity, and
moderate PK profile.
Received 28 July 2008
Revised 7 October 2008
Accepted 28 October 2008
Available online 5 November 2008
Keywords:
Gout
Xanthine oxidoreductase
XO
Ó 2008 Elsevier Ltd. All rights reserved.
Hyperuricemia
Triazole derivatives
CYP3A4
Structure–activity relationship
Gout is a painful disease that is mainly caused by the deposition
of monosodium urate crystals in joints. Various factors are sug-
gested to contribute to the sustained elevation of the uric acid
(UA) level in blood, termed hyperuricemia. Normally, UA is dis-
solved in blood. As a result of hyperuricemia, insoluble UA forms
microscopic crystals in the capillary vessels of joints. These crystals
cause inflammation and sharp pain, which is termed acute gouty
arthritis or acute gout, and result in a significant degradation in
the quality of life of patients. Moreover, some epidemiologic stud-
ies suggest that hyperuricemia is an independent risk factor for
cardiovascular diseases such as myocardial infarction and cerebral
infarction.1 There are two types of drugs for controlling serum
UA—xanthine oxidoreductase (XO) inhibitors and uricosuric
agents. XO inhibitors block the terminal step in UA biosynthesis.2
The only clinically available XO inhibitor is Allopurinol (1) that
has been widely used in clinical practice because it is commonly
tolerated as compared to uricosuric agents (Fig. 1). However, in
patients with renal insufficiency, the plasma half life of oxipurinol,
which is the major active metabolite of allopurinol, is prolonged.
This is because oxipurinol is mainly removed by renal excretion.
Moreover, as a consequence of its structural similarities to purine
compounds, rare but severe hypersensitivity to Allopurinol (1)
has been reported.3 Thus, we have initiated the development of a
novel safe XO inhibitor with non-purine isosteres that is removed
by nonrenal excretion.
Thus far, we have investigated many compounds reported to
exhibit XO inhibitory activities, such as Febuxostat (2) and Y-700
(3) (Fig. 1). We focused on the structural similarities of these XO
inhibitors and prepared various types of acidic compounds; we
found oxanilide-type compound 4 that exhibited weak XO inhibi-
tory activity4 (Fig. 2). We then considered introducing a heterocy-
clic ring moiety for activity reinforcement. Compound 4 was
modified to 4-pyridyl compound 5 that exhibited strong in vitro
COOH
COOH
S
OH
N
NC
O
N
NC
O
N
N
N
N
N
H
* Corresponding author. Tel.: +81 48 625 0071; fax: +81 48 625 0072.
E-mail addresses: takahiro@fujiyakuhin.co.jp, sato-ty@nifty.com (T. Sato).
Present address: Faculty of Pharmaceutical Sciences, Josai International Univer-
sity, 1 Gumyo, Togane-shi, Chiba 283-8555, Japan.
Febuxostat (2)
Y-700 (3)
Allopurinol (1)
Figure 1. Structures of Allopurinol 1, Febuxostat 2, and Y-700 3.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.122

T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 184–187
185
Table 1
O
H
N
H
N
H
N
SAR on the pyridyl moiety substituent.
Cl
O N
2
OH
N
HN
O
O
5
N
R1
O
O
O2N
O
4
N
in vitro 15 nM
in vivo -3.2% (0.3 mg/kg)
in vitro 7.3 µM
Compound R1
In vitro
In vivo (%)5 at
0.3 mg/kg
Inhibition of CYP3A4
Figure 2. Oxanilide compound and urea-type compound.
IC50 (nM)4
(%)8 at 10
lM
XO inhibitory activity; however, Compound 5 did not decrease ser-
um UA level in vivo5 (Fig. 2). It was assumed that a urea moiety
was metabolized into inactive form in vivo; therefore, we tried to
convert this urea moiety into a stable cyclic structure. Compound
6 with triazole moiety exhibited the strongest in vitro activity
and considerable in vivo activity (Fig. 3). Diaryl triazole derivatives
have already been reported to exhibit XO inhibitory activity.6
These reported compounds exhibit serum UA-lowering activity
using spider monkeys, but the doses were as high as 5 mg/kg/
day.7 We failed to detect their potent serum UA-lowering activi-
ties.8 The acute oral toxicity of 6 was very low in mice; however,
6 potently inhibited human cytochrome P450 (CYP) 1A2, 2C9,
2C19, 2D6, and 3A4. Especially, inhibition of CYP3A4 was most
6
13
14
3.7
5900
33.7
NT
80.9
N
18.2
NT
N
>10,000
NT
N
15
16
17
10
39.1
40.6a
NT
7.7
7.1
N
N
N
Me
Cl
potent (80.9%; 10 l
M),9 which was suggestive of causing a serious
10
drug–drug interaction. Some nitro compounds are afraid of having
toxicities such as carcinogenicity and mutagenicity. Moreover, the
plasma concentration of 6 was very low. The maximum drug con-
centration in plasma (Cmax) was 13.8 ng/mL (1 mg/kg po).10 Thus,
we modified 6.
The general synthesis of the diaryltriazole derivative 12 for
determining the structure–activity relationship (SAR) is shown in
Scheme 1.6 The condensation of isonicotinic hydrazide 8 and imino
ether 10 produced acylamidrazone 11, which was thermally
cyclized to yield 12.
>10,000
>10,000
6.4
OMe
Cl
18
NT
10.9
N
Me
a
Test compound was administered orally at 1 mg/kg; NT, not tested.
First, we attempted to resolve the most serious problem per-
taining to the inhibition of CYP enzymes (Table 1). CYP3A4 is the
most abundant CYP in adult human liver and small intestine and
oxidizes numerous clinically important drugs among CYP en-
zymes,11 and the SAR trends for inhibition of other CYP enzymes
are typically similar to that for CYP3A4.12 We therefore selected
CYP3A4 as an index of inhibitory effect on CYP enzymes. We
expected that this activity was mainly caused by the 4-pyridyl
moiety.12 Therefore, we synthesized and examined 3-pyridyl and
2-pyridyl compounds ( 13 and 14); however, these compounds
did not inhibit the XO activity. We found that the 4-pyridyl moiety
was essential for the XO inhibitory activity; therefore, we at-
tempted the introduction of some substituent in position 2 (and
6) of the 4-pyridyl moiety (Table 1). As a result, we could reduce
the CYP3A4 inhibitory activity by the substitution of groups such
N
N
HN
H
N
H
N
N
O N
2
O N
2
O
N
O
O
5
6
in vitro 15 nM
in vivo -3.2% (0.3 mg/kg)
in vitro 3.7 nM
in vivo 33.7% (0.3 mg/kg)
Figure 3. Modification to triazole compound.
a
COOH
CONHNH
2
1
1
R
R
8
7
NH
N
HN
NH
2
3
2
3
H
N
b
c
d
1
CN
R
R
1
2
3
2
3
R
OMe
R
R
R
R
R
N
N
R
R
H
O
9
10
11
12
1
R : substituted or unsubstituted pyridyl
Scheme 1. Reagents and conditions: (a) NH2NH2, 1,10-carbonyldiimidazole, THF, rt; (b) NaOMe, MeOH, rt; (c) MeOH, reflux; (d) 200 °C.

186
T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 184–187
Table 2
Some studies suggested that low aqueous solubility leads to
SAR at the 3-position on the benzene ring.
poor absorption from gut. We assumed that a low drug concentra-
tion in blood for these series of compounds was a result of their
low aqueous solubility. Therefore, we modified the alkyl chain of
the alkoxy group of 19 to hydrophilic groups (Table 3). Regrettably,
phenol-type (22), carboxylic-type (23), and hydroxyl-type (24)
compounds did not reduce UA level in vivo. However, the
methyl-capped ether-type compound (25) retained the in vivo
activity. Therefore, we fabricated polyethers of various lengths,
which had alkyl-capped hydroxyl groups at the end of the alkyl
chain (26–31). As a result, we obtained three polyether compounds
(27, 28, and 30) exhibiting potent in vivo activity. The pharmaco-
kinetic (PK) profiles of these polyethers were greatly improved
over that of 19 (Table 4). The best compound (28) had the most
potent serum UA-lowering activity (52% decrease) and moderate
PK profile (Cmax 712 ng/mL, t1/2 0.97 h, 3 mg/kg, po) without
N
HN
1
R
N
N
Me
O
Compound R1
In vitro IC50
(nM)
In vivo (%)5 at
0.3 mg/kg
Inhibition of CYP3A4
(%)9 at 10
4
lM
15
19
20
21
NO2
CN
COOEt 20
10
17
39.1
46.0
42.4
NT
7.7
16.8
NT
COOH >10,000
NT
NT, not tested.
as methyl (15) or chloro groups (16) at position 2 without greatly
reducing the XO inhibitory activity. On the other hand, the XO
inhibitory activity of the methoxy-substituted compound (17)
and the 2,6-disubstituted compound (18) disappeared.
Next, the nitro group of compound 15 was replaced with
other groups (Table 2). The same electron-withdrawing groups
such as cyano (19) and carboethoxy (20) groups retained
in vitro activity, however, carboxyric acid compound (21) which
was expected to be the metabolite of 20 did not exhibit potent
XO inhibitory activity. Cyano compound 19 exhibited more
potent in vivo activity than the nitro group. However, the Cmax
value of 19 was very low (4.5 ng/mL, 1 mg/kg, po) likewise 15
(7.8 ng/mL, 1 mg/kg, po).10
CYP3A4 inhibition (ꢀ6.7%; 10
lM).
In conclusion, a series of pyridyl-triazole-phenyl-type com-
pounds have been synthesized for the development of a novel safe
XO inhibitor with a non-purine structure. In the course of SAR
studies, we have found the pharmacophore of these compounds.
The extent of CYP3A4 inhibition is decreased by introducing sub-
stituents in position 2 of the 4-pyridyl moiety. The nitro group
was replaced with the cyano group. Further, the PK profile is im-
proved by the replacement of the alkoxy substituents by alkyl-
capped polyether groups. These optimizations led to the best com-
pound 28 possessing potent in vivo serum UA-lowering activity
and moderate PK profile without CYP3A4 inhibition.
Table 3
SAR at the 4-position on the benzene ring.
N
HN
N
NC
1
O
N
R
Me
Compounds
R1
In vitro IC50 (nM)4
In vivo (%)5 at 0.3 mg/kg
Me
19
22
17
43
46.0
Me
H
ꢀ8.2
23
24
25
26
27
28
29
30
110
40
64
24
24
31
26
21
NT
COOH
OH
10.9
36.5
27.0
40.0
52.0
10.1
40.7
OMe
OMe
OMe
O
O
OMe
O
O
O
O
Me
Cl
O
31
60
27.9
O
NT, not tested.

T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 184–187
187
Table 4
Comparison of 19 and polyether compounds.
N
HN
N
NC
N
R1
Me
O
R1
In vitro IC50
(nM)
In vivo (%)5
Inhibition of CYP3A4 (%)9
Cmax (ng/mL)10
at 3 mg/kg
t1/2 (h)
4
10
Compounds
at 0.3 mg/kg
46.0
at 10
lM
19
27
28
17
24
31
21
16.8
16.7
360.1
712.2
684.4
0.37
0.45
0.97
0.52
OMe
40.0
52.0
40.7
16.5
ꢀ6.7
NT
O
O
O
OMe
Cl
30
O
NT, not tested.
sinus 6 h after the administration. The blood was allowed to clot for 1 h at room
temperature and centrifuged. The serum UA level was measured by the
phosphotungstic acid method.
Acknowledgment
The authors are grateful to Junichiro Uda for valuable
discussions.
6. Baldwin, J. J.; Kasinger, P. A.; Novello, F. C.; Sprague, J. M. J. Med. Chem. 1975, 18,
895.
7. Duggan, D. E.; Noll, R. M.; Baer, J. E.; Novello, F. C.; Baldwin, J. J. J. Med. Chem.
1975, 18, 900.
8. UA-lowering activities were less than 26% at 0.3 mg/kg, po5 (unpublished data).
9. The effects of the test compounds on the CYP3A4-mediated hydroxylation of
testosterone were examined using human liver microsome. The amount of 6b-
hydroxytestosterone produced in the reaction was determined by HPLC
analysis.
References and notes
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A. Prescribers’ J. 1997, 37, 29.
10. The test compounds were orally administered to rats, and their blood samples
were collected from jugular veins at predetermined times. All blood samples
were centrifuged to obtain plasma. Aliquots of plasma samples were mixed
with acetonitrile containing an appropriate internal standard and centrifuged.
Aliquots of the supernatant were measured by the LC–MS system.
11. Dresser, G. K.; Spence, J. D.; Bailey, D. G. Clin. Pharmacokinet. 2000, 38, 41.
12. (a) Miwatashi, S.; Arikawa, Y.; Kotani, E.; Miyamoto, M.; Naruo, K.; Kimura, H.;
Tanaka, T.; Asahi, T.; Ohkawa, S. J. Med. Chem. 2005, 48, 5966; (b)
Venkatakrishnan, K.; von Moltke, L. L.; Greenblatt, D. J. Clin. Pharmacokinet.
2000, 38, 111.
4. The IC50 values for the XO inhibitory activity of the test compounds were
determined as follows: bovine milk XO (2 mU/mL) was incubated with 15 lM
xanthine in a 50 mM phosphate buffer (pH 7.4) with or without the test
compound at 25 °C. Uric acid formation was determined by the increase in
absorbance at 292 nm using
calculated from 0 to 2.5 min.
a spectrophotometer. The initial rate was
5. The serum UA-lowering activity was measured as follows: the test compounds
were administered orally to rats, and their blood was drawn from the orbital