Discovery of 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole, FYX-051-a xanthine oxidoreductase inhibitor for the treatment of hyperuricemia

Article abstract of DOI:10.1016/j.bmcl.2009.08.091

Our previous study identified 2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4]triazol-3-yl]-benzonitrile (2) as a safe and potent xanthine oxidoreductase (XOR) inhibitor for the treatment of hyperuricemia. Here, we synthesized a series of 3,5-dipyridyl-1,2,4-triazole derivatives and, in particular, examined their in vivo activity in lowering the serum uric acid levels in rats. As a result, we identified 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) to be one of the most potent XOR inhibitors; it exhibited an extremely potent in vivo activity, weak CYP3A4-inhibitory activity and a better pharmacokinetic profile than compound 2. Compound 39 is currently being evaluated in a phase 2 clinical trial.

Full text of DOI:10.1016/j.bmcl.2009.08.091

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6225–6229
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Bioorganic & Medicinal Chemistry Letters
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Discovery of 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole, FYX-051-a
xanthine oxidoreductase inhibitor for the treatment of hyperuricemia
b
b
b
c
Takahiro Sato a, , Naoki Ashizawa , Koji Matsumoto , Takashi Iwanaga , Hiroshi Nakamura ,
*
Tsutomu Inoue a, Osamu Nagata b
a Research Laboratories 1, Research Department, Medical R&D Division, Fuji Yakuhin Co., Ltd, 3936-2 Sashiougi, Nishi-ku, Saitama-shi, Saitama 331-0047, Japan
b Research Laboratories 2, Research Department, Medical R&D Division, Fuji Yakuhin Co., Ltd, 636-1 Iidashinden, Nishi-ku, Saitama-shi, Saitama 331-0068, Japan
c Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane-shi, Chiba 283-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2009
Revised 26 August 2009
Accepted 28 August 2009
Available online 2 September 2009
Our previous study identified 2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4]-
triazol-3-yl]-benzonitrile (2) as a safe and potent xanthine oxidoreductase (XOR) inhibitor for the treat-
ment of hyperuricemia. Here, we synthesized a series of 3,5-dipyridyl-1,2,4-triazole derivatives and, in
particular, examined their in vivo activity in lowering the serum uric acid levels in rats. As a result, we
identified 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) to be one of the
most potent XOR inhibitors; it exhibited an extremely potent in vivo activity, weak CYP3A4-inhibitory
activity and a better pharmacokinetic profile than compound 2. Compound 39 is currently being evalu-
ated in a phase 2 clinical trial.
Keywords:
Gout
Xanthine oxidoreductase inhibitor
XO
Ó 2009 Elsevier Ltd. All rights reserved.
Hyperuricemia
Triazole derivatives
CYP3A4
Structure–activity relationships
Hyperuricemia is a metabolic disorder in which blood uric acid
(UA) levels are elevated for a sustained period of time; it is caused
by various genetic and environmental factors.1 In a normal person,
UA is dissolved in the blood; however, in a person with hyperuri-
cemia, insoluble UA forms microscopic crystals in the capillary ves-
sels of joints. These crystals cause joint inflammation and sharp
pain which are symptoms of acute gout; this condition signifi-
cantly degrades the quality of life of patients.
Some epidemiological studies have suggested that hyperurice-
mia is an independent risk factor for cerebral infarction and cardio-
vascular diseases such as myocardial infarction.2 Thus, the
amelioration of hyperuricemia is of great importance for prevent-
ing the occurrence of gout and cardiovascular diseases. Two types
of drugs are used for controlling the blood UA levels—xanthine oxi-
doreductase (XOR) inhibitors and uricosuric agents. XOR inhibitors
are ideal for treating hyperuricemia because XOR catalyzes the ter-
minal step in UA biosynthesis,3 and patients lacking only XOR
(xanthinuria type 1) do not exhibit any severe disorders except
for xanthine urolithiasis.4 Allopurinol (1), the only clinically avail-
able XOR inhibitor until recently, has been widely used in clinical
practice because it is generally well tolerated by patients (Fig. 1).
However, in patients with renal impairment, the use of allopu-
rinol (1) is restricted because the plasma half-life of oxipurinol, a
major active metabolite of allopurinol (1) that is mainly eliminated
via renal excretion, becomes prolonged. Moreover, rare but severe
cases of hypersensitivity to allopurinol (1), which is a purine
analogue, have been reported.5 Therefore, there has been a consid-
erable demand for a novel and safe XOR inhibitor. Recently, one
such novel inhibitor—febuxostat—that was approved by the Euro-
pean Medicines Evaluation Agency (EMEA) and the Food and Drug
Administration (FDA), has attracted worldwide attention. We also
have been attempting to develop a novel and safe XOR inhibitor
with nonpurine isosteres that can be eliminated by nonrenal excre-
tion. In our previous research, we identified certain 4-pyridyltriaz-
ole derivatives that can potently inhibit the XOR in vitro6 and
reduce the serum UA levels in vivo;7 for example, at a dose of
0.3 mg/kg, compound 2 decreased the serum UA levels of rats by
52% (Fig. 2).8
OH
N
N
N
H
N
Allopurinol (1)
* Corresponding author. Tel.: +81 48 625 0071; fax: +81 48 625 0072.
E-mail address: takahiro@fujiyakuhin.co.jp (T. Sato).
Figure 1. Structure of allopurinol (1).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.091

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T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6225–6229
N
N
N
HN
HN
N
N
N
O
Me
Me
MeO
O
O
CN
CN
2
3
IC50 = 31 nM in vitro
IC50 = 17 nM in vitro
52 2.9 % (0.3 mg/kg) in vivo
46 1.8% (0.3 mg/kg) in vivo
Cmax = 712 ng/mL (3 mg/kg)
Cmax = 4.5 ng/mL (1 mg/kg)
Figure 2. Potent XOR inhibitors identified in our previous study.
HN N
N
N
N
4
IC50 = 170 nM in vitro
Cmax = 4.50 µg/mL (3 mg/kg)
Figure 3. XOR inhibitor with high Cmax value.
Table 1
3,5-Dipyridyl-1,2,4-triazole compounds with XOR-inhibitory activity
HN N
N
2
R
N
1
R
Compound
R1
R2
In vitro
In vivo (%)7
at 0.3 mg/
kg, 6 h
Inhibition of
CYP3A4 (%)12
IC50 (nM)6
at 10
lM
4
5
H
H
170
ꢀ6.8 30.9a
ꢀ7.7 22.6a
18.5
N
130
10.4
N
6
7
H
31% inhibition
NT
20.3
24.7
N
(10
lM)
Me
28
ꢀ0.4 5.9
N
8
Me
310
26.1 12.0
15.3
N
Me
a
Test compound was administered orally at a dose of 1 mg/kg; NT: Not tested.
a
COOH
CONHNH2
R1
R1
10
9
NH
H
N
N
NH
OMe
c
HN
b
d
R1
R1
CN
R2
R2
R2
N
H
R2
N
O
14
11
12
13
R1, R2: substituted or unsubstituted 3- or 4-pyridyl
Scheme 1. Reagents and conditions: (a) NH2NH2, 1,10-carbonyldiimidazole, THF, room temperature (rt); (b) NaOMe, MeOH, rt; (c) MeOH, reflux; (d) 200 °C.

T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6225–6229
6227
Table 2
SAR analysis of 3-(3-pyridyl)-5-(4-pyridyl)-1,2,4-triazole compounds
Table 3 (continued)
Compound R1
R2
In vitro IC50 In vivo (%)7 at
(nM)6
0.3 mg/kg, 6 h
N
HN
N
N
2
N
R
1
R
25
H
4.9
36.3 8.2
Me
Me
R1
R2
In vitro
In vivo (%)7
at 0.3 mg/kg, 6 h
O
O
Compound
IC50 (nM)6
CN
CN
5
15
H
H
H
CN
130
38
ꢀ7.7 22.6a
ꢀ4.0 12.5
3
Me
17
46.0 1.8
ꢀ0.4 5.9
26.1 12.0
Me
Me
Me
Me
O
O
16
17
H
24
13.4 7.4b
13.0 6.7
Me
Me
Me
170
7
H
28
N
Me
Me
Me
O
18
CN
590
ꢀ6.1 5.2b
a
Test compound was administered orally at a dose of 1 mg/kg.
Test compound was administered orally at a dose of 3 mg/kg.
b
8
Me
310
N
Me
Table 3
Comparison of the in vitro and in vivo activities of mono-substituted 4-pyridyl
compounds and their methyl-substituted derivatives
Table 4
N
HN
SAR analysis of 3-(3-methyl-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole compounds
2
R
N
N
N NH
1
R
N
N
N
Compound R1
R2
In vitro IC50 In vivo (%)7 at
R1
(nM)6
0.3 mg/kg, 6 h
Me
Compound R1
In vitro
In vivo (%)7 at Cmax g/mL)9
(l
IC50 (nM)6 0.3 mg/kg, 6 h at 3 mg/kg
19
20
H
4.7
0.9 22.8
7
8
26
27
H
Me
Cl
28
310
89
ꢀ0.4 5.9
26.1 12.0
32.9 6.0
41.1 1.3
1.24a
NT
4.07
3.97
Cl
Cl
NO2
NO2
CN
59
Me
28
29
170
59
19.2 5.8
8.0 8.4
NT
NT
S
Me
30
19.8 8.6
Ph
Me
O
30
31
63
46
2.3 8.7
4.5 7.1
1.44
NT
Me
21
22
H
3.3
19.6 15.1
OMe
OMe
Me
Me
NO2
S
a
Test compound was administered orally at a dose of 1 mg/kg; NT: Not tested.
Me
17
19.3 5.5
The pharmacokinetic profile of compound 2, such as the maxi-
NO2
mum blood concentration (Cmax 712 ng/mL (3 mg/kg))9 and the
half-life in the blood (t1/2, 0.97 h),9 can be improved further.
In light of such issues, we performed pharmacokinetic assays on
other compounds that exhibited XOR-inhibitory activity. This led
us to the identification of a 3,5-dipyridyl-1,2,4-triazole compound
23
24
H
3.7
10
33.7 5.7
Me
Me
O
O
4
,
possessing a high Cmax value (Fig. 3).
Certain 3,5-dipyridyl-1,2,4-triazole compounds with XOR-inhi-
NO2
NO2
bition properties have been described previously,10 and they
exhibited serum UA-lowering activity at a dose of 5 mg/kg/day.11
In our assay, they lowered the UA in rats by 26% or less at a dose
of 0.3 mg/kg (Table 1).7
On the other hand, we found that these compounds possess
good pharmacokinetic potential with an adequate Cmax value. The
Me
39.1 2.4
Me
Me

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T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6225–6229
Table 5
SAR analysis of 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole compounds
N NH
N
N
N
R1
NC
Compound
R1
In vitro IC50 (nM)6
In vivo (%)7 at 0.3 mg/kg, 6 h
Inhibition of CYP3A4 (%)12 at 10
lM
Cmax (l
g/mL)9 at 3 mg/kg
27
32
Me
OMe
59
640
41.1 1.3
2.3 3.2a
4.7
NT
3.97
NT
Me
33
34
35
160
130
53
ꢀ3.7 11.5
1.9 6.1
NT
NT
NT
NT
S
Me
Me
O
NT
Me
Me
S
ꢀ1.4 10.6
0.20
36
37
38
39
Ph
Cl
CN
H
37
39
18
5.3
4.2 8.1
NT
3.6
NT
NT
5.34
NT
33.6 5.1
32.0 14.1
51.1 1.2
18.6
4.62
a
Test compound was administered orally at a dose of 3 mg/kg; NT: Not tested.
CYP3A4-inhibitory activities of these compounds were relatively
low although an unsubstituted 4-pyridyl compound tends to inhi-
bit CYP3A4.8,13
Therefore, we subsequently examined the activities of different
4,4-dipyridyltriazole compounds with the 3-methyl-4-pyridyl-
triazole moiety in common (Table 4).
Considering the abovementioned characteristics, we modified
these compounds in order to enhance their in vivo activities.
Scheme 1 shows the general procedure used to synthesize 3,5-
dipyridyl-1,2,4-triazole derivative 14 for determining the struc-
ture–activity relationship (SAR).10 Acylamidrazone (13) was syn-
thesized by the condensation of isonicotinic hydrazide (10) and
iminoether (12), and it was then thermally cyclized to yield com-
pound 14.
Compounds containing less bulky steric groups exhibited po-
tent UA-lowering activity (8, 26, and 27). On the other hand, com-
pounds containing bulky substituents were weak despite their
potent in vitro activity (29, 30, and 31).
The compound containing a cyano group at position 2 of the 4-
pyridyl moiety (27) exhibited marked in vivo activity in lowering
the serum UA levels and it had a high Cmax value. Therefore, the
methyl group in the pyridyl moiety of compound 27 was replaced
with various substituents (Table 5).
The substitution of the methyl group by electron-donating
groups (32, 33, 34, and 35) and bulky groups (36) decreased the
in vivo activity. Compounds containing less bulky steric and elec-
tron-withdrawing groups (37 and 38) exhibited more than 30%
UA-lowering activity. Unexpectedly, the mono-substituted R1 com-
pound (39) exhibited the most potent activity both in vitro and
in vivo. Therefore, we examined various mono-substituted 4,4-
dipyridyltriazole compounds (Table 6).
First, the in vitro and in vivo activities of 3- and 4-dipyridyl-
triazole compounds with relatively low CYP3A4-inhibitory activity
were examined (Table 2).
Although the in vitro activity increased noticeably by the intro-
duction of cyano (15) and isobutoxy (16) groups at position 6 of the
3-pyridyl moiety, the in vivo activity did not. The introduction of
methyl (17) and cyano (18) groups at position 2 of the 4-pyridyl
moiety did not yield beneficial effects. We found that the presence
of a methyl group at position 2 of the 4-pyridyl moiety tended to
enhance the in vivo activity despite their less-potent in vitro
activities (Table 3).8 Compounds 16 and 17 provided similar results
(Table 2).
These mono-substituted compounds exhibited highly potent
in vitro activity; however, except 39, none of these compounds
exhibited potent in vivo activity.
Table 6
SAR analysis of mono-substituted 4,4-dipyridyltriazole compounds
N
NH
N
N
N
R1
Compound
R1
In vitro IC50 (nM)6
In vivo (%)7 at 0.3 mg/kg, 6 h
Inhibition of CYP3A4 (%)12 at 10
lM
Cmax (l
g/mL)9 at 3 mg/kg
7
Me
CN
Cl
Br
F
28
ꢀ0.4 5.9
51.1 1.2
26.5 25.4a
ꢀ6.2 6.3
ꢀ0.2 2.1
ꢀ0.4 12.6
24.7
18.6
54.1
NT
40.2
NT
1.24a
4.62
4.36
NT
NT
NT
39
40
41
42
43
5.3
7.9
9.9
48
Ph
6.8
a
Test compound was administered orally at a dose of 1 mg/kg; NT: Not tested.

T. Sato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6225–6229
6229
Table 7
Comparison of in vitro and in vivo activities of compound 39 and allopurinol (1)
Supplementary data
Compound
In vitro IC50 (nM)6
In vivo (%)7 at 0.3 mg/kg
6 h
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.08.091.
2 h
39
1
5.3
2300
59.9 1.4
35.9 4.0
51.1 1.2
ꢀ3.3 2.7
References and notes
1. Yamamoto, T. Nippon Rinsho 2008, 66, 636.
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5. (a) Hande, K. R.; Noone, R. M.; Stone, W. J. Am. J. Med. 1984, 76, 47; (b) Bradlow,
A. Prescribers’ J. 1997, 37, 29.
As compared to allopurinol (1), 39 exhibited more potent
in vitro and in vivo activities and had a more sustained in vivo ef-
fect than allopurinol (1) (Table 7).
These potent and more sustained effects of 39 have been con-
firmed by a crystallographic analysis of XOR-39 complex. The cya-
no group of compound 39 has been reported to play an important
role in the binding activity between 39 and XOR. This is attribut-
able to the formation of a hydrogen bond between Asn 768 of
XOR and the cyano group of compound 39, as revealed by X-ray
crystal structure analysis (PDB code 1V97).14
6. The IC50 values for the XOR-inhibitory activity of the test compounds were
determined as follows: bovine milk XOR (2 mU/mL) was incubated with 15 lM
xanthineina 50 mMphosphatebuffer(pH7.4)withorwithoutthetest compound
at 25 °C. UA formation was determined by the increase in absorbance at 292 nm
using a spectrophotometer. The initial rate was calculated from 0 to 2.5 min.
7. 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
sinus 2 or 6 h after the administration. The blood was allowed to clot for 1 h at
room temperature and centrifuged. The serum UA levels were measured by the
phosphotungstic acid method. Data are represented as the means SD of at
least three rats.
Compound 39 exhibited a weak CYP3A4-inhibitory activity
(18.6%); its Cmax and bioavailability were as high as 4.62 lg/mL
8. Sato, T.; Ashizawa, N.; Iwanaga, T.; Nakamura, T.; Matsumoto, T.; Inoue, T.;
Nagata, O. Bioorg. Med. Chem. Lett. 2009, 19, 184.
(3 mg/kg) and 69.6%, respectively. Moreover, the t1/2 value of 39
was greater (19.7 h) than that of compound 2 (0.97 h). Since 39
is mainly excreted in the urine as triazole N1- and N2-glucuronides
in monkeys and humans,15 it is expected to be a safe drug in
patients with renal impairment.
In conclusion, we modified a series of 3,5-pyridyl-1,2,4-triazole
compounds in order to improve their in vivo activities. The SAR
analysis of the methyl-substituted compounds confirmed the
important role of the cyano substituent in the expression of the
in vivo activity. The optimization of the series of compounds led
to the identification of the mono-substituted compound 39
(FYX-051). This compound exhibits extremely potent effect in
lowering the serum UA levels in vivo and it has good pharmacoki-
netic properties; moreover, it exhibits a weak CYP3A4-inhibitory
activity. It is expected to be a beneficial drug for patients with
hyperuricemia.
9. 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.
10. Baldwin, J. J.; Kasinger, P. A.; Novello, F. C.; Sprague, J. M. J. Med. Chem. 1975, 18, 895.
11. Duggan, D. E.; Noll, R. M.; Baer, J. E.; Novello, F. C.; Baldwin, J. J. J. Med. Chem.
1975, 18, 900.
12. The effects of the test compounds on the CYP3A4-mediated hydroxylation of
testosterone were examined at a concentration of 10
lM using human liver
microsome. The amount of 6b-hydroxytestosterone produced in the reaction
was determined by HPLC analysis.
13. (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.
14. Okamoto, K.; Matsumoto, K.; Hille, R.; Eger, B. T.; Pai, E. F.; Nishino, T. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 7931.
15. Nakazawa, T.; Miyata, K.; Omura, K.; Iwanaga, T.; Nagata, O. Drug Metab. Dispos.
2006, 34, 1880.