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
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
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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.