Metabolism investigation leading to novel drug design: Orally active prostacyclin mimetics. Part 4

Article abstract of DOI:10.1016/j.bmcl.2005.04.076

A metabolism study of FR181157 (1) led to the discovery of new oxazole derivatives as active metabolites. The metabolite 6 with an epoxy ring exhibited high anti-aggregative potency with an IC₅₀ of 5.8 nM and potent binding affinity for the human recombinant IP receptor with a K_i value of 6.1 nM and selectivity for human IP receptor over all other members of the human prostanoid receptor family.

Full text of DOI:10.1016/j.bmcl.2005.04.076

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3284–3287
Metabolism investigation leading to novel drug design:
Orally active prostacyclin mimetics. Part 4
Kouji Hattori,a,* Fujiko Takamura,b Akira Tanaka,a Hisashi Takasugi,a
Kiyoshi Taniguchi,a Mie Nishio,c Satoshi Koyama,c Jiro Sekic and Kazuo Sakanea
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima,
Yodogawa-ku, Osaka 532-8514, Japan
bBiopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd,
2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
cMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
Received 18 February 2005; revised 11 April 2005; accepted 22 April 2005
Abstract—A metabolism study of FR181157 (1) led to the discovery of new oxazole derivatives as active metabolites. The metabolite
6 with an epoxy ring exhibited high anti-aggregative potency with an IC50 of 5.8 nM and potent binding affinity for the human
recombinant IP receptor with a Ki value of 6.1 nM and selectivity for human IP receptor over all other members of the human pro-
stanoid receptor family.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Metabolism study
In the previous article, we described the design, synthe-
sis, and pharmacology of a new diphenyloxazole deriva-
tive with a cyclohexene ring FR181175 (1) as a PGI2
agonist.1 Tables 1 and 2 show the profiles of 1 and the
related compound 2, which has the cyclopentene ring in-
stead of the cyclohexene ring. 1 and 2 had similar po-
tency and affinity for IP receptors in vitro; however, 1
had 30-fold more potent in vivo activity, such as in the
D-GalN/LPS-induced rat hepatic injury model.2 Com-
pound 2 exhibited 3-fold improved PK properties over
1, particularly bioavailability and duration time in rats.
These results suggested that active metabolites of 1 may
exist with resultant improvement of in vivo potency.
After exploring the metabolites of 1 using rat liver
microsomes, we found the active metabolite M-4, which
exhibited 10-fold improved potency for the IP receptor.
Herein, we describe a stereoselective synthesis and the
biological activity of the active metabolite M-4.
The time course of metabolite formation for 1 in rat
liver microsomes is shown in Figure 1.3 When 1
(10 lM) was incubated with rat liver microsomes
(1 mg protein/ml), four metabolites were formed and
named, M-1, M-2, M-3, and M-4, according to the elu-
tion order on HPLC. M-1, M-2, and M-4 were isolated
by HPLC, PGI2 agonist activities of these metabolites
were determined. M-1 and M-2 were slightly less potent
than the parent compound; however, M-4 exhibited
dramatically improved activity toward inhibition of
ADP-induced aggregation in human platelets. The
chemical structures of these metabolites were deter-
mined by LC/MS/MS analysis and are shown in Scheme
1. Based on the LC/MS analysis, M-1, M-2, and M-4
had an [M+H]+ ion at m/z 482, 16 Da larger than that
of 1, and the fragmentation patterns of these metabolites
suggested that the addition of an oxygen atom had
occurred on the cyclohexene moiety. The identification
of M-1, M-2, and M-4 was confirmed by comparing
their MS/MS spectra and HPLC retention times with
those of the corresponding synthetic authentic
compounds. We also explored the metabolite of 2 and
identified the metabolites with similar structures. The
re-synthesized epoxide derivatives did not exhibit
Keywords: Metabolism; Prostacyclin mimetic; IP receptor.
*
Corresponding author. Tel.: +81 6 6390 1220; fax: +81 6 6304
5435; e-mail: kouji_hattori@po.fujisawa.co.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.076

K. Hattori et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3284–3287
Table 1. Pharmacological profiles of 1 and 2
3285
In vitro
Function assay: IC50 (nM)
In vivo
Binding assay: Ki (nM)
Effective dose in rat hepatic injury model (mg/kg, po)
Human
Rat
Human
(S)-1
(S)-2
60 5.2
56 1.9
1200 170
2800 40
54 0.36
41 0.24
0.032
1.0
Table 2. Pharmacokinetic profiles of 1 and 2 in rats
po (fasted), n = 3
Cmax (ng/ml)
iv, n = 3
CLtot (ml/min/kg)
Ba (%)
Dose (mg/kg)
AUC (ng h/ml)
t1/2b (h)
(S)-1
(S)-2
0.32
0.32
16.4 0.88
45.2 29.6
147 14.3
425 45.7
6.6 0.33
17 3.3
17.7 0.33
9.19 0.16
50
78
Figure 1. Rat microsome-induced metabolite formation from 1.
Scheme 1.

3286
K. Hattori et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3284–3287
Scheme 2. Reagents: (a) HC(OMe)3, cat. p-TsOH, CH2Cl2; (b) AcBr, CH2Cl2; (c) K2CO3, MeOH; (d) TBAF, THF, (e) ethyl bromoacetate, K2CO3,
DMF; (f) NaOH, EtOH.
improved PGI2 potency of inhibition of ADP-induced
aggregation using human and rat platelets.
the standard conditions with 1 mol% of AD-mix-b, with
a better matching pair for the substrate, the enantio-
merically pure diol 5 was obtained in 90% yield. On
the other hand, under the conditions with AD-mix-a
with mismatching pair, the diols 4 and 5 with 4:1 a ratio
were obtained in 65% yield. The previous work indicated
that AD-mix-a is effectively covering the a-face of the
cyclohexene 8 and selective approach from the b-face
of 8 should result.1 After separation of 4 and 5 by col-
umn chromatography, the diols were smoothly con-
verted to the epoxide using the method involving a
cyclic acetoxonium intermediate,5 followed by the stan-
dard method to give the corresponding products 6 and
7, in 66% and 70% yield, respectively.
3. Chemistry
The synthetic strategy for the optically active epoxide is
illustrated in Scheme 2. We planned to prepare these
compounds by controlling the stereochemistry at the
epoxy part on the cyclohexene ring. The required opti-
cally active epoxides were obtained via dihydroxylation
of the olefin 3 with the Sharpless AD reagent and OsO4.4
The optical active intermediate 3 was produced by the
previously reported asymmetric synthesis.1 Asymmetric
dihydroxylation of 3 by OsO4 provided the mixture of
the diol 4 and 5 with a 1:3 ratio in 95% yield. Under
4. Biological activity
Table 3. Function assay of the epoxide derivativesa
The metabolite 6 possesses potent PGI2 agonist activity
and especially excellent selectivity over all other human
PG receptors. Table 3 shows the results of the functional
assay by measuring the inhibition of ADP-induced
platelet aggregation using human, rat, dog, and monkey
platelet-rich plasma. The epoxide 6 was 10-fold more
potent than mother compound 1 in human and 4-fold
more potent in rat. The diastereomer 7 was 5-fold more
Compound
Function assay: IC50 (nM)
Human
Rat
300 28
NT
Dog
Monkey
6
7
1
5.8 1.2
12
60 5.2
1470 376
NT
1300 180
12 1.3
NT
188 11
1200 170
a Results are the average of two or three experiments.
Table 4. Affinity of 6 and 1 to eight prostanoid receptors
Compound
Human Ki (nM)a
IP
DP
2900
>1000
FP
TP
930
>1000
EP1
EP2
1400
>1000
EP3
EP4
6
1
6.1 0.11
54 0.36
>10,000
>1,000
>10,000
>1,000
3200
6800
7200
1020
a Ki determination are averages of at two or three experiments. Competitive binding assay based on the displacement of [3H]-Iloprost for human IP
receptor, [3H]-PGD2 for human DP receptors, [3H]-PGF2 for human FP receptors, [3H]-SQ29548 for human TP receptor, and [3H]-PGE2 for
human EP1–4 receptors.

K. Hattori et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3284–3287
3287
Table 5. Pharmacokinetic profile of 6a
po (fasted), n = 3
Cmax (ng/ml)
67.9 10.9
iv, n = 3
Ba (%)
Dose (mg/kg)
AUC (ng h/ml)
168.2 36.3
t1/2b (h)
CLtot (ml/min/kg)
18.6 0.63
Rat
1
26.9 3.2
20
a Results are shown as means SE.
potent. The newly designed 6 showed excellent affinity
for the IP receptor with Ki = 6.1 nM and excellent selec-
tivity over other human prostanoid receptors (receptor
selectivity >100) in Table 4.6 6 exhibited powerful po-
tency in vivo in the D-GalN/LPS-induced rat hepatic in-
jury model, comparable to 1. Table 5 shows the
pharmacokinetic profiles of 6, which displayed moderate
oral bioavailability in rat.
References and notes
1. Hattori, K.; Tabuchi, S.; Okitsu, O.; Taniguchi, K. Bioorg.
Med. Chem. Lett. 2003, 13, 4277.
2. Louis, H.; LeMoine, O.; Peny, M. O.; Gulbis, B.; Nisol, F.;
Goldman, M.; Deviere, J. Gastroenterology 1997, 112, 935.
3. In vitro metabolism: incubation mixtures (0.5 ml) contained
rat liver microsomes (1 mg/ml), 10 lM 1, 2 mM NADP,
10 mM glucose-6-phosphate, 5 mM MgCl2, 0.5 U glucose-
6-phosphate dehydrogenase, and 100 mM phosphate buffer
(pH 7.4). After preincubation at 37 °C for 5 min, the
reaction was started by addition of 1. Incubations were
carried out at 37 °C for 10–40 min and the reactions were
stopped by adding 0.1 ml of 1 N HCl and diethyl ether. The
mixtures were shaken for 10 min and centrifuged at 1700g
for 5 min. The organic phase (3 ml) was removed and
evaporated to dryness. The residue was dissolved in 100 ll
methanol and analyzed by HPLC. For the purpose of
isolation and purification of metabolites, large-scale incu-
bation of 1 (200 lM; 110 ml incubation) was carried out
with rat liver microsomes (2 mg/ml) for 40 min. The diethyl
ether extracts were evaporated to dryness and dissolved in
methanol for isolation by HPLC.
5. Conclusion
We have explored the metabolites of 1 using rat liver
microsomes and identified the active metabolites M-1,
M-2, and M-4. The potent metabolite M-4 was a mix-
ture of 6 and 7, which were re-synthesized and evalu-
ated. The isomer 6 exhibited the strongest potency for
the IP receptor of all oxazole derivatives we had pro-
duced and also excellent selectivity over the other eight
PG receptors. These results indicate that the active
metabolite of 1 may contribute to enhancement of the
potency in vivo. Furthermore, 6 emerged as a new can-
didate as an orally active prostacyclin mimetic with high
IP receptor selectivity.
4. Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G.
A.; Hartung, J.; Jeong, K.-S.; Kwong, H.-L.; Morikawa,
K.; Wang, Z.-M.; Xu, D.; Zhang, X.-L. J. Org. Chem. 1992,
57, 2768.
5. Kolb, H. C.; Sharpless, K. B. Tetrahedron 1992, 48, 10515.
6. (a) Abramovitz, A. M.; Adam, M.; Boie, Y. Biochem.
Biophys. Acta 2000, 1483, 285–293; (b) Negishi, M.;
Sugimoto, Y.; Ichikawa, A. Biochem. Biophys. Acta 1995,
1259, 109.
Acknowledgment
We express our thanks to Dr. David Barrett for his crit-
ical reading of the manuscript.