Metabolism investigation leading to novel drug design 2: Orally active prostacyclin mimetics. Part 5

Article abstract of DOI:10.1016/j.bmcl.2006.06.033

A metabolism study of FK788 (2) led to the discovery of new diphenylcarbamoyl derivatives as prostacyclin mimetics without the PG skeleton. We designed and evaluated PGI₂ mimetics based on blocking the main metabolic pathway of FK788. The new compound 7c was found to be equipotent to FK788 towards PGI₂ agonist activity and metabolically more stable than FK788.

Full text of DOI:10.1016/j.bmcl.2006.06.033

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4475–4478
Metabolism investigation leading to novel drug design 2:
Orally active prostacyclin mimetics. Part 5
Fujiko Takamura,a Akira Tanaka,b Hisashi Takasugi,b Kiyoshi Taniguchi,b
Mie Nishio,c Jiro Sekic and Kouji Hattorib,*
aBiopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa Pharmaceutical Co., Ltd,
2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
bMedicinal Chemistry 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 25 March 2006; revised 1 June 2006; accepted 12 June 2006
Available online 7 July 2006
Abstract—A metabolism study of FK788 (2) led to the discovery of new diphenylcarbamoyl derivatives as prostacyclin mimetics
without the PG skeleton. We designed and evaluated PGI2 mimetics based on blocking the main metabolic pathway of FK788.
The new compound 7c was found to be equipotent to FK788 towards PGI2 agonist activity and metabolically more stable than
FK788.
Ó 2006 Elsevier Ltd. All rights reserved.
In the previous paper, we have described the design, syn-
thesis and pharmacology of a new diphenylcarbamoyl
derivative with a tetrahydronaphthalene skeleton as a
PGI2 agonist.1 FK788: 2 has been selected as a
promising therapeutic candidate, and exhibited the same
potency as prostacyclin analogues and also excellent
selectivity over other human prostanoid receptors. After
a single oral dosing of 12.5 lg/body 2 in healthy
volunteers, the maximum plasma concentration (Cmax),
the area under the plasma concentration–time curve
agonists with suitable pharmacokinetics properties for
further development (Fig. 1).
The time courses of metabolite formation from 2 in rat
and human liver microsomes are shown in Figure 2.4
When 2 was incubated with rat and human liver micro-
somes, five metabolites were formed and named, M-1,
M-2, M-3, M-4 and M-5, according to the elution order
on HPLC. M-2 was the main metabolite in both rat and
human liver microsomes, as shown in Figure 2. The chem-
ical structures of M-2, M-4 and M-5 were confirmed by
comparing their MS/MS spectra and HPLC retention
times with those of the corresponding authentic
(AUC) and the elimination half-life (t1/2
) were
156.9 pg/mL, 327 pg h/mL and 1.5 h, respectively.
Despite the observation that 2 was well absorbed from
intestine (calculated bioavailability: 55%),2 its elimina-
tion half-life was relatively short and insufficient for
once daily dosing. Metabolic studies indicated there
were three primary metabolic sites for 2.3 Accordingly,
we set out to block these sites of metabolism in order
to obtain more efficacious compounds. We disclose
herein a series of tetrahydronaphthalene-based PGI2
HO2C
O
OH
Ph
O
N
O
Ph
HO2C
O
HO
Keywords: Prostacyclin; FK788; PG; PGI2; Metabolism.
OH
1: Prostacyclin
*
Corresponding author at present address: Chemistry Research
Laboratories, Astellas Pharmaceutical Co. Ltd, 21, Miyukigaoka,
Tsukuba-shi, Ibaraki 305-8585, Japan. Tel.: +81 29 863 7179; fax:
+81 29 852 2971; e-mail: kouji.hattori@jp.astellas.com
2: FK788
Figure 1.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.033

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F. Takamura et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4475–4478
Figure 2. Rat and human microsome-induced metabolite formation from FK788: 2.
R1
R2
O
Inhibition of ADP-induced
aggregation of human and
O
OH
OH
Ph
Rat liver microsomes
O
N
O
N
Ph
rat platelets
IC50 (nM)
Ph
Human Rat
HO2C
O
HO2C
R3
O
18
35
1400
2490
2
M-2
M-4
M-5
M-2: R1=OH, R2=H, R3=H
M-4: R1=H, R2=OH, R3=H
M-5: R1=H, R2=H, R3=OH
1290 >10000
198 >10000
2: FK-788
Scheme 1. Metabolite of FK788 by rat liver microsomes.
O
Cl
NHAc
Br
a, b, c
N
R1
R2
+
R1
R2
70 - 80% yield
3
4
5
R1
OH
O
OH
OH
OH
O
N
Ref 1
d, e
60 - 80% yield
O
OR
O
R2
EtO2C
HO2C
6
7
Scheme 2. Reagents: (a) CuCl, K2CO3, xylene; (b) 1 N-NaOH, EtOH; (c) triphosgen, pyridine, CH2Cl2; (d) 5, pyridine (e) 1 N-NaOH, EtOH.
Table 1. SAR of the diphenylcarbamoyl derivatives
compounds in Scheme 1.5 PGI2 activity of M-2 towards
inhibition of ADP-induced aggregation in human or rat
platelets was of similar potency compared to the parent
compound. On the other hand, M-4 and M-5 showed
R1
O
O N
OH
70-fold and10-fold less activity. After intravenousadmin-
istration of FK788 in rats, M-2 and glucuronide of M-2
were found in the bile at 12% and 33% of the administered
dose, respectively. Since M-2 and glucuronide of M-2
were rapidly excreted in the bile, plasma concentration
of M-2 after oral administration of FK788 would be
low.3 From the results of this metabolism study, it could
be considered that blockage of the main metabolic
pathway p-oxidation of the benzene moiety would be
effective to improve the metabolic stability of 2 without
affecting the potency.
HO2C
O
R2
Compound R1
R2
Functional assay:
IC50 (nM)
CLint
(mL/min/kg)b
a
Human Rat
Rat
2(FK788)
H
H
18
13
49
59
1400
4580
6490
31.9
10.3
12.3
7a
7b
7c
7d
7e
7f
p-F
p-Br
p-F
m-F
o-F
p-F
p-F
H
H
p-F
>10000 4.6
>10000 13.5
m-F 14
o-F
o-F
63
22
NT
5110
44.8
11.8
Diphenylcarbamoyl derivatives in which a proton was
replaced by halogen atoms for this study were prepared
as shown in Scheme 2. Copper-catalyzed amination
reactions6 of aryl halides with N-acyl aryl amines
produced di-aryl amines. Hydrolysis of the products
7g
m-F 25
>10000 11.7
a Results are average of two or three experiments.
b Compounds were incubated at 37 °C for 60 min with rat liver
microsomes in the presence of the NADPH-generating system.

F. Takamura et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4475–4478
4477
with 4 N NaOH solution, followed by treatment with
triphosgen in pyridine, gave the corresponding diph-
enylcarbamoyl chlorides 5. Treatment of optically active
diol 61 with 5 in pyridine led to smooth conversion to
the desired (2R)-hydroxy carbamoyl derivative, followed
by hydrolysis of ester with 1 N NaOH solution to afford
the desired compounds 7 in 60–80% overall yield from 6.
human and rat platelet rich plasma, and the metabolic
rate using rat liver microsomes was examined for the
novel diphenylcarbamoyl derivatives. p-Fluoro 7a and
m-difluoro 7d maintained similar PGI2 activity, while
p-bromo 7b, p-difluoro 7c, and o-difluoro 7e exhibited
approximately 3-fold less activity than the parent com-
pound 2. The metabolic rate of substitution at the
para-position of the phenyl ring was slower than that
of the parent compound, and particularly, p-difluoro
7c was metabolically the most advantageous among all
compounds.
Table 1 shows the results of biological evaluation of the
prepared derivatives. Functional assay by measuring
inhibition of ADP-induced platelet aggregation using
Figure 3. PK profiles of 2, 7a, and 7c by rat cassette-dosing.
Table 2. Pharmacokinetic profiles in rats
Compound
po (fasted)
iv
Dose (mg/kg)
Cmax (ng/mL)
AUC (ng/mL h)
t1/2 b (h)
CLtot (mL/min/kg)
F(%)
2
10
10
10
282
434
575
601
961
1.8
1.6
3.0
38.0
32.9
30.3
9.6
19.2
29.5
7a
7c
1607
-30 ~ -15
(min)
0
24h
IP agonist D-GalN
LPS
Blood
##
20000
15000
10000
12000
10000
8000
6000
4000
2000
0
##
*
**
**
5000
0
7c: mg/kg
2: mg/kg
##
: p<0.01 vs Normal, *: p,0.05 vs control, ** p,0.01 vs control
Figure 4. Effect of 2 and 7c in rat hepatic injury model by D-GalN/LPS, N = 15.

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F. Takamura et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4475–4478
Figure 3 and Table 2 show the pharmacokinetic profiles
after administration of a mixture of 7a, 7c and 2 at a
dose of 10 mg/kg each to rats by cassette-dosing.7 The
rank order of elimination half-lives after intravenous
administration was 7c > 7a > 2 and also 7c displayed a
higher plasma concentration level after oral administra-
tion compared to 2, which is explained by the smaller
in vitro clearance of 7c. Enhanced metabolic stability
by blockage of specific metabolic sites of 2 resulted in
improved bioavailability.
References and notes
1. Hattori, K.; Tanaka, A.; Okitsu, O.; Tabuchi, S.; Tanigu-
chi, K.; Nishio, M.; Koyama, S.; Higaki, M.; Seki, J.;
Sakane, K. Bioorg. Med. Chem. Lett. 2005, 15, 3091.
2. The bioavailability (F) was calculated from the following
equation: F = Qh/Qh + (Dose/AUC*BP), where Qh and
BP, respectively, are liver blood flow in humans (1500 mL/
min/70 kg) and blood-to-plasma ratio of FK788 (0.57).
3. Glucuronidation of FK788 was not observed in rat excre-
ment after intravenous administration of FK788, which is
explained by being stable towards glucuronidation in vitro.
4. 14C-FK788 (10 lmol/L) was incubated at 37 °C with rat
and human liver microsomes (1 mg protein/mL)
in the presence of a NADPH-generating system. Incubation
mixtures were analyzed by radio-HPLC and the concen-
trations of formed metabolites were determined.
Next, we examined a rat hepatic injury model with D-ga-
lactosamine hydrochloride (D-GalN) and lipopolysac-
charide (LPS), as shown in Figure 4.8 The compounds
7c and 2 exhibited a similar protection effect at a dose
of 3.2 mg/kg, even though 7c had more than 7-fold less
potent in vitro activity of rat.9 This result suggested that
the improved bioavailability of 7c leads to the resultant
improvement of in vivo potency.
5. FK788 was incubated at 37 °C with rat liver microsomes in
the presence of a NADPH-generating system. M-2, M-4
and M-5 in incubation mixtures were isolated by HPLC
and their chemical structures were determined by LC/MS/
MS and NMR analysis.
In this communication, we have explored the metabo-
lites of 2 using rat and human liver microsomes and
identified the major metabolite, M-2. Blockage of oxida-
tion at the para-position of the benzene moiety greatly
improved the metabolic stability of 2. Among the new
derivatives, compound 7c was found to have improved
bioavailability and duration time than 2. These results
indicate that 7c was a potential new candidate with po-
tent PGI2 activity and preferable pharmacokinetic
profile.
6. Weston, P. E.; Adkins, H. J. Am. Chem. Soc. 1928, 50, 859.
7. A dosing solution containing 7a, 7c and 2 was prepared in
PEG400 and the dose of each compound was 10 mg/kg.
After administration of the dosing solution to male rats, the
blood was collected from cannulated femoral artery. The
blood samples were centrifuged to separate plasma. The
plasma samples were analyzed by LC/MS/MS for determi-
nation of plasma concentrations of 7a, 7c and 2.
8. (a) Rahman, T. M.; Hodgson, H. J. F. Int. J. Exp. Path.
2000, 81, 145; (b) Jonker, A. M.; Dijkhuis, F. W. J.; Kroese,
F. G. M.; Hardonk, M. J.; Grond, J. Hepatology 1990, 11,
622; (c) Galanos, C.; Freudenberg, M. A.; Reutter, W.
Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 5939.
9. The compounds 2 and 7c were orally administered to rats 15–
30 min prior to treatment with D-GalN (300 mg/kg)/LPS
(0.32 lg/kg), and after 24 h, the blood was collected with a
syringe from the abdominal artery. The separated plasma
samples were examined for ALT levels by auto-analyzer.
Acknowledgment
We express our thanks to Dr. David Barrett for his
critical reading of the manuscript.