Synthesis and antihypertensive activity of pyrimidin-4(3H)-one derivatives as losartan analogue for new angiotensin II receptor type 1 (AT1) antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.12.116

The discovery, in vitro and in vivo studies of the highly potent AT1 antagonist 12a (BR-A-657, Fimasartan) are presented. A series of pyrimidin-4(3H)-one derivatives as losartan analogue were synthesized and evaluated for a novel class of AT₁ receptor antagonists. Among them, 12a containing thioamido moiety displayed both high in vitro functional antagonism and binding affinity [IC₅₀ = 0.42 and 0.13 nM, respectively] and inhibited strongly in vivo AngII-induced pressor response in pithed rats with an ED₅₀ of 0.018 mg/kg. Moreover, in vivo evaluation in furosemide-treated rat and conscious renal hypertensive rat models and the pharmacokinetic study showed that 12a is a highly potent and orally active AT1 selective antagonist having stronger in vivo potency than losartan.

Full text of DOI:10.1016/j.bmcl.2011.12.116

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1649–1654
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antihypertensive activity of pyrimidin-4(3H)-one derivatives
as losartan analogue for new angiotensin II receptor type 1 (AT1) antagonists
Tae Woo Kim a, Byoung Wook Yoo b, Joon Kwang Lee b, Ji Han Kim b, Kyung-Tae Lee c,d
,
Yong Ha Chi b, , , Jae Yeol Lee a, ,
⇑
⇑
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b Central Research Institute, Boryung Pharm. Co. Ltd, 1122-3, Shingil-Dong, Danwon-Gu, Ansan-Si, Kyungki-Do 425-839, Republic of Korea
c Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
d Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery, in vitro and in vivo studies of the highly potent AT1 antagonist 12a (BR-A-657, Fimasartan)
are presented. A series of pyrimidin-4(3H)-one derivatives as losartan analogue were synthesized and
evaluated for a novel class of AT1 receptor antagonists. Among them, 12a containing thioamido moiety
displayed both high in vitro functional antagonism and binding affinity [IC50 = 0.42 and 0.13 nM, respec-
tively] and inhibited strongly in vivo AngII-induced pressor response in pithed rats with an ED50 of
0.018 mg/kg. Moreover, in vivo evaluation in furosemide-treated rat and conscious renal hypertensive
rat models and the pharmacokinetic study showed that 12a is a highly potent and orally active AT1 selec-
tive antagonist having stronger in vivo potency than losartan.
Received 31 October 2011
Revised 9 December 2011
Accepted 24 December 2011
Available online 2 January 2012
Keywords:
Losartan analogue
Pyrimidin-4(3H)-one
Angiotensin II receptor antagonist
Antihypertensive activity
Fimasartan (BR-A-657)
Ó 2011 Elsevier Ltd. All rights reserved.
Hypertension is a condition with a well understood pathophysi-
ology, which correspondingly led to the development of diverse
pharmaceuticals working at various physiological pathways such
as diuretics, beta blockers, angiotensin converting enzyme inhibi-
tors, calcium channel antagonists, angiotensin receptor antagonists,
and more recently renin inhibitors.1 Among them, angiotensin II
receptor (in particular Type 1: AT1) antagonists are positioned as
highly effective class of drugs for the treatment of hypertension as
they are safe with few side effects.2 Development of majority of
AT1 antagonists are based on modifications of the heterocyclic part
of losartan (Fig. 1), which is the first clinically used selective and
non-peptidic AT1 antagonist.3 Replacement of imidazole ring of
losartan with five-membered, six-membered, fused and acyclic
moieties have resulted in the identification of potent AT1 selective
antagonists such as telmisartan and valsartan, etc.4–6
development odyssey of 12a including the brief structure–activity
relationships (SAR) of pyrimidin-4(3H)-one derivatives tethered
with biphenyl tetrazole at its 3-position, and its pharmacological
characterization.
The synthesis of new pyrimidin-4(3H)-one derivative is shown
in Scheme 1. Pyrimidin-4(3H)-one 3 was prepared in two steps from
a nitrile according to a procedure described by Ellingboe and his col-
laborators.14 Conversion of nitrile 1 into amidine hydrochloride 2
was accomplished by the treatment of methanol saturated with
HCl gas at 0 °C, followed by the charge of NH3 gas. Condensation
of 2 with diethyl acylsuccinate (n = 1) or diethyl 2-acylglutarate
(n = 2) in the presence of sodium ethoxide provided pyrimidin-
4(3H)-one 3. Coupling of 3 with commercially available 4-[20-(N-
triphenylmethyltetrazol-5-yl)phenyl]benzyl bromide (4) in the
presence of lithium hydride in EtOAc–DMF (9:1) led to a mixture
of regioisomers favoring the N-alkylated 5 over the O-alkylated
product (ratio = 98:2), which was easily separated by column
chromatography due to the less polarity of O-alkylated product.
N-alkylated compound 5 was treated with 2.5 N HCl in methanol
at reflux to afford the desired pyrimidin-4(3H)-one derivative 6 con-
taining ester group at C-5. Saponification of 5 with 10% sodium
hydroxide in ethanol solution at room temperature gave the free
carboxylic acid 7. Removal of the trityl group of 7 using 2.5 N HCl
in methanol at reflux afforded the desired pyrimidin-4(3H)-one
derivative 8 containing free carboxyl group at C-5. Coupling of 7
(for n = 1) with amine in the presence of DCC/NMM/HOBt in CHCl3
In line with this approach, we attempted to replace the imidaz-
ole ring of losartan with pyrimidin-4(3H)-one fragment and dis-
covered a high potent and AT1-selective antagonist 12a (Fig. 1:
BR-A-657, Fimasartan), which has been approved for the treatment
of hypertension by the Korean Food and Drug Administration in
September 2010.7–13 Herein, we describe the discovery and early
⇑
Corresponding authors. Tel.: +82 2 961 0726; fax: +82 2 966 3701 (J.Y.L.);
tel.: +82 31 491 2271; fax +82 31 491 5340 (Y.H.C.).
E-mail addresses: yhchi@boryung.co.kr (Y.H. Chi), ljy@khu.ac.kr (J.Y. Lee).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.116

1650
T. W. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1649–1654
Cl
N
O
N
N
OH
NH
N
N
N
N
CO2H
N
N
HO2C
N
N
NH
N
N
Losartan
Telmisartan
Valsartan
R2
R3
O
N
N
N
S
R1
N
N
O
N
N
N
N
NH
NH
N
N
Pyrimidin-4(3H)-one derivatives
12a (BR-A-657, Fimasartan)
tethered with the biphenyl tetrazole
Figure 1. Chemical structures of losartan, telmisartan, valsartan, pyrimidin-4(3H)-one derivatives, and 12a (BR-A-657, Fimasartan).
R2 CO2Et
( )n
R2 CO2Et
( )n
R2
N
( )n
OH
NH
a
b
N
N
N
R1 CN
R1
.HCl
O
R1
Br
N
H
O
R1
N
O
R1
O
NH2
1
2
3 (n = 1-2)
c
d
for 5
+
N
N
N
N
N
N
N
N
N
N
R3
R3
5: R3 = CPh3
7: R3 = CPh3
e
e
N
N
N
N
6: R3 = H
8: R3 = H
N
CPh3
4
f for 7 (n = 1)
R2
R3
N
R2
R3
N
N
R4
N
R4
S
O
R1
N
O
R1
N
O
N
g
for 9
N
N
N
N
N
N
N
N
N
R5
R5
11: R5 = CPh3
12: R5 = H
9: R5 = CPh3
10: R5 = H
e
e
Scheme 1. Reagents and conditions: (a) HCl (gas), MeOH, 0 °C, then NH3 (gas); (b) NaOEt, diethyl acylsuccinate (n = 1) or diethyl 2-acylglutarate (n = 2), EtOH; (c) LiH, EtOAc–
DMF (9:1); (d) 10% NaOH (aq.), MeOH, rt; (e) 2.5 N HCl (aq.), MeOH, reflux; (f) HOBt, NMM, DCC, amine, CHCl3; (g) Lawesson’s reagent, toluene, reflux.
provided amide derivative 9.15 Finally, the removal of the trityl
group of tetrazole 9 using the same condition provided the pyrimi-
din-4(3H)-one derivative 10 containing amido group at C-5. Thiona-
tion of 9 with Lawesson’s reagent in toluene at reflux condition gave

T. W. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1649–1654
1651
Table 1
Biological activity data of selected pyrimidin-4(3H)-one derivatives
R2
N
R3
O
N
R1
N
N
NH
N
R1
R2
R3
Antagonism IC50 (nM)a
Binding assay IC50 (nM)b
ED50 (mg/kg)
d
Entry
6a
6b
6c
6d
8a
8b
8c
n-Bu
n-Bu
n-Bu
Et
n-Bu
n-Pr
n-Bu
Me
Me
n-Pr
Me
Me
Me
Me
CH2CO2Et
4.63
2.51
44.49
30.74
2.63
66.20
130.70
NT
NT
0.108
0.066
NT
NT
0.130
NT
CH2CH2CO2Et
CH2CH2CO2Et
CH2CH2CO2Et
CH2CO2H
CH2CO2H
CH2CH2CO2H
431.00 (3.65%)c
NT
27.13
9.43
306.50 (27.93%)c
0.094
O
Me
10a
10b
n-Bu
n-Bu
Me
Me
2.37
71.67%
NT
0.018
0.089
CH2
CH2
CH2
N
Me
O
O
Et
30.77
N
Et
Et
10c
10d
n-Pr
Me
Me
4.69
1.58
NT
NT
N
Et
O
O
n-Bu
59.24 (76.74%)c
0.056
CH2
CH2
N
N
10e
10f
n-Bu
n-Bu
Me
Me
2.06
2.08
59.28
17.00
0.043
0.023
MeO2C
O
CH2
N
H2NOC
O
10g
10h
n-Bu
n-Bu
Me
Me
2.46
3.59
63.26 (71.67%)c
0.063
NT
CH2
N
N
O
CH2
19.00
N
Me
O
10i
10j
n-Bu
n-Bu
Me
Me
2.76
1.75
9.00
0.038
0.018
CH2
N
N
N
S
O
O
O
23.00
CH2
O
Me
CH2
10k
10l
n-Bu
n-Bu
Me
Me
1.03
3.36
24.00
58.49
0.029
Me
O
CH2CH2
N
0.063
MeO2C
(continued on next page)

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T. W. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1649–1654
Table 1 (continued)
d
Entry
R1
R2
R3
Antagonism IC50 (nM)a
4.06
Binding assay IC50 (nM)b
28.00
ED50 (mg/kg)
O
O
10m
n-Bu
Me
0.204
CH2CH2
N
N
S
10n
10o
10p
n-Bu
n-Bu
n-Bu
Me
Me
Me
5.22
5.86
9.62
60.01
70.52
64.70
0.143
0.206
0.164
CH2CH2
O
O
O
CH2CH2
CH2CH2
N
N
O
Me
CH2CH2
N
10q
n-Bu
Me
13.10
72.56
0.228
O
Me
S
Me
12a (fimasartan)
n-Bu
n-Bu
n-Bu
Me
Me
Me
0.42
1.31
0.13
6.27
0.018
0.023
CH2
N
Me
S
S
Et
12b
CH2
CH2
N
Et
12c
6.11
8.04
0.034
0.336
N
Losartan
61.12
80.00
a
Antagonism of AngII-induced contraction is expressed as IC50 value, which is the concentration of compound which inhibits AngII-induced contractions (100% standard)
in the rabbit thoracic aortic rings by 50%.
b
AngII receptor (AT1)-binding assay. IC50 value is the concentration of compound which inhibits [125I] AngII (0.05 nM) binding to AT1 receptor by 50%.
c
% inhibition of [125I] AngII (0.05 nM) binding to AT1 receptor at 10
lM of compound as a preliminary test.
d
Antagonism of AngII-induced pressor response. ED50 value is the iv administered dose of compound which inhibits AngII-induced pressor response (100% standard) in the
pithed rat model by 50%.
Table 2
Pharmacokinetic parameters (mean S.D) of 12a (BR-A-657-K, Fimasartan) in rats (n = 7) obtained after oral administration of fimasartan (dose, 3 mg/kg)
AUC (ng h/mL)
678.4 224.5
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
Cl/F (mL/min/kg)
Vss/F (L/kg)
241.6 150.8
0.1 0.0
5.3 2.2
84.0 30.0
37.3 15.5
thioamide derivative 11, which was treated with 2.5 N HCl in meth-
anol to give pyrimidin-4(3H)-one derivative 12 containing thioam-
ido group at C-5.
potency) of AT1 receptor in a functional assay. Analysis on the
SAR of compounds containing ester and acid moieties- 6b and 8a
to 6c–d and 8b demonstrated that n-butyl group at R1 and methyl
group at R2 appeared to be favorable to improve in vitro potency,
respectively. In particular, compound containing N,N-dim-
ethylethanethioamido group at R3 position 12a showed sub-nano-
molar potency, which is ca. 145-fold potent than that of the
positive control losartan.
In the first stage to establish in vitro hypotensive effect of syn-
thetic pyrimidin-4(3H)-one derivatives using thoracic aorta ring,
we prepared the descending thoracic aorta which was dissected
from New Zealand white rabbits according to the standard
protocol16–18 and this aorta ring was exposed to AngII (3 nM) for
obtaining maximum value of AngII-induced constriction from a
concentration–contractile response curve. After a resting period,
the aorta ring was treated with the above synthetic compound
and after incubating for 30 min, it was exposed to AngII and then
IC50 value of compound to inhibit the AngII-induced maximum
constriction by 50% was obtained from a concentration–contractile
response curve.19 Among them, compounds exhibiting plausible
activities were selected and listed in Table 1. Losartan was taken
as a positive control drug in the assays. The assay results showed
that most of our compounds except compound 6c–d, 8b and 10q
exhibited potent antagonistic activity (single-digit nanomolar
In the second experimental stage, the synthetic compounds
were also evaluated for their activity to competitively inhibit
[
125I] AngII binding to the AT1 receptor by a conventional ligand-
binding assay using a rat adrenal cortex as described previ-
ously.20,21 The binding affinity is expressed as % inhibition value
at 10
(Table 1), which is the concentration of compound which inhibits
125I] AngII (0.05 nM) binding to AT1 receptor by 50%. Apparent
lM of compound as a preliminary test and/or as IC50 value
[
analysis on the SAR demonstrated that pyrimidin-4(3H)-one deriv-
atives containing thioamido group at R3 position 12a–12c gener-
ally appeared to improve the binding affinity to AT1 receptor

T. W. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1649–1654
1653
than those containing ester, acid, and amide group in our series. In
Acknowledgments
addition, compounds containing thioamido group displayed the
high correlation with the above functional antagonism, whereas
the other compounds did not. Compound 12a also showed sub-
nanomolar binding affinity (IC50 = 0.13 nM) which is ca. 615-fold
potent than losartan (IC50 = 80.00 nM). This affinity correlates well
with its most potent functional antagonism with IC50 value of
0.42 nM in our series. In addition, 12a had no significant effect
This study was sponsored by Boryung Pharmaceutical Co. Ltd,
Seoul, Republic of Korea, and was supported by a grant from the
Korea Health 21 R&D Project, Ministry of Health & Welfare, Repub-
lic of Korea (A070001).
References and notes
on KCl (60 mM)- or noradrenaline (1
(The data is not shown).
lM)-induced contractions
1. Casimiro-Garcia, A.; Filzen, G. F.; Flynn, D.; Bigge, C. F.; Chen, J.; Davis, J. A.;
Dudley, D. A.; Edmunds, J. J.; Esmaeil, N.; Geyer, A.; Heemstra, R. J.; Jalaie, M.;
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VanMeel, J. C. A.; Wienen, W.; Hauel, N. H. J. Med. Chem. 1993, 36, 4040.
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I. J.; Yu, K. S. J. Cardiovasc. Pharmacol. 2012, 59, 84.
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Based on the results of in vitro functional antagonism and
AngII-binding assay, the pithed rat model was applied to evaluate
in vivo hypotension effect of selected pyrimidin-4(3H)-one deriva-
tives using a Gould pressure transducer coupled to a Grass poly-
graph for measuring arterial blood pressure and heart rate.20,22,23
The conscious normotensive rat was treated with AngII (100 ng/
kg, iv) three times at 15 min intervals to establish a reproducible
basal pressor response. Each compound was intravenously admin-
istered and then AngII (100 ng/kg, iv) was administered. ED50 value
of each compound to inhibit AngII-induced maximal pressor
response by 50% was obtained from
a dose-pressure curve
(Table 1). All of tested compounds showed sub-milligram/kg dose
to inhibit AngII-induced pressor response by 50% and also
were more efficacious than losartan. In particular, pyrimidin-
4(3H)-one derivatives containing amide or thioamido group at R3
position 10a, 10j and 12a showed potent inhibitory activities
(ED50 = 0.018 mg/kg) which is ca. 19-fold potent than losartan
(ED50 = 0.336 mg/kg).
10. Shin, K. H.; Kim, T. E.; Kim, S. E.; Lee, M. G.; Song, I. S.; Yoon, S. H.; Cho, J. Y.;
Jang, I. J.; Shin, S. G.; Yu, K. S. J. Cardiovasc. Pharmacol. 2011, 58, 492.
11. Yi, S.; Kim, J. W.; Kim, T. E.; Kim, J.; Jun, Y. K.; Choi, J.; Yoon, S. H.; Cho, J. Y.;
Song, S. H.; Shin, S. G.; Jang, I. J.; Yu, K. S. Int. J. Clin. Pharmacol. Ther. 2011, 49,
321.
Based on these results, 12a was converted into potassium salt
(BR-A-657-K, Fimasartan)24 and further evaluated in two vivo
models: furosemide-treated rats and conscious spontaneously
hypertensive rats as preclinical studies. In furosemide-treated rats,
a high-renin model,25,26 the orally administered 12a (10 mg/kg)
caused immediate decreases in mean arterial pressure, and the ef-
fect was sustained for at least 8 h. Compound 12a produced no ob-
servable alteration in the basal heart rate at these doses (The data
is not shown). In conscious spontaneously hypertensive rats,27,28
the orally administered 12a (10 mg/kg) elicited a significant de-
crease in mean arterial blood pressure with rapid onset. The onset
of the antihypertensive effects was rapid and progressive, occur-
ring 0.5–5 h. The maximal decrease in blood pressure at 10 mg/
kg was reached at 5 h (75%) and the effect was maintained for 24 h.
With regard to toxicity study, maximum tolerated doses
(MTDs) of 12a for single oral administration were higher than
2,000 mg/kg in mice and rats. Pharmacokinetic study of 12a were
performed in male Sprague–Dawley rats (n = 7) at a dose of 3 mg/
kg and its pharmacokinetic parameters were summarized in Table
2.13 In these studies, most of 12a was excreted in feces as an
unchanged parent drugs after oral administration. Absorption
and excretion pattern observed after repeated dose administration
for 21 days in rats revealed that there was no accumulation
tendency.
In summary, a series of pyrimidin-4(3H)-one derivatives as a
losartan analogue were synthesized and biologically evaluated
for a novel class of non-peptidic AT1 receptor antagonists. The bio-
isosteric replacement of the imidazole part of losartan with pyrim-
idin-4(3H)-one fragment led to the of a highly potent and orally
active AT1 selective antagonist 12a (BR-A-657, Fimasartan) having
excellent in vivo potency in rats compared to losartan. The study
results revealed that the bioisosteric pyrimidin-4(3H)-one ring
bearing thioamido group may provide an extra binding interaction
in angiotensin II receptor which attribute to their higher potency
and stronger efficacy than losartan. The structural novelty and
excellent pharmacological properties of 12a (BR-A-657, Fimasar-
tan) made it a new medicine for antihypertensive therapy after
clinical trials.7–13
12. Yi, S.; Kim, T. E.; Yoon, S. H.; Cho, J. Y.; Shin, S. G.; Jang, I. J.; Yu, K. S. J. Cardiovasc.
Pharmacol. 2011, 57, 682.
13. Shin, B. S.; Kim, T. H.; Paik, S. H.; Chi, Y. H.; Lee, J. H.; Tan, H. K.; Choi, Y.; Kim,
M.; Yoo, S. D. Biomed. Chromatogr. 2011, 25, 1208.
14. Ellingboe, J. W.; Nikaido, M.; Bagli, J. F. European Patent 539086 A2, 1993.
15. Kaldor, S. W.; Hammond, M.; Dressman, B. A.; Labus, J. M.; Chadwell, F. W.;
Kline, A. D.; Heinz, B. A. Bioorg. Med. Chem. Lett. 1995, 5, 2021.
16. Robertson, M. J.; Cunoosamy, M. P.; Clark, K. L. Br. J. Pharmacol. 1992, 106, 166.
17. Almansa, C.; Gomez, L. A.; Cavalcanti, F. L.; Arriba, A. F.; Rodriguez, R.; Carceller,
E.; Garcia-Rafanell, J.; Forn, J. J. Med. Chem. 1996, 39, 2197.
18. Xu, J. Y.; Zeng, Y.; Ran, Q.; Wei, Z.; Bi, Y.; He, Q. H.; Wang, Q. J.; Hu, S.;
Zhang, J.; Tang, M. Y.; Hua, W. Y.; Wu, X. Mi. Bioorg. Med. Chem. Lett. 2007,
17, 2921.
19. The descending thoracic aorta was quickly removed from New Zealand White
rabbits (2.0–2.5 kg; Samtako, South Korea) and cleaned of fats and connective
tissues. Rings of 3–4 mm in length were suspended in 10-mL organ baths filled
with an oxygenated (95% O2 and 5% CO2) and pre-warmed (37 °C) physiological
Krebs bicarbonate solution of the following composition (mM): NaCl 118.0, KCl
4.7, MgSO4 1.2, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25 and glucose 11.0 (pH 7.4).
Isometric contraction was measured on physiograph (Grass model-7
polygraph, Grass Instrument Company, Quincy, Massachusetts, USA) with
force transducer (Grass FT03, Grass instruments, Quincy, Massachusetts, USA).
They were stretched to a 2 g of resting tension and allowed to equilibrate for
60 min during which time they were washed repeatedly with Krebs
bicarbonate solution and the tension readjusted. The tissues were exposed to
AngII to verify responsiveness to a vasoconstrictor. Following washings and
recovery of the basal tension, the first control cumulative concentration–
contractile response curve for AngII was obtained. The tissues were left to
recover baseline tension with repeated washing. After test substances were
applied, each tissue was incubated for 30 min and then exposed to AngII or
vasoconstrictors again to obtain the second cumulative concentration–
contractile response curve.
20. Yanagisawa, H.; Amemiya, Y.; Kanazaki, T.; Shimoji, Y.; Fujimoto, K.; Kitahara,
Y.; Sada, T.; Mizuno, M.; Ikeda, M.; Miyamoto, S.; Furukawa, Y.; Koike, H. J. Med.
Chem. 1996, 39, 323.
21. Rat adrenal membranes from male Sprague–Dawley rats (200–250 g) were
dissected out after cervical dislocation and rinsed with cold sucrose buffer
(0.2 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4). The medulla was
removed and the cortices were homogenized by use of a Potter homogenizer.
The homogenate was spun at 3,000 g for 10 min. The supernatant was
centrifuged at 10,000 g for 15 min and at 100,000 g for 60 min. The final
supernatant was discarded and the pellets were stored at ꢀ20 °C. The stored
pellets were suspended in incubation buffer (50 mM Tris–HC1, 10 mM MgC12,
0.25% BSA, pH 7.4) before performing experiments. [125I]AngII were added to
the experiments protein (60–70 lg protein/assay) and incubated for 60 min at
25 °C. AngII and test compounds were dissolved in dimethylsulfoxide. The
concentrations of test compounds were 3 ꢁ 10ꢀ12–10ꢀ6 M. The incubation was
terminated by rapid filtration through Whatman GF/B filter. Filters were

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T. W. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1649–1654
washed twice in cold incubation buffer. The radioactivity bound to the filter
was counted in a -counter. Binding data were analysed by a non-linear least-
25. Wong, P. C.; Hart, S. D.; Chiu, A. T.; Herblin, W. F.; Carini, D. J.; Smith, R. D.;
c
Wexler, R. R.; Timmermans, P. B. M. W. M. J. Pharmacol. Exp. Ther. 1991, 259,
square curve fitting method using SigmaPlot 8.0 (SPSS Inc.). IC50 was
calculated.
861.
26. The arterial catheter was connected to a Gould pressure transducer coupled to
22. Kubo, K.; Inada, Y.; Kohara, Y.; Sugiura, Y.; Ojima, M.; Itoh, K.; Furukawa, Y.;
Nishikawa, K.; Naka, T. J. Med. Chem. 1993, 36, 1772.
a Grass polygraph, and mean arterial pressure and heart rate were
continuously monitored. Animals were treated with furosemide (10 mg/kg
subcutaneously) at 20 and 2 h before the experiment. Food and water intake
was withdrawn for 24 h after starting furosemide treatment. After blood
pressure had stabilized for about 60 min, experiments were initiated by
administering 12a (10 mg/kg) orally. Arterial pressure and heart rate were
monitored at 20, 40, 60 min and every 30 min intervals thereafter to 8 h.
27. Kusumoto, K.; Igata, H.; Ojima, M.; Tsuboi, A.; Imanishi, M.; Yamaguchi, F.;
Sakamoto, H.; Kuroita, T.; Kawaguchi, N.; Nishigaki, N.; Nagaya, H. Eur. J.
Pharmacol. 2011, 669, 84.
28. After acclimatization for 10 weeks, SHRs were preheated in a chamber at 37 °C
for 10 min, then placed in plastic restrainers. A cuff with a pulse sensor was
attached to the tail. SHRs were allowed to habituate to this procedure for
7 days before experiments were performed. Adult male SHRs that had mean
blood pressure of less than 140 or more than 220 were excluded from this
study. SBP, DBP, MBP and HR values were recorded on a CODA-6 SYSTEM
(CODA-6, Kent Scientific, Torrington, CT) with heating and were averaged from
at least three consecutive readings obtained from each rat. Compound 12a
(10 mg/kg) was administered orally after stabilization. The blood pressure
measurements were conducted at 30 min pre dose and at 30 min, 1.5, 3, 5, 8
and 24 h following treatment.
23. In the pithed rat model, endogenously generated AngII can enhance
sympathetic neurotransmission by acting on AngII type 1 (AT1) receptors
that are located on sympathetic nerve terminals. The arterial catheter was
connected to a Gould pressure transducer coupled to a Grass polygraph for
measuring blood pressure and heart rate. After blood pressure was stabilized
about 60 min, AngII (100 ng/kg, iv) was treated three times at 15 min intervals
to establish
a reproducible basal pressor response. Test substance was
intravenously administered and then AngII was administered. Maximal
inhibition of AngII-pressor response was determined.
24. Spectral data of 12a (BR-A-657-K, fimasartan potassium trihydrate): 1H NMR
(500 MHz, DMSO-d6) d 7.54–7.27 (4H, m), 7.07–6.94 (4H, m), 5.23 (2H, s), 3.78
(1H, s), 3.44 (3H, s), 3.40 (3H, s), 2.61 (2H, t, J = 7.5 Hz), 2.16 (3H, s), 1.58–1.55
(2H, m), 1.30–1.26 (2H, m), 0.82 (3H, t, J = 7.5 Hz); 13C NMR (125 MHz, DMSO-
d6) d 199.91, 162.56, 161.40, 159.85, 159.29, 141.63, 140.56, 134.90, 133.28,
131.19, 130.69, 130.00, 127.97, 127.35, 126.09, 119.72, 46.08, 44.97, 42.14,
38.90, 34.07, 28.94, 22.48, 22.38, 14.40; IR (KBr) 3300 (broad), 2920, 1647,
1538, 1359, 1105, 754 cmꢀ1; UV kmax 262 nm; Low-resolution MS (ESI) m/z 500
[MꢀH]; Anal. Calcd for C27H36KN7O4S: C, 54.61; H, 6.11; N, 16.51; S, 5.40.
Found: C, 54.63; H, 6.08; N, 16.66; S, 5.47.