Synthesis and pharmacological activity of the metabolites of Pratosartan

Article abstract of DOI:10.1248/cpb.54.782

Three hydroxylated metabolites of 2-propyl-3-[2′-(1H-tetrazol-5-yl) biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (Pratosartan), which is a selective angiotensin II receptor antagonist, were synthesized in confirmation of their struct

Full text of DOI:10.1248/cpb.54.782

782
Chem. Pharm. Bull. 54(6) 782—787 (2006)
Vol. 54, No. 6
Synthesis and Pharmacological Activity of the Metabolites of Pratosartan
Motoharu SONEGAWA,* Hiroaki SUZAKA, Akira TOMIYAMA, and Tsuyoshi TOMIYAMA
Kotobuki Research Laboratories, Kotobuki Seiyaku Company, Ltd.; 6351 Sakaki-Machi, Nagano 389–0697, Japan.
Received November 4, 2005; accepted February 28, 2006; published online March 2, 2006
Three hydroxylated metabolites of 2-propyl-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-
3H-cycloheptimidazol-4-one (Pratosartan), which is a selective angiotensin II receptor antagonist, were synthe-
sized in confirmation of their structures and in studies of their pharmacological properties. An MTPA ester of
1
the human main metabolite was identified with the synthesized compound by comparing H-NMR spectra, MS
spectra, and HPLC retention time. The structure of the human main metabolite was confirmed to be (S)-(ꢁ)-2-
(1-hydroxypropyl)-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one
((S)-(ꢁ)-1). Also, the rat main metabolites were confirmed to be 8-hydroxylated compound (2) and 5-hydroxy-
lated compound (3). These metabolites showed lower antagonistic activity than that of the parent compound.
Key words Pratosartan; metabolite; improved Mosher’s method; absolute configuration
In the previous paper,1) we reported the design and synthe- Chemistry
sis of Pratosartan (Chart 1), which is an orally active an-
Metabolite 1 was synthesized as shown in Chart 2. Protec-
giotensin II (AII) antagonist, exhibiting selective and potent tion of 2-propyl-3H-cycloheptimidazol-4-one 43,4) with
antagonistic activity to AT1 subtype. Furthermore, long dura- chloromethyl methyl ether (MOMCl) gave 5, which was
tion was shown in various animal models in vivo experiment. treated with N-bromosuccinimide (NBS) to give the bromi-
Clinical trials of Pratosartan are now in progress as an anti- nated product 6. The bromo group was converted to an ace-
hypertensive agent.
toxy group using sodium acetate to yield 7. Removal of the
Clarification of the possible metabolites and their thera- MOM group and subsequent ester exchange of the acetate
peutic or toxic effects during the development process of new gave the corresponding alcohol 9. Hydrogenation of 9 pro-
drugs is mandatory. For this purpose, we have tried to syn- vided 10, and it was alkylated with 115) to afford the desired
thesize possible metabolites in human and rat to identify intermediate compound 12. N-Alkylation of 3H-cyclohepti-
their structures and activities. In a pharmacokinetic study of midazol-4-one in the presence of K2CO3 in N,N-dimethyl-
Pratosartan, three hydroxylated metabolites were observed as formamide (DMF) gave an N3-alkylated compound prefer-
major metabolites in human and rat after oral administration. entially similarly to the biphasic reaction condition6) and the
These structures were characterized on the basis of MS and regioisomer was separated easily by silica gel column chro-
NMR spectra. Metabolite 1 isolated from human urine was matography. The N-benzylic proton of compound 12 and 20
estimated as a hydroxylated metabolite at the propyl side was observed at lower field characteristically (0.4—0.5 ppm)
chain of Pratosartan. A similar hydroxlated metabolite was compared to their regioisomers, which was caused by the
also reported for Losartan.2) Metabolite 2 and 3 isolated from anisotropic effect of the C4-carbonyl group. For the purpose
rat bile were estimated as 8- and 5-hydroxylated metabolites of optical resolution, the racemic trityl compound 12 was
on the cycloheptane ring of Pratosartan, respectively. In this converted to a-methoxy-a-(trifluoromethyl)phenylacetate
paper, we describe the synthesis of these metabolites to iden- (MTPA) 13 with (ꢀ)-MTPA-Cl.7) Each diastereomer could
tify their structures and to study their pharmacological prop- be separated by silica gel column chromatography. Hydroly-
erties.
sis of the isolated single isomer 13a gave an optically active
hydroxy intermediate, and finally (ꢀ)-1 was obtained by the
successive deprotection of MTPA and trityl groups. Enan-
tiomer (ꢁ)-1 was obtained from 13b in the same manner.
In order to confirm the structure, the human main metabo-
lite was led to MTPA ester (Chart 3). The human main
metabolite was protected with trityl chloride to give trityl
compound 12, and then it was converted to MTPA esters 13b
and 13c as a single isomer with (ꢀ)-MTPA-Cl and (ꢁ)-
MTPA-Cl, respectively.
The metabolite 2 was prepared as shown in Chart 4. The
hydroxy group of compound 148) was oxidized by manganese
dioxide to give aldehyde. Alkylation of aldehyde with al-
lyltrimethylsilane gave the corresponding alcohol,9) which
was protected using tert-butyldimethylchlorosilane to yield
15. An intramolecular cyclization was accomplished by
the Kulinkovich-type reaction.10) Reaction of 15 with a
TiCl(OiPr)3/iPrMgCl reagent resulted in a tandem intramole-
cular nucleophilic acyl substitution and intramolecular car-
Chart 1. Structure of 1—3
∗ To whom correspondence should be addressed. e-mail: ktb-g121@kotobuki-pharm.co.jp
© 2006 Pharmaceutical Society of Japan

June 2006
783
Chart 2. Preparation of (R)-(ꢀ)-1 and Metabolite (S)-(ꢁ)-1
Chart 3. Preparation of 13b and 13c
Chart 4. Preparation of Metabolite 2
bonyl addition reaction to give 16. Ring expansion was tion of the olefin were carried out concurrently with catalytic
achieved using ferric chloride in pyridine,11) followed by palladium carbon under hydrogen to give 19. Alkylation of
treatment with sodium acetate to yield dihydrocycloheptimi- 19 with 11 gave the corresponding biphenyl compound 20,
dazolone 18. Deprotection of the N-benzyl group and reduc- which was deprotected with tetrabutylammonium fluoride

784
Vol. 54, No. 6
Chart 5. Preparation of Metabolite 3
Chart 6. Dd Values (ppm) Obtained by d13aꢁd13b
Chart 7. Dd Values (ppm) Obtained by d13cꢁd13b
and aqueous acetic acid to give metabolite 2.
Table 1. Potencies of Pratosartan and Its Metabolites on Angiotensin II-
Induced Contraction in Isolated Rabbit Thoracic Aorta
Metabolite 3 was prepared by the method shown in Chart
5. The tetrazole moiety of Pratosartan was protected using
trityl chloride to give 22, which was treated with lithium di-
isopropylamide and then MoO5·Py·HMPA (MoOPH) to
yield a-hydroxy compound 23.12) Finally, compound 23 was
deprotected with 10% hydrochloric acid to afford metabolite
3.
Compounds
pA2
(S)-1
(R)-1
2
3
7.35
5.79
8.83
9.32
9.75
Pratosartan
Results and Discussion
The chemical structure of the human main metabolite was
confirmed by comparison of the MTPA ester 13 and it was
shown to be identical with the synthetic compound 13b on compound.
1
the basis of HPLC, MS and H-NMR spectroscopy. As a re-
Furthermore, the enantiomer (R)-1 shows obviously lower
sult, the human main metabolite was confirmed to be (ꢁ)-1. activity. It is considered that the hydroxylation to this hy-
On the other hand, rat main metabolites were confirmed to be drophobic part of Pratosartan caused loss of the activity. One
2 and 3 by direct comparison with synthetic compounds on of the rat main metabolites, 2 shows one order lower activity
the basis of HPLC, MS and 1H-NMR spectroscopy.
in comparison with the parent compound. On the other hand,
The human main metabolite gave a single isomer 13b and another rat main metabolite 3 shows the most potent activity
13c with (ꢀ)-MTPA-Cl and (ꢁ)-MTPA-Cl, respectively among the synthesized metabolites (1—3). As a whole, these
(Chart 3). Therefore, the human main metabolite was shown hydroxylated metabolites were found to have lower antago-
as an optically active compound. Accordingly, the absolute nistic activity in rabbit aorta than the parent compound as ob-
configuration of metabolite 1 was determined by the im- served in other sartans.4,16,17)
proved Mosher’s method.13,14) As shown in Chart 6, proton
signals at the N-benzylic position and the 8-position in com- Conclusion
pound 13b were observed at higher fields compared to those
Three hydroxylated metabolites of Pratosartan were syn-
of compound 13a (Dd: positive), while the signals due to 2- thesized. Metabolites 1 and 3 were synthesized from the syn-
and 3-positions of the propyl group in 13b were observed at thetic intermediate of Pratosartan, or Pratosartan itself, re-
lower fields compared to those of 13a (Dd: negative). On the spectively. Metabolite 2 was synthesized from another imida-
basis of this evidence, the absolute configuration of 13b was zole compound by the ring expansion reaction. The human
determined to be (S)-13. Also, the identical absolute configu- main metabolite was confirmed to be (S)-1 on the basis of
1
ration was shown by comparison with 13b and 13c (Chart 7). HPLC retention time, MS and H-NMR spectroscopic data
From these results, the human main metabolite was deter- using the improved Mosher’s method. Also, the rat main
mined to be (S)-1.
metabolites were determined to be 2 and 3. All these metabo-
The synthesized metabolites were evaluated for antagonis- lites showed lower antagonistic activity than that of the par-
tic activity against angiotensin II-induced contraction in iso- ent compound.
lated rabbit thoracic aorta.15) The obtained pA2 values of
Experimental
these compounds are listed in Table 1 in comparison with the
Melting points were determined on Yamato melting point apparatus and
data of the parent compound. The human main metabolite
(S)-1 shows lower activity in comparison with the parent
are uncorrected. Infrared (IR) spectra were recorded on a Hitachi 270-30.
High performance liquid chromatography (HPLC) was carried out on Hitach

June 2006
785
chromatography over silica gel eluting CHCl3–EtOH (10 : 1 by volume), to
give title compound as thin yellow oil (2.67 g, 80.5% yield). 1H-NMR
(CDCl3) d: 1.11 (3H, t, Jꢃ7.2 Hz), 2.00—2.24 (2H, m), 5.09 (1H, t-like),
5.56 (1H, br s), 7.21 (1H, dd, Jꢃ8.8, 10.8 Hz), 7.38 (1H, d, Jꢃ12.0 Hz), 7.56
(1H, dd, Jꢃ8.8, 12.0 Hz), 7.97 (1H, d, Jꢃ10.8 Hz). MS m/z: 204 (Mꢀ), 175
(B.P.), 147, 119, 92. IR (neat) cmꢁ1: 3358, 3070, 1620, 1572, 1515.
655A-11 using solvents and columns (silica 60, 25 cmꢂ4.6 mmF, n-
hexane/EtOAcꢃ95/5) with flow rate of 1.0 ml/min, and peaks were observed
using an UV detector (260 nm). Specific optical rotation [a]2D0 was measured
by HORIBA SEPA-200 using a cell (50 mꢂ5 mmF). Proton nuclear mag-
netic resonance (1H-NMR) spectra were measured at 400 MHz on a JEOL
EX-400 Fourier-transform NMR spectrometer. Chemical shifts are quoted in
part per million (ppm) with tetramethylsilane as an internal standard. Cou-
pling constants (J) are given in Hz. The following abbreviations are used: s,
singlet; d, doublet; t, triplet; q, quartet; br s, broad singlet; dd, doublet of
doublet; m, multiplet. Mass spectra (MS) were taken on a Hitachi M-80B
spectrometer and ESI-MS spectra (MS (ESI)) were taken on a Finnigan TSQ
700 spectrometer. Elemental analyses were within 0.4% of theoretical values
and were determined by Hitachi 026 CHN analyzer. Rf values and prepara-
tive TLC were done on silica gel 60F254 (Merck). For column chromatogra-
phy, silica gel (Merck, Kieselgel 60, 70—230 mesh) was used.
2-(1-Hydroxypropyl)-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one
(10) To a solution of 9 (2.00 g, 9.80 mmol) in MeOH (20 ml) was added
5% Pd on carbon (containing 50% water, 0.20 g) and the mixture was stirred
at 40 °C for 14 h under an atmosphere of hydrogen (1 atm). After filtration of
the catalyst, the filtrate was concentrated in vacuo. The residue was sub-
jected to chromatography over silica gel eluting EtOAc, to give title com-
pound as colorless oil (1.86 g, 91.2% yield). 1H-NMR (CDCl3) d: 1.00 (3H,
t, Jꢃ7.2 Hz), 1.79—2.05 (6H, m), 2.71—2.89 (2H, m), 2.95—3.01 (2H, m),
4.82 (1H, t, Jꢃ4.4 Hz). MS m/z: 208 (Mꢀ), 179 (B.P.), 147. IR (neat) cmꢁ1
:
3330, 2926, 1626, 1548, 1455, 1311, 1230.
1(or 3)-Methoxymethyl-2-propyl-1(or 3)H-cycloheptimidazol-4-one
(5) To a stirred solution of 2-propyl-3H-cycloheptimidazol-4-one (4)
(10.0 g, 53.0 mmol) in N,N-dimethylformamide (DMF) (30 ml) was added
NaH (2.98 g, 50% dispersion in mineral oil, 62.0 mmol) at 0 °C. The reac-
tion mixture was stirred for 0.5 h. To this mixture was added chloromethyl
methyl ether (5.0 ml) at 0 °C, and then allowed to warm to room tempera-
ture, and stirred for a further 2 h. After the addition of saturated aqueous
NH4Cl, the reaction mixture was extracted with EtOAc. The organic layer
was washed with H2O and then brine, dried over MgSO4. The organic layer
was concentrated in vacuo. The residue was subjected to chromatography
over silica gel eluting n-hexane–EtOAc (3 : 1 by volume), to give title com-
2-(1-Hydroxypropyl)-3-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-yl-
methyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (12) To a mix-
ture of 10 (1.80 g, 8.64 mmol), 5-(4ꢄ-bromomethylbiphenyl-2-yl)-1-trityl-
1H-tetrazole (11) (5.30 g, 9.51 mmol) and DMF (36 ml) was added K2CO3
(1.31 g, 9.51 mmol). The reaction mixture was stirred for 14 h at room tem-
perature, and then poured into ice-water (200 g). The mixture was extracted
with EtOAc, and the extract was washed with H2O and then brine, dried over
Na2SO4. The organic layer was concentrated in vacuo. The residue was sub-
jected to chromatography over silica gel eluting n-hexane–EtOAc (1 : 1 by
volume), to give title compound as colorless solid (3.66 g, 61.9% yield). mp:
1
1
69—71 °C. H-NMR (CDCl3) d: 0.84 (3H, t, Jꢃ7.2 Hz), 1.73—1.78 (2H,
pound as thin yellow oil (7.50 g, 60.9% yield). H-NMR (CDCl3) d: 1.06
m), 1.84—1.87 (4H, m), 2.60—2.63 (2H, m), 2.97—3.00 (2H, m), 4.51 (1H,
m), 5.52 (2H, s), 6.82 (2H, d, Jꢃ7.6 Hz), 6.93—6.95 (5H, m), 7.05 (2H, d,
Jꢃ7.6 Hz), 7.24—7.36 (10H, m), 7.41—7.49 (3H, m), 7.85 (1H, d,
Jꢃ7.6 Hz). MS (ESI): m/z 685 [Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 3450, 2950, 1638,
1449.
(3H, t, Jꢃ7.2 Hz), 1.82—1.99 (2H, m), 2.90 (2H, t, Jꢃ7.6 Hz), 3.39 (3H, s),
6.18 (2H, s), 6.96 (1H, dd, Jꢃ8.8, 11.2 Hz), 7.15—7.36 (2H, m), 7.72 (1H,
d, Jꢃ11.2 Hz). MS m/z: 232 (Mꢀ), 217, 189 (B.P.). IR (neat) cmꢁ1: 2956,
1578, 1467, 1120.
2-(1-Bromopropyl)-1(or 3)-methoxymethyl-1(or 3)H-cycloheptimida-
zol-4-one (6) A mixture of 5 (7.40 g, 31.9 mmol), N-bromosuccinimide
(6.24 g, 35.1 mmol), 2,2ꢄ-azobis(isobutyronitrile) (0.01 g), DMF (0.1 ml) and
CCl4 (80 ml) was refluxed for 2 h. After cooling, the reaction mixture was
filtered and the filtrate was concentrated in vacuo. The residue was subjected
to chromatography over silica gel eluting n-hexane–EtOAc (3 : 1 by volume),
1-[8-Oxo-1-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-
1,4,5,6,7,8-hexahydrocycloheptimidazol-2-yl]propyl a-Methoxy-(a-tri-
fluoromethyl)phenylacetate (13a, b) A solution of (S)-(ꢀ)-a-methoxy-
a-(trifluoromethyl)phenylacetyl chloride (1.00 g, 3.96 mmol) in CH2Cl2
(10 ml) was added dropwise to 12 (2.71 g, 3.96 mmol) and triethylamine
(0.55 ml, 3.96 mmol) in CH2Cl2 (30 ml) at 0 °C. The reaction mixture was al-
lowed to warm to room temperature and stirred for 14 h, and then poured
into ice-water (100 g). The mixture was extracted with CH2Cl2 and the ex-
tract was washed with brine, dried over Na2SO4. The organic layer was con-
centrated in vacuo. The residue was subjected to chromatography over silica
gel eluting n-hexane–EtOAc (8 : 1 by volume), to give 13a as colorless solid
(1.51 g, 42.4% yield), and then eluting n-hexane–EtOAc (6 : 1 by volume), to
1
to give title compound as thin yellow oil (8.90 g, 89.8% yield). H-NMR
(CDCl3) d: 0.94 (3H, t, Jꢃ7.2 Hz), 1.82—1.99 (2H, m), 3.39 (3H, s), 4.76
(1H, t, Jꢃ7.6 Hz), 6.18 (2H, s), 6.96 (1H, dd, Jꢃ8.8, 11.2 Hz), 7.15—7.36
(2H, m), 7.72 (1H, d, Jꢃ11.2 Hz). MS m/z: 312 (Mꢀꢀ2), 300 (Mꢀ), 297,
295, 230 (B.P.). IR (neat) cmꢁ1: 3070, 1617, 1569, 1269.
1-(1(or 3)-Methoxymethyl-8-oxo-1,8-dihydro-cycloheptimidazol-2-
yl)propyl Acetate (7) To a stirred solution of 6 (8.80 g, 28.3 mmol) in
DMF (40 ml) was added sodium acetate (6.96 g, 84.8 mmol) at room temper-
ature. The reaction mixture was stirred at 50 °C for 7 h. After cooling, the re-
action mixture was poured into ice-water (100 g), and extracted with EtOAc.
The organic layer was washed with H2O and then brine, dried over Na2SO4.
The organic layer was concentrated in vacuo. The residue was subjected to
chromatography over silica gel eluting n-hexane–EtOAc (1 : 1 by volume), to
give title compound as thin yellow oil (7.03 g, 85.6% yield).1H-NMR
(CDCl3) d: 1.00 (3H, t, Jꢃ8.0 Hz), 2.10 (3H, s), 2.10—2.21 (2H, m), 3.40
(3H, s), 5.84 (1H, t, Jꢃ7.2 Hz), 6.00 (1H, d, Jꢃ10.0 Hz), 6.73 (1H, d,
Jꢃ10.0 Hz), 6.96 (1H, d, Jꢃ12.4 Hz), 7.14—7.27 (2H, m), 7.74 (1H, d,
Jꢃ11.2 Hz). MS m/z: 290 (Mꢀ), 275, 215, 187 (B.P.). IR (neat) cmꢁ1: 2968,
2932, 1734, 1632, 1236.
1-(8-Oxo-1,8-dihydro-cycloheptimidazol-2-yl)propyl Acetate (8) To a
stirred solution of 7 (6.00 g, 20.7 mmol) in EtOH (35 ml) was added satu-
rated ethanolic HCl (5 ml) at room temperature. The reaction mixture was
stirred for 0.5 h at ambient temperature. The reaction mixture was concen-
trated in vacuo, and residue was diluted with H2O. The mixture was neutral-
ized with saturated aqueous NaHCO3 and extracted with EtOAc (3ꢂ100 ml).
The combined extracts layer was washed with brine, dried over Na2SO4. The
organic layer was concentrated in vacuo. The residue was subjected to chro-
matography over silica gel eluting EtOAc, to give title compound as thin yel-
low solid (4.00 g, 78.3% yield). mp: 158—160 °C. 1H-NMR (CDCl3) d: 1.01
(3H, t, Jꢃ7.2 Hz), 2.16 (3H, s), 2.12—2.25 (2H, m), 6.04 (1H, t, Jꢃ7.2 Hz),
7.07 (1H, dd, Jꢃ8.8, 10.8 Hz), 7.29 (1H, d, Jꢃ12.0 Hz), 7.44 (1H, dd,
Jꢃ8.8, 12.0 Hz), 7.97 (1H, d, Jꢃ10.8 Hz). MS m/z: 246 (Mꢀ), 203 (B.P.),
175. IR (KBr) cmꢁ1: 3034, 1743, 1617, 1563, 1515, 1236.
1
give 13b as colorless solid (1.32 g, 37.0% yield). 13a: mp: 78—80 °C. H-
NMR (CDCl3) d: 0.53 (3H, t, Jꢃ7.2 Hz), 1.50—1.59 (1H, m), 1.85—2.00
(5H, m), 2.70 (2H, m), 3.03 (2H, m), 3.51 (3H, s), 5.33 (1H, d, Jꢃ16.0 Hz),
5.61—5.65 (1H, m), 6.14 (1H, d, Jꢃ16.0 Hz), 6.92 (2H, d, Jꢃ8.0 Hz),
6.93—7.00 (5H, m), 7.09 (2H, d, Jꢃ8.0 Hz), 7.22—7.55 (18H, m), 7.86
(1H, d, Jꢃ7.6 Hz). MS (ESI): m/z: 901 [Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 2962,
1
1743, 1644, 1449, 1233, 1167. 13b: mp: 80—81 °C. H-NMR (CDCl3) d:
0.61 (3H, t, Jꢃ7.2 Hz), 1.66 (1H, m), 1.80—2.00 (5H, m), 2.70 (2H, m),
2.96 (2H, m), 3.52 (3H, s), 5.23 (1H, d, Jꢃ16.0 Hz), 5.65—5.68 (1H, m),
6.04 (1H, d, Jꢃ16.0 Hz), 6.88 (2H, d, Jꢃ8.0 Hz), 6.95—6.98 (5H, m), 7.08
(2H, d, Jꢃ8.0 Hz), 7.25—7.54 (18H, m), 7.85 (1H, dd, Jꢃ2.0, 7.6 Hz). MS
(ESI): m/z 901 [Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 2920, 1740, 1647, 1449, 1257,
1117.
(S)-(ꢁ)-2-(1-Hydroxypropyl)-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-yl-
methyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one ((S)-(ꢁ)-1) To
a stirred solution of 13b (0.90 g, 1.00 mmol) in tetrahydrofuran (THF) (6 ml)
was added MeOH (6 ml) and 3 M aqueous NaOH (9 ml) and stirred for 14 h
at room temperature. The reaction mixture was concentrated in vacuo, and
added water (10 ml). The mixture was extracted with chloroform (2ꢂ20 ml).
The combined extracts layer was washed with brine, dried over MgSO4. The
organic layer was concentrated in vacuo. The residue was dissolved with
THF (1.2 ml). To this solution was added acetic acid (3 ml) and H2O
(1.2 ml). The reaction mixture was stirred for 14 h at room temperature, and
then concentrated in vacuo. The residue was subjected to chromatography
over silica gel eluting CHCl3–MeOH (10 : 1 by volume), to give title com-
pound as colorless solid (0.38 g, 86.0% yield). mp: 93—94 °C. [a]2D0
ꢃ
ꢁ2.8° (cꢃ1.0, MeOH). 1H-NMR (CDCl3) d: 0.93 (3H, t, Jꢃ7.2 Hz), 1.80—
1.90 (6H, m), 2.65 (2H, m), 2.83 (2H, m), 4.58 (1H, t, Jꢃ6.8 Hz), 5.34 (1H,
d, Jꢃ16.8 Hz), 5.89 (1H, d, Jꢃ16.8 Hz), 6.89 (2H, d, Jꢃ8.0 Hz), 7.00 (2H, d,
Jꢃ8.0 Hz), 7.34 (1H, s), 7.35 (1H, d, Jꢃ8.0 Hz), 7.45 (1H, t, Jꢃ8.0 Hz),
2-(1-Hydroxypropyl)-3H-cycloheptimidazol-4-one (9) To a solution
of 8 (4.00 g, 16.2 mmol) in MeOH (30 ml) was added 28% NaOMe (6.3 ml,
MeOH solution, 32.5 mmol). The reaction mixture was stirred for 20 min at
room temperature, and concentrated in vacuo. The residue was subjected to

786
Vol. 54, No. 6
7.54 (1H, t, Jꢃ8.0 Hz), 7.91 (1H, d, Jꢃ8.0 Hz). MS (ESI): m/z 443 [Mꢀ1]ꢀ.
Anal. Calcd for C25H26N6O2: C, 67.86; H, 5.92; N, 18.99. Found: C, 67.80;
H, 6.03; N, 18.93. IR (KBr) cmꢁ1: 3358, 2926, 1638, 1533, 1464, 756.
(R)-(ꢂ)-2-(1-Hydroxypropyl)-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-yl-
methyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one ((R)-(ꢂ)-1)
The title compound was prepared using a procedure similar to that described
(S)-(ꢁ)-1 from the corresponding ester 13a as colorless solid. mp: 93—
95 °C. [a]2D0ꢃꢁ2.8° (cꢃ1.0, MeOH). 1H-NMR (CDCl3) d: 0.86 (3H, t,
Jꢃ7.2 Hz), 1.78—1.94 (6H, m), 2.64 (2H, t, Jꢃ6.4 Hz), 2.83 (2H, m), 4.58
(1H, t, Jꢃ6.8 Hz), 5.40 (1H, d, Jꢃ16.4 Hz), 5.83 (1H, d, Jꢃ16.4 Hz), 6.93
(2H, d, Jꢃ8.0 Hz), 7.09 (2H, d, Jꢃ8.0 Hz), 7.36 (1H, s), 7.42 (1H, d,
Jꢃ8.0 Hz), 7.50 (1H, t, Jꢃ8.0 Hz), 7.57 (1H, t, Jꢃ8.0 Hz), 7.99 (1H, d,
Jꢃ8.0 Hz). MS (ESI): m/z 443 [Mꢀ1]ꢀ. Anal. Calcd for C25H26N6O2: C,
67.86; H, 5.92; N, 18.99. Found: C, 67.93; H, 6.00; N, 18.80. IR (KBr)
cmꢁ1: 3358, 2926, 1638, 1533, 1464, 756.
action mixture was poured into a 1 N hydrochloric acid and extracted with
EtOAc. The organic layer was washed with H2O and the brine, dried over
Na2SO4. The organic layer was concentrated in vacuo. The residue was dis-
solved in MeOH (110 ml). To the solution was added sodium acetate (18.6 g,
227 mmol) and refluxed for 1 h. The reaction mixture was concentrated in
vacuo. To the residue was added H2O and the mixture was extracted with
EtOAc. The organic layer was washed with brine, dried over Na2SO4. The
organic layer was concentrated in vacuo. The residue was subjected to chro-
matography over silica gel eluting n-hexane–EtOAc (4 : 1 by volume), to
give title compound as colorless oil (0.91 g, 10.0% yield). 1H-NMR (CDCl3)
d: ꢁ0.05 (3H, s), 0.12 (3H, s), 0.83 (9H, s), 0.94 (3H, t, Jꢃ7.2 Hz), 1.74
(2H, m), 2.61 (2H, t, Jꢃ7.2 Hz), 2.73—2.78 (1H, m), 2.83—2.89 (1H, m),
4.89—5.00 (1H, m), 5.63 (2H, s), 6.10—6.14 (1H, m), 6.38—6.44 (1H, m),
6.97—7.00 (2H, m), 7.22—7.30 (3H, m). MS m/z: 353 (Mꢀꢁ57), 285, 91
(B.P.). IR (neat) cmꢁ1: 1641, 1602, 1458, 1398, 1383, 1251.
8-(tert-Butyldimethylsilanyloxy)-2-propyl-5,6,7,8-tetrahydro-3H-cy-
cloheptimidazol-4-one (19) To a solution of 18 (0.91 g, 2.22 mmol) in
MeOH (9.1 ml) was added 5% Pd on carbon (containing 50% water, 0.10 g)
and the mixture was stirred for 48 h under an atmosphere of hydrogen (1
atm) at room temperature. After filtration of the catalyst, the filtrate was con-
centrated in vacuo. The residue was subjected to chromatography over silica
gel eluting EtOAc, to give title compound as colorless solid (0.53 g, 74.0%
1-[8-Oxo-1-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-
1,4,5,6,7,8-hexahydrocycloheptimidazol-2-yl]propyl a-Methoxy-(a-tri-
fluoromethyl)phenylacetate (13c, b) To a stirred solution of the human
main metabolite (11.7 mg 26 mmol) in CH2Cl2 1 ml was added trityl chloride
(8.1 mg 29 mmol) and triethyl amine (5 ml, 36 mmol) and the mixture was
stirred at room temperature for 18 h. The reaction mixture was concentrated
in vacuo, purified by preparative TLC (EtOAc/n-hexaneꢃ1 : 1), to give the
trityl compound 12 (17.7 mg) Rf 0.44 (EtOAc/n-hexaneꢃ1 : 1). To a stirred
solution of 12 (7.4 mg 11 mmol) in CH2Cl2 0.25 ml was added (R)-(ꢁ)-a-
methoxy-a-(trifluoromethyl)phenylacetyl chloride (40 ml, 214 mmol) and tri-
ethyl amine (40 ml, 287 mmol) and the mixture was stirred at room tempera-
ture for 18 h. The reaction mixture was purified directly by preparative TLC
(EtOAc/n-hexaneꢃ1 : 3), to give 13c (10.6 mg) as colorless amorphous. Rf
1
yield). H-NMR (CDCl3) d: 0.07 (3H, s), 0.19 (3H, s), 0.88 (9H, s), 0.98
(3H, t, Jꢃ7.2 Hz), 1.72—1.82 (4H, m), 2.12—2.18 (1H, m), 2.29—2.32
(1H, m), 2.57—2.87 (4H, m), 4.95—4.97 (1H, m), 9.62 (1H, br s). MS m/z:
307 (Mꢀꢁ15), 265 (Mꢀꢁ57), 191, 149, 75 (B.P.). mp: 142—144 °C. IR
(KBr) cmꢁ1: 3250, 2920, 1737, 1617, 1548, 1521, 1461, 1392, 1242.
8-(tert-Butyldimethylsilanyloxy)-2-propyl-3-[2ꢀ-(1-trityl-1H-tetrazol-
5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-
one (20) To a mixture of 19 (0.53 g, 1.64 mmol), 11 (1.10 g, 1.97 mmol)
and DMF (4.2 ml) was added K2CO3 (0.46 g, 3.33 mmol). The reaction mix-
ture was stirred for 14 h at room temperature, and then poured into H2O. The
mixture was extracted with EtOAc, and the extract was washed with H2O
and then brine, dried over Na2SO4. The organic layer was concentrated in
vacuo. The residue was subjected to chromatography over silica gel eluting
n-hexane–EtOAc (1 : 1 by volume), to give title compound as colorless solid
(0.60 g, 45.7% yield). 1H-NMR (CDCl3) d: ꢁ0.05 (3H, s), 0.15 (3H, s), 0.85
(9H, s), 0.87 (3H, t, Jꢃ7.2 Hz), 1.65—1.72 (3H, m), 1.99—2.05 (1H, m),
2.10—2.17 (2H, m), 2.47—2.52 (3H, m), 3.08—3.14 (1H, m), 5.07—5.09
(1H, m), 5.39 (1H, d, Jꢃ16.0 Hz), 5.52 (1H, d, Jꢃ16.0 Hz), 6.74 (2H, d,
Jꢃ8.4 Hz), 6.93 (6H, d, Jꢃ7.2 Hz), 7.05 (2H, d, Jꢃ8.4 Hz), 7.22—7.36
(10H, m), 7.42—7.50 (2H, m), 7.89 (1H, d, Jꢃ7.6 Hz). MS (ESI) m/z: 800
[Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 2920, 1641, 1461, 1383, 1251.
1
0.74 (EtOAc/n-hexaneꢃ1 : 3). H-NMR (CDCl3) d: 0.51 (3H, t, Jꢃ7.2 Hz),
1.49—1.60 (2H, m), 1.83—2.00 (4H, m), 2.70 (2H, m), 3.02 (2H, m), 3.50
(3H, s), 5.33 (1H, d, Jꢃ16.0 Hz), 5.61—5.65 (1H, m), 6.15 (1H, d,
Jꢃ16.0 Hz), 6.92 (2H, d, Jꢃ8.0 Hz), 6.93—6.99 (5H, m), 7.09 (2H, d,
Jꢃ8.0 Hz), 7.24—7.60 (18H, m), 7.86 (1H, d, Jꢃ7.6 Hz). MS (ESI): m/z: 901
[Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 2960, 1745, 1644, 1448, 1230, 1166. Compound
13b was obtained with (S)-(ꢀ)-a-methoxy-a-(trifluoromethyl)phenylacetyl-
chloride in same manner as colorless amorphous. 1H-NMR, MS and IR data
was identified with that of 13b already obtained.
3-Benzyl-5-[1-(tert-butyldimethylsilanyloxy)but-3-enyl]-2-propyl-3H-
imidazole-4-carboxylic Acid Ethyl Ester (15) To a solution of 3-benzyl-
5-hydroxymethyl-2-propyl-3H-imidazole-4-carboxylic acid ethyl ester (14)
(6.05 g, 20.0 mmol) in CHCl3 (60 ml) was added manganese(IV) oxide
(52.2 g, 600 mmol). The reaction mixture was stirred at room temperature
for 1.5 h. and was filtrated. The filtrate was concentrated in vacuo, and the
residue was diluted with CH2Cl2 (80 ml). To the solution was added
allyltrimetylsilane (3.5 ml, 22.0 mmol). Titanium(IV) chloride (2.2 ml,
20.1 mmol) was added dropwise to this solution at ꢁ78 °C. Stirring was
continued for 7 h at ꢁ78 °C to ambient temperature. The reaction mixture
was poured into H2O and extracted with CH2Cl2. The extract was washed
with brine, and dried over MgSO4, and concentrated in vacuo. The reside
was dissolved with DMF (40 ml). To this solution was added imidazole
(2.72 g, 40.0 mmol) and tert-butyldimethylsilyl chloride (4.22 g, 28.0 mmol)
at room temperature and stirred for 2 h. H2O was added to the reaction mix-
ture and the mixture was extracted with EtOAc. The organic layer was
washed with H2O and brine, dried over Na2SO4. The organic layer was con-
centrated in vacuo. The residue was subjected to chromatography over silica
gel eluting n-hexane–EtOAc (4 : 1 by volume), to give title compound as col-
8-Hydroxy-2-propyl-3-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-yl-
methyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one
(21) To
a
stirred solution of 20 (1.80 g, 2.25 mmol) in THF (9 ml) was added tetra-
butylammonium fluoride (4.5 ml, 1 M in THF, 4.50 mmol) at room tempera-
ture. The reaction mixture was allowed to warm to 50 °C and stirred for 3 h.
After the addition of saturated aqueous NH4Cl, the reaction mixture was ex-
tracted with EtOAc. The organic layer was washed with H2O and brine,
dried over Na2SO4. The organic layer was concentrated in vacuo. The
residue was subjected to chromatography over silica gel eluting n-
hexane–EtOAc (4 : 1 by volume), to give title compound as colorless oil
1
(1.34 g, 87.0% yield). H-NMR (CDCl3) d: 0.89 (3H, t, Jꢃ7.2 Hz), 1.39—
1.41 (1H, m), 1.69 (2H, m), 1.83—1.97 (3H, m), 2.32—2.35 (1H, m), 2.50
(2H, t, Jꢃ7.2 Hz), 2.62—2.66 (2H, m), 4.90—4.93 (1H, m), 5.46 (2H, dd,
Jꢃ4.8, 16.0 Hz), 6.78 (2H, d, Jꢃ8.4 Hz), 6.92—6.95 (6H, m), 7.07 (2H, d,
Jꢃ7.6 Hz), 7.24—7.36 (10H, m), 7.42—7.50 (2H, m), 7.89 (1H, m). MS
(ESI) m/z: 685 [Mꢀ1]ꢀ. IR (neat) cmꢁ1: 2998, 2950, 1641, 1464, 1215.
8-Hydroxy-2-propyl-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-
5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (2) To a stirred solution
of 21 (1.34 g, 1.96 mmol) in THF (5 ml) was added acetic acid (3 ml) and
H2O (1 ml). The reaction mixture was stirred for 17 h at room temperature,
and then concentrated in vacuo. The residue was subjected to chromatogra-
phy over silica gel eluting CHCl3–MeOH (10 : 1 by volume), to give title
1
orless oil (7.62 g, 83.4% yield). H-NMR (CDCl3) d: ꢁ0.04 (3H, s), ꢁ0.01
(3H, s), 0.84 (9H, s), 0.89 (3H, t, Jꢃ7.2 Hz), 1.27 (3H, t, Jꢃ7.2 Hz), 1.65
(2H, m), 2.46—2.50 (1H, m), 2.65 (2H, t, Jꢃ7.6 Hz), 2.67—2.70 (1H, m),
4.22 (2H, q, Jꢃ7.2 Hz), 4.98—5.07 (2H, m), 5.40—5.43 (1H, m), 5.51 (2H,
q, Jꢃ16.6 Hz), 5.80—5.87 (1H, m), 6.90 (2H, d, Jꢃ7.4 Hz), 7.21—7.30
(3H, m). MS m/z: 399 (Mꢀꢁ57), 267, 91 (B.P.). IR (neat) cmꢁ1: 1698, 1461,
1383, 1278, 1248.
3-Benzyl-8-(tert-butyldimethylsilanyloxy)-2-propyl-7,8-dihydro-3H-
cycloheptimidazol-4-one (18) To a mixture of 15 (10.35 g, 22.7 mmol),
chlorotriisopropoxytitanium(IV) (45.4 ml, 1 M in n-hexane, 45.4 mmol) and
THF (450 ml) was added isopropylmagnesium chloride (90.8 ml, 2 M in THF,
182 mmol) at ꢁ60 °C. The reaction mixture was allowed to warm to room
temperature slowly and stirred for 20 h. After the addition of saturated aque-
ous NH4Cl, the reaction mixture was extracted with EtOAc. The organic
layer was washed with H2O and brine, dried over Na2SO4. The organic layer
was concentrated in vacuo. The residue was dissolved with DMF (100 ml).
To the stirred solution was added pyridine (4.1 ml, 51.0 mmol) and ferric(III)
chloride (8.10 g, 49.9 mmol) at room temperature and stirred for 1 h. The re-
1
compound as colorless solid (0.74 g, 85.3% yield). mp: 192—194 °C. H-
NMR (CD3OD) d: 0.97 (3H, t, Jꢃ7.2 Hz), 1.66 (2H, m), 1.87—1.95 (1H,
m), 2.17—2.20 (3H, m), 2.62—2.73 (3H, m), 2.83—2.95 (1H, m), 4.97—
5.01 (1H, m), 5.97 (2H, s), 6.99 (2H, d, Jꢃ8.4 Hz), 7.11 (2H, d, Jꢃ8.4 Hz),
7.56—7.60 (2H, m). 7.67—7.79 (2H, m). MS (ESI) m/z: 443 [Mꢀ1]ꢀ. Anal.
Calcd for C25H26N6O2: C, 67.86; H, 5.92; N, 18.99. Found: C, 68.04; H,
6.14; N, 19.08. IR (KBr) cmꢁ1: 3400, 1641, 1464, 1215.
2-Propyl-3-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-
tetrahydro-3H-cycloheptimidazol-4-one (22) To a suspension of 2-

June 2006
787
4.8; MgSO4·7H2O, 1.3; KH2PO4, 1.2; CaCl2·2H2O, 1.2; NaHCO3, 24.2 and
glucose, 5.5 mM) maintained at 37 °C and bubbled with a 95% O2ꢀ5% CO2
gas. Under the resting tension of 1.5 g, isometric tension changes were
recorded on a polygraph (Nihon Kohden, Tokyo, Japan) through a force dis-
placement transducer (T-7-30-240; Orientec, Tokyo, Japan). After a 1.5 h
equilibration period, the cumulative concentration–response curve for AII
was constructed by the method of stepwise addition of the agonist. Then,
AII was washed out repeatedly for 1 h. Tissues were incubated with various
concentrations of compounds for 20 min and the concentration–response
curve for AII was again obtained. Responses were expressed as a percentage
of the maximum response in the first concentration–response curve for AII.
To measure the potency of the antagonist, the pA2 values were determined
from Schild plots using the least squares method. The effects of test com-
pound on the contractile responses of rabbit aorta induced by KCl, norepi-
nephrine and serotonin were also examined.
propyl-3-[2ꢄ-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-
cycloheptimidazol-4-one (Pratosartan) (2.00 g, 4.69 mmol) and THF (20 ml)
was added trityl chloride (1.57 g, 5.63 mmol) and triethyl amine (0.78 ml)
and the mixture was stirred at room temperature for 18 h. The reaction mix-
ture was concentrated in vacuo and to the residue saturated aqueous NH4Cl
was added. The mixture was extracted with EtOAc (2ꢂ50 ml). The com-
bined extracts layer was washed with H2O and then brine, dried over
Na2SO4. The organic layer was concentrated in vacuo. The residue was sub-
jected to chromatography over silica gel eluting n-hexane–EtOAc (1 : 1 by
volume), to give title compound as colorless powder (3.03 g, 96.6% yield).
1H-NMR (CDCl3) d: 0.88 (3H, t, Jꢃ7.6 Hz), 1.63—1.72 (4H, m), 1.80—
1.93 (4H, m), 2.48 (2H, t, Jꢃ7.8 Hz), 2.62 (2H, t, Jꢃ6.0 Hz), 3.00 (2H, t,
Jꢃ6.4 Hz), 5.45 (2H, s), 6.78 (2H, d, Jꢃ8.4 Hz), 6.93 (6H, d, Jꢃ8.4 Hz),
7.06 (2H, d, Jꢃ8.4 Hz), 7.23—7.36 (10H, m), 7.42—7.50 (2H, m), 7.88
(1H, d, Jꢃ8.0 Hz). MS (ESI) m/z: 669 [Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 2908, 1634,
1464, 744, 696.
Acknowledgements We thank Professor S. Ikegami, Teikyo University,
for his generous and helpful discussions. We also thank Dr. Akira Ohno for
his technical assistance; Miss Noriko Koide for in vitro testing.
5-Hydroxy-2-propyl-3-[2ꢀ-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-yl-
methyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (23) A solution
of 22 (0.67 g, 1.00 mmol) in THF (5 ml) was added dropwise to lithium di-
isopropylamide (1.7 ml, 0.9 M in pentane/THF, 1.5 mmol) at ꢁ78 °C. The re-
action mixture was stirred for 15 min at ꢁ78 °C. To this mixture was added
MoOPH (0.65 g. 1.50 mmol) and then allowed to warm at ꢁ25 °C. The reac-
tion mixture was stirred for 30 min at ꢁ25 °C. To this reaction mixture was
added saturated aqueous Na2SO3 (50 ml) and the allowed to warm to room
temperature. The reaction mixture was extracted with EtOAc (2ꢂ50 ml).
The combined extracts layer was washed with H2O and then brine, dried
over Na2SO4. The organic layer was concentrated in vacuo. The residue was
subjected to chromatography over silica gel eluting n-hexane–AcOEt (1 : 1
by volume), to give title compound as colorless powder (0.38 g, 55.5%
References
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1
yield). H-NMR (CDCl3) d: 0.90 (3H, t, Jꢃ7.2 Hz), 1.48—1.75 (3H, m),
1.76—2.00 (1H, m), 2.00—2.23 (1H, m), 2.30—2.45 (1H, m), 2.51 (2H, t,
Jꢃ7.8 Hz), 2.85—3.15 (2H, m), 4.12 (1H, d, Jꢃ2.1 Hz), 4.20—4.35 (1H,
m), 5.35 (1H, d, Jꢃ16.0 Hz), 5.58 (1H, d, Jꢃ16.0 Hz), 6.77 (2H, d,
Jꢃ8.0 Hz), 6.93 (6H, d, Jꢃ7.2 Hz), 7.08 (2H, d, Jꢃ8.0 Hz), 7.22—7.40
(10H, m), 7.42—7.50 (2H, m), 7.90 (1H, d, Jꢃ7.2 Hz). MS (ESI) m/z: 685
[Mꢀ1]ꢀ. IR (KBr) cmꢁ1: 3450, 2930, 1630, 744, 699.
5-Hydroxy-2-propyl-3-[2ꢀ-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-
5,6,7,8-tetrahydro-3H-cycloheptimidazo-4-one (3) To a solution of 23
(0.15 g, 0.22 mmol) in THF (2.5 ml) was added 10% HCl (1.5 ml) at room
temperature and stirred for 14 h. The reaction mixture was neutralized with
10% aqueous NaOH and extracted with CHCl3 (3ꢂ20 ml). The combined
extracts layer was washed with H2O and then brine, dried over Na2SO4. The
organic layer was concentrated in vacuo. The residue was subjected to chro-
matography over silica gel eluting CHCl3–EtOH (1 : 3 by volume), to give
title compound as colorless powder (0.60 g, 62.2% yield). mp: 74—76 °C.
1H-NMR (DMSO-d6) d: 0.85 (3H, t, Jꢃ7.2 Hz), 1.50—1.75 (4H, m), 1.85—
2.15 (2H, m), 2.45—2.55 (3H, m), 2.87 (2H, t, Jꢃ6.2 Hz), 4.29 (1H, dd,
Jꢃ4.0, 10.4 Hz), 5.49 (1H, d, Jꢃ16.8 Hz), 5.64 (1H, d, Jꢃ16.8 Hz), 6.93
(2H, d, Jꢃ8.0 Hz), 7.04 (2H, d, Jꢃ8.0 Hz), 7.48 (1H, d, Jꢃ7.8 Hz), 7.51
(1H, d, Jꢃ8.0 Hz), 7.58—7.65 (2H, m). MS (ESI): m/z 443 [Mꢀ1]ꢀ. Anal.
Calcd for C25H26N6O2: C, 67.86; H, 5.92; N, 18.99. Found: C, 67.67; H,
6.04; N, 18.95. IR (KBr) cmꢁ1: 3400, 2926, 1630, 1467, 1377, 756.
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Effects on AII Contractile Responses in Isolated Rabbit Aorta Tho-
racic aorta was isolated from male New Zealand white rabbits (2.7—3.2 kg,
Tokyo Laboratory Animals Science Inc., Tokyo, Japan). The aorta was
cleaned off connective tissue and adherent fat and cut into 3 mm ring seg-
ments. Opened ring preparations were used in the present study. The vascu-
lar endothelium was removed by gently rubbing the intimal surface of the
blood vessel with cotton wool. Preparations were mounted vertically in
organ baths containing 10 ml of Krebs-Ringer solution (NaCl, 120.3; KCl,