Synthesis and structure-activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity

Article abstract of DOI:10.1016/j.bmc.2009.10.013

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.

Full text of DOI:10.1016/j.bmc.2009.10.013

Bioorganic & Medicinal Chemistry 17 (2009) 8234–8240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of cassiarin A as potential
antimalarials with vasorelaxant activity
a
a
a
a
a
Hiroshi Morita a, , Yuichiro Tomizawa , Jun Deguchi , Tokio Ishikawa , Hiroko Arai , Kazumasa Zaima ,
Takahiro Hosoya a, Yusuke Hirasawa a, Takayuki Matsumoto a, Katsuo Kamata a, Wiwied Ekasari b,
Aty Widyawaruyanti b, Tutik Sri Wahyuni b, Noor Cholies Zaini b, Toshio Honda a
*
a Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan
b Faculty of Pharmacy, Airlangga University, Jalan Dharmawangsa Dalam, Surabaya 60286, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful
antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be
valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothe-
lium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria.
Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere
to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1
showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series
of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthe-
sized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce
cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity
and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom with-
out any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy
derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibi-
tion of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials
with different mode of actions.
Received 21 September 2009
Revised 6 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Keywords:
Cassiarin A
Cassia siamea
Plasmodium falciparum
Antimalarial activity
Vasorelaxant activity
Nitric oxide
SAR
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
have potential as adjunctive therapy early during the course of se-
vere malaria.8,9
Malaria caused by Plasmodium falciparum is a major parasitic
infection disease in the world and continues to cause morbidity
and mortality on a large scale in tropical countries.1 According to
WHO, it is a threat to over 2 billion people living in areas of high
incidence.2 A major contributor to malarial morbidity and mortal-
ity is almost certainly the increasing resistance of malaria parasites
to available drugs.3 Such a situation has heralded the need for
alternative antiplasmodial therapy. Antimalarial potential of drugs
derived from plants has been proven by examples such as quinine
from Cinchona species and artemisinin from Artemisia annua.4,5
Nitric oxide (NO), a highly diffusible cellular mediator involved
in a wide range of biological effects, has been implicated as one of
Recently we have isolated two new chromone alkaloids, cassia-
rins A 1 and B 2 from the leaves of Cassia siamea (Leguminosae),10
which has been used in traditional Indonesian medicine for the
treatment of fevers caused by malaria (Chart 1).11 Cassiarin A 1
is a promising antimalarial drug, although mode of action of cassi-
arin A on P. falciparum has not been known. Since 1 shows power-
ful in vitro antimalarial activity against P. falciparum and in vivo
activity against mouse malaria, P. berghei,12 the potent antimalarial
activity of 1 has stimulated medicinal chemists to pursue deriva-
O
O
HO
O
the agents to counteract malaria infection.6 NO and
L-Arg (the sub-
strate for NO synthase) reducing cytoadherence of pRBC to vascu-
lar endothelium are low in clinical severe malaria.7 Agents that can
improve endothelial NO production and endothelial function may
N
COOMe
N
cassiarin B (2)
cassiarin A (1)
* Corresponding author. Tel./fax: +81 3 5498 5778.
E-mail address: moritah@hoshi.ac.jp (H. Morita).
Chart 1. Structures of cassiarins A (1) and B (2).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.013

H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
8235
tives of 1, which may provide valuable leads for novel drugs. In
HO
O
N
R1O
O
N
(i), (ii)
addition, we evaluated vasorelaxant activity against rat aortic rings
whether 1 possessed potential to improve endothelial NO produc-
tion. In this article, we report the syntheses of the cassiarin A deriv-
atives and evaluate their in vitro antimalarial activity as well as
vasorelaxant activity against rat aortic rings.
1
1a-f
HO
O
O
O
2. Results and discussion
2.1. Chemistry
(iii)
O
N
R2
As outlined in Scheme 1, cassiarin A 1 was prepared in eight
steps through 5-acetonyl-7-hydroxy-2-methyl chromone 10 from
3 by modified biomimetic methods as described previously.13
Under the literature conditions,14 5,7-dihydroxy-2-methyl-4H-
chromen-4-one 5 was prepared in a large scale from the commer-
cially available material, 2,4,6-trihydroxyacetophenone 3. With
assistance of the intramolecular H-bonding between C-5 hydroxyl
and C-4 carbonyl, selective protection of C-7 hydroxyl group of
chromone 5 was achieved using MOMCl and DIPEA in CH2Cl2 to
give MOM ether 6 in 83% yield. Then, treatment of chromone 6
with PhN(Tf)2 in the presence of NaH at 0 °C gave the correspond-
ing triflate 7 in 97% yield. Sonogashira coupling15 of triflate 7 with
in situ generated propyne gave alkyne 8 in 85% yield.10c Conversion
from 8 into 5-acetonyl-7-hydroxy-2-methylchromone 10, which is
a potential biogenetic precursor for cassiarins, was carried out by
treatment with a catalytic amount of AgNO3 and 3 equiv of TFA
in 61% yield, and then acidic deprotection of MOM ether 9 in 96%
yield. Cassiarin A 1 was easily prepared in good yield (91%) by
treating the chromone 10 with AcONH4 in AcOH.10b
We prepared ester derivatives 1a–c and ether derivatives 1d–f
from 1 in good yields by use of appropriate acid chloride and alkyl
iodide or bromide, respectively (Scheme 2). The nitrogen-substi-
tuted derivatives 10a–c were synthesized from 5-acetonyl-7-hy-
droxy-2-methylchromone 10 by appropriate amines in AcOH.
Dehydroxy derivative 11 was synthesized by way of trifluorometh-
anesulfonate 1g (Scheme 3), followed by reductive deoxygenation
with TES in the presence of Pd(PPh3)2Cl2.
O
10
10a-c
Scheme 2. Synthesis of 1a–f. Reagents and conditions: (i) appropriate acid chloride
(or Ac2O), pyridine, CH2Cl2; (ii) appropriate alkyl iodide (or bromide), K2CO3,
acetone, reflux; (iii) appropriate amine, AcOH, reflux.
derivatives at C-7 (Table 1), the acetyl and methyl ether derivatives
1a and 1d exhibited relatively potent antimalarial effect with high
selectivity index. There was a tendency that the ester derivatives
showed more potent than the ether ones. Derivatives with bulky
substituents such as benzoyl and benzyl showed less effective
against P. falciparum 3D7. However, the excellent activity was
not consistent with the activity of a dehydroxy derivative 11. The
design of nitrogen-substituted derivatives 10a–c built in this study
and cassiarin B 2, did not afford improved antimalarial effects with
respect to the parent cassiarin A (Table 1). Interestingly the N-Ph
derivative 10c still showed potent antimalarial activity and good
selective index. Significant antimalarial effect for the parent cassi-
arin A 1, diminished depending on size of the substitution both of
the O-alkyl (R1), O-acyl (R1), and N-alkyl (R2) side chain.
2.3. Vasorelaxant activity in ex vivo
It has been shown that the endothelium plays an important role
in controlling vascular tone by releasing NO,16 which is widely
known to inhibit platelet and leukocyte adhesion to endothelium
through its regulatory effect on adhesion molecule expression.7 Re-
cently, NO has been reported to be protective against P. falciparum
infection by inhibiting cytoadherence, and emphasize the thera-
peutic potential of NO in the treatment of severe malaria.7–9 The
present widely used artemisin derivatives have vascular effects.17
Artemisin causes relaxation of precontracted rat aortic rings which
is partly mediated by NO.17
2.2. Antimalarial activity in vitro
All compounds synthesized were tested for their antiparasitic
activity on the chloroquine (CQ)-sensitive P. falciparum strain
3D7. In parallel, cytotoxicity of the compounds was determined
on human MCF7 cell line. The results are expressed as IC50 values
representing the drug concentration required to inhibit the growth
of parasites and human cells with selectivity index, SI (IC50 for
MCF7)/(IC50 for 3D7) (Table 1). Among the ester and ether
When phenylephrine (PE, 3 Â 10À7 M) was applied to thoracic
aortic rings with endothelium after achieving a maximal response,
cassiarin A 1 showed vasorelaxant actions at 3 Â 10À5 M and
MOMO
O
O
HO
OH
O
AcO
OAc
HO
O
O
MOMO
HO
O
O
(iv)
(i)
(ii)
(iii)
OTf
OH
OAc O
OH
OH
3
4
5
6
7
HO
O
N
O
MOMO
O
MOMO
O
O
(vii)
(viii)
(v)
(vi)
O
O
O
O
10
1
8
9
Scheme 1. Synthesis of 1 and 10. Reagents and conditions: (i) acetic anhydride, pyridine; (ii) LiH, THF, reflux, then aq HCl 0 °C, and aq Na2CO3, reflux; (iii) chloromethyl
methyl ether, DIPEA; (iv) PhN(Tf)2, NaH; (v) 1-bromo-1-propene, n-BuLi, then Pd(PPh3)2Cl2, CuI, THF-i-Pr2NH; (vi) AgNO3, TFA, ClCH2CH2Cl, À24 °C, then aq NaOAc, reflux;
(vii) 2 N aq HCl, MeOH, reflux; (viii) AcONH4, AcOH, reflux.

8236
H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
HO
O
N
TfO
O
N
H
O
N
(i)
(ii)
1
1g
11
Scheme 3. Synthesis of 1g and 11. Reagents and conditions: (i) Tf2O, pyridine, CH2Cl2, rt; (ii) TES, Pd(PPh3)2Cl2, DMF, reflux.
Table 1
about antimalarial and vasorelaxant activities which may be med-
iated by NO production from endothelium, are necessary to devel-
op a novel antimalarial drug with controlling vascular tone by
releasing NO, which may inhibit cytoadherence of P. falciparum
infection.
Antimalarial activity of cassiarin derivatives 1, 2, 1a–g, 10a–c, and 11
1
O
8
9
8a
4a
O
O
N
R1O
H
O
N
7
2
3
6
4
5
N
10
3. Experimental section
R2
11
12
1 and 1a-g
2 and 10a-c
11
M)
3.1. General experimental procedures
Compd
R1 or R2
IC50 3D7a
(lM)
IC50 MCF7b
(l
SIc
IR spectra on a JASCO FTIR-230 spectrometer. Mass spectra
were obtained with a Micromass LCT spectrometer. 1H and 13C
NMR spectra were recorded on a Bruker AV 400 spectrometer
and chemical shifts were referenced to the residual solvent peaks
(dH 3.31 and dC 49.0 for methanol-d4). Analytical HPLC was carried
out on with a Shimadzu CLASS-M10A Series HPLC with a photodi-
CQ
1
0.011
0.023
1.2
36.1
3281
>4348
>83
>24
>83
H
>100
>100
>100
>100
87.8
1a
1b
1c
1d
1e
1f
1g
2
10a
10b
10c
11
COMe
COPr
COPh
Me
4.1
1.2
3.6
>24
Bu
Bn
Tf
3.0
76.7
72.2
>26
4.8
ode array detector. Separations were done with a 5 lm,
14.9
0.41
22.0
23.3
4.2
4.6 mm  250 mm, column (Waters, SunFire), (method A): flow
rate 0.5 mL/min, 50% MeOH with 0.1% TFA; (method B): 60% MeOH
with 0.1% TFA. The purity of derivatives by HPLC (methods A or B)
was higher than 95%.
>100
>100
>100
>100
>100
>100
>244
>4.5
>4.2
>23
>1112
>18
(CH2)3–COOMe
Me
Bu
Ph
0.090
5.4
3.2. Synthesis
a
In this assay, 3D7 is CQ-sensitive Plasmodium falciparum strain. The standard
drug chloroquine (CQ) served as positive control for CQ-sensitive P. falciparum 3D7
3.2.1. 5, 7-Dihydroxy-2-methyl-4H-chromen-4-one (5)
To a stirred solution of 3 (3.0 g, 17.9 mmol) in pyridine (6 mL) at
room temperature was added Ac2O (7.6 mL, 68.8 mmol) and the
reaction mixture was stirred for 4 h. The reaction was quenched
by addition of satd aq NaHCO3 and then extracted with ethyl ace-
tate. The combined organic phases were successively washed with
1 M HCl and dried over anhydrous Na2SO4, and concentrated in
vacuo to give a crude product of 4.
After working up, a solution of crude product of 4 (6.3 g) in THF
(150 mL) was treated with LiH (650 mg, 81.3 mmol) and the mix-
ture was stirred at 60 °C for 4 h. To this, aq HCl was added drop-
wise at 0 °C. After stirring for 1 h at 0 °C, the reaction mixture
was adjusted to pH 10 with satd aq Na2CO3 and stirred for a further
2 h at reflux. The mixture was extracted with ethyl acetate. The
combined organic phases were washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. Purification with
column chromatography (toluene/ethyl acetate = 1/1) gave a pale
yellow solid (5, 2.3 g, 67% from 3). Spectroscopic data were corre-
sponding to those of the article.13
strain.
b
The cytotoxicity is evaluated against MCF7 cells. Vincristine exhibited an IC50
value of 2.4 lM against the human MCF7 cell line.
c
SI is selectivity index (IC50 MCF7/IC50 3D7).
1 Â 10À5 M (Fig. 1), whereas cassiarin B 2 was found to have no
vasorelaxant effect. All compounds synthesized were tested for
vasorelaxant activity against rat aorta (Fig. 2). Derivatives with
bulky substituents such as benzoyl 1c, butyl 1e, and benzyl 1f were
found to be less potent than 1, although those with smaller substit-
uents, such as acetyl 1a and propionyl 1b, still showed potent vaso-
dilator effect. Methyl ether derivative 1d showed more potent
vasorelaxant effect than 1. However, the excellent activity could
not be observed for a dehydroxy derivative 11. The nitrogen-
substituted derivatives 10a–c including 2 did not show vasorelax-
ant activity (Fig. 2). There has been shown to be slightly different
tendency as antimalarial activity.
The activity of 1d-induced vasorelaxation was observed in a
concentration-dependent manner (Fig. 3). The vasorelaxant effects
of 1 and 1d were attenuated by endothelium removal or pretreat-
ment with a NO synthase inhibitor, NG-monomethyl-
L
-arginine
3.2.2. 5-Hydroxy-7-(methoxymethoxy)-2-methyl-4H-chromen-
4-one (6)
(L
-NMMA, 10À4 M).18 Our results suggested that the vasorelaxant
effect was attributed to its actions on the endothelial cells to
release NO.
To a stirred solution of 5,7-dihydroxy-2-methyl-chromen-4-one
(5, 920 mg, 4.79 mmol) in CH2Cl2 (15 mL) was added DIPEA
(1.0 mL, 5.74 mmol) at 0 °C under Ar. After the solution was stirred
for 10 min, MOMCl (0.47 mL, 4.95 mmol) was added at the same
temperature. The reaction mixture was allowed to warm to room
temperature and stirred for 1.5 h. The reaction was quenched with
satd aq NaHCO3 and then extracted with ethyl acetate. The
combined organic phases were washed with 1 M HCl, water, and
In conclusion, we synthesized and evaluated a new series of
cassiarin derivatives. Among them, natural cassiarin A 1 exhibited
potent antimalarial activity and a high selectivity index against
cytotoxicity of human cells in vitro. In addition, we found 1 and
methyl ether derivative 1d showed vasorelaxant activity against
rat aorta. Further studies on 1, such as mode of action mechanisms

H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
8237
Figure 1. Typical recording of the vasorelaxation effects of 1 and 2 on the rat aortic rings precontracted with 3 Â 10À7 M PE: (a) and (b) for cassiarin A 1 (3 Â 10À5 M and
1 Â 10À5 M, respectively). (c) For cassiarin B 2 (3 Â 10À5 M). (d) Pretreatment with
L
-NMMA with endothelium (+E). (e) Without endothelium (ÀE).
brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
Purification with column chromatography (hexane/ethyl
acetate = 3/1) gave a pale yellow solid (6, 1.0 g, 83%). Spectroscopic
data were corresponding to those of the article.13
it became clear. After being stirred for an additional 1 h, the reac-
tion was quenched with satd aq NH4Cl. THF was evaporated, and
the residue was diluted with water and extracted with ethyl
acetate. The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, and concentrated. Purification with
column chromatography (hexane/ethyl acetate = 3/1) gave a pale
yellow solid (7, 610 mg, 97%). Spectroscopic data and correspond-
ing to those of the article.13
3.2.3. 7-(Methoxymethoxy)-2-methyl-4-oxo-4H-chromen-5-yl
trifluoromethanesulfonate (7)
To a stirred solution of 6 (400 mg, 1.70 mmol) in THF (10 mL)
was added NaH (60%, 120 mg, 2.54 mmol) at 0 °C under Ar. The
suspended yellow solution was stirring for 10 min, and then a solu-
tion of PhNTf2 (910 mg, 2.54 mmol) in THF (2 mL) was added. The
resulting mixture was allowed to warm to room temperature after
Figure 3. Time course and dose dependency of vasorelaxation effects of 1d on the
rat aortic rings precontracted with 3 Â 10À7 M PE. Values are the mean SE (n = 3).
Each relaxation response is expressed as a percentage of the contraction induced by
PE. ***P <0.001 versus vehicle.
Figure 2. Vasorelaxation effects of 1, 2, 1a–g, 11, and 10a–c (3 Â 10À5 M) on the rat
aortic rings precontracted with 3 Â 10À7 M PE. Values are the mean SE (n = 3).

8238
H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
3.2.4. 7-(Methoxymethoxy)-2-methyl-5-(prop-1-ynyl)-4H-
chromen-4-one (8)
(1H, d, J = 1.9 Hz), 7.05 (1H, br s); 13C NMR (400 MHz, CD3OD) d
19.9, 21.0, 23.4, 105.2, 105.5, 110.8, 115.3, 115.9, 139.2, 152.4,
153.0, 155.4, 156.3, 163.0, 170.4; HRESIMS m/z 214.0865 [calcd
for C13H12O2N (MÀAc+H)+, 214.0863].
1-Bromo-1-propene (1.05 mL, 12.2 mmol) was dissolved in THF
(18 mL). After cooling to À78 °C, to the solution was added n-BuLi
(1.5 M in hexane, 10.9 mL, 16.3 mmol). The resulting mixture was
stirred at À78 °C for 1 h. Water (0.3 mL, 16.3 mmol) was added,
and the temperature was allowed to rise to 0 °C where the mixture
was further stirred for 30 min. To the mixture was added a solution
of triflate 7 (1.5 g, 4.08 mmol) in THF (12 mL), Pd(PPh3)2Cl2
(143 mg, 0.20 mmol), CuI (77.5 mg, 0.408 mmol), and i-Pr2NH
(12 mL). The resulting mixture was stirred at room temperature
for 2.5 h. The reaction was quenched by addition of satd aq NH4Cl.
After separation, the water layer was extracted with ethyl acetate.
The combined organic phases were washed with brine, dried over
anhydrous Na2SO4, and concentrated. Purification with column
chromatography (hexane/ethyl acetate = 3/1) gave a pale yellow
solid (8, 900 mg, 85%). Spectroscopic data were corresponding to
those of the article.13
3.2.9. Butyroylcassiarin A (2,5-dimethylpyrano[2,3,4-ij]isoquin-
olin-8-yl butyrate) (1b)
To a stirred solution of 1 (5.0 mg, 23
lmol) in DCM (200
lL) and
pyridine (100 L) at 0 °C was added butyryl chloride (12
l
lL) and the
solution was allowed to warm to room temperature. The reaction
mixture was stirred for 1.5 h. The reaction was quenched by addition
of satd aq NaHCO3 and then extracted with CHCl3. The combined or-
ganic phases were dried over anhydrous Na2SO4, and concentrated
in vacuo. Purification with column chromatography (CHCl3/
MeOH = 49/1) gave a pale yellow solid (1b, 6.1 mg, 97%). IR (neat)
1573, 1622, 1659, 1762, 2871, 2936, 2970 cmÀ1
;
1H NMR
(400 MHz, CD3OD) d 1.06 (3H, dd, J = 7.3, 7.3 Hz), 1.78 (3H, m),
2.25 (3H, d, J = 0.7 Hz), 2.43 (3H, d, J = 0.4 Hz), 2.60 (2H, dd, J = 7.3,
7.3 Hz), 6.17 (1H, d, J = 0.7 Hz), 6.77 (1H, d, J = 1.9 Hz), 6.94 (1H, d,
J = 1.9 Hz), 7.01 (1H, s); 13C NMR (400 MHz, CD3OD) d 13.9, 19.3,
19.9, 23.7, 36.9, 104.9, 105.9, 110.6, 115.2, 116.0, 139.2, 152.6,
153.8, 155.2, 156.3, 162.3, 173.1; ESIMS 284 (M+H)+. HRESIMS m/z
284.1281 [calcd for C17H18O3N (M+H)+, 284.1281].
3.2.5. 7-(Methoxymethoxy)-2-methyl-5-(2-oxopropyl)-4H-
chromen-4-one (9)
To a solution of 8 (50 mg, 0.19 mmol) and AgNO3 (1.5 mg,
5 mol %) in (CH2Cl)2 (7.5 mL) was added dropwise trifluoroacetic
acid (TFA) (45
l
L, 0.56 mmol) at À24 °C under Ar. The orange solu-
tion was stirred for 1 h at this temperature, and then allowed to
warm up. Water was added, and the mixture was extracted with
ethyl acetate. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated. Purification with column
chromatography (hexane/ethyl acetate = 3/2) gave a pale yellow
solid (7, 33 mg, 61%). Spectroscopic data were corresponding to
those of the article.13
3.2.10. Benzoylcassiarin A (2,5-dimethylpyrano[2,3,4-ij]isoquin-
olin-8-yl benzoate) (1c)
To a stirred solution of 1 (10.0 mg, 46
lmol) in pyridine (500
lL)
at 0 °C was added benzoyl chloride (27
lL) and the solution was al-
lowed to warm to room temperature. The reaction mixture was stir-
redfor1 h. ThereactionwasquenchedbyadditionofsatdaqNaHCO3
and then extracted with CHCl3. The combined organic phases were
dried over anhydrous Na2SO4, and concentrated in vacuo. Purifica-
tion with column chromatography (hexane/ethyl acetate = 1/2)
gave a pale yellow solid (1c, 14.0 mg, 94%). IR (neat) 1573, 1620,
3.2.6. 7-Hydroxy-2-methyl-5-(2-oxopropyl)-4H-chromen-4-one
(10)
1657, 2921, 2953, 3063 cmÀ1 1H NMR (400 MHz, CD3OD) d 2.22
;
A mixture of 9 (110 mg, 0.24 mmol) and 2 N aq HCl (0.5 mL) in
MeOH (10 mL) was refluxed for 4 h. MeOH was removed and water
was added. The mixture was extracted with ethyl acetate. The or-
ganic layer were washed with brine, dried over Na2SO4, and con-
centrated. Purification with flash chromatography (CHCl3/
MeOH = 19/1) gave a pale yellow solid (10, 89 mg, 96%). Spectro-
scopic data were corresponding to those of the article.13
(3H, d, J = 0.8 Hz), 2.42 (3H, d, J = 0.4 Hz), 6.14 (1H, d, J = 0.8 Hz),
6.86 (1H, d, J = 1.9 Hz), 6.97 (1H, s), 7.04 (1H, d, J = 1.9 Hz), 7.54
(1H, dd, J = 8.0, 8.0 Hz), 7.69 (1H, m), 8.16 (2H, dd, J = 8.0, 8.0 Hz);
13C NMR (400 MHz, CD3OD) d 19.9, 23.8, 104.9, 106.0, 110.6, 115.2,
116.0, 129.9, 129.9, 130.4, 131.2, 131.2, 135.1, 139.2, 152.5, 153.8,
155.2, 156.3, 162.2, 166.0; ESIMS 318 (M+H)+. HRESIMS m/z
318.1125 [calcd for C20H16O3N (M+H)+, 318.1125].
3.2.7. Cassiarin A (1)
3.2.11. Cassiarin A methyl ether (8-methoxy-2,5-dimethylpyrano-
To a stirred solution of 10 (10 mg, 0.036 mmol) in AcOH (2 mL)
at room temperature was added AcONH4 (30 mg) and the reaction
mixture was stirred for 48 h at reflux. The solvent was removed
and satd aq NaHCO3 was added. The mixture was extracted with
CHCl3. The organic layer was dried over anhydrous Na2SO4 and
concentrated. Purification with flash chromatography (CHCl3/
MeOH = 9/1) gave a pale yellow solid (1, 8.4 mg, 91%). Spectro-
scopic data were corresponding to those of the article.10a
[2,3,4-ij]isoquinoline) (1d)
To a stirred solution of 1 (4.5 mg, 21
room temperature were added K2CO3 (30 mg) and MeI (10
l
mol) in acetone (2 mL) at
l
L) and
the reaction mixture was refluxed for 5 h. The solvent was
removed and the crude products was purified by flash chromatogra-
phy (CHCl3/MeOH = 9/1) gave a pale yellow solid (1d, 2.7 mg, 56%).
IR (neat) 1575, 1624, 1663, 2849, 2925 cmÀ1 1H NMR (400 MHz,
;
CD3OD) d 2.21 (3H, d, J = 0.8 Hz), 2.41 (3H, s), 3.88 (3H, s), 6.09 (1H,
d, J = 0.8 Hz), 6.57 (1H, d, J = 2.1 Hz), 6.67 (1H, d, J = 2.1 Hz), 6.94
(1H, s); 13C NMR (400 MHz, CD3OD) d 18.5, 22.1, 54.8, 98.2, 98.2,
103.8, 112.0, 113.5, 138.4, 150.5, 155.0, 160.3, 163.1; HRESIMS m/z
228.1023 [calcd for C14H14O2N (M+H)+, 228.1019].
3.2.8. Acetylcassiarin A (2,5-dimethylpyrano[2,3,4-ij]isoquinolin-
8-yl acetate) (1a)
To a stirred solution of 1 (2.0 mg, 9.39
lmol) in pyridine
(100 L) at room temperature was added Ac2O (100
l
lL) and the
reaction mixture was stirred for 4 h. The reaction was quenched
by addition of satd aq NaHCO3 and then extracted with CHCl3.
The combined organic phases were dried over anhydrous Na2SO4,
and concentrated in vacuo. Purification with column chromatogra-
phy (CHCl3/MeOH = 9/1) gave a pale yellow solid (1a, 2.1 mg, 87%).
IR (neat) 1572, 1621, 1659, 1762, 2871, 2936, 2970 cmÀ1; 1H NMR
(400 MHz, CD3OD) d 2.27 (3H, d, J = 0.7 Hz), 2.32 (3H, s), 2.45 (3H,
d, J = 0.5 Hz), 6.22 (1H, d, J = 0.7 Hz), 6.84 (1H, d, J = 1.9 Hz), 6.70
3.2.12. Cassiarin A butyl ether (8-butoxy-2,5-dimethylpyrano-
[2,3,4-ij]isoquinoline) (1e)
To a stirred solution of 1 (10.0 mg, 46
at room temperature were added K2CO3 (32 mg) and 1-iodobutane
(27 L) and the reaction mixture was refluxed for 24 h. The solvent
lmol) in acetone (1.5 mL)
l
was removed and the crude product was purified by flash chroma-
tography (CHCl3/MeOH = 9/1) to give a pale yellow solid (1e,
9.3 mg, 67%). IR (neat) 1575, 1623, 1659, 2871, 2936, 2958 cmÀ1
;

H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
8239
1H NMR (400 MHz, CD3OD) d 1.01 (3H, dd, J = 7.3, 7.3 Hz), 1.50 (2H,
m), 1.78 (2H, m), 2.19 (3H, d, J = 0.8 Hz), 2.38 (3H,s), 4.02 (2H,dd,
J = 6.4, 6.4 Hz), 6.03 (1H, d, J = 0.8 Hz), 6.50 (1H, d, J = 2.1 Hz), 6.58
(1H, d, J = 2.1 Hz), 6.85 (1H, s); 13C NMR (400 MHz, CD3OD) d 14.2,
19.9, 20.3, 23.8, 32.4, 69.2, 100.1, 100.1, 105.5, 113.5, 114.8,
140.0, 152.1, 153.3, 156.5, 161.4, 163.9; ESIMS 270 (M+H)+. HRE-
SIMS m/z 270.1490 [calcd for C17H20O2N (M+H)+, 270.1489].
NMR (400 MHz, CD3OD) d 2.07 (3H, d, J = 0.5 Hz), 2.27 (3H, s),
5.59 (1H, s), 6.64 (1H, d, J = 1.9 Hz), 6.69 (1H, d, J = 1.9 Hz), 7.04
(1H, d, J = 0.5 Hz), 7.48 (2H, m), 7.73 (3H, m); 13C NMR (400 MHz,
CD3OD) d 20.6, 21.0, 98.5, 105.5, 109.0, 116.0, 129.0, 129.0, 132.0,
132.2, 132.2, 137.5, 138.6, 142.4, 150.0, 157.9, 168.4, 176.8; HRE-
SIMS m/z 290.1179 [calcd for C19H16O2N (M+H)+, 290.1176].
3.2.17. 2,5-Dimethylpyrano[2,3,4-ij]isoquinolin-8-yl trifluorome-
thanesulfonate (1g)
3.2.13. Cassiarin A benzyl ether (8-(benzyloxy)-2,5-dimethylpy-
rano[2,3,4-ij]isoquinoline) (1f)
To a stirred solution of 1 (7.8 mg, 37
lmol) in pyridine (120 lL)
To a stirred solution of 1 (10.0 mg, 46
at room temperature were added K2CO3 (40 mg) and BnBr (34
l
mol) in acetone (2.0 mL)
L)
at 0 °C was added Tf2O (25 L) and the reaction mixture was stir-
l
l
red for 3 h. The reaction was quenched by addition of satd aq NaH-
CO3 and then extracted with CHCl3. The combined organic phases
were dried over anhydrous Na2SO4, and concentrated in vacuo.
Purification with column chromatography (hexane/ethyl ace-
tate = 1/1) gave a pale yellow solid (1g, 9.2 mg, 73%). IR (neat)
and the reaction mixture was refluxed for 24 h. The solvent was re-
moved and the crude products was purified by flash chromatogra-
phy (CHCl3/MeOH = 18/1) to give a pale yellow solid (1f, 9.1 mg,
64%). IR (neat) 1573, 1622, 1659, 2925, 3037, 3067 cmÀ1 1H
;
NMR (400 MHz, CD3OD) d 2.19 (3H, s), 2.39 (3H, s), 5.13 (2H, s),
6.06 (1H, s), 6.61 (1H, d, J = 2.1 Hz), 6.71 (1H, d, J = 2.1 Hz), 6.88
(1H, s), 7.38 (5H, m); 13C NMR (400 MHz, CD3OD) d 19.9, 23.8,
71.4, 100.4, 100.8, 105.6, 113.7, 114.9, 128.7, 128.7, 129.1, 129.6,
129.6, 138.0, 140.0, 152.2, 153.6, 156.7, 161.5, 163.5; HRESIMS
m/z 304.1336 [calcd for C20H18O2N (M+H)+, 304.1332].
1571, 1617, 1659, 2921, 2958, 3093 cmÀ1 1H NMR (400 MHz,
;
CD3OD) d 2.28 (3H, s), 2.47 (3H, s), 6.23 (1H, s), 6.94 (1H, d,
J = 2.1 Hz), 7.11 (1H, s), 7.18 (1H, d, J = 2.1 Hz); 13C NMR
(400 MHz, CD3OD) d 19.8, 24.0, 103.4, 105.6, 110.3, 115.2, 116.8,
118.5, 139.1, 152.3, 152.5, 155.6, 162.3; HRESIMS m/z 346.0358
[calcd for C14H11O4NF3S (M+H)+, 346.0355].
3.2.14. 2,4,5-Trimethylpyrano[2,3,4-ij]isoquinolin-8-(4H)-one (10a)
3.2.18. 7-Dehydroxycassiarin A (2,5-dimethylpyrano[2,3,4-ij]-
To a stirred solution of 10 (5.0 mg, 22
lmol) in AcOH (1.0 mL) at
isoquinoline) (11)
room temperature was added aq 40% MeNH2–MeOH (100
lL) and
To a solution of 1g (21 mg, 61 lmol) and Pd(PPh3)Cl2 (4.2 mg) in
the resulting mixture was heated at reflux for 1.5 h. The reaction
was quenched by addition of satd aq NaHCO3 and then extracted
with CHCl3. The combined organic phases were dried over anhy-
drous Na2SO4, and concentrated in vacuo. Purification with column
chromatography (CHCl3/MeOH = 8/2) gave a pale yellow solid (10a,
DMF (1.0 mL) at room temperature was added TES (140 mL) and
the resulting mixture was stirred at reflux under Ar for 30 min.
The catalyst was removed by filtration and the filtrate was concen-
trated under reduced pressure to give a crude product, which was
purified by flash chromatography (hexane/toluene = 2/1) to give a
pale yellow solid 11 (12 mg, 99%). IR (neat) 1573, 1619, 1655,
3.5 mg, 72%). IR (neat) 1443, 1593, 1658 cmÀ1 1H NMR (400 MHz,
;
CD3OD) d 2.38 (3H, s), 2.43 (3H, d, J = 0.5 Hz), 3.61 (3H, s), 6.31 (1H,
d, J = 2.0 Hz), 6.44 (1H, d, J = 2.0 Hz), 6.47 (1H, br s), 6.74 (1H, br s);
13C NMR (400 MHz, CD3OD) d 20.6, 20.7, 35.9, 97.4, 105.7, 108.1,
108.5, 116.0, 136.8, 142.4, 148.6, 156.9, 167.4, 177.9; HRESIMS
m/z 228.1022 [calcd for C14H14O2N (M+H)+, 228.1019].
2857, 2921, 3060 cmÀ1 1H NMR (400 MHz, CD3OD) d 2.24 (3H,
;
s), 2.43 (3H, s), 6.15 (1H,s), 6.98 (1H, d, J = 8.2 Hz), 7.02 (1H, s),
7.20 (1H, d, J = 8.2 Hz) 7.54 (1H, dd, J = 8.2, 8.2 Hz); 13C NMR
(400 MHz, CD3OD) d 19.9, 23.8, 105.9, 109.4, 115.1, 118.0, 118.5,
133.3, 138.6, 152.8, 152.9, 155.4, 161.9; ESIMS 198 (M+H)+. HRE-
SIMS m/z 198.0913 [calcd for C13H12ON (M+H)+, 198.0913].
3.2.15. 4-Butyl-2,5-dimethylpyrano[2,3,4-ij]isoquinolin-8-(4H)-
one (10b)
3.3. Antiplasmodial activity
To a stirred solution of 10 (5.0 mg, 22
lmol) in AcOH (1.0 mL) at
room temperature was added n-butylamine (100
l
L) and the
Human malaria parasites were cultured according to the meth-
od of Trager and Jensen.19 The antimalarial activity of the isolated
compounds was determined by the procedure described by Bud-
imulja et al.20 In brief, stock solutions of the samples were pre-
pared in DMSO (final DMSO concentrations of <0.5%) and were
diluted to the required concentration with complete medium
(RPMI 1640 supplemented with 10% human plasma, 25 mM HEPES
and 25 mM NaHCO3) until the final concentrations of samples in
resulting mixture was heated at reflux for 24 h. The reaction was
quenched by addition of satd aq NaHCO3 and then extracted with
CHCl3. The combined organic phases were dried over anhydrous
Na2SO4, and concentrated in vacuo. Purification with column chro-
matography (CHCl3/MeOH = 5/1) gave a pale yellow solid (10b,
3.5 mg, 60%). IR (neat) 1354, 1467, 1595, 1653, 2872, 2932,
2958 cmÀ1 1H NMR (400 MHz, CD3OD) d 1.06 (3H, dd, J = 7.3,
;
7.3 Hz), 1.51 (2H, m), 1.72 (2H, m), 2.41 (3H, s), 2.50 (3H, s), 4.09
(2H, dd, J = 8.3, 8.3 Hz), 6.42 (1H, d, J = 2.0 Hz), 6.53 (1H, s), 6.54
(1H, d, J = 2.0 Hz), 6.84 (1H, s); 13C NMR (400 MHz, CD3OD) d
12.6, 18.7, 19.2, 19.3, 30.1, 48.6, 96.0, 104.3, 107.1, 107.9, 115.8,
135.7, 140.7, 147.4, 155.9, 166.7, 175.2; ESIMS 270 (M+H)+. HRE-
SIMS m/z 270.1488 [calcd for C17H20O2N (M+H)+, 270.1489].
culture plate wells were 10; 1; 0.1; 0.01; 0.001 lg/mL. The malarial
parasite P. falciparum 3D7 clone was propagated in a 24-well cul-
ture plates. Growth of the parasite was monitored by making a
blood smear fixed with MeOH and stained with Giemsa stain.
The antimalarial activity of each compound was expressed as an
IC50 value, defined as the concentration of the compound causing
50% inhibition of parasite growth relative to an untreated control.
The percentage of growth inhibition was expressed according to
following equation: Growth inhibition% = 100 À [(test parasitemia/
3.2.16. 2,5-Dimethyl-4-phenylpyrano[2,3,4-ij]isoquinolin-8-
(4H)-one (10c)
To a stirred solution of 10 (5.0 mg, 22
lmol) in AcOH (1.0 mL) at
control parasitemia) Â 100]. Chloroquine: IC50 0.011
lM.
room temperature was added PhNH2 (100
lL) and the resulting
mixture was heated at reflux for 5 h. The reaction was quenched
by addition of satd aq NaHCO3 and then extracted with CHCl3.
The combined organic phases were dried over anhydrous Na2SO4,
and concentrated in vacuo. Purification with column chromatogra-
phy (CHCl3/MeOH = 5/1) gave a pale yellow solid (10c, 3.5 mg,
3.4. Cytotoxic activity
MCF7 (human breast adenocarcinoma) cell line was seeded
onto 96-well microtiter plates at 5 Â 103 cells per well. Cells were
preincubated for 24 h at 37 °C in a humidified atmosphere of 5%
56%). IR (neat) 1478, 1545, 1597, 1656, 2924, 3059 cmÀ1 1H
;

8240
H. Morita et al. / Bioorg. Med. Chem. 17 (2009) 8234–8240
CO2. Different concentrations of each compound (10
added to the cultures, and then the cells were incubated at 37 °C
for 48 h. On the third day, 15 L MTT solution (5 mg/mL) was
added into each well of the cultured medium. After further 2 h of
incubation, 100 L of 10% SDS–0.01 N HCl solution was added to
each well and the formazan crystals in each well were dissolved
by stirring with a pipette. The optical density measurements were
made using a micropipette reader (Benchmark Plus microplate
spectrometer, BIO-RAD) equipped with a two wavelengths system
(550 and 700 nm). In each experiment, three replicate of wells
were prepared for each sample. The ratio of the living cells was
determined based on the difference of the absorbance between
those of samples and controls. These differences are expressed in
percentage and cytotoxic activity was indicated as an IC50 value.
lL) were
Acknowledgments
l
This work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and a grant from the Open Research
Center Project. We acknowledge the Faculty of Pharmacy, Air-
langga University for antimalarial activity and financial support.
l
References and notes
1. (a) Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M. Angew. Chem., Int. Ed. 2003,
42, 5274; (b) Greenwood, B.; Mutabingwa, T. Nature 2002, 415, 670; (c) Wyler,
D. J. Clin. Infect. Dis. 1993, 16, 449.
2. Taylor, W. R. J.; White, N. J. Drug Safety 2004, 27, 25.
3. (a) Gelb, M. H.; Hol, W. G. J. Science 2002, 297, 343; (b) Talisuna, A. O.; Bloland,
P.; D’Alessandro, U. Clin. Microbiol. Rev. 2004, 17, 235; (c) Gelb, M. H. Curr. Opin.
Chem. Biol. 2007, 11, 440.
Vincristine: IC50 2.4 lM.
4. Delhaes, L.; Benoit-Vical, F.; Camus, D.; Capron, M.; Meunier, B. Drugs 2003, 6,
674.
3.5. Vasodilation assay21
5. (a) Muhammad, I.; Samoylenko, V. Expert Opin. Drug. Discov. 2007, 2, 1065; (b)
Dunbar, D. C.; Li, X.-C.; Muhammad, I. Biomater. Aquat. Terr. Org. 2006, 207.
6. Ghigo, D.; Todde, R.; Ginsburg, H.; Costamagna, C.; Gautret, P.; Bussolino, F.;
Ulliers, D.; Giribaldi, G.; Deharo, E.; Gabrielli, G.; Pescarmona, G.; Bosia, A. J. Exp.
Med. 1995, 182, 677.
7. (a) Sobolewski, P.; Gramaglia, I.; Frangos, J.; Intaglietta, M.; van der Heyde, H. C.
Trends Parasitol. 2005, 21, 415; (b) Yeo, T. W.; Lampah, D. A.; Gitawati, R.; Tjitra,
E.; Kenangalem, E.; McNeil, Y. R.; Darcy, C. J.; Granger, D. L.; Weinberg, J. B.;
Lopansri, B. K.; Price, R. N.; Duffull, S. B.; Celermajer, D. S.; Anstey, N. M. J. Exp.
Med. 2007, 204, 2693; (c) Serirom, S.; Raharjo, W. H.; Chotivanich, K.;
Loareesuwan, S.; Kubes, P.; Ho, M. Am. J. Pathol. 2003, 162, 1651.
8. Weinberg, J. B.; Lopansri, B. K.; Mwaikambo, E.; Granger, D. L. Curr. Opin. Infect.
Dis. 2008, 21, 468.
9. Yeo, T. W.; Lampah, D. A.; Gitawati, R.; Tjitra, E.; Kenangalem, E.; McNeil, Y. R.;
Darcy, C. J.; Granger, D. L.; Weinberg, J. B.; Lopansri, B. K.; Price, R. N.; Duffull, S.
B.; Celermajer, D. S.; Anstey, N. M. J. Infect. Dis. 2008, 198, 602.
10. (a) Morita, H.; Oshimi, S.; Hirasawa, Y.; Koyama, K.; Honda, T.; Ekasari, W.;
Indrayanto, G.; Zaini, N. C. Org. Lett. 2007, 9, 3691; (b) Oshimi, S.; Tomizawa, Y.;
Hirasawa, Y.; Honda, T.; Widyawaruyanti, A.; Rudyanto, M.; Ekasari, W.;
Indrayanto, G.; Zaini, N. C.; Morita, H. Bioorg. Med. Chem. Lett. 2008, 18, 3761;
(c) Rudyanto, M.; Tomizawa, Y.; Morita, H.; Honda, T. Org. Lett. 2008, 10, 1921;
(d) Oshimi, S.; Deguchi, J.; Hirasawa, Y.; Ekasari, W.; Wahyuni, T. S.; Zaini, N. C.;
Shirota, O.; Morita, H. J. Nat. Prod. 2009, 72, 1899–1901.
11. (a) Mbatchi, S. F.; Mbatchi, B.; Banzouzi, J. T.; Bansimba, T.; Nsonde Ntandou, G.
F.; Ouamba, J. M.; Berry, A.; Benoit-Vical, F. J. Ethnopharmacol. 2006, 104, 168;
(b) Sanon, S.; Ollivier, E.; Azas, N.; Mahiou, V.; Gasquet, M.; Ouattara, C. T.;
Nebie, I.; Traore, A. S.; Esposito, F.; Balansard, G.; Timon-David, P.; Fumoux, F. J.
Ethnopharmacol. 2003, 86, 143.
12. Ekasari, W.; Widyawaruyanti, A.; Zaini, N. C.; Syafruddin, D.; Honda, T.; Morita,
H. Heterocycles 2009, 78, 1831.
13. Yao, Y. S.; Yao, Z. J. J. Org. Chem. 2008, 73, 5221.
14. Murti, V. V. S.; Seshadri, T. R. Proc. Indian Acad. Sci. Sect. A 1949, 30A, 107.
15. Sonogashira, K.; Tohda, Y.; Hagihara, N. A. Tetrahedron Lett. 1975, 16, 4467.
16. Muller, B.; Kleschyov, A. L.; Gyorgy, K.; Stoclet, J.-C. Physiol. Res. 2000, 49,
19.
17. Sofola, O. A.; Raji, I.; Ladipo, C.; Coker, H. A. B. Niger. Q. J. Hosp. Med. 2008, 18,
50.
18. Wang, Y. X.; Poon, C. I.; Pang, C. C. J. Pharmacol. Exp. Ther. 1993, 267, 1091.
19. Trager, W.; Jensen, J. B. Science 1976, 193, 673.
20. Budimulja, A. S.; Syafruddin, T. P.; Wilariat, P.; Marzuki, S. Mol. Biochem.
Parasitol. 1997, 84, 137.
21. Morita, H.; Iizuka, T.; Choo, C. Y.; Chan, K. L.; Takeya, K.; Kobayashi, J. Bioorg.
Med. Chem. Lett. 2006, 16, 4609.
A male Wistar rat weighting 260 g was sacrificed by bleeding
from carotid arteries under an anesthetization. A section of the
thoracic aorta between the aortic arch and the diaphragm was re-
moved and placed in oxygenated, modified Krebs–Henseleit solu-
tion (KHS: 118.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO3,
1.8 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgSO4, and 11.0 mM glu-
cose). The aorta was cleaned of loosely adhering fat and connective
tissue and cut into ring preparations 3 mm in length. The tissue
was placed in a well-oxygenated (95% O2, 5% CO2) bath of 5 mL
KHS solution at 37 °C with one end connected to a tissue holder
and the other to a force–displacement transducer (Nihon Kohden,
TB-611T). The tissue was equilibrated for 60 min under a resting
tension of 1.0 g. During this time the KHS in the tissue bath was re-
placed every 20 min.
After equilibration, each aortic ring was contracted by treatment
with 3 Â 10À7 M PE. The presence of functional endothelial cells was
confirmed by demonstrating relaxation to 10À5 M acetylcholine
(ACh), and aortic ring in which 80% relaxation occurred, were
regarded as tissues with endothelium. When the PE-induced con-
traction reached a plateau, each sample (1 Â 10À6 M À 3 Â 10À5 M)
was added.
Data are expressed as mean SEM. Statistical comparisons be-
tween time–response curves were made using a one-way analysis
of variance (ANOVA), with Bonferroni’s correction for multiple
comparisons being performed post-hoc (P <0.05 being considered
significant).
These animal experimental studies were conducted in accor-
dance with the Guiding Principles for the Care and Use of Labora-
tory Animals, Hoshi University and under the supervision of the
Committee on Animal Research of Hoshi University, which is
accredited by the Ministry of Education, Science, Sports Culture,
and Technology of Japan.