Synthesis and positive inotropic evaluation of N-(1-oxo-1,2,4,5-tetrahydro- [1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing piperazine and 1,4-diazepane moieties

Article abstract of DOI:10.1016/j.bmcl.2012.05.049

Two series of N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7- yl)acetamides bearing piperazine and 1,4-diazepane moieties were synthesized and screened for their positive inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. Most of the derivatives exhibited better in vitro positive inotropic activity than the existing drug, milrinone, among which 2-(4-(4-chlorobenzyl)-1,4-diazepan-1-yl)-N-(1-oxo-1,2,4,5- tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamide 6c proved to be the most potent with 15.48 ± 0.27% increased stroke volume (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10^-5 M. The chronotropic effects of the compounds that exhibited inotropic effects were also evaluated.

Full text of DOI:10.1016/j.bmcl.2012.05.049

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4229–4232
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and positive inotropic evaluation of N-(1-oxo-1,2,4,5-tetrahydro-
[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing piperazine and
1,4-diazepane moieties
Yan Wu a, Long-Xu Ma a, Tian-Wei Niu a, Xun Cui b, , Hu-Ri Piao a,
⇑
⇑
a Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, Yanbian University
College of Pharmacy, Yanji 133000, PR China
b Yanbian University College of Medicine, Yanji 133002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing pipera-
zine and 1,4-diazepane moieties were synthesized and screened for their positive inotropic activity by
measuring left atrium stroke volume on isolated rabbit heart preparations. Most of the derivatives
exhibited better in vitro positive inotropic activity than the existing drug, milrinone, among which
2-(4-(4-chlorobenzyl)-1,4-diazepan-1-yl)-N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-
7-yl)acetamide 6c proved to be the most potent with 15.48 0.27% increased stroke volume (milrinone:
2.46 0.07%) at a concentration of 3 Â 10À5 M. The chronotropic effects of the compounds that exhibited
inotropic effects were also evaluated.
Received 21 February 2012
Revised 19 April 2012
Accepted 11 May 2012
Available online 18 May 2012
Keywords:
[1,2,4]Triazolo[4,3-a]quinolin-1(2H)-one
Positive inotropic activity
Stroke volume
Ó 2012 Elsevier Ltd. All rights reserved.
Therapeutic treatment of cardiac failure (CHF) includes, among
other measures, the use of cardiac glycosides such as digoxin or
digitalis compounds.1–3 The narrow safety margin of digitalis com-
pounds has until now represented a serious problem due to the
high frequency and severity of digitalis intoxication.4,5 The discov-
ery of amrinone led to the synthesis of a number of promising non-
sympathomimetic and non-glycoside agents for the treatment of
CHF.6 The phosphodiesterase-inhibiting agent milrinone (Fig. 1),
which has both vasodilator and inotropic properties, was approved
for the treatment of CHF more than one decade ago. Nonetheless,
the significant ventricular arrhythmias and tachycardia associated
with the elevated cAMP level also limit its clinical use.7 Similar is-
sues have been recognized for the recently developed vesnari-
none8,9 and toborinone.10,11 Therefore, the identification of novel
positive inotropic agents, which not only improve the quality of life
but also reduce the mortality of CHF patients, is still an important
challenge for medicinal chemists.12
For several years, our laboratory has been pursuing intensive
work to identify more potential positive inotropic agents with
fewer side effects. A series of 3,4-dihydro-2(1H)-quinolinone deriva-
tives were synthesized and tested for their biological activity. Among
these, the compound 2-(4-(4-(benzyloxy)-3-methylbenzyl)pip-
erazin-1-yl)-N-(1-methyl-4,5-dihydro-[1,2,4]triazolo[4,3-a] quin-
olin-7-yl)acetamide PHR0007 (Fig. 1) showed the most potent
positive inotropic activity,13 which it displays through phosphodi-
esterase III inhibition.14 In this study, to further optimize PHR0007,
we kept the [1,2,4]triazolo[4,3-a]quinoline moiety unchanged, re-
placed the methyl group at the 1-position of the triazole ring with a
carbonyl group, and changed the substituents on the piperazine
ring at the 4-position. Replacement of the piperazine ring with a
1,4-diazepan ring was also considered in order to investigate the
contribution of such a structural change on biological activity. All
newly synthesized compounds were characterized by IR, 1H
O
N
CN
O
N
N
H3CO
N
H
H3C
OCH3
O
N
H
Milrinone
Vesnarinone
H
N
O
N
N
O
N
H3C
N
N
⇑
PHR0007
H3C
Corresponding authors. Tel.: +86 433 2435132; fax: + 86 433 2435004 (X.C.);
tel.: +86 433 2435003; fax: + 86 433 2435004 (H.-R. P.).
E-mail addresses: cuixun@ybu.edu.cn (X. Cui), piaohuri@yahoo.com.cn (H.-R.
Figure 1. Cardiotonic agents used for the treatment of congestive heart failure
Piao).
(CHF) and the previously reported compound PHR0007.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.049

4230
Y. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4229–4232
H2N
H2N
H2N
b
a
N
N
H
O
N
H
S
N
NH
O
3
1
2
H
N
N
N
N
O
H
N
R
N
N
Cl
5a-p
NH
c
d
O
O
N
N
NH
H
N
N
O
4
O
R
N
N
NH
6a-e
O
Scheme 1. Synthetic scheme for the synthesis of compound 5a–p and 6a–e. Reagents and conditions: (a) P2S5, Et3N, CH3CN; (b) Ethyl carbazate, cyclohexanol, reflux, N2, 6 h;
(c) ClCH2COCl, CH2Cl2, rt; (d) monosubstituted piperazines or 1,4-diazepanes, K2CO3, acetone, reflux, 10 h.
NMR, 13C NMR, MS and elemental analysis, and their positive ino-
tropic activities were evaluated by measuring their effects on left
atrium stroke volume in isolated rabbit heart preparations.
Compounds5a–p, 6a–eweresynthesizedby therouteoutlinedin
Scheme 1, and their structures and inotropic activities are displayed
in Table 1.15 Compound 1 was synthesized according to previously
described methods.16 Sulfurization of 6-amino-3,4-dihydroquino-
lin-2(1H)-one 1 with phosphorous pentasulfide in refluxing acetoni-
trile in the presence of triethylamine gave the corresponding thione
2. Cyclization of 2 with ethyl carbazate in refluxing cyclohexanol
under nitrogen atmosphere afforded the desired triazolone
compound 3 in moderate yield,17 followed by the acylation of the
amino group with 2-chloroacetyl chloride in dichloromethane at
room temperature to provide the corresponding amide 4. Nucleo-
philic-substitution of 4 with various monosubstituted piperazines
and 1, 4-diazepans in refluxing acetone in the presence of potassium
carbonate afforded compounds 5a–p and 6a–e in high yields.18
As shown in Table 1, most (15) of the 21 compounds tested dis-
played inotropic effects on isolated rabbit heart preparations, of
which 11 compounds exhibited more potent effects than milrinone
(2.46 0.07%, 3 Â 10À5 M). For 5a–m, chlorinated compounds 5d,
5e, and 5f showed good activity, but the dichloro-substituted
derivative 5k showed no potency. Among the bromo- and fluoro-
substituted compounds, only one compound (2-fluorinated 5a,
Table 1
Positive inotropic activity of the test compounds
H
H
N
N
N
N
N
O
N
O
R
N
R
N
N
N
NH
NH
O
O
5a-p
6a-e
Compound
R
Increased stroke volumea (%)
1.82 0.01
—
—
4.01 0.10
3.46 0.03
3.09 0.06
—
—
—
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
6a
6b
6c
6d
6e
–CH2C6H4(o-F)
b
–CH2C6H4(m-F)
–CH2C6H4(p-F)
–CH2C6H4(o-Cl)
–CH2C6H4(m-Cl)
–CH2C6H4(p-Cl)
–CH2C6H4(o-Br)
–CH2C6H4(m-Br)
–CH2C6H4(p-Br)
–CH2C6H5
–CH2C6H3(2,6-Cl2)
–CH2C6H4(p-CH3)
–CH2C6H4(p-OCH3)
–CH2CH=CHC6H5
3.56 0.05
—
5.48 0.07
1.46 0.04
9.40 0.12
2.22 0.02
2.26 0.01
8.98 0.07
6.41 0.08
15.48 0.27
9.07 0.16
6.75 0.07
2.46 0.04
–CH2C6H3(4-C6H4(4-Cl)CH2–, 3-OCH3)
–CH2C6H3(4-C6H5CH2-, 3-OCH3)
–CH2C6H4(o-Cl)
–CH2C6H4(m-Cl)
–CH2C6H4(p-Cl)
–CH2C6H5
–CH2C6H4(p-CH3)
Milrinone
a
The concentration for the test sample is 3 Â 10À5 M.
b
None or negative stroke volume increase.

Y. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4229–4232
4231
A
Control
B
Control
6c(30µmol/L)
6b(30µmol/L)
5 j(30µmol/L)
30
25
20
15
10
5
5n(30µmol/L)
20
6e(30µmol/L)
5e(30µmol/L)
Milrinone(30µmol/L)
***
Milrinone(30µmol/L)
15
10
5
***
0
0
-5
-5
0
5
10
15
20
25
30
0
10
20
30
Fraction number
Fraction number
C
D
Control
Control
20
15
10
5
6d(30µmol/L)
5 l(30µmol/L)
20
15
10
5
6a(30µmol/L)
5d(30µmol/L)
Milrinone(30µmol/L)
5 f(30µmol/L)
Milrinone(30µmol/L)
***
***
0
0
-5
-5
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction numbe
Figure 2. Effects of milrinone and compounds 5d, 5e, 5f, 5j, 5l, 5n, 6a, 6b, 6c 6d and 6e on stroke volume in beating rabbit atria (1.5 Hz). Values are means SE. ⁄⁄⁄P <0.001
versus control.
1.82 0.01%) exhibited weak activity. Unsubstituted and para-
methyl substituted derivatives (5j, 3.56 0.05%; 5l, 5.48 0.07%)
E
Control
exhibited more potent activity than milrinone, while para-meth-
oxy substituted derivative 5m exhibited slightly weaker activity
with 1.46 0.04% increased stroke volume. The compounds 5o
and 5p bearing a 4-benzyloxy-3-methoxybenzyl group at the 4-po-
sition of the piperazine ring displayed moderate activity, with
2.22 0.02% and 2.26 0.01% increased stroke volume, respec-
tively. Furthermore, the cinnamyl substituted derivative 5n
(9.40 0.12%) presented the most favorable activity in this series,
which might be attributed to hydrophobic interactions of cinnamyl
moiety with conjugate structure. The series of derivatives 6a–e,
bearing 4-substituted 1,4-diazepanes, displayed higher potency
than the piperazine series. In particular, the 4-chlorinated deriva-
tive 6c showed the most potent activity in this study by a
15.48 0.27% increased stroke volume, which was 5-fold more po-
tent than 5f in the piperazine series. Similar results were obtained
for the inotropic activity of 6a, 6b, 6d and 6e, in which 2-chlori-
nated 6a (8.98 0.07%) was 2.3-fold more potent, 3-chlorinated
6b (6.41 0.08%) was 1.8-fold more potent, unsubstituted 6d
(9.07 0.16%) was 2.6-fold more potent, and para-methyl 6e
(6.75 0.07%) was 1.2-fold more potent than the corresponding
compounds in the piperazine series.
20
15
10
5
5n(10µmol/L)
5n(30µmol/L)
5n(100µmol/L)
Milrinone(10µmol/L)
Milrinone(30µmol/L)
Milrinone(100µmol/L)
***
0
-5
0
5
10
15
20
25
30
Fraction number
F
Control
25
20
15
10
5
6c(10µmol/L)
6c(30µmol/L)
6c(100µmol/L)
Milrinone(10µmol/L)
Milrinone(30µmol/L)
Milrinone(100µmol/L)
***
Based on these facts, some preliminary remarks on structure–
activity relationship can be drawn from the results of bioactivities.
For the series 5 and 6, the latter showed much more stronger activ-
ity, generally. The results obtained indicated that the 1,4-diazepan
ring was important for the activity of these compounds, for which
further investigation should be considered. For the series 5, it could
be found that different substituent groups on the phenyl ring of the
benzyl group at the 4-position of the piperazine have different effect
on activity. Herein some halogen substituted derivatives (5a–i and
0
-5
0
5
10
15
20
25
30
Fraction number
Figure 3. Concentration-response curves of compounds 5n, 6c and milrinone on
stroke volume in beating rabbit artia (1.5 Hz). Values are means SE. ⁄⁄⁄P <0.001
versus control.

4232
Y. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4229–4232
Table 2
References and notes
Changes of heart rate caused by compounds in isolated rabbit heart preparations
1. Thomas, R.; Gray, P.; Andrews, J. Adv. Drug Res. 1990, 19, 313.
2. Smith, J. R.; Gheorghiade, M.; Goldstein, S. Coronary Artery Dis. 1993, 4, 16.
3. Smith, T. W. N. Engl. J. Med. 1988, 318, 358.
4. Becker, D. E. Anesth Prog. 2007, 54, 178.
5. Hallberg, P.; Lindbäck, J.; Lindahl, B.; Stenestrand, U.; Melhus, H. Eur. J. Clin.
Pharmacol. 2007, 63, 959.
6. Takafumi, F.; Shuji, T.; Toyoki, M.; Tetsumi, H.; Takumi, S.; Michiaki, T.; Youichi,
Y. J. Med. Chem. 1992, 35, 3607.
7. Packer, M.; Carver, J. R.; Rodeheffer, R. J.; Ivanhoe, R. J.; DiBianco, R.; Zeldis, S.
M.; Hendrix, G. H.; Bommer, W. J.; Elkayam, U.; Kukin, M. L. N. Engl. J. Med.
1991, 325, 1468.
8. Cohn, J. N.; Goldstein, S. O.; Greenberg, B. H.; Lorell, B. H.; Bourge, R. C.; Jaski, B.
E.; Gottlieb, S. O.; McGrew, F.; DeMets, D. L.; White, B. G. N Engl. J. Med. 1810,
1998, 339.
Compound
Mean SEa
Mean SEb
5n
6a
6b
6c
6d
6e
99.97 0.04
118.73 0.07
109.86 0.12
120.67 0.12
99.52 0.09
111.36 0.18
99.89 0.05
118.36 0.10
120.84 0.08
121.68 0.09
90.01 0.16c
111.86 0.16
a
b
c
Control.
Data after using the test samples.
P <0.01 versus control.
9. Nikpour, M.; Sadeghian, H.; Saberi, M. R.; Nick, R. S.; Seyedi, S. M.; Hosseini, A.;
Parsaee, H.; Bozorg, A. T. Bioorg. Med. Chem. 2010, 18, 855.
10. Fedman, M. D.; Pak, P. H.; Wu, C. C.; Haber, H. L.; Heesch, C. M.; Bergin, J. D.;
Powers, E. R.; Cowart, T. D.; Ohnson, W.; Feldman, A. M.; Kass, D. A. Circulation
1996, 93, 474.
11. Macgowan, G. A. Expert Opin. Invest. Drugs 2000, 9, 1109.
12. Neubauer, S. N. Eng. J. Med. 2007, 356, 1140.
13. Zhang, C. B.; Cui, X.; Hong, L.; Quan, Z. S.; Piao, H. R. Bioorg. Med. Chem. Lett.
2008, 18, 4606.
14. Lan, Y.; Piao, H. R.; Cui, X. Drug Disc. Ther. 2009, 6, 272.
15. The method of measuring left atrium stroke volume was described
previously.19,20 The features of CHF are cardiac dilatation, poor contractility
of cardiac muscle, decreased ejection fraction, and depression of left
ventricular pressure maximum alleosis. Therefore, macroscopic measurement
of the variance in left atrium stroke volume can be used to estimate the
positive inotropic effects of the compounds synthesized. Milrinone (Shuzhou
Unite Pharmaceutical Co., Dongwu Road, Shuzhou), DMSO (Sigma–Aldrich
Chemical Co., St. Louis, MO, USA) were used. All other reagents were of
analytical gradused. Atria were obtained from New Zealand white rabbits, and
the mean atrial weight was 183.6 6.8 mg. Hearts were removed from rabbits
5k) were designed and synthesized, different halogen atoms con-
tributed to the activity in the order of Cl > F > Br, generally. Compar-
ing the derivatives with different Cl-substitution positions on the
benzyl ring, their activity order was o-Cl > m-Cl > p-Cl > 2, 6-2Cl.
The electron-donor derivatives were also designed and prepared,
containing CH3 and OCH3. Their activity order was p-CH3 > p-
OCH3. For the series 6, comparing the derivatives with different
substitutents on the benzyl ring, their activity order was p-
Cl > H > o-Cl > m-Cl > p-CH3. From these results, we can see that
the effects of the substituent’s position on the benzene ring to the
biological activity showed no clear regularity, for which more this
kind of compounds need to be designed and synthesized for further
investigation.
The dynamics of the test compounds were also investigated in
perfused beating rabbit atria. We found that compounds 5j and
5f (Fig. 2 A and C) did not show a desirable biological dynamic pro-
file, because they decreased the stroke volume with time. Nine
compounds possessing inotropic effects (5d, 5e, 5l, 5n, 6a, 6b, 6c,
6d, and 6e) showed desirable biological dynamic profiles com-
pared to milrinone (Fig. 2 A, B, C and D), of which compound 6c
displayed excellent inotropic effects with the highest increased
stroke volume (Fig. 2 A).
We next tested the dose–activity relationships of the most effec-
tive compounds, 5n and 6c, at concentrations of 1 Â 10À5, 3 Â 10À5
and 1 Â 10À4 M. Both compounds showed maximal effects at
3 Â 10À5 M, and less activity at the higher dose of 1 Â 10À4 M, as
shown in Fig. 3 E and F. Compounds 5n, 6a, 6b, 6c, 6d, and 6e were
also investigated for their chronotropic effects in beating atria. As
shown in Table 2, no significantly increased heart rates (P >0.05)
were observed for 5n, 6a, 6b, 6c and 6e at the same concentration.
Unfortunately, however, heart rate was changed by compound 6d.
In summary, we report herein the design and synthesis of
novel N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7
-yl)acetamides, and demonstrate their positive inotropic activities.
The compounds were based on the structure of compound
PHR0007, modified to bear 4-substituted piperazine and 1,4-dia-
zepane moieties. Among these compounds, 5n and 6c in particu-
lar exhibited promising cardiovascular properties and potent
activities compared with milrinone. The results suggested that
their favourable activity was due to the 1,4-diazepane ring in
the 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-1(2H)-one scaffold
rather than the piperazine ring, and further development of such
compounds may be of interest. Other biological tests, including
in vivo evaluation, coronary vasodilating tests and studies on
the possible mechanism of action, are currently ongoing in our
laboratory.
and the left atria dissected free.
A calibrated transparent atrial cannula
containing two small catheters was inserted into the left atrium. The
cannulated atrium was transferred to an organ chamber and perfused
immediately with N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid
(HEPES) buffer solution by means of a peristaltic pump (1.25 mL/min) at
34 °C.21 The composition of the buffer was as follows (in mM): 118 NaCl, 4.7
KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 10.0 glucose, 10.0 HEPES (adjusted to pH
7.4 with 1 M NaOH) and 0.1% bovine serum albumin (BSA). Soon after the
perfused atrium was set up, transmural electrical field stimulation with a
luminal electrode was started at 1.5 Hz (duration, 0.3–0.5 ms, voltage 30 V).
The changes in the atrial stroke volume were monitored by reading the lowest
level of the water column in the calibrated atrial cannula during the end
diastole. The atria were perfused for 60 min to stabilize the stroke volume. The
atrial beat rate was fixed at 1.5 Hz, the left atrium stroke volume was recorded
at 2-min intervals, and the stimulus effect of the sample was recorded after a
circulation of the control group. Every circulation was 12 min. The compounds
were investigated using the single dose technique at
a concentration of
3 Â 10À5 M. Samples were dissolved in DMSO and diluted with the HEPES
buffer to 0.1% DMSO. The biological evaluation data for these compounds were
expressed in means of increased stroke volume percentage as shown in Table
1. Heart rate measurements for those selected compounds were carried out in
isolated rabbit hearts by recording the electrocardiogram in the volume
conduction model. In order to assess differences, repeated measurements were
compared by means of an ANOVA test followed by the Bonferroni’s multiple-
comparison test. Statistical significance was defined as P <0.05 and the data is
presented as means SE.
16. Yang, K.; Sun, L. P.; Liu, J. Y.; Cui, X.; Hong, L.; Piao, H. R. Bioorg. Med. Chem. Lett.
2010, 20, 4464.
17. Zhang, C. B.; Piao, H. R.; Quan, Z. S. Chem. Res. Appl. 2002, 5, 618.
18. Preparation of 6c: A mixture of 4 (224 mg, 1.0 mmol), 1-(4-chlorobenzyl)-1,4-
diazepane (278 mg, 1.0 mmol), K2CO3 (54 mg, 0.5 mmol) in acetone (10 ml)
was stirred under reflux for 6 h and concentrated under reduced pressure. The
resulting residue was purified by chromatography (CH2Cl2/CH3OH 30:1) to
afford 6c (428 mg, 92%) as white solid. Mp: 160–162 °C; IR (KBr) cm–1: 3276
(NH), 1716, 1687 (C@O) ; 1H NMR (CDCl3, 300 MHz, ppm): 1.77–1.81 (m, 2H,
CH2), 2.66–2.75 (m, 8H, CH2), 2.79 (t, J = 6.5 Hz, 2H, CH2), 2.83 (t, J = 6.5 Hz, 2H,
CH2), 3.22 (s, 2H, CH2), 3.57 (s, 2H, CH2), 7.17–8.25 (m, 7H, Ar-H), 9.30 (s, 1H,
NH), 9.67 (s, 1H, NH). 13C NMR (CDCl3, 75 MHz , ppm): d 168.00, 151.62,
143.18, 136.70, 133.99, 131.24, 128.72, 127.53, 127.07, 125.86, 118.17, 117.14,
116.33, 76.06, 75.64, 75.22, 60.83, 60.70, 54.89, 53.77, 53.62, 52.85, 26.67,
24.88, 20.30; ESI-MS (m/z): 467 [M+H]+; Anal. Calcd for C25H27ClN6O2: C,
61.73; H, 5.83; N, 18.00. Found: C, 61.66; H, 5.93; N, 18.12.
Acknowledgment
19. Cho, K. W.; Kim, S. H.; Kim, C. H.; Seul, K. H. Am. J. Physiol. 1995, 268, 1129.
20. Lee, S. J.; Kim, S. Z.; Cui, X.; Kim, S. H.; Lee, K. S.; Chung, Y. J.; Cho, K. W. Am. J.
Physiol. Heart Circ. Physiol. 2000, 278, H208.
21. Cui, X.; Wen, J. F.; Jin, J. Y.; Xu, W. X.; Kim, S. Z.; Kim, S. H.; Lee, H. S.; Cho, K. W.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002, 282, R1477.
This work was supported by the National Science Foundation of
China (81160381).