Synthesis and positive inotropic evaluation of [1,2,4]triazolo[3,4-a] phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted piperazine moieties

Article abstract of DOI:10.1016/j.bmcl.2014.02.040

Four series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a] phthalazine derivatives bearing substituted piperazine moieties were synthesized and evaluated for their positive inotropic activity by measuring the left atrium stroke volume in isolated rabbit-heart preparations. Several compounds were developed and showed favorable activities compared to the standard drug milrinone, with (4-([1,2,4]triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)(p- tolyl)methanone (5g) being identified as the most potent with an increased stroke volume of 19.15 ± 0.22% (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10^-5 M. A preliminary study of mechanism of action revealed that 5g displayed its positive inotropic effect may be related to the PDE-cAMP-PKA signaling pathway. Compounds exhibiting inotropic effects were also evaluated in terms of the chronotropic effects.

Full text of DOI:10.1016/j.bmcl.2014.02.040

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1737–1741
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and positive inotropic evaluation of [1,2,4]triazolo
[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives
bearing substituted piperazine moieties
a,
Long-Xu Ma a, , Bai-Ri Cui c, , Yan Wu a, Jia-Chun Liu a, Xun Cui b, Li-Ping Liu b, , Hu-Ri Piao
⇑
⇑
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 133000, PR China
c Department of Orthopedics, Yanbian University Affiliated Hospital, 133000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing
substituted piperazine moieties were synthesized and evaluated for their positive inotropic activity by
measuring the left atrium stroke volume in isolated rabbit-heart preparations. Several compounds were
developed and showed favorable activities compared to the standard drug milrinone, with (4-
([1,2,4]triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)(p-tolyl)methanone (5g) being identified as the
most potent with an increased stroke volume of 19.15 0.22% (milrinone: 2.46 0.07%) at a concentra-
tion of 3 Â 10–5 M. A preliminary study of mechanism of action revealed that 5g displayed its positive
inotropic effect may be related to the PDE-cAMP-PKA signaling pathway. Compounds exhibiting inotropic
effects were also evaluated in terms of the chronotropic effects.
Received 10 December 2013
Revised 2 February 2014
Accepted 14 February 2014
Available online 3 March 2014
Keywords:
[1,2,4]Triazolo[3,4-a]phthalazine
Tetrazolo[5,1-a]phthalazine
Cinnamylpiperazine
Positive inotropic activity
Stroke volume
Ó 2014 Elsevier Ltd. All rights reserved.
Congestive heart failure (CHF) is a major health problem and a
leading cause of death throughout the world.1 Cardiac glycosides
such as digoxin are frequently prescribed for the treatment of
CHF.2 The narrow safety margins associated with the use of digi-
talis compounds have always been recognized as a serious problem
because of the high frequency and severity of digitalis intoxica-
tion.3 The discovery of amrinone, which was approved for the
treatment of CHF over a decade ago as a phosphodiesterase-
inhibiting agent, led to the synthesis of a number of promising
non-sympathomimetic and non-glycoside agents for the treatment
of CHF (Fig. 1).4 The clinical use of this compound, however, has
been limited because it can lead to tachycardia through elevated
cAMP levels, as well as the occurrence of significant ventricular
arrhythmias.5 Vesnarinone is a novel inotropic drug that operates
via a unique and complex mechanism of action.6 Although vesnari-
none operates via a different mechanism of action to amrinone,
similar problems have been reported in terms of its clinical
application,7,8 and new positive inotropic agents with fewer side
OCH3
N
CN
O
N
N
N
H
H3C
Milrinone
O
N
N
N
N
N
H3CO
N
N
OCH3
O
N
H
Compound 1
Vesnarinone
Figure 1. Cardiotonic agents used for the treatment of congestive heart failure
(CHF) and the previously reported compound 1.
effects that operate via a unique mechanism of action are still
needed.9
In our previous report, we described the design and synthesis of
[1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine
derivatives bearing a substituted-benzylpiperazine moiety, and
compound 1 (Fig. 1) was identified as the most potent of these
compounds by providing an increased stroke volume of
⇑
Corresponding authors. Tel.: +86 433 2435130; fax: +86 433 2435004 (L.-P.L.);
tel.: +86 433 2435003; fax: +86 433 2435004 (H.-R.P.).
E-mail addresses: liuliping@ybu.edu.cn (L.-P. Liu), piaohuri@yahoo.com.cn
(H.-R. Piao).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.02.040
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1738
L.-X. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1737–1741
O
O
Cl
Cl
Cl
N
NH
NH
d
N
N
b
N
N
c
N
R
N
5a-i
O
a
N
N
N
4
N
N
3
2
e
O
N
O
N
f
O
N
N
6a-h
N
N
R
O
1
O
Cl
Cl
N
g
N
N
N
N
h
N
N
R
N
N
i
NHNH2
9a-i
N
N
N
N
N
8
R= 5a, 6a, 9a,10a: p-F
5b, 6b, 9b, 10b: m-Cl
5c, 6c, 9c, 10c: p-Cl
5d, 6d, 9d, 10c: m-Br
5e, 6e, 9e, 10e: p-Br
5f, 6f, 9f, 10f: H
7
O
N
N
5g, 6g, 9g, 10g: p-CH3
N
N
N
5h, 6h, 9h, 10h: m-OCH3
N
N
10a-h
R
5i, 9i: p-OCH3
Scheme 1. Synthetic scheme for the synthesis of compounds 5a–i, 6a–h, 9a–i and 10a–h. Reagents and conditions: (a) NH2NH2ÁH2O/ethanol, reflux 12 h; (b) POCl3/TEA,
reflux 4 h; (c) NH2NHCOH/N-BuOH, reflux 8 h; (d) monosubstituted piperazines, K2CO3, acetone, reflux, 48 h; (e) monosubstituted piperazines, K2CO3, acetone, reflux, 48 h. (f)
NH2NH2ÁH2O/ethanol, reflux 4 h; (g) NaNO2/HCl; (h) monosubstituted piperazines, K2CO3, acetone, reflux, 12 h; (i) monosubstituted piperazines, K2CO3, acetone, reflux, 12 h.
O
O
R
N
N
Table 1
R
N
N
Positive inotropic activity of the test compounds
N
Compound
R
Increased stroke volumea (%)
N
N
X
N
N
N
X
N
N
5a
5b
5c
5d
5e
4-F
3-Cl
4-Cl
3-Br
4-Br
H
2.91 0.04
2.56 0.08
13.33 0.11
8.49 0.07
3.65 0.09
5a-i X= C
9a-i X= N
6a-h X= C
10a-h X= N
Figure 2. Structure-based design of the target compounds.
b
5f
—
5g
5h
5i
6a
4-CH3
3-OCH3
4-OCH3
4-F
19.15 0.22
0.95 0.06
3.68 0.04
—
12.02 0.20% (milrinone: 2.46 0.07%) at
a concentration of
3 Â 10–5 M.10 As part of our ongoing work towards the develop-
ment of novel inotropic agents we also reported the discovery of
a series of compounds bearing cinnamylpiperazine moieties that
effectively enhanced the positive inotropic activity of some of
these derivatives.11 With this in mind, and in an attempt to further
optimize the inotropic properties of compound 1, we designed a
series of new compounds based on compound 1 where the
[1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine
ring moieties were left unchanged. In contrast, the decision was ta-
ken to replace the methyl group at the 4-position of the piperazine
ring in compound 1 with a carbonyl group connected to an (E)-but-
2-enal group, and changes were also made to the substituents on
the phenyl ring to investigate their contribution to the activity.
Based on these changes, four novel series of compounds, including
5a–i, 6a–h, 9a–i, and 10a–h, were synthesized and characterized,
and their positive inotropic activities subsequently evaluated by
measuring the left atrium stroke volume in isolated rabbit heart
preparations.
6b
6c
6d
6e
6f
6g
6h
9a
9b
9c
9d
9e
9f
9g
9h
9i
10a
10b
10c
10d
10e
10f
10g
10h
Milrinone
3-Cl
4-Cl
—
—
—
—
3-Br
4-Br
H
4-CH3
3-OCH3
4-F
—
14.87 0.23
4.13 0.06
4.73 0.10
0.23 0.02
2.05 0.03
—
7.49 0.08
—
13.46 0.16
1.94 0.11
9.39 0.11
—
3-Cl
4-Cl
3-Br
4-Br
H
4-CH3
3-OCH3
4-OCH3
4-F
3-Cl
4-Cl
—
—
—
—
3-Br
4-Br
H
4-CH3
3-OCH3
Compounds 5a–i, 6a–h, 9a–i, and 10a–h were synthesized
according to previously described methods,12–14 as shown in
Scheme 1. The structures of these compounds are depicted in
Figure 2. Isoindoline-1,3-dione (1) was reacted with hydrazine
hydrate in ethanol to afford 2,3-dihydrophthalazine-1,4-dione
(2), which was heated at reflux in phosphorus oxychloride (POCl3)
8.09 0.17
2.74 0.15
5.35 0.10
2.46 0.04
The concentration for the test sample is 3 Â 10À5 M.
a
b
None or negative stroke volume increase.

L.-X. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1737–1741
1739
Control
5g(30μmol/L)
A
B
35
30
25
20
15
10
5
30
25
20
15
10
5
Control
6g(30μmol/L)
9e(30μmol/L)
5c(30μmol/L)
Milrinone(30μmol/L)
5d(30μmol/L)
5e(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 number
Control
C
D
Control
9a(30μmol/L)
9g(30μmol/L)
15
10
5
10g(30μmol/L)
10h(30μmol/L)
Milrinone(30μmol/L)
25
20
15
10
5
9i (30μmol/L)
10f(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 number
Control
E
F
10
6
5a(30μmol/L)
10
6
6h(30μmol/L)
Control
Milrinone(30μmol/L)
5b(30μmol/L)
5i (30μmol/L)
Milrinone(30μmol/L)
***
***
2
2
-2
-2
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
Figure 3. Effects of milrinone and compounds 5a, 5b, 5c, 5d, 5e, 5g, 5i, 6g, 6h, 9a, 9e, 9g, 9i, 10f, 10g and 10h on stroke volume in beating rabbit atria (1.5 Hz). Values are
means SE. ⁄⁄⁄P < 0.001 versus control.
Control
G
H
Control
Milrinone(10μmol/L)
Milrinone(30μmol/L)
Milrinone(100μmol/L)
5g(10μmol/L)
5g(30μmol/LL)
5g(100μmol/L)
25
20
15
10
5
25
20
15
10
5
6g(10μmol/L)
6g(30μmol/L)
6g(100μmol/L)
Milrinone(10μmol/L)
Milrinone(30μmol/L)
Milrinone(100μmol/L)
***
***
0
0
-5
-5
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
Figure 4. Concentration–response curves of compounds 5g, 6g and milrinone on stroke volume in beating rabbit artia (1.5 Hz). Values are means SE. ⁄⁄⁄P < 0.001 versus
control.

1740
L.-X. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1737–1741
I
J
30
20
30
Control
Control
H89(1.0μmol/L)
Nifedipine(1.0μmol/L)
20
10
H89(1.0μmol/L)
+5g(30μmol/L)
Nifedipine(1.0μmol/L)
+5g(30μmol/L)
10
0
0
***
***
-10
-20
-30
-40
-10
-20
-30
-40
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
Control
Control
K
L
forskolin(30μmol/L)
IBMX(30μmol/L)
forskolin(30μmol/L)
IBMX(30μmol/L)
50
40
30
20
10
0
+5g(30μmol/L)
50
40
30
20
10
0
+5g(30μmol/L)
forskolin(30μmol/L)
IBMX(30μmol/L)
***
***
-10
-10
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
Figure 5. Role of phosphodiesterase (PDE)-cAMP-PKA pathway in the 5g induced increase in atrial pulse pressure and stroke volume. ⁄⁄⁄P < 0.001 compared with the control
period.
to give 1,4-dichlorophthalazine (3).15,16 The subsequent reaction of
compound 3 with formylhydrazine in n-butyl alcohol afforded
compound 4,17 which was reacted via a nucleophilic aromatic sub-
stitution reaction with a variety of different monosubstituted
piperazines in refluxing acetone in the presence of potassium car-
bonate to give compounds 5a–i and 6a–h. The reaction of com-
pound 3 with hydrazine hydrate in ethanol gave compound
7, which was reacted with sodium nitrite in 30% hydrochloric acid
to afford compound 8. The subsequent reaction of 8 with a variety
of different monosubstituted piperazines in refluxing acetone in
the presence of potassium carbonate gave compounds 9a–i and
10a–h in high yields.18 The structures of all of the synthesized
compounds were confirmed by IR, 1H NMR, 13C NMR, and MS
analysis.
Table 1 shows the inotropic activity data for the 34 test
compounds synthesized in the current study.19 Sixteen of the com-
pounds showed significantly increased inotropic effects on isolated
rabbit heart preparations compared to milrinone (2.46 0.07%,
3 Â 10À5 M), with compound 5g being the most potent of these
compounds with an increased stroke volume of 19.15 0.22%.
For compounds 5a–i those which bear electron-withdrawing sub-
stituents on their phenyl ring, the 4-fluorinated and 3-chlorinated
derivatives (compounds 5a and 5b, respectively) displayed moder-
ate activity, with increased stroke volumes of 2.91 0.04% and
2.56 0.08%, respectively. The 4-chlorinated, 3-brominated, and
4-brominated derivatives (5c–e) showed better activities than mil-
rinone, whereas the unsubstituted derivative 5f was inactive. Com-
pounds bearing electron-donating substituents on their phenyl
ring presented a range of different activities, with the 4-methyl
derivative 5g (19.15 0.22%) affording the highest activity of this
series. In contrast, the 3-methoxy derivative 5h displayed weaker
activity, with an increased stroke volume of 0.95 0.06%, whereas
the 4-methoxy derivative 5i exhibited moderate activity
(3.68 0.04%). For the series (6a–h), the 4-methyl derivative 6g
and 3-methoxy derivative 6h exhibited good activity with an in-
creased stroke volume of 14.87 0.23% and 4.13 0.06%.
For the tetrazolo[5,1-a]phthalazine series of derivatives 9a–i
and 10a–h, the 4-fluorinated, 4-brominated, 4-methyl, and
4-methoxy derivatives (9a, 9e, 9g and 9i) displayed higher activi-
ties than the standard drug milrinone. In contrast, the 3-chlori-
nated and 3-methoxy derivatives (9b and 9h) exhibited slightly
weaker activities, with increased stroke volumes of 0.23 0.02%
and 1.94 0.11%, respectively. The 3-brominated and unsubstitut-
ed derivatives (9d and 9f), however, were inactive. For the series
(10a–h), compounds bearing electron-withdrawing substituents
on their phenyl ring exhibited no activity. Pleasingly, the unsubsti-
tuted, 4-methyl, and 3-methoxy derivatives (10f–h) showed higher
activities than milrinone, with increased stroke volumes of
8.09 0.17%, 2.74 0.15% and 5.35 0.10%, respectively.
Based on an analysis of the activities of the synthesized
compounds, the following structure activity relationships (SARs)
were obtained. Comparing the activities across the four different
series of compounds, a general positive inotropic activity order of
series 5 > series 6 > series 9 > series 10 was obtained, which indi-
cated that the replacement of the triazole moiety with a tetrazole
did not have a positive impact on the activity of the compounds.20
Relative to the substituted (E)-3-phenyl-1-(piperazin-1-yl)prop-2-
en-1-one, the substituted phenyl(piperazin-1-yl)methanone gen-
erally exhibited higher levels of inhibitory activity. In terms of
the effects of the different substituent groups, compounds bearing
electron-donating substituents had a much greater impact on the
activity than those bearing electron-withdrawing substituents,
although the position of the substituent was not fixed.
The dynamics of the synthesized compounds were also evalu-
ated in perfused beating rabbit atria, and the results revealed that
5a, 5b, 5e, 5i, 6h, 9a, 9e, 9g, 9i, 10f, 10g, and 10h exhibited similar

L.-X. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1737–1741
1741
5. 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. Eng. J. Med. 1991,
325, 1468.
6. Cavusoqlu, E.; Frishman, W. H.; Klapholz, M.; Card, J. Fail 1995, 1, 249.
7. 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. Eng. J. Med. 1998,
339, 1810.
8. 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.
9. Neubauer, S. N. Eng. J. Med. 2007, 356, 1140.
10. Wu, Y.; Sun, L. P.; Ma, L. X.; Che, J.; Song, M. X.; Cui, X.; Piao, H. R. Chem. Biol.
Drug Des. 2013, 81, 591.
atrial dynamic profiles to milrinone with good increased stroke
volumes (Fig. 3). Compounds 5c, 5d, 5g, and 6g afforded the most
desirable of all of the atrial dynamic profiles (Fig. 3A and B), where
the stroke volumes gradually increased as the time progressed. The
dose–activity relationships of compounds 5g and 6g were also
determined at concentrations of 1 Â 10À5
,
3 Â 10À5
,
and
1 Â 10À4 M, respectively, with the maximum effects being ob-
served at a concentration of 3 Â 10À5 M, and the lower potency
being observed at the higher dose of 1 Â 10À4 M, as shown in
Figure 4G and H.
11. Wu, Y.; Ma, L. X.; Niu, T. W.; Meng, F. L.; Cui, X.; Piao, H. R. Arch. Pharm. Chem.
Life Sci. 2012, 345, 980.
A preliminary investigation of the mechanism of action of com-
pound 5g on the atrial dynamics was conducted (Fig. 5). It is well
known that when the L-type Ca2+ channel on the cell membrane
opens, an influx of Ca2+ ions can be actuated via the activation of
protein kinase A (PKA) to generate a positive inotropic effect on
the myocardium when the concentration of cAMP in the myocar-
dial cell increases. The results of the current study clearly indicate
that 5g exerts its positive inotropic effect on the rabbit atrium. The
effects of compound 5g may be blocked therefore not only by the
L-type Ca2+ channel blocker nifedipine but also by the PKA inhibi-
tor H-89 (Fig. 5I and J). These results indicate that 5g generates its
positive inotropic effect on the myocardium by increasing the
intracellular concentration of cAMP and promoting the influx of
Ca2+ ions. Furthermore, the fact that adenylate cyclase activator
forskolin led to a significant increase in the atrial stroke volume
accumulated by 5g, but failed to change the effect of the nonselec-
tive phosphodiesterase (PDE) inhibitor IBMX-increased atrial
stroke volume (Fig. 5K and L), indicated that 5g induced its positive
inotropic effect may be related to the PDE-cAMP-PKA signaling
pathway in the isolated perfused beating rabbit atria.
In summary, [1,2,4]triazolo[3,4-a]phthalazine and tetrazol-
o[5,1-a]phthalazine derivatives bearing substituted piperazine
moieties have been synthesized for the first time and their positive
inotropic activities evaluated. Compounds 5g and 6g, in particular,
exhibited promising cardiovascular properties and potent activi-
ties compared with milrinone. Compound 5g is most likely a phos-
phodiesterase inhibitor that is capable of increasing the
concentration of intracellular cAMP and activating PKA. The posi-
tive inotropic action of this compound may be related to the
PDE-cAMP-PKA signal transduction pathway. The results of this
study suggest that compounds of this type should be developed
further and subjected to a range of different biological tests includ-
ing in vivo evaluation. We are currently evaluating the coronary
vasodilating abilities of these compounds in our laboratory.
12. Gale, K. Epilepsia 1992, 33, 3.
13. Liu, X. K.; Cui, X.; Hong, L.; Sun, L. P.; Quan, Z. S.; Piao, H. R. Eur. J. Med. Chem.
2009, 44, 3027.
14. Löscher, W. Biochem. Pharmacol. 1979, 28, 1397.
15. Zhang, C. B.; Cui, X.; Hong, L.; Quan, Z. S.; Piao, H. R. Bioorg. Med. Chem. Lett.
2008, 18, 4606.
16. Li, J. Y.; Cui, X.; Liu, X. K.; Hong, L.; Quan, Z. S.; Piao, H. R. Arch. Pharm. 2008, 341,
794.
17. Wu, Y.; Ma, L. X.; Niu, T. W.; Cui, X.; Piao, H. R. Bioorg. Med. Chem. Lett. 2012, 22,
4229.
18. Preparation of 5g: A mixture of 4 (1.0 mmol), monosubstituted piperazine
(2.0 mmol), K2CO3 (0.5 mmol) in acetone (10 ml) was stirred under reflux for
48 h and concentrated under reduced pressure. The resulting residue was
purified by chromatography (CH2Cl2/CH3OH 50:1) to afford 5g (301 mg, 81%)
as white solid. Mp: 232–233 °C; IR (KBr) cmÀ1: 1650 (C@O), 1569, 1508, 1440
(C@N); 1H NMR (CDCl3, 300 MHz, ppm): d 2.32 (s, 3H, CH3), 3.41–3.65 (m, 4H,
(CH2)2), 3.66–3.85 (m, 4H, (CH2)2), 7.17–8.92 (m, 8H, Ar-H), 8.94 (s, 1H, N@CH).
13C NMR (CDCl3, 75 MHz, ppm): d 169.99, 157.11, 141.40, 139.43, 138.47,
132.48, 131.42, 129.64, 128.37, 126.39, 125.36, 123.72, 123.24, 119.51, 50.06,
49.69, 20.57; HRMS (MALDI) calcd for
395.1596 (M+Na).
C21H20N6ONa: 395.1591, found:
19. The method of measuring left atrium stroke volume was described
previously.21,22 The features of CHF are cardiac dilatation, poor contractility
of cardiac muscle, decreased ejection fraction, and depression of left
ventricular pressure maximum alleosis. Therefore, the macroscopic
measurement of the varianc previously 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. The atrium was perfused 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.23 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 10–5 M. Samples were dissolved in DMSO and diluted
with the HEPES buffer to an appropriate volume. The biological evaluation data
for these compounds were expressed in means of increased stroke volume
percentage as shown in Table 1. 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.
Acknowledgment
This work was supported by the National Science Foundation of
China (81160381).
20. Song, M. X.; Zheng, C. J.; Deng, X. Q.; Sun, L. P.; Wu, Y.; Hong, L.; Li, Y. J.; Liu, Y.;
Wei, Z. Y.; Jin, M. J.; Piao, H. R. Eur. J. Med. Chem. 2013, 60, 376.
References and notes
21. Cho, K. W.; Kim, S. H.; Kim, C. H.; Seul, K. H. Am. J. Physiol. 1995, 268, 1129.
22. 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.
23. 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.
1. Fromm, R. E., Jr.; Varon, J.; Gibbs, L. R. J. Emerg. Med. 1995, 13, 7l.
2. Smith, J. R.; Gheorghiade, M.; Goldstein, S. Coron. Artery Dis. 1993, 4, 16.
3. Becker, D. E. Anesth. Prog. 2007, 54, 178.
4. Takafumi, F.; Shuji, T.; Toyoki, M.; Tetsumi, H.; Takumi, S.; Michiaki, T.; Youichi,
Y. J. Med. Chem. 1992, 35, 3607.