Synthesis of 2-(4-substitutedmethylpiperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamides and their positive inotropic evaluation

Article abstract of DOI:10.1016/j.ejmech.2008.08.006

In an attempt to search for more potent positive inotropic agents, a series of 2-(4-substitutedmethylpiperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamides were synthesized and their positive inotropic activities were evaluated by measuring left atrium stroke volume on isolated rabbit heart preparations. Several compounds showed favorable activities compared with the standard drug, milrinone, among which 2-(4-(4-methylbenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide 4e showed the most potent activity with the 5.09 ± 0.00% increased stroke volume (milrinone 1.67 ± 0.64%) at a concentration of 1 × 10^-5 M in our in vitro study. The chronotropic effects of those compounds having significant inotropic effects were also evaluated in this work.

Full text of DOI:10.1016/j.ejmech.2008.08.006

European Journal of Medicinal Chemistry 44 (2009) 3027–3031
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of 2-(4-substitutedmethylpiperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-
benzo[b][1,4]oxazin-7-yl)acetamides and their positive inotropic evaluation
,
Xue-Kun Liu a, Xun Cui b, Lan Hong b, Liang-Peng Sun a, Zhe-Shan Quan a, 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,
Jilin Province, PR China
b College of Medicine, Yanbian University, Yanji, 133000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2008
Received in revised form 18 August 2008
Accepted 19 August 2008
Available online 29 August 2008
Inanattemptto searchformorepotentpositiveinotropicagents, aseriesof2-(4-substitutedmethylpiperazin-
1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamides were synthesized and their positive
inotropic activities were evaluated by measuring left atrium stroke volume on isolated rabbit heart
preparations. Several compounds showed favorable activities compared with the standard drug, milrinone,
among which 2-(4-(4-methylbenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)
acetamide 4e showed the most potent activity with the 5.09 ꢀ 0.00% increased stroke volume (milrinone
1.67 ꢀ 0.64%) at a concentration of 1 ꢁ 10ꢂ5 M in our in vitro study. The chronotropic effects of those
compounds having significant inotropic effects were also evaluated in this work.
Keywords:
3,4-Dihydro-3-oxo-2H-benzo[b][1,4]oxazine
Positive inotropic activity
Stroke volume
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
of life but also reduce the mortality of CHF patients, is still an
important challenge for medicinal chemists [9].
Cardiac glycosides, like digoxin, have consistently been one of
the most frequently prescribed cardiotonics used for the treatment
of congestive heart failure (CHF) for many years and are the only
medications used without increasing the CHF patient’s mortality up
to now, but their high toxicity and narrow therapeutic window still
limit their clinical application as positive inotropic agents [1]. Other
alternative synthetic replacements, like the phosphodiesterase-
inhibiting agent milrinone [2], recently developed vesnarinone
[3,4] and toborinone [5], ultimately exert their inotropic effects via
an increase in intracellular calcium and consequently result in
strong arrhythmogenic effects that caused increased mortality
when used to CHF patients. Because of overall deleterious effects on
long-term survival in CHF, this class of drugs should now be
considered most suitable for short-term use in acute episodes of
decompensated heart failure [6]. Levosimendan, which exerts its
positive inotropic effect by both increasing the sensitivity of the
myofilament to intracellular calcium and inhibiting phosphodies-
terase, could reduce mortality in the short-term treatment of CHF,
but it is uncertain whether it will reduce the mortality in long-term
treatment [7,8]. Therefore, due to the lack of a desirable inotropic
agent for the treatment of cardiac failure so far, the development of
novel positive inotropic agents with approved therapeutic prop-
erties in the treatment of CHF, which not only improve the quality
In our previous work to search for more potent positive
inotropic agents having less side effects, a series of 2-(4-sub-
stitutedpiperazin-1-yl)-N-(3,4-dihydro-2(1H)-quinolinone-6-yl)a-
cetamides was synthesized and tested for their biological activity,
among which the compound 2-(4-benzylpiperazin-1-yl)-N-(3,4-
dihydro-2(1H)-quinolinone-6-yl)acetamide PHR9612 showed
moderate positive inotropic activity [10]. In our present continual
research to further optimize the compound PHR9612, we replaced
the 3,4-dihydro-2(1H)quinolinone moiety with the 3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazine and changed the substituents on the
piperazine ring at the 4-positon, simultaneously, in which either
substituted benzyl groups or (2-substituted naphthalen-6-
yl)methyl groups were introduced, in order to preliminarily
investigate the contribution of such a structural change to the
biological activity. The compounds synthesized were characterized
by IR, NMR, MS and elemental analysis and their positive inotropic
activities were evaluated by measuring their effects on left atrium
stroke volume in isolated rabbit heart preparations using milri-
none, a marked drug, as a reference compound for a comparison.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 3a–c and 4a–k is presented in
Scheme 1. Compound 1 was synthesized through cyclization and
* Corresponding author. Tel.: þ86 433 2660503; fax: þ86 433 2659795.
E-mail address: piaohuri@yahoo.com.cn (H.-R. Piao).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.08.006

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X.-K. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3027–3031
H
N
O2N
O
H2N
O
O
O2N
OH
c
a
b
Cl
O
N
H
O
N
H
O
N
H
O
NH2
2
1
e
d
H
N
H
N
O
O
O
N
N
N
O
R
N
O
N
H
O
N
H
O
R
3a-c
4a-k
Scheme 1. Synthetic scheme for the synthesis of compounds 3a–d and 4a–k. Reagents and conditions: (a) ClCH2COCl/acetonitrile/TEBA/Na2CO3, (b) H2/Pd/C/MeOH, (c) ClCH2COCl/
Cl2CH2, (d) monosubstituted piperazines/Na2CO3/MeOH, (e) 1-((2-substitutedbenzyloxy)naphthalen-6-yl)methylpiperazines/Na2CO3/MeOH.
reduction according to the previously described methods by using
commercially available 2-amino-5-nitrophenol as a starting mate-
rial. Catalytic hydrogenation method was used to reduce the nitro
group instead of the method of using iron powder in hydrochloride,
and the yield was increased from 75% to 98% [11]. The resulting
amino group at the 7-position of 1 was acylated with 2-chloroacetyl
chloride according to the reported method in dichloromethane to
provide corresponding amide 2 in excellent yield [10]. Nucleo-
philic-substitution reaction of 2 with various monosubstituted
piperazines in refluxing methanol in the presence of sodium
carbonate afforded corresponding compounds 3a–c and 4a–k in
moderate yield.
On the other hand, we investigated the dynamics of the test
compounds in perfused beating rabbit atria and found that four
compounds possessing inotropic effects (4c, 4e, 4f and 4j) showed
desirable biological dynamic profiles compared with that of milri-
none, although the lower potency was observed for 6j (1.10 ꢀ 0.22%,
Fig. 1C). It was found that the stroke volumes of 4c and 4e were
gradually increased as the time progressed (Fig. 1A), but more
desirable dynamic property was observed for 4f whose stroke
volume was kept unchanged after rising to a certain extent (Fig.1B).
As shown in Table 2, compounds 4c, 4e, and 4f were also
investigated for their chronotropic effects in a beating atria and no
significant increased heart rate (P > 0.05) was observed for
compound 4f at the same concentration. Compounds 4c and 4e,
however, showed the changed heart rates unfortunately, for which
in vivo study was required in order to further investigate their
chronotropic effects. Compound 4f, due to its more promising
cardiovascular property, is now undergoing further biological tests
including in vivo evaluation, coronary vasodilating test, and
possible mechanism of action study in order to be selected as
a candidate for further clinical trials.
2.2. Biological evaluation
As shown in Table 1, 4 compounds out of the 15 test compounds
showed inotropic effects on isolated rabbit heart preparations.
Compounds 4c, 4e, and 4f exhibited more potent effects while 4j
showed slightly lower potency compared to milrinone
(1.67 ꢀ 0.64%, 1 ꢁ10ꢂ5 M), among which compound 4e showed the
most potency with 5.09 ꢀ 0.00% increased stroke volume. In
contrast to the previously evaluated PHR9612 with a potency which
was weaker compared with milrinone (no data), three compounds
showed significantly increased inotropic activities through the
structure modification of replacing 3,4-dihydro-2(1H)-quinolinone
moiety with 3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazine, but none
of those compounds possessing (2-substituted naphthalen-6-yl)
methyl groups at 4-position of the piperazine ring showed any
inotropic activities. It seems that a suitable length of molecule is
required for the activity, while for the compounds 3a–c an
extremely extended molecular length was caused by inleting
a naphthalene ring that was further connected with substituted
benzyloxy group at 2-position of the naphthalene. As for the rela-
tionship between inotropic activity and the different substituents
(R) on the phenyl ring of the benzyl group at the 4-position of the
piperazine for 4a–k, we couldn’t find any clear regularity for the
contribution of the substituents R to the biological effect, but those
compounds having electron-donating substituents on phenyl ring
appeared to have more potent effects although 4d and 4g didn’t
show any potency. Interestingly, for the halogen substituted
derivatives, bromo- and fluoro-substituted compounds (4c, 4j)
showed good activities except 4b, but those chloro-substituted
compounds (4h, 4i, and 4k) showed no potency. In addition, the
effects of the substituent’s position on the benzene ring to the
efficancy also showed no regularity, for which more compounds
need to be designed and synthesized for further investigation.
3. Experimental section
3.1. Chemistry
Melting points were determined in open capillary tubes and are
uncorrected. Reaction courses were monitored by TLC on silica gel
Table 1
Positive inotropic activity of the test compounds
Compound
R
Increased stroke volume (%)a
b
3a
H
–
3b
3c
3d
4-OCH3
4-CH3
3-Cl
–
–
–
4a
H
–
4b
4-F
–
4c
4d
4e
4f
4g
4h
4i
2-F
4.29 ꢀ 0.00
3,4-OCH2O-
4-CH3
3-OCH3
4-OCH3
4-Cl
–
5.09 ꢀ 0.00
3.23 ꢀ 0.00
–
–
–
3-Cl
4j
2-Br
2,6-(Cl)2
1.43 ꢀ 0.00
4k
Milrinone
–
1.67 ꢀ 0.64
a
The concentration for the test sample is 1 ꢁ10ꢂ5 M.
b
None or negative stroke volume increases.

X.-K. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3027–3031
3029
precoated F254 Merck plates and developed plates were examined
with UV lamps (254 nm). IR spectra were recorded (in KBr) on a FT-
IR1730. 1H NMR spectra were measured on Bruker AV-300 spec-
trometer, using TMS as internal standard. Mass spectra were
measured on an HP1100LC (Agilent Technologies, USA). Elemental
analyses for C, H, and N were within ꢀ0.4% of the theoretical values
and were performed on a 204Q CHN Rapid Analyzer (Perkin–Elmer,
USA). The major chemicals were purchased from Aldrich and Fluka
Companies. Monosubstituted piperazines were synthesized by the
method reported [12].
Valuesare means SE.
6
5
A
4
3
2
1
Control
4c (10.0μmol/L)
4e (10.0μmol/L)
3.1.1. 7-Amino-2H-benzo[b][1,4]oxazin-3(4H)-one (1)
0
A
suspension of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one
(4.0 g, 0.021 mol), 5% palladium on charcoal (0.4 g), and absolute
ethanol (50 mL) was stirred at room temperature under atmo-
spheric pressure of hydrogen until the absorption of hydrogen
ceased. The catalyst was filtered off and the solvent was evaporated
to afford 1 in 98% yield; mp 211–213 ꢃC; 1H NMR (DMSO-d6,
-1
0
5
10
15
20
25
30
Fraction number
300 MHz)
3H, Ar–H), 10.27 (s, 1H, –CONH).
d 4.41 (s, 2H, –COCH2), 4.88 (s, 2H, –NH2), 6.17–6.57 (m,
6
5
B
3.1.2. 2-Chloro-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-
7-yl)acetamide (2)
4
To a stirred solution of 1 (2.0 g, 0.012 mol) in dichloromethane
was added dropwise a solution of 2-chloroacetyl chloride (1.49 g,
0.013 mol) in 20 mL of dichloromethane (2 h). The resulting yellow
solid was collected by filtration at the pump to afford 2 in 99% yield;
m.p. 306–308 ꢃC. IR (KBr) cmꢂ1: 3350 (NH), 1707 (C]O), 1695
3
2
1
(C]O). 1H NMR (CDCl3)
d 2.67 (s, 3H, –CH3), 2.97 (m, 4H, –CH2CH2),
Control
4f (10.0μmol/L)
0
4.10 (s, 2H, –CH2), 7.56–7.63 (m, 3H, Ar–H); MS m/z 241 (M þ 1).
Anal. Calcd for C10H9ClN2O3: C, 49.91; H, 3.77; N, 11.64. Found: C,
50.02; H, 3.73; N, 11.82.
-1
0
5
10
15
Fraction number
20
25
30
3.1.3. General procedure for compounds 3a–d and 4a–k
A mixture of 2 (0.24 g, 0.001 mol), monosubstituted piperazine
(0.002 mmol), and sodium carbonate anhydrous in refluxing
methanol was stirred for 24 h. The solvent was evaporated under
reduced pressure and the residue was dissolved in dichloro-
methane, washed with water and brine, dried over MgSO4, and the
solvent was removed under reduced pressure. The resulting residue
was purified by silica gel column chromatography (dichloro-
methane/methanol, 40:1). The yield, melting point and spectral
data of each compound are given below.
6
5
C
Control
4j (10.0μmol/L)
Milrinone (10.0μmol/L)
4
3
2
3.1.4. 2-(4-(2-Benzyloxynaphthalen-6-yl)piperazin-1-yl)-N-(3,4-
dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (3a)
Yield 49%; m.p. 189–190 ꢃC. IR (KBr) cmꢂ1: 3451 (NH), 1691
1
0
(C]O), 1686 (C]O). 1H NMR (CDCl3)
d 2.51–2.56 (m, 8H, –CH2),
3.51 (s, 2H, –CH2), 4.01 (s, 2H, –CH2), 4.57 (s, 2H, –CH2), 5.18 (s, 2H,
–CH2), 6.32–7.66 (m,14H, Ar–H), 8.19 (s,1H, –NH), 8.86 (s, 1H, –NH).
MS m/z 536 (M þ 1). Anal. Calcd for C32H32N4O4: C, 71.62; H, 6.01; N,
10.44. Found: C, 71.62; H, 6.03; N, 10.58.
-1
0
5
10
15
Fraction number
20
25
30
Fig. 1. Effects of milrinone and compounds 4c, 4e, 4f, and 4j on stroke volume in
beating rabbit atria (1.5 Hz). Values are mean ꢀ SE.
3.1.5. 2-(4-(2-(4-Methoxybenzyloxy)naphthalen-6-yl)piperazin-
1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)
acetamide (3b)
Yield 46%; m.p. 190–191 ꢃC. IR (KBr) cmꢂ1: 3456 (NH), 1691
(C]O),1687 (C]O). 1H NMR (CDCl3)
d 2.58–2.63 (m, 8H, –CH2), 3.12
Table 2
(s, 3H, –CH3), 3.50 (s, 2H, –CH2), 3.73 (s, 2H, –CH2), 4.38 (s, 2H, –CH2),
4.60 (s, 2H, –CH2), 6.74–7.89 (m, 16H, Ar–H), 8.77 (s, 1H, –NH), 9.11
(s, 1H, –NH). MS m/z 567 (M þ 1). Anal. Calcd for C33H34N4O5: C,
69.95; H, 6.05; N, 9.89. Found: C, 70.01; H, 6.03; N, 9.92.
Changes of heart rate caused by compounds in isolated rabbit heart preparations
Compound
Mean ꢀ SEa
146.80 ꢀ 0.09
152.20 ꢀ 0.42
134.30 ꢀ 0.24
Mean ꢀ SEb
4c
4e
6f
139.10 ꢀ 0.77c
142.20 ꢀ 0.37c
134.80 ꢀ 0.23
3.1.6. 2-(4-(2-(4-Methylbenzyloxy)naphthalen-6-yl)piperazin-1-yl)
-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (3c)
Yield 43%; m.p. 195–196 ꢃC. IR (KBr) cmꢂ1: 3452 (NH), 1692
a
Control.
b
Data after using the test samples.
P < 0.01 vs. control.
(C]O), 1681 (C]O). 1H NMR (CDCl3)
d 2.38 (s, 3H, –CH3), 2.55–2.61
c

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X.-K. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3027–3031
(m, 8H, –CH2), 3.50 (s, 2H, –CH2), 3.67 (s, 2H, –CH2), 4.57 (s, 2H,
–CH2), 5.14 (s, 2H, –CH2), 6.28–7.74 (m, 16H, Ar–H), 8.36 (s, 1H,
–NH), 8.62 (S, 1H, –NH). MS m/z 551 (M þ 1). Anal. Calcd for
C33H34N4O4: C, 71.98; H, 6.22; N, 10.17. Found: C, 71.81; H, 6.23; N,
10.22.
6.35–7.58 (m, 7H, Ar–H), 8.85 (s, 1H, –NH), 9.11 (s, 1H, –NH). MS m/z
411 (M þ 1). Anal. Calcd for C22H26N4O4: C, 64.37; H, 6.38; N, 13.65.
Found: C, 64.42; H, 6.36; N, 13.45.
3.1.14. 2-(4-(4-Methoxybenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4g)
3.1.7. 2-(4-(2-(3-Chlorobenzyloxy)naphthalen-6-yl)piperazin-1-
yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)
acetamide (3d)
Yield 48%; m.p. 175–176 ꢃC. IR (KBr) cmꢂ1: 3465 (NH), 1694
(C]O), 1688 (C]O). 1H NMR (CDCl3)
d 2.29–2.35 (m, 8H, –CH2),
2.93 (s, 2H, –CH2), 3.35 (s, 2H, –CH2), 3.93 (s, 3H, –CH3), 4.36 (s, 2H,
–CH2), 6.14–7.53 (m, 7H, Ar–H), 9.05 (s, 1H, –NH), 9.99 (s, 1H, –NH).
MS m/z 411 (M þ 1). Anal. Calcd for C22H26N4O4: C, 64.37; H, 6.38; N,
13.65. Found: C, 64.52; H, 6.31; N, 13.85.
Yield 47%; m.p. 203–204 ꢃC. IR (KBr) cmꢂ1: 3458 (NH), 1692
(C]O),1687 (C]O). 1H NMR (CDCl3)
d 2.56–2.61 (m, 8H, –CH2), 3.13
(s, 2H, –CH2), 3.67 (s, 2H, –CH2), 4.51 (s, 2H, –CH2), 5.14 (s, 2H,
–CH2), 7.31–7.84 (m,13H, Ar–H), 8.36 (s,1H, –NH), 8.84 (s, 1H, –NH).
MS m/z 571 (M þ 1). Anal. Calcd for C32H31ClN4O4: C, 67.30; H, 5.47;
N, 9.81. Found: C, 67.43; H, 5.58; N, 9.98.
3.1.15. 2-(4-(4-Chlorobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4h)
Yield 47%; m.p. 174–176 ꢃC. IR (KBr) cmꢂ1: 3466 (NH), 1698
3.1.8. 2-(4-Benzylpiperazin-1-yl)-N-(3,4-dihydro-3-oxo-2H-
benzo[b][1,4]oxazin-7-yl)acetamide (4a)
(C]O), 1690 (C]O). 1H NMR (CDCl3)
d 2.31–2.36 (m, 8H, –CH2),
3.39 (s, 2H, –CH2), 3.93 (s, 2H, –CH2), 5.19 (s, 2H, –CH2), 6.14–7.56
(m, 7H, Ar–H), 8.76 (s, 1H, –NH), 8.89 (s, 1H, –NH). MS m/z 415
(M þ 1). Anal. Calcd for C21H23ClN4O3: C, 60.79; H, 5.59; N, 13.50.
Found: C, 60.92; H, 5.62; N, 13.75.
Yield 50%; m.p. 203–204 ꢃC. IR (KBr) cmꢂ1: 3467 (NH), 1690
(C]O), 1682 (C]O). 1H NMR (CDCl3)
d 2.43–2.48 (m, 8H, –CH2),
3.47 (s, 2H, –CH2), 3.80 (s, 2H, –CH2), 4.56 (s, 2H, –CH2), 6.28–7.27
(m, 8H, Ar–H), 7.84 (s, 1H, –NH), 8.16 (s, 1H, –NH). MS m/z 381
(M þ 1). Anal. Calcd for C21H24N4O3: C, 66.30; H, 6.36; N, 14.73.
Found: C, 66.29; H, 6.38; N, 14.42.
3.1.16. 2-(4-(3-Chlorobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4i)
Yield 52%; m.p. 185–187 ꢃC. IR (KBr) cmꢂ1: 3469 (NH), 1697
3.1.9. 2-(4-(4-Fluorobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-oxo-
2H-benzo[b][1,4]oxazin-7-yl)acetamide (4b)
(C]O), 1691 (C]O). 1H NMR (CDCl3)
d 2.69–2.74 (m, 8H, –CH2),
3.50 (s, 2H, –CH2), 3.94 (s, 2H, –CH2), 4.36 (s, 2H, –CH2), 7.08–7.74
(m, 7H, Ar–H), 9.48 (s, 1H, –NH), 10.01 (s, 1H, –NH). MS m/z 415
(M þ 1). Anal. Calcd for C21H23ClN4O3: C, 60.79; H, 5.59; N, 13.50.
Found: C, 60.82; H, 5.64; N, 13.85.
Yield 51%; m.p. 202–203 ꢃC. IR (KBr) cmꢂ1: 3462 (NH), 1696
(C]O), 1682 (C]O). 1H NMR (CDCl3)
d 2.42–2.47 (m, 8H, –CH2),
2.08 (s, 2H, –CH2), 3.58 (s, 2H, –CH2), 4.56 (s, 2H, –CH2), 6.29–7.35
(m, 7H, Ar–H), 8.27 (s, 1H, –NH), 8.76 (s, 1H, –NH). MS m/z 399
(M þ 1). Anal. Calcd for C21H23FN4O3: C, 63.30; H, 5.82; N, 14.06.
Found: C, 63.29; H, 5.82; N, 14.31.
3.1.17. 2-(4-(2-Bromobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4j)
Yield 51%; m.p. 164–166 ꢃC. IR (KBr) cmꢂ1: 3464 (NH), 1696
3.1.10. 2-(4-(2-Fluorobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4c)
(C]O), 1687 (C]O). 1H NMR (CDCl3)
d 2.48–2.53 (s, 8H, –CH2), 3.11
(s, 2H, –CH2), 3.54 (s, 2H, –CH2), 4.42 (s, 2H, –CH2), 6.14–7.59 (m, 7H,
Ar–H), 8.38 (s, 1H, –NH), 9.03 (s, 1H, –NH). MS m/z 459 (M þ 1).
Anal. Calcd for C21H23BrN4O3: C, 54.91; H, 5.05; N, 12.20. Found: C,
55.16; H, 5.04; N, 12.45.
Yield 52%; m.p. 185–186 ꢃC. IR (KBr) cmꢂ1: 3462 (NH), 1695
(C]O), 1684 (C]O). 1H NMR (CDCl3)
d 2.59–2.65 (m, 8H, –CH2),
3.56 (s, 2H, –CH2), 3.77 (s, 2H, –CH2), 4.60 (s, 2H, –CH2), 7.08–7.66
(m, 7H, Ar–H), 8.76 (s, 1H, –NH), 9.11 (s, 1H, –NH). MS m/z 399
(M þ 1). Anal. Calcd for C21H23FN4O3: C, 63.30; H, 5.82; N, 14.06.
Found: C, 63.28; H, 5.81; N, 14.35.
3.1.18. 2-(4-(2,6-Dichlorobenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4k)
Yield 45%; m.p. 182–184 ꢃC. IR (KBr) cmꢂ1: 3463 (NH), 1696
3.1.11. 2-(4-(Benzo[d][1,3]dioxol-5-yl-methyl)piperazin-1-yl)-N-
(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4d)
Yield 48%; m.p. 179–180 ꢃC. IR (KBr) cmꢂ1: 3462 (NH), 1693
(C]O), 1688 (C]O). 1H NMR (CDCl3)
d 2.45–2.50 (s, 8H, –CH2), 2.99
(s, 2H, –CH2), 3.67 (s, 2H, –CH2), 4.58 (s, 2H, –CH2), 6.57–7.78 (m,
6H, Ar–H), 8.65 (s, 1H, –NH), 9.12 (s, 1H, –NH). MS m/z 449 (M þ 1).
Anal. Calcd for C21H23Cl2N4O3: C, 56.13; H, 4.94; N, 12.47. Found: C,
56.22; H, 4.91; N, 12.65.
(C]O), 1680 (C]O). 1H NMR (CDCl3)
d 2.39–2.44 (m, 8H, –CH2),
3.51 (s, 2H, –CH2), 4.01 (s, 2H, –CH2), 4.62 (s, 2H, –CH2), 5.95 (s, 2H,
–CH2), 6.29–8.20 (m, 6H, Ar–H), 8.57 (s, 1H, –NH), 8.85 (s, 1H, –NH).
MS m/z 425 (M þ 1). Anal. Calcd for C22H24N4O5: C, 62.25; H, 5.70;
N, 13.20. Found: C, 62.10; H, 5.71; N, 13.47.
3.2. Evaluation of positive inotropic effects in vitro
The method of measuring left atrium stroke volume was
adopted for the biological evaluation of the compounds synthe-
sized in the present work. The features of congestive heart failure
are cardiac dilatation, poor contractility of cardiac muscle,
decreased ejection fraction, and depression of left ventricular
pressure maximum alleosis. Therefore, the macroscopic measure-
ment of the variance of left atrium stroke volume can be used to
estimate the positive inotropic effects of the compounds
synthesized.
3.1.12. 2-(4-(4-Methylbenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4e)
Yield 47%; m.p. 193–194 ꢃC. IR (KBr) cmꢂ1: 3468 (NH), 1690
(C]O), 1685 (C]O). 1H NMR (CDCl3)
d 2.31–2.37 (m, 8H, –CH2),
2.95 (s, 2H, –CH2), 3.37 (s, 2H, –CH2), 3.88 (s, 3H, –CH3), 4.37 (s, 2H,
–CH2), 6.15–7.47 (m, 7H, Ar–H), 8.79 (s, 1H, –NH), 9.27 (s, 1H, –NH).
MS m/z 395 (M þ 1). Anal. Calcd for C22H26N4O3: C, 66.99; H, 6.64;
N, 14.20. Found: C, 66.92; H, 6.66; N, 14.46.
3.1.13. 2-(4-(3-Methoxybenzyl)piperazin-1-yl)-N-(3,4-dihydro-3-
oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide (4f)
3.2.1. Drugs and chemicals
The following drugs and chemicals were used in this biological
evaluation test: milrinone (Shuzhou Unite Pharmaceutical Co.,
Dongwu Road, Shuzhou), DMSO (Sigma–Aldrich Chemical Co., St.
Louis, MO). All other reagents were of analytical grade.
Yield 51%; m.p. 182–183 ꢃC. IR (KBr) cmꢂ1: 3463 (NH), 1695
(C]O),1687 (C]O). 1H NMR (CDCl3)
d
2.61–2.67 (m, 8H, –CH2), 3.19
(s, 2H, –CH2), 3.55 (s, 2H, –CH2), 3.95 (s, 3H, –CH3), 4.62 (s, 2H, –CH2),

X.-K. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 3027–3031
3031
3.2.2. Methods
Acknowledgement
The experiments were carried out in an isolated, perfused atrial
preparation that was prepared by using the method described
previously [13,14]. Thus, the atrium was perfused with N-2-
hydroxyethyl piperazine-N0-2-ethanesulfonic acid (HEPES) buffer
solution by means of a peristaltic pump (1.25 mL/min) [15]. 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 tech-
nique at a concentration of 1 ꢁ10ꢂ5 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
as 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 electrocar-
diogram 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 mean ꢀ SE.
This work was supported by the National Natural Science
Foundation of China (No. 30560177) and the Natural Science
Foundation of Jilin Province of China (No. 20060567).
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