Synthesis of 2-(4-substituted benzyl-1,4-diazepan-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.bmcl.2010.06.046

Herein we describe the discovery of compound 3g, a potent positive inotropic agent compared with the standard drug, milrinone. Compound 3g was developed from a series of 2-(4-substitutedbenzyl-1,4-diazepan-1-yl)-N-(3,4- dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl) acetamides found in an evaluation of inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. Several compounds showed favorable activities, but 3g was the most potent, with 7.68 ± 0.14% increased stroke volume (milrinone 2.38 ± 0.05%) at 1 × 10^-5 M in our in vitro study. The chronotropic effects of compounds having significant inotropic effects were also evaluated.

Full text of DOI:10.1016/j.bmcl.2010.06.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4464–4467
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 2-(4-substituted benzyl-1,4-diazepan-1-yl)-N-(3,4-dihydro-3-oxo-
2H-benzo[b][1,4]oxazin-7-yl)acetamides and their positive inotropic evaluation
a,
Kun Yang a, Liang-Peng Sun a, Ji-Yong Liu a, Xun Cui b, , Hu-Ri Piao
*
*
a Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133000, PR China
b Yanbian University College of Medicine, 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:
Herein we describe the discovery of compound 3g, a potent positive inotropic agent compared with the
standard drug, milrinone. Compound 3g was developed from a series of 2-(4-substitutedbenzyl-1,4-dia-
zepan-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl) acetamides found in an evaluation of
inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. Several
compounds showed favorable activities, but 3g was the most potent, with 7.68 0.14% increased stroke
volume (milrinone 2.38 0.05%) at 1 ꢀ 10ꢁ5 M in our in vitro study. The chronotropic effects of com-
pounds having significant inotropic effects were also evaluated.
Received 5 January 2010
Revised 17 May 2010
Accepted 8 June 2010
Available online 15 June 2010
Keywords:
3,4-Dihydro-3-oxo-2H-
benzo[b][1,4]oxazine
Positive inotropic activity
1,4-Diazepane
Ó 2010 Elsevier Ltd. All rights reserved.
Stroke volume
The development of innovative positive inotropic agents with
approved therapeutic properties for the treatment of congestive
heart failure (CHF) remains a great challenge in medicinal chemis-
try.1 During our previous studies that aimed to discover more
potent positive inotropic agents with fewer side effects, a series
of 2-(4-substitutedpiperazin-1-yl)-N-(3,4-dihydro-2(1H)-quinoli-
none-6-yl)acetamides, as a vesnarinone2 analog, were synthesized
and tested for their biological activity. Among these, the compound
2-(4-benzylpiperazin-1-yl)-N-(3,4-dihydro-2(1H)-quinolinone-6-yl)-
acetamide PHR9612 showed moderate positive inotropic activity.3,4
Replacement of the carbon atom at the 4-position of PHR9612
with an oxygen atom yielded 2-(4-(3-methoxybenzyl)piperazin-
1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-7-yl)acetamide
(PHRL0010), which possessed significant inotropic effects,5 and
which is now undergoing further biological tests (Fig. 1).
In the present study, to further optimize compound PHRL0010,
we replaced the piperazine ring with a 1,4-diazepane ring. We also
varied the substituents on the phenyl ring of the benzyl group to
preliminarily investigate the contribution of such a structural
change on biological activity. The compounds synthesized were
characterized by IR, NMR, MS, and elemental analysis. The positive
inotropic activity was evaluated by measuring the left atrium
stroke volume on isolated rabbit heart preparations.
The synthesis of compounds 3a–j and 4a–j is presented in
Scheme 1. Compound 1 was synthesized through cyclization
and reduction according to previously described methods by
using commercially available 2-amino-5-nitrophenol as a starting
material.6 A catalytic hydrogenation method was used to reduce
the nitro group instead of using iron powder in hydrochloric acid,
and the yield was increased from 75% to 98%. The resulting amino
group of compound 1 was acylated with 2-chloroacetyl chloride
in dichloromethane to provide amide 2 in excellent yield. A
nucleophilic-substitution reaction of 2 with various monosubsti-
tuted 1,4-diazepanes7 in refluxing acetone in the presence of
potassium carbonate afforded the corresponding compounds
3a–j and 4a–j in moderate yields.8
The method for measuring left atrium stroke volume was de-
scribed previously.9,10 The features of CHF are cardiac dilatation,
poor contractility of cardiac muscle, decreased ejection fraction,
and depression of left ventricular maximum pressure. Therefore,
macroscopic measurement of the variance in left atrium stroke vol-
ume can be used to estimate the positive inotropic effects of the
compounds synthesized. Milrinone (Suzou Unite Pharmaceutical
Company, Suzou, China) and DMSO (Sigma–Aldrich Chemical Com-
pany, St. Louis, MO, USA) were used; all other reagents were of
analytical grade. Atria were obtained from New Zealand white rab-
bits, and the mean atrial weight was 182.5 6.8 mg. In brief, hearts
were removed from rabbits and the left atria dissected free. A cal-
ibrated transparent atrial cannula containing two small catheters
was inserted into the left atrium. The cannulated atrium was trans-
ferred to an organ chamber and perfused immediately with N-2-
hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES) buffer
* Corresponding authors. Tel.: +86 433 2660584; fax: +86 433 2659795 (X.C.);
tel.: +86 433 2660503; fax: +86 433 2659795 (H.-R.P.).
E-mail addresses: cuixun@ybu.edu.cn (X. Cui), piaohuri@yahoo.com.cn (H.-R.
Piao).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.046

K. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4464–4467
4465
O
H
N
N
N
N
N
O
O
H3CO
N
H
OCH3
Vesnarinone
PHR9612
O
N
H
H
N
O
N
N
O
H3CO
N
H
O
PHRL0010
Figure 1. Cardiotonic agents used for the treatment of CHF and previous lead compounds.
O
H2N
c
O
NO2
OH
NO2
b
a
O
N
H
O
NH2
N
H
1
H
N
O
Cl
O
N
H
O
e
2
d
H
H
N
O
N
O
N
N
H3CO
R
O
R
N
O
O
N
H
N
O
N
H
O
3a-j
4a-j
Scheme 1. Synthetic scheme for the synthesis of compounds 3a–j and 4a–j. Reagents: (a) ClCH2COCl, acetonitrile, TEBA, Na2CO3; (b) H2/Pd, MeOH; (c) ClCH2COCl, CH2Cl2;
(d) 1-substitutedbenzyl-1,4-diazepanes, KI, K2CO3, acetone; (e) 1-(4-(substitutedbenzyloxy)-3-methoxybenzyl)-1,4-diazepanes, KI, K2CO3, acetone.
solution with a peristaltic pump (1.25 mL/min) at 34 °C.11 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 (ad-
justed to pH 7.4 with 1 M NaOH), and 0.1% bovine serum albumin
(BSA). Soon after the perfused atrium was set up, transmural elec-
trical field stimulation with a luminal electrode was started at
1.5 Hz (duration, 0.3–0.5 ms; voltage, 30 V). Changes in atrial
stroke volume were monitored by reading the lowest level of the
water column in the calibrated atrial cannula during end diastole.
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 test compound or milrinone
infused for 36 min after a control period of 12 min.
activities of the same substituted derivatives in the piperazine ser-
ies, 2-Br derivative was 0.9-fold and 2-F derivative was 2.6-fold
more potent than milrinone at the same concentrations, respec-
Table 1
Positive inotropic activity of the test compounds
Compound
R
Increased stroke
volumea (%)
b
3a
3b
4-CH3
H
—
—
3c
3d
4-OCH3
4-Cl
3.22 0.06
—
3e
2-Cl
—
3f
3g
3-OCH3
2-Br
—
Compounds were tested at 1 ꢀ 10ꢁ5 M. Samples were dissolved
in DMSO and diluted with HEPES buffer to 0.1% DMSO. Data are
expressed as the mean of increased stroke volume percentage
(Table 1). Repeated measurements were compared by an ANOVA
test followed by Bonferroni’s multiple-comparison test. Statistical
significance was defined as P < 0.05 and data are presented as
means SE.
Table 1 shows the inotropic activity data for the 20 test com-
pounds. Four compounds (3c, 3g, 3h, and 4d) showed significantly
increased inotropic effects on isolated rabbit heart preparations
compared with milrinone (2.38 0.05%, 1 ꢀ 10ꢁ5 M). Compounds
3g and 3h clearly exhibited more potent effects compared with
our lead PHRL0010 and milrinone; as 2-Br and 2-F derivatives, 3g
was 3.2-fold and 3h was 2.6-fold more potent (7.68 0. 14% and
6.05 0.11%, 1 ꢀ 10ꢁ5 M) than milrinone. Compared with the
7.68 0.14
3h
2-F
6.05 0.11
3i
3j
4a
2,6-(Cl)2
3,4-(OCH3)2
2-Cl
—
—
—
4b
H
—
4c
4d
4-OCH3
4-Cl
—
2.87 0.08
4e
4f
4g
4-CH3
3-OCH3
2-Br
—
—
—
4h
2-F
—
4i
4j
2,6-(Cl)2
3,4-(OCH3)2
—
—
PHRL0010
Milrinone
3.96 0.15
2.38 0.05
a
Concentration for the test sample is 1 ꢀ 10ꢁ5 M.
b
None or negative stroke volume increases.

4466
K. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4464–4467
tively.5 This indicated that replacing the piperazine ring with the
1,4-diazepane did not significantly affect the activity. Most com-
pounds from the series 4a–j were not active (except 4d), so a suit-
able length of the molecule may be required for the activity. As for
the relationship between inotropic activity and different substitu-
tions on the phenyl ring of the benzyl group for compounds 3a–j,
we found that, among halo-substituted compounds, bromo and
fluoro derivatives 3g and 3h had significant activity, but not the
chloro or dichloro analogs (3d, 3e, and 3i). Among the methoxy-
substituted derivatives, only the 4-methoxy derivative (3c) had
activity (3-methoxy or 3,4-dimethoxy derivatives (3f and 3j) were
inactive). Thus, the substituents on the phenyl ring seemed to sig-
nificantly affect the biological activity. A clear pattern was not ob-
served for the relationship between the inotropic activity and the
different substituents for these derivatives.
3 ꢀ 10ꢁ6
,
1 ꢀ 10ꢁ5
,
and 3 ꢀ 10ꢁ5 M, and for milrinone at
1 ꢀ 10ꢁ6, 3 ꢀ 10ꢁ6, 1 ꢀ 10ꢁ5, 3 ꢀ 10ꢁ5, and 1 ꢀ 10ꢁ4 M (Fig. 2D
for 3h, Fig. 2E for 3g, and Fig. 2F for milrinone). Compound 3g dis-
played stronger potency than milrinone at all tested concentra-
tions with a maximal effect at 3 ꢀ 10ꢁ5 M, and 3h showed
maximal effect at 1 ꢀ 10ꢁ5 M. Compounds 3c, 3g, 3h, and 4d were
also investigated for their chronotropic effects in beating atria, and
no significant increase in heart rates (P > 0.05) was observed for 3c,
3g, and 3h at the same concentration (Table 2). However, com-
pound 4d unfortunately resulted in changed heart rates. Further
in vivo studies are therefore required for these compounds to fur-
ther investigate their chronotropic effects.
In conclusion, based on the structure of compound PHRL0010,
we designed and synthesized two series of 2-(4-substitutedbemz-
yl-1,4-diazepan-1-yl)-N-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxa-
zin-7-yl)acetamides with the aim of identifying more potent
compounds that increase cardiac contractility without increasing
heart rate. Compounds 3g and 3h exhibited the most promising
cardiovascular profiles compared with the lead compound
PHRL0010 and milrinone (3.2-fold more potent at 1 ꢀ 10ꢁ5 M for
3g). Further modification of compound 3g is in progress.
We also investigated the dynamics of the test compounds (3c,
3g, 3h, and 4d) in the perfused beating atria of rabbits and found
that compound 3g showed a similar atrial dynamic profile to that
of milrinone (Fig. 2B). Much more desirable atrial dynamic profiles
were measured for compounds 3c and 3h (Fig. 2A and C). We also
investigated the preliminary dose-dependency for 3g and 3h at
7
A
B
Control
3c (10mmol/L)
6
14
12
10
8
Control
Milrinone (10μmol/L)
3g (10μmol/L)
5
PHRL0010 (10μmol/L)
Milrinone (10μmol/L)
4
3
6
2
4
1
2
0
0
-2
-1
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
14
12
10
8
C
Control
3h (10μmol/L)
PHRL0010 (10μmol/L)
Milrinone (10μmol/L)
D 12
Control
3h (3μmol/L)
3h (10μmol/L)
3h (30μmol/L)
10
8
6
6
4
4
2
2
0
0
-2
-2
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Fraction number
Fraction number
Control
12
20
Control
Milrinone (3μmol/L)
Milrinone (10μmol/L)
Milrinone (30μmol/L)
3g (3μmol/L)
3g (10μmol/L)
3g (30μmol/L)
F
E 18
Milrinone (1μmol/L)
Milrinone (3μmol/L)
Milrinone (10μmol/L)
Milrinone (30μmol/L)
Milrinone (100μmol/L)
10
8
16
14
12
10
8
6
4
6
4
2
2
0
0
-2
-2
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
30
Fraction number
Fraction number
Figure 2. Effects of milrinone and compounds 3c (A), 3g (B), and 3h (C) on stroke volume in beating rabbit atria (1.5 Hz). Concentration–response curves of 3g (E) and 3h (D)
on isolated rabbit heart preparations. Values are means SE.

K. Yang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4464–4467
4467
Table 2
3. Piao, H. R.; Quan, Z. S.; Xu, M. X.; Deng, D. W. Chin. J. Med. Chem. 1999, 9, 79.
4. Piao, H. R.; Quan, Z. S.; Jiang, Y. Z.; Xu, M. X.; Deng, D. W. Chin. J. Modern Appl.
Pharm. 2000, 3, 206.
5. Liu, X. K.; Cui, X.; Hong, L.; Sun, L. P.; Quan, Z. S.; Piao, H. R. Eur. J. Med. Chem.
2009, 44, 3027.
6. Piao, Z. T.; Guan, L. P.; Zhao, L. M.; Piao, H. R.; Quan, Z. S. Eur. J. Med. Chem. 2008,
43, 1216.
7. Liao, X. C. Chin. J. Pharm. 1993, 1, 21.
Changes in heart rate caused by compounds on isolated rabbit heart preparations
Compound
Mean SEa
Mean SEb
3c
3g
3h
4d
92.52 0.17
102.31 0.07
105.33 0.12
89.51 0.09
89.37 0.08
98.64 0.24
102.88 0.19
80.02 0.15c
8. Preparation of 3g: A mixture of 2 (0.24 g, 1.0 mmol), 1-(2-bromobenzyl)-
1,4-diazepane (0.54 g, 2.0 mmol), and KI/K2CO3 in refluxing acetone was
stirred for 12 h. The solvent was evaporated in vacuo and the residue was
dissolved in dichloromethane, 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
(dichloromethane/methanol, 30:1 v/v) to afford 3g (0.26 g, 56%). Mp
a
b
c
Control.
Data after using the test samples.
P < 0.01 versus control.
Acknowledgments
185–186 °C. 1H NMR (CDCl3, 300 MHz):
(m, 8H), 3.28 (s, 2H), 3.77 (s, 2H), 4.62 (s, 2H), 6.76–7.56 (m, 7H), 8.61 (s,
1H), 9.30 (s, 1H). MS m/z 473 (M+1). IR (KBr) cmꢁ1
3462 (NH), 1691
(C@O), 1683 (C@O). Anal. Calcd for 22H25BrN4O3: C, 55.82; H, 5.32; N,
d 1.85–1.91 (m, 2H), 2.78–2.92
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 30560177) and the Natural Science
Foundation of Jilin Province of China (20060567).
:
C
11.88. Found: C, 55.78; H, 5.34; N, 11.74.
9. Cho, K. W.; Kim, S. H.; Kim, C. H.; Seul, K. H. Am. J. Physiol. 1995, 268,
1129.
10. 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.
11. 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.
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