Synthesis of new triazole acetamides with inotropic effects

Article abstract of DOI:10.1016/j.bmcl.2012.11.078

A series of new triazole acetamides 5a-w were synthesized and evaluated for their positive inotropic activity of left atrium stroke volume on isolated rabbit-heart preparations. The majority of the derivatives presented favorable in vitro activity compared with the reference drug, milrinone. Among them triazole acetamide 5a was identified as the most potent with 20.29 ± 0.18% increased stroke volume (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10^-5 M. The chronotropic effects of the compounds having inotropic effects were also evaluated.

Full text of DOI:10.1016/j.bmcl.2012.11.078

Bioorganic & Medicinal Chemistry Letters 23 (2013) 757–760
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of new triazole acetamides with inotropic effects
Yan Wu a, Long-Xu Ma a, Liang-Peng Sun a, Ming-Xia Song 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 133000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new triazole acetamides 5a–w were synthesized and evaluated for their positive inotropic
activity of left atrium stroke volume on isolated rabbit-heart preparations. The majority of the derivatives
presented favorable in vitro activity compared with the reference drug, milrinone. Among them triazole
acetamide 5a was identified as the most potent with 20.29 0.18% increased stroke volume (milrinone:
2.46 0.07%) at a concentration of 3 ꢀ 10–5 M. The chronotropic effects of the compounds having inotro-
pic effects were also evaluated.
Received 16 October 2012
Revised 17 November 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Triazole acetamides
Positive inotropic activity
Stroke volume
Milrinone
3,4-Dihydro-2(1H)-quinolinone
Over the past decades, chemical modification of vesnarinone
(1)1,2 with 3,4-dihydro-2(1H)-quinolinone (DHQO) have been the
subject of great interest in our laboratory, leading to the identifica-
tion of several new analogues (2–5, Fig. 1)3–5 displaying good
activities as positive inotropic agents. Our recent work on
DHQOs disclosed that the incorporation of triazole ring with 1-
chlorophenyl group to the 1,2-position of 3,4-dihydro-2(1H)-quin-
olinone resulted in the discovery of several novel DHQO analogues
(6, Fig. 1)6 with significant inotropic activity on isolated rabbit
heart preparations. These promising results indicated that this sub-
class of DHQO analogues containing chlorophenyl triazole ring
raises the appealing possibility for future structural optimization
using 6 as a starting point. Our continuing interest in the search
for new positive inotropic agents promoted us to investigate the
effect on the replacement of cinnamyl group on the piperazine ring
with various substituted benzyl and alkyl groups for the positive
O
H
H
N
N
N
N
N
N
N
O
N
O
H3CO
R2
N
R
N
N
N
NH
OCH3
N
N
O
R1
H
O
1
, Vesnarinone
3
2
H
H
N
H
N
N
N
N
N
N
N
R
O
O
N
O
N
N
N
N
N
N
N
NH
N
R
O
6
4
5
Cl
Figure 1. Chemical structure of vesnarinone (1) and our reported compounds (2–6) with inotropic activity.
⇑
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. Piao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.078

758
Y. Wu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 757–760
H2N
H2N
H2N
N
N
N
a
b
c
Cl
N
H
O
N
H
S
N
3 3'
,
2
1
H
N
H
N
Cl
O
R
N
O
N
N
N
N
N
Cl
N
d
Cl
5a-r
: m-Cl
5s-w: o-Cl
4, 4'
5a-w
Scheme 1. Synthetic scheme for the synthesis of compound 5a–w. Reagents and conditions: (a) P2S5, Et3N, CH3CN; (b) RCONHNH2, cyclohexanol, reflux, N2, 6 h; (c)
ClCH2COCl, CH2Cl2, rt; (d) monosubstituted piperazines, K2CO3, acetone, reflux, 10 h.
(1H)-one (1) with phosphorous pentasulfide in refluxing acetoni-
Table 1
trile in the presence of triethylamine gave the corresponding
thione 2. Cyclization of 2 with 3-chlorobenzhydrazide and 2-chlo-
robenzhydrazide in refluxing cyclohexanol under nitrogen atmo-
sphere afforded the desired triazole compounds 3 and 3’ in
moderate yields,9 followed by the acylation of the amino group
with 2-chloroacetyl chloride in dichloromethane at room temper-
ature to provide the corresponding amides 4 and 40. Nucleo-
philic-substitution of 4 and 40 with various monosubstituted
piperazines in refluxing acetone in the presence of potassium car-
bonate afforded 5a–w in high yield,10 respectively. All of the com-
pounds synthesized were characterized by IR, 1H NMR, 13C NMR,
MS and elemental analysis.
As shown in Table 1, most compounds tested showed inotropic
effects on isolated rabbit heart preparations. Among them, 11 com-
pounds exhibited more potent effects compared to milrinone
(2.46 0.07%, 3 ꢀ 10ꢁ5 M) as reference drug. Interestingly, 5a with
o-flurobenzyl group was the most potent with 20.29 0.18% in-
creased stroke volume. Also, the substituted influence of benzyl
group on inotropic activity was found. The fluorinated and chlori-
nated compounds showed better activity than the brominated
compounds. For fluoro substituted compound 5a–c, the order of
inotropic activity is o > p > m, while chloro substituted compound
5d–f is m > p > o. However, no activity was detected for the di-
chloro-substituted derivatives 5k, 5l. In bromo substituted series,
only 4-brominated derivative 5i exhibited weak activity
(2.31 0.05%). The inotropic activity of 5j with benzyl group, 5m
with p-methylbenzyl, and 5n with p-methoxybenzyl substituted
derivatives are 8.07 0.08%, 4.25 0.06%, 16.84 0.15%, respec-
tively, showing more potent activity than milrinone. For alkyl
substituted compounds 5o–r, the length of the alkyl group seems
to have a significant impact on the activity. The activity decreased
gradually with the alkyl chain length increased.
Positive inotropic activity of the test compounds
H
N
N
R
N
O
N
N
N
Cl
5a-r
: m-Cl
: o-Cl
5s w
-
Compound
R
Increased stroke volumea (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
–C6H4(o-F)
–C6H4(m-F)
–C6H4(p-F)
–C6H4(o-Cl)
–C6H4(m-Cl)
–C6H4(p-Cl)
–C6H4(o-Br)
–C6H4(m-Br)
–C6H4(p-Br)
–C6H5
–C6H3(2,4-Cl2)
–C6H3(3,4-Cl2)
–C6H4(p-CH3)
–C6H4(p-OCH3)
–C3H7
20.29 0.18
6.33 0.08
16.49 0.22
0.80 0.09
15.75 0.34
7.22 0.13
b
—
—
2.31 0.05
8.07 0.08
—
—
4.25 0.06
16.84 0.15
2.33 0.04
0.80 0.02
—
–C4H9
–C5H11
–C7H15
—
5s
5t
–C6H4(o-F)
–C6H4(p-F)
–C6H4(m-Cl)
–C6H5
2.39 0.06
4.58 0.05
2.18 0.05
15.62 0.16
9.12 0.05
2.46 0.04
5u
5v
5w
Milrinone
–C6H4(p-OCH3)
a
The concentration for the test sample is 3 ꢀ 1 0ꢁ5 M.
b
Inaddition, the dynamics of the tested compounds in perfused
beating rabbit atria were investigated, and found that 5m and 5t
exhibited similar atrial dynamic profiles to milrinone with good in-
creased stroke volume (Fig. 2 A and F). More desirable atrial dy-
namic profiles were measured for 5a–c, 5e–f, 5j, 5n, 5v, and 5w
(Fig. 2 A, B, C, and D). It was found that the stroke volumes were
gradually increased as the time progressed, in which 5a displayed
excellent inotropic effects with the highest increased stroke vol-
ume (Fig. 2A). The dose-activity relationships 5a and 5n were also
determined at concentrations of 1 ꢀ 10ꢁ5, 3 ꢀ 10ꢁ5, and 1 ꢀ
10ꢁ4 M, respectively, showing maximal effects at the concentration
None or negative stroke volume increase.
inotropic activity to delineate structure-activity relationship (SAR)
of 6 and furfure improve their inotropic potency. Herein, the detail
of synthesis, inotropic activity and preliminary SAR studies of these
new congeners are described.
Compounds 5a–w were synthesized according to our previously
reported methods7 as presented in Scheme 1, and their structures
and inotropic activities are displayed in Table 1.8 The sulfurization
of the commercially available 6-amino-3,4-dihydroquinolin-2

Y. Wu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 757–760
759
Control
Control
A
B
40
35
30
25
20
15
10
5
30
5a (30µmol/L)
5j (30µmol/L)
5m(30µmol/L)
Milrinone(30µmol/L)
5n (30µmol/L)
5f (30µmol/L)
25
20
15
10
5
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
Control
C
D
30
25
20
15
10
5
30
25
20
15
10
5
5c (30µmol/L)
5e (30µmol/L)
5b(30µmol/L)
Milrinone(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
30
25
20
15
10
5
20
15
10
5
E
Control
F
Control
5w (30µmol/L)
5v (30µmol/L)
5t (30µmol/L)
Milrinone(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
Figure 2. Effects of milrinone and compounds 5a, 5b, 5c, 5e, 5f, 5j, 5m, 5n, 5t, 5v, and 5w on stroke volume in beating rabbit atria (1.5 Hz). Values are means SE. ⁄⁄⁄P <0.001
versus control.
Control
G
H
25
20
15
10
5
25
20
15
10
5
Control
5a (10µmol/L)
5a (30µmol/L)
5a (100µmol/L)
Milrinone(10µmol/L)
Milrinone(30µmol/L)
Milrinone(100µmol/L)
5n (10µmol/L)
5n (30µmol/L)
5n(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 3. Concentration-response curves of compounds 5a, 5n and milrinone on stroke volume in beating rabbit artia (1.5 Hz). Values are means SE. ⁄⁄⁄P <0.001 versus
control.

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Y. Wu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 757–760
Table 2
6. Wu, Y.; Ma, L. X.; Niu, T. W.; Meng, F. L.; Cui, X.; Piao, H. R. Arch. Pharm. Chem.
Changes of heart rate caused by compounds in isolated rabbit heart preparationsa
Life Sci. 2012, 345, 980.
7. Liu, J. Y.; Yu, H. L.; Quan, Z. S.; Piao, H. R. Bioorg. Med. Chem. Lett. 2009, 19, 2392.
8. The method of measuring left atrium stroke volume was described
previously.11,12 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 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. 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 ꢀ 10ꢁ5 M. Samples were
dissolved in DMSO and diluted with the HEPES buffer to 0.1% DMSO.13 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.
b
c
Compound
Mean SE
Mean SE
7a
7c
7e
7n
7v
130.70 0.00
118.28 0.11
113.38 0.26
121.19 0.23
119.84 0.08
130.30 0.01
118.37 0.12
93.33 0.12d
120.26 0.20
119.47 0.11
The concentration for the test sample is 3 ꢀ 1.0ꢁ5 M.
Control.
a
b
c
Data after using the test samples.
P <0.01 versus control.
d
of 3 ꢀ 10ꢁ5 M, and lower potency at the higher dose of 1 ꢀ 10ꢁ4 M,
as shown in Figure 3 G and H.
Next, the chronotropic activity of 5a, 5c, 5e, 5n, and 5v in beat-
ing atria was studied. As seen from Table 2, no significantly in-
creased heart rates (P >0.05) were observed for 5a, 5c, 5n and 5v
at the same concentration, while 5e adversely changed the heart
rates.
In summary, new triazole acetamides were synthesized and
evaluated for their positive inotropic activity. It was found that
the compound 5a was the most potent (8.2-fold more active than
milrinone) since modification of DHQO in our laboratory in 2008.
It is also suggested that further modifications of such compounds
may be of interest. The biological tests including in vivo evaluation,
coronary vasodilating tests and studies into the possible mecha-
nism of action are currently ongoing in our laboratory.
Acknowledgement
9. Yang, K.; Sun, L. P.; Liu, J. Y.; Cui, X.; Hong, L.; Piao, H. R. Bioorg. Med. Chem. Lett.
2010, 20, 4464.
10. Preparation of 5a: A mixture of 4a (372 mg, 1.0 mmol), 1-(2-fluorobenzyl)
piperazine (194 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 7a (496 mg, 93%) as white solid. Mp: 104–106 °C; IR (KBr) cmꢁ1: 3376
(NH), 1696 (C@O); 1H NMR (CDCl3, 300 MHz, ppm): 2.59–2.66 (m, 8H, CH2),
3.07–3.29 (m, 4H, CH2), 3.49 (s, 2H, CH2), 3.63 (s, 2H, CH2), 6.84–7.78 (m, 11H,
Ar-H), 9.22 (s, 1H, NH). 13C NMR (CDCl3, 75 MHz, ppm): d 168.51, 161.06,
152.03, 138.02, 132.46, 132.07, 130.22, 130.02, 129.67, 128.98, 127.05, 125.86,
125.53, 123.97, 119.96, 119.37, 117.95, 63.61, 56.88, 55.79, 53.33, 27.92, 24.86.
ESIMS (m/z): 531 (M+1). Anal. Calcd for C29H28ClFN6O: C, 65.59; H, 5.31; N,
15.83. Found: C, 65.53; H, 5.36; N, 15.89.
11. 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.
12. Cho, K. W.; Kim, S. H.; Kim, C. H.; Seul, K. H. Am. J. Physiol. 1995, 268, 1129.
13. 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).
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