Synthesis and inotropic evaluation of 1-substituted-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)piperidine-4-carboxamides

Article abstract of DOI:10.1016/j.bmcl.2009.03.089

A series of 1-substituted-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl) piperidine-4-carboxamides has been synthesized and evaluated for positive inotropic activity by measuring left atrium stroke volume in isolated rabbit-heart preparation

Full text of DOI:10.1016/j.bmcl.2009.03.089

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2392–2395
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and inotropic evaluation of 1-substituted-N-(4,5-dihydro-1-methyl-
[1,2,4]triazolo[4,3-a]quinolin-7-yl)piperidine-4-carboxamides
a,
Ji-Yong Liu a, Hai-Ling Yu b, Zhe-Shan Quan a, Xun Cui b, , Hu-Ri Piao
*
*
a Changbai Mountain Key Laboratory of Natural Resources and Functional Molecules, Affiliated Ministry of Education, Yanbian University College of Pharmacy, Yanji 133000, PR China
b Yanbian University College of Preclinical 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 1-substituted-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl) piperidine-4-car-
boxamides has been synthesized and evaluated for positive inotropic activity by measuring left atrium
stroke volume in isolated rabbit-heart preparations. Some of these derivatives exhibited favorable activ-
ity compared with the standard drug, milrinone, among which 1-(2-fluorobenzyl)-N-(4,5-dihydro-1-
methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)piperidine-4-carboxamide 6a was the most potent, increasing
stroke volume by 11.92 0.35% (milrinone: 6.36 0.13%) at 1 ꢀ 10ꢁ4 M.
Received 11 January 2009
Revised 5 March 2009
Accepted 23 March 2009
Available online 26 March 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
[1,2,4]Triazolo[4,3-a]quinolines
Positive inotropic activity
Isonipecotic acid
Stroke volume
Cardiac glycosides like digoxin are some of the most frequently
prescribed cardiotonics used for the treatment of congestive heart
failure (CHF). Unlike other CHF drugs, they do not increase mortal-
ity, but their high toxicity and narrow therapeutic window still
limit their clinical use as positive inotropic agents.1 The discovery
of amrinone led to the synthesis of a number of agents with prom-
ise for the treatment of CHF as non-sympathomimetic, non-glyco-
side agents.2 The phosphodiesterase (PDE)-inhibiting agent,
milrinone (Fig. 1), has both vasodilator and inotropic properties
and was approved for the treatment of CHF more than a decade
ago. Nevertheless, the significant ventricular arrhythmias and
tachycardia associated with elevated cAMP levels also limit the
clinical use of milrinone,3 as well as a newer agent, vesnarinone.4,5
Therefore, newer positive inotropic agents with fewer side effects
are still needed.
through phosphodiesterase III inhibition (unpublished data). To
further optimize compound 1, we replaced the piperazine ring
with a piperidine ring, changed the acetamide at the 7-position
of 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline to a carboxamide,
and changed the substituents on the corresponding phenyl rings
at the 1-position of piperidine-4-carboxamide, and then measured
the effects on inotropic activity. The compounds were character-
ized by IR, 1H NMR, MS, and elemental analysis, and inotropic
activities were evaluated by measuring changes in left atrium
stroke volume in isolated rabbit-heart preparations.
Compounds 6a–p, 7a–o were synthesized as outlined in Scheme
1. Compound 2 was synthesized through sulfurization and cycliza-
tion reactions according to previously described methods with
commercially available 6-amino-3,4-dihydroquinolin-2(1H)-one
as a starting material.7 Isonipecotic acid 3 was used to prepare
For many years, we have been searching for compounds with
favorable positive inotropic activities with fewer side effects in a
series of 3,4-dihydro-2(1H)-quinolinone derivatives. At the outset
tert-butyl 4-(chlorocarbonyl)piperidine-1-carboxylate 4 as re-
ported.8,9 Thus 3 was reacted with (Boc)2O in dioxame to afford
1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid, followed by
halogenation with thionyl chloride in dichloromethane at 40–
50 °C to provide 4 in high yield. Compound 2 was acylated with
4 in dichloromethane at room temperature to afford the corre-
sponding amide, followed by deprotection with dry HCl gas to give
5 as a hydrochloride salt.10 The nucleophilic substitution reaction
of 5 with substituted benzyl chlorides or 4-substituted benzyl-
oxy-3-methoxy benzyl chlorides in refluxing acetone in the pres-
ence of potassium carbonate and triethylamine afforded
compounds 6a–p and 7a–o.11
of our studies,
a series of N-(4,5-dihydro-[1,2,4]triazolo[4,3-
a]quinolin-7-yl)-2-(piperazine-1-yl)acetamide derivatives was
synthesized and tested for their biological activity, among which
the compound N-(1-benzyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quin-
olin-7-yl)-2-(4-benzylpiperazin-1-yl)acetamide
1
showed the
most potent positive inotropic activity.6 Compound 1 is inotropic
* 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).
The method of measuring left atrium stroke volume was de-
scribed previously.12,13 The features of CHF are cardiac dilatation,
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.089

J.-Y. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2392–2395
2393
O
N
N
CN
O
N
H3CO
N
H
H3C
OCH3
Vesnarinone
O
N
H
Milrinone
OCH3
H
N
O
N
N
O
N
N
N
Compound 1
H3C
Figure 1. Cardiotonic agents used for CHF treatment and lead compound 1.
poor contractility of cardiac muscle, decreased ejection fraction,
and depression of left ventricular maximum pressure. Therefore,
the macroscopic measurement of the variance of left atrium stroke
volume can be used to estimate the positive inotropic effects of the
compounds synthesized. Milrinone (Shuzhou Unite Pharmaceuti-
cal Co., Dongwu Road, Shuzhou) and DMSO (Sigma–Aldrich Chem-
ical Co., St. Louis, MO) 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. Briefly, hearts
were removed from rabbits and the left atria were dissected free. A
calibrated transparent atrial cannula containing two small cathe-
ters was inserted into the left atrium. The cannulated atrium was
transferred to an organ chamber and perfused immediately with
N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid (HEPES)
buffer solution with 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, trans-
mural electrical 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 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 test
compound or milrinone was infused for 36 min after a control
period of 12 min.
Compounds were tested at 1 ꢀ 10ꢁ4 M. Samples were dissolved
in DMSO and diluted with HEPES buffer to 0.1% DMSO. Data is
O
H2N
O
H2N
a
b
Cl
OH
N
N
N
N
N
H
O
HN
Boc
H3C
c
4
3
2
HCl
HN
H
N
O
N
N
N
d
e
5
H3C
N
H
N
N
H
N
O
R
O
O
OCH3
N
N
N
N
H3C
N
N
R
6 a-p
7a-o
H3C
R:
6a 2-F
6b
6j 4-OCH3
6k 3-NO2
R: 7a 2-F
7b H
7i 4-CH3
H
j
7
3-NO2
6c 4-F
6d 4-Cl
6e 2-Br
6f 3-F
6g 3-Cl
6l 3,4-OCH2O
6m 2,4-(Cl)2
7c 4-F
7k 2-Cl
7d 4-Cl
7e 2-Br
7f 3-F
7g 3-Cl
7l 2,4-(Cl)2
7m 2-NO2
6n 3,4-(OCH3)2
6o 2-NO2
6p 2-Cl
7n 4-OCH3
7o 3,4-(OCH3)2
6h 2,6-(Cl)2
6i 4-CH3
7h 2,6-(Cl)2
Scheme 1. Reagents and conditions: (a) (i) P2S5, Et3N, CH3CN; (ii) CH3CONHNH2, n-butanol, reflux, N2; (b) (i) (Boc)2O, NaOH, dioxame; (ii) SOCl2, TEA, CH2Cl2, 40–50 °C; (c) (i)
TEA, CH2Cl2; (ii) dry HCl gas, CH2Cl2, 0–5 °C; (d) substituted benzyl chlorides, K2CO3, KI, Et3N, acetone; (e) 4-substitutedbenzyloxy-3-methoxybenzyl chlorides, K2CO3, KI,
Et3N, acetone.

2394
J.-Y. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2392–2395
Table 1
expressed as the mean of increased stroke volume percentage (Ta-
ble 1). Repeated measurements were compared by an ANOVA test
followed by Bonferroni’s multiple-comparison test. Statistical sig-
nificance was defined as P < 0.05 and the data are presented as
means SE.
Inotropic activity of compounds 6a–p, 7a–o in changing left atrium stroke volume in
isolated rabbit-heart preparations
Compound
R
Increased stroke volumea (%)
11.92 0.35*
—
—
6a
6b
6c
6d
6e
2-F
H
4-F
4-Cl
2-Br
3-F
3-Cl
2,6-Cl2
4-CH3
4-OCH3
3-NO2
3,4-OCH2O–
2,4-Cl2
3,4-(OCH3)2
2-NO2
2-Cl
2-F
H
4-F
4-Cl
b
Most (16) compounds out of 31 tested showed inotropic effects
on isolated rabbit-heart preparations (Table 1). Compounds 6a, 6g,
6k, 6m and 7a exhibited more efficacy than milrinone
(6.36 0.13%, 1 ꢀ 10ꢁ4 M), with 6a showing the strongest activity,
an 11.92 0.35% increase in stroke volume. Previous N-(4,5-dihy-
dro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)-2-(piperazin-1-yl)acetam-
ide derivatives, such as compound 1, were more efficacious than
milrinone.6 Thus replacing the 2-(piperazin-1-yl)acetamide moiety
with a piperidine-4-carboxamide removed a critical nitrogen atom
required for high-affinity binding to PDE III. Substitutions (R) with
electron-withdrawing groups on the phenyl ring of the benzyl
group at the 4-position of the piperidine in 6a–p showed good
activity, whereas compounds unsubstituted or substituted by elec-
tron-donating groups did not. One exception was 6n, which was
active (1.87 0.08%) but had electron-donating groups. Further-
more, 2-fluorinated and 3-chlorinated compounds (6a, 6g) dis-
played better activity, with 6a the most potent derivative in this
study. The position of the substituent on the phenyl ring signifi-
cantly influenced the inotropic activity, with an activity order of
o > m > p for fluoro-substituted compounds, and m > o > p for
chloro-substituted compounds. Thus the 2-fluorinated derivative
6a (11.92 0.35%) was 7.5-fold more potent than 3-fluorinated 6f
(1.56 0.09%), and the 3-chlorinated derivative 6g (9.97 0.23%)
was 20-fold more active than 2-chlorinated 6p (0.40 0.03%). Sim-
ilarly for the two nitro-substituted derivatives (6k, 6o) and two di-
chloro-substituted derivatives (6h, 6m), only the 3-nitrated 6k
(8.14 0.23%) and 2,4-dichlorated 6m (7.83 0.20%) affected
stroke volume, but 2-nitrated 6o and 2,6-dichlorated 6h did not.
—
1.67 0.18
1.56 0.09
9.97 0.23*
—
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
Milrinone
—
—
8.14 0.23*
2.96 0.11
7.83 0.20*
1.87
—
0.08
0.40 0.03
7.41 0.15*
5.19 0.12
—
5.13 0.22
—
0.18 0.14
5.56 0.21
—
—
2-Br
3-F
3-Cl
2,6-Cl2
4-CH3
3-NO2
2-Cl
2,4-Cl2
2-NO2
4-OCH3
3,4-(OCH3)2
—
—
2.78 1.63
—
6.08 0.17
—
6.36 0.13
a
Test sample concentration 1 ꢀ 10ꢁ4 M.
None or negative stroke volume increase.
p < 0.05 versus milrinone.
b
*
A18
B13
***
***
10
7
13
8
4
3
1
Control
Control
7a (100 μmol/L)
Milrinone (100 μmol/L)
-2
-7
6g (100 μmol/L)
Milrinone (100 μmol/L)
-2
-5
0
4
8
12 16 20 24 28
Fraction number
0
4
8
12 16 20 24 28
Fraction number
D
C
22
17
12
7
30
Control
6m (100 μmol/L)
***
24
18
12
6
7d (100 μmol/L)***
2
Control
6a (100 μmol/L)
6k (100 μmol/L)
Milrinone (100 μmol/L)
0
-3
-8
-6
-12
0
4
8
12 16 20 24 28
0
4
8
12 16 20 24 28
Fraction number
Fraction number
Figure 2. Effects of milrinone and compounds 6a, 6g, 6k, 6m, 7a and 7d on stroke volume in beating rabbit atria (1.5 Hz). The atrium stroke volume was recorded at 2-min
intervals. Values are means SE. ***P < 0.001 versus control.

J.-Y. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2392–2395
2395
E9
F 9
***
***
6
3
6
3
Control
Control
10 μmol/L
10 μmol/L
0
0
30 μmol/L
30 μmol/L
100 μmol/L
300 μmol/L
100 μmol/L
300 μmol/L
-3
-3
0
5
10 15 20 25 30 35
Fraction number
0
5
10 15 20 25 30 35
Fraction number
Figure 3. Concentration–response curves of compounds 6a (E) and 7a (F) on isolated rabbit-heart preparations. Values are means SE. ***P < 0.001 versus control.
For 7a–o, which possesses 4-substituted phenylmethoxy-3-
methoxybenzyl groups at the 4-position of the piperidine, there
was no clear structure–activity relationship, and only one com-
pound (2-fluorinated 7a, 7.41 0.15%) was more active than milri-
none, with compounds 7b, 7d, 7g, and 7n showing slightly weaker
activities. This series was generally weaker than 6a–p, indicating
that the length of the molecule seems to hinder PDE III binding.
Although 6m and 7d produced initial increases in stroke vol-
ume, longer treatment caused decreases in stroke volume
(Fig. 2D), as did 6p, 7g, and 7n (data not shown). Compounds 6g
and 7a exhibited similar atrial dynamic profiles to milrinone, with
increased stroke volume of 7.41 0.15% and 9.97 0.23%, respec-
tively (Fig. 2A and B).
References and notes
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Y. J. Med. Chem. 1992, 35, 3607.
3. 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.
4. 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. 1998,
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8. Bonina, F. P.; Arenare, L.; Palagiano, F.; Saija, A.; Nava, F.; Trombetta, D.; De
Caprariis, P. J. Pharm. Sci 1999, 88, 561.
We next tested the dose-dependency of the most effective com-
pounds, 6a and 7a, at 1 ꢀ 10ꢁ5 M, 3 ꢀ 10ꢁ5 M, 1 ꢀ 10ꢁ4 M, and 3 ꢀ
10ꢁ4 M. Both compounds showed maximal effects at 1 ꢀ 10ꢁ4 M,
and less activity at the higher dose (3 ꢀ 10ꢁ4 M; Fig. 3E for 6a and
Fig. 3F for 7a).
9. Yu, H. F.; Xiao, Y. P.; Liu, Q.; Chi, Y. N.; Zhang, Q. Chem. J. Chin. Univ. 2004, 25,
673.
10. Modestode, C.; Luciana, S.; Antonio, C.; Cosimo, A.; Adele, N.; Saverio, C.;
Angelo, C. Bioorg. Med. Chem. 2003, 11, 1439.
11. Preparation of 6a: A suspension of 5 (0.40 g, 1.15 mmol), 1-(chloromethyl)-2-
fluorobenzene (0.33 g, 2.28 mmol), potassium carbonate (0.32 g, 2.32 mmol),
KI (0.12 g, 0.72 mmol), triethylamine (0.78 mL, 3.45 mmol) in acetone (40 mL)
was stirred under reflux for 10 h and filtered. The filtrate was evaporated in
vacuo and the residue was dissolved in dichloromethane (50 mL). The solution
was washed with water (2 ꢀ 15 mL) and brine (20 mL), dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (dichloromethane/methanol, 15:1 v/v) to afford 6a
(0.15 g, 31%) as a white solid. Mp 135–137 °C. 1H NMR (CDCl3, 300 MHz): d
1.94–2.20 (6H, m), 2.40–2.44 (1H, m), 2.73 (3H, s), 2.94–3.06 (6H, m), 3.66 (2H,
s), 7.01–7.73 (7H, m). MS m/z 420 (M+1). IR (KBr) cmꢁ1: 3529 (NH), 1682
(C@O). Anal. Calcd for C24H26FN5O: C, 68.72; H, 6.25; N, 16.69. Found: C, 68.69;
H, 6.36; N, 16.53.
In conclusion, we designed and synthesized two series of 1-substi-
tuted-N-(4,5-dihydro-1-methyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)-
piperidine-4-carboxamides based on compound
1 to identify
compounds that improve cardiac contractility. Results show that
compounds 6a, 6g, 6k, 7a, and 7n exhibit promising cardiovascular
profiles and better activity than milrinone at the concentration of
1 ꢀ 10ꢁ4 M, and further modification of compound 1 is in progress.
Acknowledgment
12. 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.
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 Natural Science Foun-
dation of China (No. 30560177).