Design, synthesis, and negative inotropic evaluation of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or piperazine moieties

Article abstract of DOI:10.1111/cbdd.12828

In this study, four novel series of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or piperazine moieties were designed, synthesized, and evaluated for negative inotropic activity by measuring the left atrium stroke volume in isolated rabbit heart preparations. Almost all of the compounds showed an ability to moderate the cardiac workload by decreasing the heart rate and contractility. Among them, 7h was found to be the most potent with a change in stroke volume of ?48.22?±?0.36% at a concentration of 3?×?10^?5?mol/L (metoprolol: ?9.74?±?0.14%). The cytotoxicity of these compounds was evaluated using the human cervical cancer cell line HeLa, the liver cancer cell line Hep3B, and the human normal hepatic cell line LO2. A preliminary study of the mechanism of action for the compound 7h on the regulation of atrial dynamics with ATP-sensitive K⁺ channel and L-type Ca²⁺ channel blockers glibenclamide and nifedipine was performed in the isolated perfused beating rabbit atria.

Full text of DOI:10.1111/cbdd.12828

Received Date : 08-Jun-2016
Revised Date : 15-Jul-2016
Accepted Date : 21-Jul-2016
Article type
: Research Article
Design, synthesis, and negative inotropic evaluation of
4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or
piperazine moieties
Zhi-Yu Wei a,1, Bai-Ri Cui c,1, Xun Cui b, Yan-Ling Wu a, Yang Fu a, Li-Ping Liub,*, Hu-Ri Piao a,*
a Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry of
Education, Yanbian University College of Pharmacy, Yanji 133000, China
b Yanbian University College of Medicine, Yanji 133000, China
c Department of Orthopedics, Yanbian University Hospital, 133000, China
*Corresponding authors. Tel.: + 86 433 2435132; fax: + 86 433 2435026 (L.-P.L.): tel.: +86 433
2436149; fax: + 86 433 2435026 (H.-R.P.).
E-mail addresses: liulp@ybu.edu.cn (L.-P. Liu), piaohr@ybu.edu.cn (H.-R. Piao).
1 These authors contributed equally to this work
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12828
This article is protected by copyright. All rights reserved.

Abstract
In the present study, four novel series of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives
containing triazole or piperazine moieties were designed, synthesized and evaluated for
negative inotropic activity by measuring the left atrium stroke volume in isolated rabbit
heart preparations. Almost all of the compounds showed an ability to moderate the cardiac
workload by decreasing the heart rate and contractility. Among them, 7h was found to be
the most potent with a change in stroke volume of −48.22 ± 0.36% at a concentration of
3×10−5 mol/L (metoprolol: −9.74 ± 0.14%). The cytotoxicity of these compounds was
evaluated using the human cervical cancer cell line HeLa, the liver cancer cell line Hep3B and
the human normal hepatic cell line LO2. A preliminary study of the mechanism of action for
the compound 7h on the regulation of atrial dynamics with ATP-sensitive K+ channel and
L-type Ca2+ channel blockers glibenclamide and nifedipine was performed in the isolated
perfused beating rabbit atria.
Keywords: [1,2,4]triazol-5(4H)-one; triazole; piperazine; negative inotropic activity; stroke
volume.
Congestive heart failure (CHF) is a major human health problem and a leading cause of
death worldwide (1). Inotropic drugs alter the force or strength of the heart’s muscular
contractions and can be divided into two different types: negative and positive. Negative
inotropic drugs make the heart beat less strongly and positive inotropic drugs make the
heart beat more strongly. Both types of drug are used in the management of various
conditions of congestive heart failure (2,3). Cardiac glycosides, such as digoxin, have been
traditionally used as positive inotropic drugs (4,5). However, there are serious problems
with digoxin use because of the frequency and severity of digitalis intoxication (6,7). The
clinical use of the phosphodiesterase-inhibiting agent milrinone is also limited because of
significant ventricular arrhythmias and tachycardia associated with elevated cAMP levels
This article is protected by copyright. All rights reserved.

caused by milrinone (8). Similar issues have been identified with the recently developed
drugs, vesnarinone and toborinone (9,10). In the past decade, the treatment of heart failure
(HF) has undergone a fundamental shift, from treatment using short-term, hemodynamic
pharmacological measures to a long-term, corrective maintenance strategy aimed at
changing the biological properties of the diseased heart. The negative inotropic drug
metoprolol, once used as contraindication in patients of heart failure due to their negative
inotropic activity, has become a conventional treatment for HF, based on the biological
effects of long-term treatment (11-13). Therefore, the development of innovative positive
and negative inotropic agents with improved therapeutic properties and fewer side effects
for the treatment of CHF is still a great challenge for medicinal chemists (14).
In our previous work searching for lead compounds with inotropic activities, we found that
compounds A and B (Figure 1) showed weak negative inotropic activity (compound A:
−24.70%) (15,16). In a summary of our previous work (17,18), we hypothesized that the
triazole and piperazine rings were essential to the observed higher levels of affinity for the
receptor and consequently enhanced the inotropic activity. Considering that triazoles and
their derivatives have attracted significant attention for their various biological activities,
including anti-inflammatory, analgesic, hypotensive, and their effect on diastolic blood
vessels (19-21), and several medicines used in the treatment of HF contain a piperazine
moiety (22,23). In an attempt to search for more potent inotropic agents, we kept
benzylpiperazine moiety unchanged and used 4-phenyl-1H-1,2,4-triazol-5(4H)-one moiety to
take the place of 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-1(2H)-one. Then introduced a
triazole ring to replace the piperazine ring with 4-phenyl-1H-1,2,4-triazol-5(4H)-one moiety
unchanged to further optimize lead compound A. Thus, we designed and synthesized four
novel series of 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole and
piperazine moieties (Figure 1). In addition, we simultaneously changed the substituents on
the phenyl ring to investigate the contribution of such a structural change on biological
activity. The negative inotropic activities of the synthesized compounds were evaluated by
measuring the left atrium stroke volume in isolated rabbit heart preparations. Furthermore,
This article is protected by copyright. All rights reserved.

to preliminary investigate the mechanism of action on the regulation of atrial dynamics,
another series experiments were performed with ATP-sensitive K+ channel and L-type Ca2+
channel blockers glibenclamide and nifedipine in the isolated perfused beating rabbit atria.
What’s more, compounds were also evaluated for their cytotoxicity against the human
cervical cancer cell line HeLa and liver cancer cell line Hep3B, as indicated by the half
inhibitory concentration (IC50) values of the tested compounds according to the
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.
Experimental Section
Material and methods
All commercially available reagents were obtained from Sigma–Aldrich (Saint Louis, MO,
USA) and Fluka (Milwaukee, WI, USA). Melting points were determined in open capillary
tubes and are uncorrected. Reaction courses were monitored by TLC on silica gel precoated
F254 Merck plates and the developed plates were examined under UV lamps (254 nm). IR
spectra were recorded (in KBr) on a FT-IR1730. Column chromatography was undertaken
with 200-mesh silica gel (Merck). 1H NMR and 13C NMR spectra were measured on Bruker
AV-300 spectrometer at 300 MHz and 75 MHz respectively used tetramethylsilane (TMS) as
an internal standard. Chemical shifts were expressed in δ, ppm. Mass spectra were
measured on an HP1100LC (Agilent Technologies, USA). High resolution mass spectroscopy
(HRMS) was measured on a Bruker ultrafleXtreme MALDI-TOF/TOF.
General procedure for synthesis of compounds 4I-4III
A mixture of aniline 1I (0.93g, 10 mmol) or 1II (10 mmol) or 1III (10 mmol), triethyl
orthoformate 2 (2.22g, 15 mmol) and methyl hydrazinecarboxylate 3 (1.35g, 15 mmol) in
refluxing ethanol (50mL) at 78 °C was stirred for 24 h. sodium methoxide (0.81g, 15 mmol)
was added to the mixture and the resulting mixture was stirred for another 24 h. The
solvent was evaporated under reduced pressure, and the residue was dissolved in
This article is protected by copyright. All rights reserved.

dichloromethane and washed with water and brine, dried over MgSO4, and the solvent was
removed under reduced pressure to afford intermediates 4I-III in 85%-90% yields.
General procedure for synthesis of compounds 5I-5III
The key intermediates 5I-5III were synthesized by reacting intermediates 4I (1.61g, 10
mmol) or 4II (10 mmol) or 4III (10 mmol) with 1,3-dibromopropane (2.02g, 10 mmol) in the
presence of potassium carbonate and potassium iodide in refluxing acetone (50mL) at 57 °C
for 6 h. The solvent was evaporated with reduced pressure and the residue was dissolved in
dichloromethane, washed with water and brine, and dried by MgSO4. A white solid was
collected through filtration and dried in a vacuum to give the compounds 5I-III in 80%-85%
yields.
General procedure for synthesis of compounds 6a-i and 7a-i
Just as outlined in Scheme 1, nucleophilic substitution of 5I (10 mmol) or 5II (10 mmol) with
various monosubstituted piperazines (10 mmol) in refluxing acetone (20mL) at 57 °C in the
presence of KI/K2CO3 afforded 6a-i and 7a-i in 40%-60% yields. The solution was evaporated
to dryness under reduced pressure, and the residue was purified by silica gel column
chromatography with dichloromethane: methanol (200:1). The yield, melting point and
spectra data of each compound are given below.
1-(3-(4-Benzylpiperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6a)
Yield: 42%; m.p. 124–126°C. IR (KBr) cm–1: 1685 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.57-7.46 (m, 4H, Ar-H), 7.40-7.25 (m, 6H, Ar-H), 3.92 (t, J = 6.9 Hz, 2H,
CH2), 3.51 (s, 2H, CH2), 2.68-2.32 (m, 10H, (CH2)5), 2.01 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 151.75, 138.17, 134.01, 133.45, 129.58, 129.58, 129.19, 129.19, 128.18,
This article is protected by copyright. All rights reserved.

128.18, 127.45, 126.99, 121.83, 121.83, 63.06, 55.53, 53.17, 53.17, 53.12, 53.12, 44.05,
26.04. HRMS (MALDI) calcd for C22H28N5O (M + H)+: 378.2288, found: 378.2289.
1-(3-(4-(2-Chlorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6b)
Yield: 58%; m.p. 120–122°C. IR (KBr) cm–1: 1689 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.61-7.48 (m, 5H, Ar-H), 7.41-7.14 (m, 4H, Ar-H ), 3.93 (t, J = 6.9 Hz, 2H,
CH2), 3.62 (s, 2H, CH2), 2.54-2.44 (m, 10H, (CH2)5), 2.08-1.94 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 151.68, 146.53, 136.75, 134.72, 132.76, 130.49, 128.98, 128.98, 128.68,
128.34, 128.31, 128.31, 127.96, 126.87, 58.27, 58.27, 57.46, 55.30, 55.30, 51.24, 40.89,
22.19. HRMS (MALDI) calcd for C22H27ClN5O (M + H)+: 412.1899, found: 412.1906.
1-(3-(4-(3-Chlorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6c)
Yield: 47%; m.p. 116–118°C. IR (KBr) cm–1: 1685 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.59-7.47 (m, 4H, Ar-H), 7.40-7.20 (m, 5H, Ar-H), 3.91 (t, J = 6.9 Hz, 2H,
CH2), 3.44 (s, 2H, CH2), 2.68-2.29 (m, 10H, (CH2)5), 2.04-1.92 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 150.70, 146.62, 136.87, 134.83, 132.64, 132.52, 128.45, 128.45, 127.90,
127.90, 127.72, 126.96, 126.88, 126.06, 65.26, 58.21, 58.21, 55.01, 55.01, 51.40, 40.84,
22.59. HRMS (MALDI) calcd for C22H27ClN5O (M + H)+: 412.1899, found: 412.1892.
1-(3-(4-(4-Chlorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6d)
Yield: 52%; m.p. 159–161°C. IR (KBr) cm–1: 1674 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.69 (s, 1H, N=CH), 7.55-7.47 (m, 4H, Ar-H), 7.36-7.21 (m, 5H, Ar-H), 3.91 (t, J = 6.9 Hz, 2H,
CH2), 3.45 (s, 2H, CH2), 2.66-2.17 (m, 10H, (CH2)5), 2.05-1.91 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 150.68, 146.53, 136.75, 132.72, 132.49, 130.48, 130.48, 128.94, 128.94,
128.31, 128.31, 128.10, 128.10, 127.72, 65.34, 58.27, 58.27, 55.30, 55.30, 51.24, 40.89,
22.19. HRMS (MALDI) calcd for C22H27ClN5O (M + H)+: 412.1899, found: 412.1901.
This article is protected by copyright. All rights reserved.

2-((4-(3-(5-Oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-1-yl)propyl)piperazin-1-yl)methyl)be
nzonitrile (6e)
Yield: 50%; m.p. 118–120°C. IR (KBr) cm–1: 1683 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.65 (m, 1H, Ar-H), 7.62-7.38 (m, 8H, Ar-H), 3.93 (t, J = 6.8 Hz, 2H, CH2),
3.70 (s, 2H, CH2), 2.53-2.45 (m, 10H, (CH2)5), 2.12-1.92 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 151.76, 142.42, 133.98, 133.45, 132.90, 132.47, 129.99, 129.59, 129.59,
127.48, 127.46, 121.84, 121.84, 117.79, 112.96, 60.39, 55.47, 53.04, 53.04, 52.95, 52.95,
44.04, 25.97. HRMS (MALDI) calcd for C23H27N6O (M + H)+: 403.2241, found: 403.2247 (M +
H)+.
1-(3-(4-(3-Fluorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6f)
Yield: 57%; m.p. 114–116°C. IR (KBr) cm–1: 1682 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.69 (s, 1H, N=CH), 7.64-7.46 (m, 4H, Ar-H), 7.44-7.33 (m, 1H, Ar-H), 7.27 (m, 1H, Ar-H), 7.09
(m, 2H, Ar-H), 6.95 (m, 1H, Ar-H), 3.93 (t, J = 6.9 Hz, 2H, CH2), 3.50 (s, 2H, CH2), 2.51-2.21 (m,
10H, (CH2)5), 2.12-1.95 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 162.69, 150.96,
146.52, 138.77, 132.73, 128.96, 128.96, 128.42, 128.42, 128.15, 126.30, 124.70, 119.61,
114.02, 65.36, 58.20, 58.20, 55.31, 55.31, 51.20, 40.87, 22.09. HRMS (MALDI) calcd for
C22H27FN5O (M + H)+: 396.2194, found: 396.2195.
1-(3-(4-(4-Fluorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6g)
Yield: 51%; m.p. 150–152°C. IR (KBr) cm–1: 1677 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.57 (m, 2H, Ar-H), 7.53-7.45 (m, 2H, Ar-H), 7.38 (m, 1H, Ar-H), 7.28 (m,
2H, Ar-H), 7.06-6.96 (m, 2H, Ar-H), 3.92 (t, J = 7.0 Hz, 2H, CH2), 3.48 (s, 2H, CH2), 2.69-2.30
(m, 10H, (CH2)5), 2.08-1.93 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 161.68, 150.63,
146.53, 134.75, 132.72, 128.49, 128.49, 128.04, 128.04, 127.92, 115.65, 115.65, 65.34,
58.27, 58.27, 55.30, 55.30, 51.24, 40.89, 22.19. HRMS (MALDI) calcd for C22H27FN5O (M +
H)+: 396.2194, found: 396.2198.
This article is protected by copyright. All rights reserved.

1-(3-(4-(4-Methoxybenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6h)
Yield: 43%; m.p. 158–160°C. IR (KBr) cm–1: 1675 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.54 (m, 4H, Ar-H), 7.39-7.21 (m, 3H, Ar-H), 6.86 (m, 2H, Ar-H), 3.93 (t, J =
7.0 Hz, 2H, CH2), 3.81 (s, 3H, CH2), 3.46 (s, 2H, CH2), 2.76-2.17 (m, 10H, (CH2)5), 2.07-1.94 (m,
2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.69, 151.59, 135.01, 134.64, 130.49, 130.43,
130.43, 129.77, 129.77, 127.36, 121.77, 121.77, 113.95, 113.95, 61.96, 55.42, 53.21, 53.21,
52.98, 52.98, 49.06, 43.69, 25.91. HRMS (MALDI) calcd for C22H30N5O2 (M + H)+: 408.2394,
found: 408.2388.
1-(3-(4-(2,4-Dichlorobenzyl)piperazin-1-yl)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (6i)
Yield: 49%; m.p. 157–159°C. IR (KBr) cm–1: 1684 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
7.70 (s, 1H, N=CH), 7.59-7.52 (m, 2H, Ar-H), 7.48 (m, 2H, Ar-H), 7.38 (m, 3H, Ar-H), 7.20 (m,
1H, Ar-H), 3.92 (t, J = 6.4 Hz, 2H, CH2), 3.55 (s, 2H, CH3), 2.70-2.29 (m, 10H, (CH2)5), 2.06-1.93
(m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.69, 146.52, 135.77, 134.73, 134.46,
132.42, 130.35, 128.95, 128.95, 128.16, 128.16, 128.07, 126.70, 58.20, 58.20, 57.45, 55.31,
55.31, 51.20, 40.87, 22.09. HRMS (MALDI) calcd for C22H26Cl2N5O (M + H)+: 446.1509, found:
446.1494.
1-(3-(4-Benzylpiperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-one (7a)
Yield: 56%; m.p. 134–136°C. IR (KBr) cm–1: 1680 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.38 (d, J = 9.2 Hz, 2H, Ar-H), 7.88 (d, J = 9.2 Hz, 2H, Ar-H), 7.83 (s, 1H, N=CH), 7.30 (m, 5H,
Ar-H), 3.96 (t, J = 7.0 Hz, 2H, CH2), 3.52 (s, 2H, CH2), 2.69-2.31 (m, 10H, (CH2)5), 2.09-1.97 (m,
2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.49, 146.22, 143.58, 138.86, 138.67, 129.23,
129.23, 128.82, 128.82, 128.45, 128.45, 127.22, 124.41, 65.16, 58.45, 58.45, 55.27, 55.27,
51.32, 40.57, 22.72. HRMS (MALDI) calcd for C22H27N6O3 (M + H)+: 423.2139, found:
423.2138.
This article is protected by copyright. All rights reserved.

1-(3-(4-(2-Chlorobenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-one
(7b)
Yield: 56%; m.p. 118–120°C. IR (KBr) cm–1: 1677 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.38 (d, J = 9.0 Hz, 2H, Ar-H), 7.89 (d, J = 9.0 Hz, 2H, Ar-H), 7.84 (s, 1H, N=CH), 7.46 (d, J = 6.1
Hz, 1H, Ar-H), 7.35 (m, 1H, Ar-H), 7.22 (m, 2H, Ar-H), 3.95 (t, J = 6.7 Hz, 2H, CH2), 3.63 (s, 2H,
CH2), 2.54-2.25 (m, 10H, (CH2)5), 2.03 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.67,
146.55, 143.72, 138.70, 135.48, 134.45, 134.38, 131.32, 130.74, 129.63, 129.63, 126.85,
124.63, 124.63, 58.24, 58.24, 57.45, 55.11, 55.11, 51.27, 40.85, 22.39. HRMS (MALDI) calcd
for C22H26ClN6O3 (M + H)+: 457.1749, found: 457.1752.
1-(3-(4-(3-Chlorobenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-one
(7c)
Yield: 54%; m.p. 84–86°C. IR (KBr) cm–1: 1675 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ 8.38
(d, J = 9.1 Hz, 2H, Ar-H), 7.88 (d, J = 9.1 Hz, 2H, Ar-H), 7.84 (s, 1H, N=CH), 7.33 (s, 1H, Ar-H),
7.24 (m, 2H, Ar-H), 7.21 (m, 1H, Ar-H), 3.95 (t, J = 6.8 Hz, 2H, CH2), 3.47 (s, 2H, CH2),
2.49-2.41 (m, 10H, (CH2)5), 2.09-1.95 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.79,
146.12, 143.57, 138.37, 135.23, 134.65, 134.52, 131.35, 130.31, 129.92, 129.92, 126.22,
124.86, 124.86, 65.16, 58.45, 58.45, 55.27, 55.27, 51.38, 40.17, 22.09. HRMS (MALDI) calcd
for C22H26ClN6O3 (M + H)+: 457.1749, found: 457.1746.
1-(3-(4-(4-Chlorobenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-one
(7d)
Yield: 55%; m.p. 138–140°C. IR (KBr) cm–1: 1673 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.38 (d, J = 9.0 Hz, 2H, Ar-H), 7.88 (d, J = 9.0 Hz, 2H, Ar-H), 7.83 (s, 1H, N=CH), 7.28 (dd, J =
7.2, 5.6 Hz, 4H, Ar-H), 3.94 (t, J = 6.9 Hz, 2H, CH2), 3.50 (s, 2H, CH2), 2.49-2.31 (m, 10H,
(CH2)5), 2.10-1.94 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 151.12, 145.92, 139.28,
136.71, 132.67, 132.01, 130.40, 130.40, 128.32, 128.32, 125.28, 125.28, 120.88, 120.88,
This article is protected by copyright. All rights reserved.

62.20, 55.42, 53.12, 53.12, 53.03, 53.03, 44.23, 25.83. HRMS (MALDI) calcd for C22H25ClN6O3
(M + H)+: 457.1749, found: 457.1751.
2-((4-(3-(4-(4-Nitrophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)propyl)piperazin-1-yl)m
ethyl)benzonitrile (7e)
Yield: 51%; m.p. 114–116°C. IR (KBr) cm–1: 1671 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.38 (d, J = 8.9 Hz, 2H, Ar-H), 7.89 (d, J = 8.9 Hz, 2H, Ar-H), 7.85 (s, 1H, N=CH), 7.65 (m, 1H,
Ar-H), 7.53 (m, 2H, Ar-H), 7.43-7.33 (m, 1H, Ar-H), 3.95 (t, J = 6.7 Hz, 2H, CH2), 3.68 (s, 2H,
CH2), 2.92-2.32 (m, 10H, (CH2)5), 2.07-1.94 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ
150.49, 146.22, 143.50, 139.78, 138.57, 132.53, 131.76, 129.52, 129.35, 129.35, 127.92,
124.11, 124.11, 115.89, 113.15, 59.45, 58.45, 58.45, 55.27, 55.27, 51.32, 40.57, 22.72.
HRMS (MALDI) calcd for C23H26N7O3 (M + H)+: 448.2092, found: 448.2078.
1-(3-(4-(3-Fluorobenzyl) piperazin-1-yl) propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol
-5(4H)-one (7f)
Yield: 47%; m.p. 80–82°C. IR (KBr) cm–1: 1677 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ 8.38
(d, J = 9.1 Hz, 2H, Ar-H), 7.88 (d, J = 9.1 Hz, 2H, Ar-H), 7.84 (s, 1H, N=CH), 7.27 (m, 1H, Ar-H),
7.07 (m, 2H, Ar-H), 6.95 (t, J = 8.2 Hz, 1H, Ar-H), 3.95 (t, J = 6.8 Hz, 2H, CH2), 3.50 (s, 2H, CH2),
2.49-2.23 (m, 10H, (CH2)5), 2.08-1.97 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.72,
146.34, 143.89, 138.74, 135.25, 134.68, 134.42, 131.55, 130.01, 129.81, 129.81, 126.22,
124.67, 124.67, 65.18, 58.48, 58.48, 55.22, 55.22, 51.38, 40.27, 21.76. HRMS (MALDI) calcd
for C22H26FN6O3 (M + H)+: 441.2045, found: 441.2039.
This article is protected by copyright. All rights reserved.

1-(3-(4-(4-Fluorobenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-one
(7g)
Yield: 56%; m.p. 136–138°C. IR (KBr) cm–1: 1687 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.38 (d, J = 9.0 Hz, 2H, Ar-H), 7.88 (d, J = 9.2 Hz, 2H, Ar-H), 7.83 (s, 1H, N=CH), 7.56-7.03 (m,
4H, Ar-H), 4.31 (s, 2H, CH2), 3.97-3.95 (t, J = 6.3 Hz, 2H, CH2), 2.54-2.58 (m, 10H, (CH2)5),
1.34-1.37 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 161.55, 151.11, 145.88, 139.28,
133.82, 132.03, 130.64, 130.53, 125.26, 125.26, 120.86, 120.86, 115.08, 114.80, 62.16,
55.41, 53.11, 53.11, 52.97, 52.97, 44.22, 25.82. HRMS (MALDI) calcd for C22H26FN6O3 (M +
H)+: 441.2045, found: 441.2039.
1-(3-(4-(4-Methoxybenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-o
ne (7h)
Yield: 53%; m.p. 134–136°C. IR (KBr) cm–1: 1684 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δ
8.37 (d, J = 9.1 Hz, 2H, Ar-H), 7.88 (d, J = 9.1 Hz, 2H, Ar-H), 7.83 (s, 1H, N=CH), 7.22 (d, J = 8.4
Hz, 2H, Ar-H), 6.85 (d, J = 8.5 Hz, 2H, Ar-H), 3.94 (t, J = 6.9 Hz, 2H, CH2), 3.81 (s, 3H, OCH3),
3.49 (s, 2H, CH2), 2.49-2.35 (m, 10H, (CH2)5), 2.01 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz,
ppm): δ 158.71, 151.14, 145.91, 139.1028, 132.02, 130.39, 130.39, 129.93, 125.27, 125.27,
120.88, 120.88, 113.56, 113.56, 62.39, 55.45, 55.23, 53.11, 53.11, 52.92, 52.92, 44.25,
25.82. HRMS (MALDI) calcd for C23H29N6O4 (M + H)+: 453.2245, found: 453.2248.
1-(3-(4-(2,4-Dichlorobenzyl)piperazin-1-yl)propyl)-4-(4-nitrophenyl)-1H-1,2,4-triazol-5(4H)-
one (7i)
Yield: 51%; m.p. 130–132°C. IR (KBr) cm–1: 1677 (C=O). 1H NMR (CDCl3, 300 MHz, ppm): δδ
8.38 (d, J = 9.1 Hz, 2H, Ar-H), 7.89 (d, J = 9.1 Hz, 2H, Ar-H), 7.84 (s, 1H, N=CH), 7.38 (m, 2H,
Ar-H), 7.21 (m, 1H, Ar-H), 3.95 (t, J = 6.9 Hz, 2H, CH2), 3.56 (s, 2H, CH2), 2.68-2.36 (m, 10H,
(CH2)5), 2.07-1.94 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 150.49, 146.22, 143.87,
138.57, 135.53, 134.76, 134.52, 131.35, 130.31, 129.92, 129.92, 126.22, 124.86, 124.86,
This article is protected by copyright. All rights reserved.

65.16, 58.45, 58.45, 55.27, 55.27, 51.32, 40.57, 22.72. HRMS (MALDI) calcd for
C22H25Cl2N6O3 (M + H)+: 491.1360, found: 491.1354.
General procedure for synthesis of compounds 13a-j and 14a-j
Synthesis of compound 13a-j and 14a-j was depicted in Scheme 2. Esterification of
substituted benzoic acid 8a-j (10 mmol) with methanol (20mL) in the presence of sulfuric
acid afforded 9a-j, which were reacted with hydrazine hydrate (10 mmol) to give
compounds 10a-j. Compounds 12a-j was prepared by the reaction of 10a-j (8 mmol) with
potassium thiocyanate (8 mmol) in refluxing hydrochloric acid and sodium hydroxide.
Nucleophilic substitution of 5I (10 mmol) or 5III (10 mmol) with 12a-j (10 mmol) in refluxing
acetone (20 mL) in the presence of KI/K2CO3 at 57 °C for 6 h afforded 13a-j and 14a-j in high
yields 70%–90%. The solution was evaporated to dryness under reduced pressure, and the
residue was purified by silica gel column chromatography with dichloromethane: methanol
(200:1). The yield, melting point and spectra data of each compound are given below.
4-Phenyl-1-(3-((5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H)-one (13a)
Yield: 77%; m.p. 190–191°C. IR (KBr) cm–1: 3186 (NH), 1685 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.32 (s, 1H, NH), 7.97 (d, J = 7.6 Hz, 2H, Ar-H), 7.67 (d, J = 8.6 Hz, 2H, Ar-H),
7.56-7.28 (m, 7H, Ar-H and N=CH), 3.90 (t, J = 6.5 Hz, 2H, CH2), 3.23 (t, J = 5.5 Hz, 2H, CH2),
2.23-2.08 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.86, 157.65, 151.02, 137.73,
137.73, 132.75, 131.40, 129.23, 129.23, 128.91, 128.91, 128.05, 127.46, 127.46, 119.43,
119.43, 45.64, 34.21, 24.60. HRMS (MALDI) calcd for C19H19N6OS (M + H)+: 379.1336, found:
379.1339.
This article is protected by copyright. All rights reserved.

1-(3-((5-(2-Chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)
-one (13b)
Yield: 83%; m.p. 120–122°C. IR (KBr) cm–1: 3165 (NH), 1678 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.49 (s, 1H, NH), 7.77-7.34 (m, 10H, Ar-H and N=CH), 3.90 (t, J = 6.4 Hz, 2H, CH2),
3.22 (t, J = 6.4 Hz, 2H, CH2), 2.25-2.07 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.86,
157.62, 151.22, 138.75, 137.71, 137.71, 132.74, 130.01, 129.85, 128.94, 128.94, 128.94,
128.02, 127.47, 119.41, 119.41, 45.65, 35.12, 26.63. HRMS (MALDI) calcd for C19H18ClN6OS
(M + H)+: 413.0946, found: 413.0944.
1-(3-((5-(3-Chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)
-one (13c)
Yield: 88%; m.p. 154–156°C. IR (KBr) cm–1: 3172 (NH), 1679 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.07 (s, 1H, NH), 7.94 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.55-7.27 (m, 8H, Ar-H and
N=CH), 4.13 (t, J = 5.9 Hz, 2H, CH2), 3.26 (t, J = 6.4 Hz, 2H, CH2), 2.27 (m, 2H, CH2). 13C NMR
(CDCl3, 75 MHz, ppm): δ 158.86, 157.69, 150.26, 146.77, 134.83, 132.77, 132.07, 129.53,
128.91, 128.91, 128.80, 128.18, 128.18, 127.98, 126.43, 125.65, 45.20, 34.53, 27.07. HRMS
(MALDI) calcd for C19H18ClN6OS (M + H)+: 413.0946, found: 413.0939.
1-(3-((5-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)
-one (13d)
Yield: 93%; m.p. 158–160°C. IR (KBr) cm–1: 3178 (NH), 1684 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.99 (s, 1H, NH), 7.76-7.56 (m, 2H, Ar-H), 7.55-7.23 (m, 8H, Ar-H and N=CH), 4.15 (t,
J = 6.5 Hz, 2H, CH2), 3.26 (t, J = 5.6 Hz, 2H, CH2), 2.28-2.25 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 158.86, 157.65, 150.22, 146.90, 134.75, 132.40, 130.63, 129.31, 129.31,
128.95, 128.95, 128.95, 128.95, 128.12, 128.12, 128.06, 45.64, 34.21, 26.59. HRMS (MALDI)
calcd for C19H18ClN6OS (M + H)+: 413.0946, found: 413.0934.
This article is protected by copyright. All rights reserved.

4-Phenyl-1-(3-((5-(o-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H)-one
(13e)
Yield: 92%; m.p. 120–122°C. IR (KBr) cm–1: 3179 (NH), 1679 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.71 (s, 1H, NH), 7.67-7.18 (m, 10H, Ar-H and N=CH), 4.09 (t, J = 6.4 Hz, 2H, CH2),
3.26 (t, J = 6.8 Hz, 2H, CH2), 2.55 (s, 3H, CH3), 2.45-2.21 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 158.50, 157.68, 150.09, 146.95, 138.58, 136.78, 132.69, 130.08, 129.54,
129.46, 128.91, 128.91, 128.65, 128.01, 128.01, 127.96, 42.68, 35.42, 24.69, 19.12. HRMS
(MALDI) calcd for C20H21N6OS (M + H)+: 393.1492, found: 393.1495.
4-Phenyl-1-(3-((5-(m-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H)-one
(13f)
Yield: 80%; m.p. 125–127°C. IR (KBr) cm–1: 3180 (NH), 1684 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.85 (s, 1H, NH), 7.82-7.19 (m, 10H, Ar-H and N=CH), 4.11 (t, J = 5.5 Hz, 2H, CH2),
3.26 (t, J = 5.4 Hz, 2H, CH2), 2.36 (s, 3H, CH3), 2.33-2.17 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 158.56, 157.32, 151.22, 137.75, 137.75, 137.31, 131.25, 130.61, 129.14,
129.01, 128.95, 128.92, 128.92, 128.01, 124.56, 119.52, 119.52, 45.89, 34.51, 27.54, 21.83.
HRMS (MALDI) calcd for C20H21N6OS (M + H)+: 393.1492, found: 393.1493.
4-Phenyl-1-(3-((5-(p-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H)-one
(13g)
Yield: 86%; m.p. 160–162°C. IR (KBr) cm–1: 3174 (NH), 1676 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.48 (s, 1H, NH), 7.83 (d, J = 7.5 Hz, 2H, Ar-H), 7.69 (d, J = 7.9 Hz, 2H, Ar-H),
7.67-7.14 (m, 6H, Ar-H and N=CH), 3.90 (t, J = 6.4 Hz, 2H, CH2), 3.17 (t, J = 5.4 Hz, 2H, CH2),
2.35 (s, 3H, CH3), 2.27-2.05 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.82, 157.73,
151.72, 137.75, 137.75, 131.79, 129.54, 129.54, 129.54, 128.93, 128.93, 128.01, 125.72,
125.72, 119.45, 119.45, 45.89, 34.36, 26.98, 21.55. HRMS (MALDI) calcd for C20H21N6OS (M +
H)+: 393.1492, found: 393.1490.
This article is protected by copyright. All rights reserved.

1-(3-((5-(2-Bromophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)
-one (13h)
Yield: 71%; m.p. 110–112°C. IR (KBr) cm–1: 3167 (NH), 1676 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.95 (s, 1H, NH), 7.81-7.24 (m, 10H, Ar-H and N=CH), 4.08 (t, J = 6.3 Hz, 2H, CH2),
3.28 (t, J = 6.5 Hz, 2H, CH2), 2.31-2.29 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.75,
157.67, 151.52, 139.75, 137.71, 137.71, 132.14, 130.91, 129.75, 128.90, 128.90, 128.24,
128.12, 119.39, 119.39, 46.65, 34.12, 26.93. HRMS (MALDI) calcd for C19H18BrN6OS (M + H)+:
457.0441, found: 457.0445.
1-(3-((5-(3-Bromophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H-1,2,4-triazol-5(4H)
-one (13i)
Yield: 75%; m.p. 144–146°C. IR (KBr) cm–1: 3172 (NH), 1680 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.25 (s, 1H, NH), 8.01-7.25 (m, 10H, Ar-H and N=CH), 4.15 (t, J = 6.2 Hz, 2H, CH2),
3.25 (t, J = 5.9 Hz, 2H, CH2), 2.28-2.26 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.86,
157.65, 151.06, 137.77, 137.77, 132.83, 131.76, 131.43, 128.291, 128.91, 128.10, 128.02,
126.10, 122.03, 119.48, 119.48, 45.20, 37.53, 27.57. HRMS (MALDI) calcd for C19H18BrN6OS
(M + H)+: 457.0441, found: 457.0446.
1-(3-((5-(4-Methoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-phenyl-1H
-1,2,4-triazol-5(4H)-one (13j)
Yield: 87%; m.p. 149–151°C. IR (KBr) cm–1: 3173 (NH), 1681 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.48 (s, 1H, NH), 7.83-7.36 (m, 10H, Ar-H and N=CH), 3.90 (t, J = 6.4 Hz, 2H, CH2),
3.17 (t, J = 5.6 Hz, 2H, CH2), 2.35 (s, 3H, OCH3), 2.27-2.05 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 161.07, 158.89, 157.65, 151.65, 145.32, 132.58, 130.76, 130.76, 128.92,
128.92, 128.25, 128.25, 127.98, 124.91, 114.77, 114.77, 55.72, 44.09, 32.35, 29.19. HRMS
(MALDI) calcd for C20H21N6O2S (M + H)+: 409.1441, found: 409.1449.
This article is protected by copyright. All rights reserved.

4-(3-Chlorophenyl)-1-(3-((5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H)
-one (14a)
Yield: 84%; m.p. 138–140°C. IR (KBr) cm–1: 3167 (NH), 1678 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.03 (s, 1H, NH), 7.91-7.03 (m, 10H, Ar-H and N=CH), 4.11 (t, J = 6.5 Hz, 2H, CH2),
3.26 (t, J = 6.9 Hz, 2H, CH2), 2.44-2.22 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.91,
156.71, 150.17, 146.52, 134.63, 134.43, 133.50, 132.50, 131.91, 131.02, 128.94, 128.94,
127.22, 127.03, 127.03, 48.93, 34.11, 33.67. HRMS (MALDI) calcd for C19H18ClN6OS (M + H)+:
413.0946, found: 413.0951.
4-(3-Chlorophenyl)-1-(3-((5-(2-chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-tria
zol-5(4H)-one (14b)
Yield: 87%; m.p. 141–143°C. IR (KBr) cm–1: 3176 (NH), 1687 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.22 (s, 1H, NH), 7.73-7.27 (m, 9H, Ar-H and N=CH), 4.09 (t, J = 6.4 Hz, 2H, CH2), 3.29
(t, J = 6.5 Hz, 2H, CH2), 2.33 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.87, 151.39,
135.87, 135.03, 134.05, 131.99, 131.78, 131.75, 131.70, 131.41, 130.83, 127.89, 127.18,
121.35, 120.04, 44.01, 34.46, 29.03. HRMS (MALDI) calcd for C19H17Cl2N6OS (M + H)+:
447.0556, found: 447.0558.
4-(3-Chlorophenyl)-1-(3-((5-(3-chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-tria
zol-5(4H)-one (14c)
Yield: 82%; m.p. 148-149°C. IR (KBr) cm–1: 3165 (NH), 1678 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.07 (s, 1H, NH), 7.94-7.92 (m, 1H, Ar-H ), 7.77-7.73 (m, 2H, Ar-H), 7.61-7.28 (m, 6H,
Ar-H and N=CH), 4.14 (t, J = 6.4 Hz, 2H, CH2), 3.27 (t, J = 6.8 Hz, 2H, CH2), 2.38-2.18 (m, 2H,
CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.94, 156.16, 150.11, 149.47, 134.99, 134.56,
134.04, 133.29, 132.11, 130.87, 129.23, 128.17, 127.99, 126.54, 126.23, 125.71, 48.81,
34.19, 33.62. HRMS (MALDI) calcd for C19H17Cl2N6OS (M + H)+: 447.0556, found: 447.0555.
This article is protected by copyright. All rights reserved.

4-(3-Chlorophenyl)-1-(3-((5-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-tria
zol-5(4H)-one (14d)
Yield: 78%; m.p. 180–182°C. IR (KBr) cm–1: 3172 (NH), 1681 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.01 (s, 1H, NH), 7.76-7.72 (m, 3H, Ar-H), 7.62-7.28 (m, 6H, Ar-H and N=CH), 4.14 (t,
J = 6.5 Hz, 2H, CH2), 3.26 (t, J = 6.7 Hz, 2H, CH2), 2.39-2.20 (m, 2H, CH2). 13C NMR (CDCl3, 75
MHz, ppm): δ 158.06, 157.69, 150.06, 146.77, 134.51, 134.21, 133.23, 132.77, 129.93,
129.93, 128.21, 128.21, 128.10, 127.28, 127.10, 127.03, 45.20, 37.53, 27.57. HRMS (MALDI)
calcd for C19H17Cl2N6OS (M + H)+: 447.0556, found: 447.0554.
4-(3-Chlorophenyl)-1-(3-((5-(o-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H
)-one (14e)
Yield: 88%; m.p. 101–103°C. IR (KBr) cm–1: 3174 (NH), 1676 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.82 (s, 1H, NH), 7.71-7.18 (m, 9H, Ar-H and N=CH), 4.08 (t, J = 6.1 Hz, 2H, CH2), 3.27
(t, J = 6.6 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.30-2.27 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz,
ppm): δ 158.06, 157.69, 150.06, 146.77, 134.86, 134.57, 133.23, 132.77, 130.05, 129.93,
128.21, 128.10, 127.28, 127.10, 127.03, 125.64, 44.09, 32.35, 26.78, 19.75. HRMS (MALDI)
calcd for C20H20ClN6OS (M + H)+: 427.1102, found: 427.1112.
4-(3-Chlorophenyl)-1-(3-((5-(m-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4
H)-one (14f)
Yield: 75%; m.p. 158–160°C. IR (KBr) cm–1: 3168 (NH), 1683 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.83 (s, 1H, NH), 7.79-7.73 (m, 2H, Ar-H), 7.61-7.21 (m, 7H, Ar-H and N=CH), 4.09 (t,
J = 6.6 Hz, 2H, CH2), 3.25 (t, J = 6.5 Hz, 2H, CH2), 2.38 (s, 3H, CH3), 2.35-2.20 (m, 2H, CH2). 13C
NMR (CDCl3, 75 MHz, ppm): δ 158.56, 157.32, 150.22, 146.75, 134.56, 132.71, 132.47,
129.74, 128.91, 128.85, 128.64, 128.53, 128.43, 128.22, 127.47, 125.41, 43.65, 38.12, 24.63,
19.65. HRMS (MALDI) calcd for C20H20ClN6OS (M + H)+: 427.1102, found: 427.1100.
This article is protected by copyright. All rights reserved.

4-(3-Chlorophenyl)-1-(3-((5-(p-tolyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-triazol-5(4H
)-one (14g)
Yield: 85%; m.p. 168–170°C. IR (KBr) cm–1: 3168 (NH), 1680 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.89 (s, 1H, NH), 7.87-7.22 (m, 7H, Ar-H and N=CH), 4.10 (t, J = 6.5 Hz, 2H, CH2), 3.26
(t, J = 6.4 Hz, 2H, CH2), 2.37 (s, 3H, CH3), 2.30-2.27 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz,
ppm): δ 158.42, 157.73, 150.72, 146.35, 132.70, 132.48, 129.55, 129.55, 129.55, 128.52,
128.52, 127.67, 126.55, 126.41, 126.20, 125.45, 43.62, 38.23, 24.54, 19.78. HRMS (MALDI)
calcd for C20H20ClN6OS (M + H)+: 427.1102, found: 427.1095.
1-(3-((5-(2-Bromophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-(3-chlorophenyl)-1H-1,2,4-tri
azol-5(4H)-one (14h)
Yield: 87%; m.p. 140–142°C. IR (KBr) cm–1: 3176 (NH), 1682 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 7.99 (s, 1H, NH), 7.73-7.25 (m, 9H, Ar-H and N=CH), 4.07 (t, J = 6.0 Hz, 2H, CH2), 3.27
(t, J = 6.6 Hz, 2H, CH2), 2.42-2.22 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 158.56,
157.32, 150.22, 146.75, 134.71, 134.47, 134.34, 134.05, 132.91, 130.85, 129.64, 128.53,
127.43, 126.22, 126.07, 125.41, 43.65, 38.12, 26.83. HRMS (MALDI) calcd for C19H17BrClN6OS
(M + H)+: 491.0051, found: 491.0053.
1-(3-((5-(3-Bromophenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-4-(3-chlorophenyl)-1H-1,2,4-tri
azol-5(4H)-one (14i)
Yield: 72%; m.p. 142–144°C. IR (KBr) cm–1: 3167 (NH), 1680 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.21 (s, 1H, NH), 7.97 (m, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.60-7.27 (m, 7H, Ar-H and
CH), 4.13 (t, J = 5.6 Hz, 2H, CH2), 3.27 (t, J = 6.5 Hz, 2H, CH2), 2.29-2.26 (m, 2H, CH2). 13C NMR
(CDCl3, 75 MHz, ppm): δ 158.06, 157.69, 150.06, 146.77, 134.89, 134.56, 134.23, 134.02,
133.23, 132.77, 129.93, 128.21, 127.90, 127.78, 127.10, 125.03, 45.20, 37.53, 27.57. HRMS
(MALDI) calcd for C19H17BrClN6OS (M + H)+: 491.0051, found: 491.0041.
This article is protected by copyright. All rights reserved.

4-(3-Chlorophenyl)-1-(3-((5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-1H-1,2,4-t
riazol-5(4H)-one (14j)
Yield: 82%; m.p. 158–160°C. IR (KBr) cm–1: 3167 (NH), 1680 (C=O). 1H NMR (CDCl3, 300 MHz,
ppm): δ 8.01-7.18 (m, 10H, Ar-H, CH), 4.10 (t, J = 6.5 Hz, 2H, CH2), 3.26 (t, J = 6.4 Hz, 2H,
CH2), 2.37 (s, 3H, OCH3), 2.30-2.27 (m, 2H, CH2). 13C NMR (CDCl3, 75 MHz, ppm): δ 161.07,
158.56, 157.63, 150.02, 146.36, 135.32, 134.58, 130.43, 130.43, 130.43, 129.76, 128.01,
127.43, 124.91, 114.75, 114.75, 55.70, 44.10, 32.36, 29.18. HRMS (MALDI) calcd for
C20H20ClN6O2S (M + H)+: 443.1051, found: 443.1053.
Evaluation of negative inotropic effects in vitro
The means of measuring left atrium stroke volume was represented previously (24). The
characteristic of CHF are cardiac dilatation, reduced ejection fraction and poor contractility
of cardiac muscle, as well as depression of left ventricular pressure maximum alleosis. As a
result, the macroscopic measurement of the variance atrium stroke volume can be applied
to evaluate the negative inotropic effects of the compounds synthesized. Almost all
reagents were of analytical grade. Metoprolol was used as reference drug as negative
inotropic drug clinically used for the treatment of congestive heart failure. The tested left
atrium was perfused with N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid (HEPES)
buffer solution use a peristaltic pump (1.25 mL/min) at 34 ℃ (25). The constituent of the
buffer was as follows (in mmol): 4.7 KCl, 118 NaCl, 1.2 MgCl2, 2.5 CaCl2, 25 NaHCO3, 10.0
glucose, 10.0 HEPES, (adjusted to pH 7.4 with 1 mol NaOH) and 0.1% bovine serum albumin
(BSA). After the perfused atrium was build, transmural electrical field stimulation using a
luminal electrode was started at 1.5 Hz (voltage 30 V, 0.3-0.5 ms, duration). The changes in
the atrial stroke volume were monitored through reading the lowest level of the water
column in the calibrated atrial cannula during the end diastole. The tested atria were
perfused for 60 minutes to stabilize the atrial stroke volume. The tested atrial beat rate was
fixed at 1.5 Hz, the atrium stroke volume was recorded at 2 minutes intervals and the
stimulus effect of the tested sample was recorded after a circulation of the control group.
This article is protected by copyright. All rights reserved.

Every circulation was 12 minutes (26). The tested compounds were studied using the single
dose technique at a concentration of 3×10-5 mol/L. The tested samples were dissolved in
DMSO, diluted with the HEPES buffer to an appropriate volume. The biological evaluation
data of the tested compounds were presented with increased stroke volume percentage as
shown in Table 1 and 2. In the aim of assess differences, repeated measurements were
compared with an ANOVA test followed by a Bonferroni’s multiple-comparison test.
Statistical significance was defined as P<0.05 and the data is presented as means ±SE.
Evaluation of cytotoxicity on human cancer cells
The cytotoxicity test of the selected compounds with potent inotropic activity was
measured through the colorimetric MTT assay. Human cervical cancer cells (HeLa), liver
cancer cells (Hep3B) and human normal hepatic cells (LO2) suspension in DMEM medium
supplemented with 10% FBS and antimycotic was added in 96-well microplates at 1.8×104
cells/well. A variety of concentrations of the test compounds (200, 100, 50, 25, 12.5, 6.25,
3.125, 1.625 μmol/L) dissolved by distilled 10% DMSO was added to each well. After
incubation for 24h at 37℃ under 5% CO2, 2.5mg/mL of MTT solution was added to each
well. The plate was incubated for another 4h. Then, the incubation medium was aspirated,
added DMSO (100 μL) to solubilize the MTT formazan product. The absorbance at 570 and
630 nm were measured after mixing. The difference between absorbance of 570 and 630
nm was used as an index of the cell viability. The morphology of the cells was observed by
Giemsa stain under Phase contrast microscope. The cells with test compounds were used as
positive control, whereas untreated cells were used as negative controls. The concentration
of the test compound that killed 50% of the cancer cells (IC50) was calculated by GraphPad
Prism 4 software (27). The results were summarized in Table 3. All experiments were
performed in triplicate.
This article is protected by copyright. All rights reserved.

Results and discussion
Chemistry
The synthesis of the compounds 6a-i, 7a-i, 13a-j, and 14a-j is outlined in Scheme 1 and 2
and structures are shown in Figure 1. The key intermediates 5I-III were synthesized by
reacting aniline 1I-III with methyl hydrazinecarboxylate in refluxing ethanol in the presence
of triethyl orthoformate and sodium methoxide (28,29), followed by reaction with
1,3-dibromopropane in the presence of potassium carbonate and potassium iodide in
refluxing acetone. Nucleophilic substitution of 5I or 5II with various monosubstituted
piperazines in refluxing acetone in the presence of KI/K2CO3 afforded 6a-i and 7a-i in
40%-60% yields. Esterification of substituted benzoic acid 8a-j with methanol in the
presence of sulfuric acid afforded 9a-j, which was reacted with hydrazine hydrate to give
compounds 10a-j. Compounds 12a-j was prepared by the reaction of 10a-j with potassium
thiocyanate in refluxing hydrochloric acid and sodium hydroxide (30). Nucleophilic
substitution of 5I or 5III with 12a-j in refluxing acetone in the presence of KI/K2CO3 afforded
13a-j and 14a-j in 70%-90% yields. The structures of the target compounds were
characterized by IR, 1H NMR, 13C NMR, HRMS and mass spectrometry. Data are shown in the
Experimental section.
Biological activity
As shown in Tables 1 and 2, most of the tested compounds showed negative inotropic
effects in isolated rabbit heart preparations. Among them, 28 compounds exhibited more
potent effects compared with the reference drug (metoprolol: −9.74 ± 0.14% at 3×10−5 M).
Compounds 6e and 7h showed significant negative inotropic activity with stroke volume
changes of −41.56 ± 0.55% and −48.22 ± 0.36%, respectively, which are 4-fold more potent
than that of the standard drug. Some preliminary conclusions on structure–activity
relationship can be drawn from the results of bioactivities. The presence of different
substituents on the phenyl ring of the benzyl group at the 4-position of the piperazine ring
This article is protected by copyright. All rights reserved.

was found to exert a different influence on the observed negative inotropic activity, but no
clear pattern could be found for the SAR. As for the halo substituted derivatives (6b, 6c, 6d,
6f, 6g, 6i), an activity order of o > m > 2,4-Cl2 > p was observed for the chlorinated
compounds, m > p for the fluorinated compounds, the unsubstituted 6a exhibited slightly
weaker activity with -4.55 ± 0.24% increased stroke volume and para-methoxy substituted
derivative 6h exhibited more potent activity than the reference drug metoprolol (-27.65 ±
0.61%). For the series of 7, compound 7h with para-methoxy substituted showed the most
potent activity with -48.22 ± 0.36% changed stroke volume. For the halo substituted
derivatives (7b, 7c, 7d, 7f, 7g, 7i), an activity order of p > o > 2,4-Cl2 > m was observed for
the chlorinated compounds, p > m for the fluorinated compounds. The cyano substituted 7e
exhibited weak activity (-4.76 ± 0.07%). Unsubstituted derivatives 7a (-15.39 ± 0.39%)
exhibited less potent negative inotropic activity than the reference drug. For the compounds
in series 6 and 7, the nitro group at the 4-position of the phenyl ring on the triazole in series
7 significantly affected the activity compared with series 6, resulting in increased activities
for most compounds except for the m-Cl and o-CN substituted derivatives 7c, and 7e.
For the compounds in series 13 and 14, there appeared to be no significant differences in
activity between the two series of compounds. The position of the methyl-substituent on
the benzene ring was found to exert a significant influence on the activity, and an activity
order of o > m > p was found for both series. While methoxy substituted derivative 13j
exhibited slightly weaker activity with -1.83 ± 0.20% increased stroke volume and 14j
showed no activity. For halogen substituents (Cl, Br) on the phenyl ring, an activity order of
m > p > o was found for chlorinated compounds in series of 13, whereas an activity order of
m > o > p was observed for 14 series. For brominated compounds, the activity order of o > m
was obtained in both 13 and 14 series. The aforementioned results also suggest that there
are no remarkable differences for the activity between the triazole ring and the piperazine
moiety, and no clear regularity could be found for the effects of the substituents R1’ position
and physicochemical properties to the activity, indicating that the electronic effect of the
substituent on the benzene ring is not critical.
This article is protected by copyright. All rights reserved.

In addition, we investigated the dynamics of the compounds in perfused beating rabbit atria
and found that 18 compounds possessed inotropic effects (6b, 6e, 6h, 7b, 7f, 7g, 7h, 13c,
13d, 13e, 13f, 14a, 14b, 14c, 14e, 14h, 14i, and 14f) and presented desirable biological
dynamic profiles compared with that of the standard drug nifedipine (Figure 2). It was found
that the stroke volumes of the test compounds (6b, 6e, 6h, 7b, 7f, 7g, 7h, 13c, 13d, 13e, 13f,
14a, 14b, 14c and 14e) were gradually decreased as the time progressed and kept
unchanged after falling to a certain extent (Figure 2A, 2B, 2C, 2D and 2E), but less desirable
dynamic property was observed for 14h, 14i, and 14f whose stroke volume was kept
changed (Figure 2F). On the other hand, the dose–activity relationships of selected
compounds 6e and 7h were also tested at concentrations of 1×10−5 mol/L, 3×10−5 mol/L,
and 1×10−4 mol/L, respectively. Both compounds showed a maximal effect at 3×10−5 mol/L,
whereas less activity was observed at the lower 1×10−6 mol/L and higher 1×10−4 mol/L doses
(Figure 3). Fortunately, none of the test compounds showed any significant effect on heart
rates.
Investigation of the mechanism of action of compound 7h
A preliminary investigation of the mechanism of action of compound 7h on the regulation of
atrial dynamics was performed (Figure 4). It is well known that decrease of the influx of Ca2+
ions or increase of the outflow of K+ ion generates a negative inotropic effect on the
myocardium. Therefore, another series of experiments were performed with ATP-sensitive
K+ channel and L-type Ca2+ channel blockers glibenclamide and nifedipine in the isolated
perfused beating rabbit atria. As shown in Figure 4A, there was a slight decrease of the
trend line, but was not obvious after treated with K+ ion channel blocker glibenclamide.
However, the trend line decreased rapidly with the addition of 7h. It is obvious that the
negative inotropic effect of 7h was not directly related to the blocking of K+ ion channel. On
the contrary, the myocardial cell stroke was significantly decreased after treated with L-type
Ca2+ channel blocker nifedipine and no further decrease was found with the addition of 7h
(Figure 4C). As shown in Figure 4D, the similar trends of L-type Ca2+ channel blocker
This article is protected by copyright. All rights reserved.

nifedipine (Figure 4B) and nifedipine + 7h (Figure 4C) indicated that the negative inotropic
effects of 7h on the rabbit atrium might be blocked by the L-type Ca2+ channel blocker
nifedipine. As shown in Figure 4, glibenclamide failed to modulate the negative inotropic
effect of compound 7h on atrial dynamics. However, nifedipine completely abolished the
negative regulatory effect of compound 7h in the beating rabbit atria. The results
demonstrate that the negative inotropic effect of these synthesized compounds is likely
associated with the blockade of the L-type calcium channel.
Cytotoxicity
Several compounds with significant inotropic activities were evaluated for their toxicity
against human cervical cancer cells HeLa, liver cancer cells Hep3B and human normal
hepatic cells LO2 with the half inhibitory concentration (IC50) value of the tested compounds
measured by the MTT assay. IC50 values are the average of three independent experiments
running in triplicate. As shown in Table 3, these compounds were tested at multiple doses
and did not show any significant cytotoxic activity (IC50 ≥ 100 μmol/L) against two human
cancer cell lines and one human normal cell lines.
Conclusion
Thirty-eight 4-phenyl-1H-1,2,4-triazol-5(4H)-one derivatives containing triazole or piperazine
moieties were synthesized and evaluated for negative inotropic activity for the first time.
Most of the synthesized compounds showed potential negative inotropic activity. In
particular, compound 7h exhibited promising cardiovascular properties and potent negative
inotropic activity, being 4-fold more potent compared with the standard drug metoprolol.
The results of the cytotoxic tests indicated that these series of compounds did not exhibit
any remarkable cytotoxic activity. Furthermore, a preliminary study of the mechanism of
action of 7h on the atrial dynamics was also conducted. The result demonstrates that the
negative inotropic property of these compounds is likely associated with the blockade of the
This article is protected by copyright. All rights reserved.

L-type calcium channels. The work might provide an experimental basis for the development
of new candidates with potent negative inotropic activities for the clinical treatment of
chronic congestive heart failure.
Acknowledgement
The work was supported by the National Science Foundation of China (No. 81160381).
References
1. Fromm RE.Jr., Varon J., Gibbs L.R. (1995) Congestive heart failure and pulmonary edema
for the emergency physician. J Emerg Med; 13:71–87.
2. Neubauer S. (2007) The failing heart–an engine out of fuel. N Engl J Med; 356:
1140–1151.
3. Harada K., Supriatno, Yoshida H., and Sato M. (2005) Vesnarinone inhibits
angiogenesis and tumorigenicity of human oral squamous cell carcinoma cells by
suppressing the expression of vascular endothelial growth factor and interleukin-8.
Int J Oncol; 27:1489–1497.
4. Smith J.R., Gheorghiade M., Goldstein S. (1993) The current role of digoxin in the
treatment of heart failure. Coron Artery Dis; 4:16–26.
5. Smith T.W. (1988) Digitalis. Mechanisms of action and clinical use. N Engl J Med;
318:358–365.
6. Becker D.E. (2007) Cardiovascular drugs: implications for dental practice part
1-cardiotonics, diuretics, and vasodilators. Anesth Prog; 54:178–187.
This article is protected by copyright. All rights reserved.

7. Hallberg P., Lindbäck J., Lindahl B., Stenestrand U., Melhus H., RIKS-HIA group. (2007)
Digoxin and mortality in atrial fibrillation: a prospective cohort study. Eur J Clin
Pharmacol; 63:959–971.
8. 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. (1991) Effect of oral milrinone on
mortality in severe chronic heart failure. The Promise Study Research Group. N Engl J
Med; 325:1468–1475.
9. 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. (1998) A dose-dependent increase in
mortality with vesnarinone among patients with severe heart failure. Vesnarinone
Trial Investigators. N Engl J Med; 339:1810–1816.
10. Feldman M.D., Pak P.H., Wu C.C., Haber H.L., Heesch C.M., Bergin J.D., Powers E.R.,
Cowart T.D., Johnson W., Feldman A.M., Kass D.A. (1996) Acute cardiovascular effects
of OPC-18790 in patients with congestive heart failure. Time- and dose-dependence
analysis based on pressure-volume relations. Circulation; 93:474–483.
11. Gibbs C.R., Davies M.K., Lip G.Y. (2000) Management: digoxin and other inotropes,
beta blockers, and antiarrhythmic and antithrombotic treatment. B M J;
320:495–498.
12. Waagstein F., Bristow M.R., Swedberg K., Camerini F., Fowler M.B., Silver M.A., Gilbert
E.M., Johnson M.R., Goss F.G., Hjalmarson A. (1993) Beneficial effects of metoprolol
in idiopathic dilated cardiomyopathy. Metoprolol in Dilated Cardiomyopathy (MDC)
Trial Study Group. Lancet; 342:1441–1446.
13. Kukin M.L., Kalman J., Charney R.H., Levy D.K., Buchholz-Varley C., Ocampo O.N., Eng
C. (1999) Prospective, randomized comparison of effect of long-term treatment with
metoprolol or carvedilol on symptoms, exercise, ejection fraction, and oxidative
stress in heart failure. Circulation; 99:2645–2651.
14. Hong L., Gong G.H., Yu Li., Song M.X., Cui Xun., Quan Z.S. (2013) Synthesis and
negative inotropic effects evaluation of
This article is protected by copyright. All rights reserved.

7-substituted-4,5-dihydro-[1,2,4]oxadiazolo[4,3-a]quinolin-1-ones. Arch Pharm Res;
36:1338–1344.
15. Wu Y., Ma L.X., Niu T.W., Cui X., Piao H.R. (2012) Synthesis and positive inotropic
evaluation of
N-(1-oxo-1,2,4,5-tetrahydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides bearing
piperazine and 1,4-diazepane moieties. Bioorg Med Chem Lett; 22: 4229–4232.
16. Li T.T., Zhang L., Quan Z.S., Zhu H.B., Piao H.R. (2009) Synthesis and positive inotropic
evaluation of
1-(benzylamino)-3-(4,5-di-hydro[1,2,4]trizaolo[4,3-a]quinolin-7-yloxy)propan-2-ol
derivatives. Chinese Chem. Lett; 20:175–177.
17. Jiang S.M., Ye B.J., Liu X.K., Zhang T.Y., Cui X., Piao H.R. (2010) Synthesis and in-vitro
inotropic evaluation of 2-(4-substitutedbenzyl-1,4-diazepan-1-yl)-
N-(4,5-di-hydro-1-phenyl-[1,2,4]triazolo[4,3-a]quinolin-7-yl)acetamides. Arch Pharm;
10:700–705.
18. Ye B.J., Liu X.K., Jiang S.M., Cui X., Piao H.R. (2011) Synthesis of
2-(4-Substitutedbenzyl-[1,4]Diazepan-1-yl)-N-(1-Methyl-4,5-Dihydro-[1,2,4]Triazolo[4,
3-A]Quinolin-7-Yl)Acetamides as Inotropic Agents. Chem Biol Drug Des; 77:98–103.
19. Sahu J.K., Ganguly S., Kaushik A. (2013) Triazoles: a valuable insight into recent
developments and biological activities. Chin J Nat Med;11:456–465.
20. Calderone V., Giorgi I., Livi O., Martinotti E., Martelli A., Nardi A. (2005) 1,4-and
2,4-substituted-1,2,3-triazoles as potential potassium channel activators. VII Farmaco;
60:367–375.
21. Koparir M., Orek C., Parlak A.E., Söylemez A., Koparir P., Karatepe M., Dastan S.D.
(2013) Synthesis and biological activities of some novel aminomethyl derivatives of
4-substituted-5-(2-thienyl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones. Eur J Med Chem;
63:340–346.
22. Ma L.X., Cui B.R., Wu Y., Liu J.C., Cui X., Liu L.P., Piao H.R. (2014) Synthesis and positive
inotropic evaluation of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo
This article is protected by copyright. All rights reserved.

[5,1-a]phthalazine derivatives bearing substituted piperazine moieties. Bioorg Med
Chem Lett; 24:1737–1741.
23. Zhang C.B., Cui X., Hong L., Quan Z.S., Piao H.R. (2008) Synthesis and positive
inotropic activity of
N-(4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-7-yl)-2-(piperazin-1-yl)acetamide
derivatives. Bioorg Med Chem Lett; 18:4606–4609.
24. Löscher W. (1979) 3-Mercaptopropionic acid: convulsant properties, effects on
enzymes of the gamma-aminobutyrate system in mouse brain and antagonism by
certain anticonvulsant drugs, aminooxyacetic acid and gabaculine. Biochem
Pharmacol; 28:1397–1407.
25. Li J.Y., Cui X., Liu X.K., Hong L., Quan Z.S., Piao H.R. (2008) Synthesis and positive
inotropic evaluation of 2-(4-(4-substituted
benzyloxy)-3-methoxybenzyl)-1,4-diazepan-1-yl)-N-(4,5-dihydro-1-methyl[1,2,4]triazo
lo[4,3-a]quinolin-7-yl)-acetamides. Arch Pharm; 341:794–799.
26. Wu Y., Sun L.P., Ma L.X., Che J., Song M.X., Cui X., Piao H.R. (2013) Synthesis and
biological evaluation of [1,2,4]triazolo[3,4-a]phthalazine and
tetrazolo[5,1-a]phthalazine derivatives bearing substituted benzylpiperazine moieties
as positive inotropic agents. Chem Biol Drug Des; 8:591–599.
27. Li J.R., Li D.D., Wang R.R., Sun J., Dong J.J., Du Q.R., Fang F., Zhang W.M., Zhu H.L.
(2014) Design and synthesis of thiazole derivatives as potent FabH inhibitors with
antibacterial activity. Eur J Med Chem; 75:438–447.
28. Jiang Y., Cao Y., Zhang J., Zou Y., Chai X., Hu H., Zhao Q., Wu Q., Zhang D., Jiang Y., Sun
Q. (2011) Design, synthesis and antifungal evaluation of
1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(
4H)-one, Eur J Med Chem; 46:3135-3141.
29. Shao N., Wang C., Huang X.H., Xiao D., Palani A., Aslanian R., Shih N.Y. (2006) Efficient
one-pot formation of 4-N-substituted 2,4-dihydro-3H-1,2,4-triazolin-3-ones from
This article is protected by copyright. All rights reserved.

primary amines using N’-(ethoxymethylene)hydrazinecarboxylic acid methyl ester,
Tetrahedron Lett; 47:6743-6746.
30. Agrawal R., Pancholi S.S. (2011) Synthesis, characterization and evaluation of
antimicrobial activity of a series of 1,2,4-triazoles. Der Pharma Chemica; 3:32–40.
Figure 1. Structures of the previously reported compound A, B and target compounds.
This article is protected by copyright. All rights reserved.

Figure 2. Effects of the test compounds 6b, 6e, 6h, 7b, 7f, 7g, 7h, 13c, 13d, 13e, 13f, 14a, 14b, 14c,
14e, 14h, 14i, 14f and metoprolol on stroke volume in beating rabbit atria (1.5 Hz). Values are means
± SE. ***P < 0.001 vs. control.
Control
A
Control
B
6b (30 μmol/L)
6e (30 μmol/L)
6h (30 μmol/L)
7b (30 μmol/L)
7h (30 μmol/L)
7f (30 μmol/L)
Metoprolol (30 μmol/L)
40
0
40
0
Metoprolol *(3*0*μmol/L)
***
-40
-80
-40
-80
0
10
20
30
0
10
20
30
Fraction number
Fraction number
Control
Control
D
C
13e (30 μmol/L)
13f (30 μmol/L)
14a (30 μmol/L)
Metoprolol (30 μmol/L)
7g (30 μmol/L)
40
40
0
13c (30 μmol/L)
13d (30 μmol/L)
Metoprolol (30 μmol/L)
***
***
0
-40
-80
-40
-80
0
10
20
30
0
10
20
30
Fraction number
Fraction number
Control
Control
F
E
14h (30 μmol/L)
14i (30 μmol/L)
14f (30 μmol/L)
14b (30 μmol/L)
14c (30 μmol/L)
14e (30 μmol/L)
Metoprolol (*30**μmol/L)
30
0
40
0
Metoprolol *(3*0*μmol/L)
-30
-60
-40
-80
0
10
20
30
0
10
20
30
Fraction number
Fraction number
This article is protected by copyright. All rights reserved.