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

Article abstract of DOI:10.1111/cbdd.12101

Two series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted benzylpiperazine moieties have been synthesized and evaluated for their positive inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. The majority of the derivatives exhibited better in vitro activity than the existing drug, milrinone, and 6-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)tetrazolo[5,1-a]phthalazine. 8 m in particular was identified as the most potent with an increased stroke volume of 12.02 ± 0.20% (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10^-5 m. The chronotropic effects of the compounds that exhibited good potency were also evaluated.

Full text of DOI:10.1111/cbdd.12101

Chem Biol Drug Des 2013
Research Article
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
Yan Wu1, Liang-Peng Sun1, Long-Xu Ma1,
treatment of CHF (2). Although the phosphodiesterase-
inhibiting agent milrinone, which possesses both vasodila-
tor and inotropic properties, was approved for the treat-
ment of CHF more than one decade ago, the significant
Jian Che1, Ming-Xia Song1, Xun Cui2,* and
Hu-Ri Piao1,*
1Key Laboratory of Natural Resources of Changbai
ventricular arrhythmias and tachycardia associated with
Mountain & Functional Molecules, Ministry of Education,
Yanbian University College of Pharmacy, Yanji, 133000,
China
the elevated cAMP levels resulting from its use have
limited its clinical application (3). Similar issues have been
encountered with the recently developed vesnarinone
(4,5) and toborinone (6). Therefore, the identification of
novel positive inotropic agents that can not only improve
the quality of life but also reduce the mortality of CHF
patients still represents an important challenge for medici-
nal chemists.
2Yanbian University College of Medicine, Yanji, 133000,
China
*Corresponding authors: Hu-Ri Piao,
piaohuri@yahoo.com.cn; Xun Cui, cuixun@ybu.edu.cn
Two series of [1,2,4]triazolo[3,4-a]phthalazine and tet-
razolo[5,1-a]phthalazine derivatives bearing substituted
benzylpiperazine moieties have been synthesized and
evaluated for their positive inotropic activity by mea-
suring left atrium stroke volume on isolated rabbit
heart preparations. The majority of the derivatives
exhibited better in vitro activity than the existing drug,
milrinone, and 6-((4-(4-methoxyphenyl)piperazin-1-yl)
methyl)tetrazolo[5,1-a]phthalazine. 8 m in particular
was identified as the most potent with an increased
stroke volume of 12.02 0.20% (milrinone: 2.46
0.07%) at a concentration of 3 9 10–5 M. The chrono-
tropic effects of the compounds that exhibited good
potency were also evaluated.
In our previous work, we reported that one of the 3,4-dihy-
dro-2(1H)-quinolinone derivatives, PHR9612, exhibited
moderate inotropic activity (7). The introduction of triazole
ring at the first and second position of the 3,4-dihydro-2
(1H)-quinolinone, as in PHR0007, resulted in a remarkable
increase in the biological activity (Figure 1) (8), as indicated
by the phosphodiesterase III inhibition (9). In a summary of
our previous work associated with the effects of modifica-
tions to PHR0007 (10,11), we hypothesized that the triaz-
ole and piperazine rings were essential to the observed
higher levels of affinity for the receptor and consequently
enhanced the positive inotropic activity. In the current
report, we describe the further modifications that we have
made to PHR0007. These modifications were focused on
preserving the three-membered ring and replacing the
Key words: [1,2,4]triazolo[3,4-a]phthalazine, positive inotropic
activity, stroke volume, tetrazolo[5,1-a]phthalazine
4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline moiety with
a
Received 7 September 2012 and accepted for publication 10
November 2012
[1,2,4]triazolo[3,4-a]phthalazine or tetrazolo[5,1-a]phthal-
azine ring. Furthermore, we wanted to directly introduce
the piperazine ring at the 4-position of the phthalazine ring
without an acetamide linkage while simultaneously investi-
gating variations to the phenyl ring substituents in the ben-
zyl group (Figure 2). Thus, two novel series of [1,2,4]
triazole and tetrazole-fused phthalazine derivatives bearing
substituted benzylpiperazine moiety were designed and
synthesized. A total of 26 compounds were synthesized
with all of the target compounds being characterized by
IR, 1H NMR, MS, and elemental analysis. The inotropic
activities of the compounds were also evaluated by mea-
suring changes in the left atrium stroke volume in isolated
rabbit heart preparations.
The therapeutic treatment of cardiac failure (CHF) cur-
rently includes, among other measures, the use of car-
diac glycosides such as digoxin and digitalis compounds.
Until recently, the narrow safety margins associated with
the use of digitalis compounds have been highlighted as
serious problems because of the high frequency and
severity of digitalis intoxication (1). The discovery of
amrinone led to the synthesis of a number of promising
non-sympathomimetic and non-glycoside agents for the
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12101
1

Wu et al.
H
N
Synthesis of 1,4-dichlorophthalazine (3)
N
N
Compound 2 (1.6 g, 1 mmol) was dissolved in phospho-
rus oxychloride (30 mL) and heated with stirring under
reflux for 4 h. The solvent was then removed in vacuo,
and the resulting residue dissolved in dichloromethane
(30 mL) with rapid stirring. Water (30 mL) was then added
to the resulting mixture, and the organic layer was
separated. The aqueous layer was then extracted with
dichloromethane, and the combined organic layers were
dried over MgSO4, filtered, and evaporated. The resulting
residue was purified by silica gel column chromatography
with ethyl acetate and petroleum ether (1:8) to afford 3 in
85% yield.
CN
O
N
O
N
H
O
N
N
H
H3C
PHR9612
H
Milrinone
O
N
N
N
O
N
H3C
N
H3C
PHR0007
Figure 1: Milrinone and the previously reported compounds.
N
R
N
R
Synthesis of 6-chloro-[1,2,4]triazolo[3,4-a]
phthalazine (4)
N
N
N
N
N
N
A solution of 3 and formylhydrazine (1.2 eq) in n-butyl
alcohol was heated at reflux for 2–3 h. The resulting
mixture was then evaporated to dryness under reduced
pressure, and the residue was purified by silica gel column
chromatography with dichloromethane and methanol
(40:1) to afford 4 in 80% yield.
N
N
N
5a-m
8a-m
N
N
Figure 2: Designed target compounds.
Experimental Section
General procedure for the synthesis of compounds
5a–m
Material and methods
Melting points were determined in open capillary tubes
and were uncorrected. The chemical reactions were
monitored by thin layer chromatography (TLC) on silica
gel precoated F254 Merck plates, and the developed
plates were visualized by UV light (254 nm). Column
chromatography was performed with Merck 200 mesh
silica gel. IR spectra were recorded (in KBr) on a FT-
IR1730. 1H NMR spectra were measured on a Bruker
AV-300 spectrometer (Bruker), using trimethylsilane (TMS)
as an internal standard. Mass spectra were measured on
an HP1100LC (Agilent Technologies, Santa Clara, CA,
USA). Elemental analyses for C, H, and N were recorded
on a 204Q CHN Rapid Analyzer (Perkin-Elmer, Waltham,
MA, USA) and found to be within Æ0.4% of the theoret-
ical values. The majority of the chemicals used in the
current study were purchased from the Sigma-Aldrich (St.
Louis, MO, USA) and Fluka (Milwaukee, WI, USA) chemi-
cal companies.
A mixture of 4 (1.0 mmol), monosubstituted piperazine
(2.0 mmol), and anhydrous potassium carbonate in ace-
tone was heated at reflux with stirring for 48 h. The solvent
was then evaporated under reduced pressure, and the
resulting residue dissolved in dichloromethane. The dichlo-
romethane solution was then washed sequentially with
water and brine, dried over MgSO4, and distilled to dry-
ness under reduced pressure. The resulting residue was
purified by silica gel column chromatography with dichlo-
romethane and methanol (30:1). The yield, melting point,
and spectral data of each compound are given below.
6-(4-(2-fluorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5a)
Yield: 81%; m.p. 156–158 °C. IR (KBr) per cm: 1612,
1508, 1454 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.81–
2.82 (m, 4H, (CH2)2), 3.43–3.46 (m, 4H, (CH2)2), 3.76 (s,
2H, CH2), 7.17–8.62 (m, 8H, Ar-H), 8.87 (s, 1H, N=CH). MS
m/z: 363 (M + 1). Anal. Calcd. for C20H19FN6: C, 66.28; H,
5.28; N, 23.19. Found: C, 66.32; H, 5.31; N, 23.22.
Synthesis of 2,3-dihydrophthalazine-1,4-dione (2)
Isoindoline-1,3-dione (1) (2.98 g, 2 mmol) was dissolved in
ethanol (30 mL) over a period of 30 min and hydrazine
hydrate (2 mmol) was added to the resulting solution in a
drop-wise manner with cooling from an ice-bath. The
reaction mixture was stirred at 70 °C for 3 h and then
cooled to 0 °C in an ice-bath, resulting in the formation of
a white precipitate. The reaction mixture was then filtered,
and the filter cake collected and dried in a vacuum at
ambient temperature to give the desired compound 2 in
90% yield.
6-(4-(3-fluorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5b)
Yield: 86%; m.p. 158–160 °C. IR (KBr) per cm: 1610,
1508, 1450 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.75–
2.76 (m, 4H, (CH2)2), 3.43–3.45 (m, 4H, (CH2)2), 3.63 (s,
2H, CH2), 7.27–8.63 (m, 8H, Ar-H), 8.86 (s, 1H, N=CH). MS
m/z: 363 (M + 1). Anal. Calcd. for C20H19FN6: C, 66.28; H,
5.28; N, 23.19. Found: C, 66.30; H, 5.32; N, 23.23.
2
Chem Biol Drug Des 2013

Synthesis and Biological Evaluation of [1,2,4]Triazolo[3,4-a]phthalazine and Tetrazolo[5,1-a]phthalazine
6-(4-(4-fluorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5c)
–2.63 (m, 4H, (CH2)2), 3.33–3.36 (m, 4H, (CH2)2), 3.52 (s,
2H, CH2), 7.16–8.51 (m, 8H, Ar-H), 8.76 (s, 1H, N=CH).
MS m/z: 423 (M + 1). Anal. Calcd. for C20H19BrN6: C,
56.75; H, 4.52; N, 19.85. Found: C, 56.78; H, 4.59; N,
19.86.
Yield: 88%; m.p. 166–168 °C. IR (KBr) per cm: 1616,
1508, 1450 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.64–
2.66 (m, 4H, (CH2)2), 3.33–3.36 (m, 4H, (CH2)2), 3.63 (s,
2H, CH2), 6.92–8.52 (m, 8H, Ar-H), 8.76 (s, 1H, N=CH). MS
m/z: 363 (M + 1). Anal. Calcd. for C20H19FN6: C, 66.28; H,
5.28; N, 23.19. Found: C, 66.33; H, 5.31; N, 23.22.
6-(4-(3-bromobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5i)
Yield: 88%; m.p. 179–181 °C. IR (KBr) per cm: 1585,
1
6-(4-(2-chlorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5d)
1510, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.63
–2.65 (m, 4H, (CH2)2), 3.34–3.41 (m, 4H, (CH2)2), 3.52 (s,
2H, CH2), 7.13–8.52 (m, 8H, Ar-H), 8.76 (s, 1H, N=CH).
MS m/z: 423 (M + 1). Anal. Calcd. for C20H19BrN6: C,
56.75; H, 4.52; N, 19.85. Found: C, 56.80; H, 4.60; N,
19.88.
Yield: 80%; m.p. 152–153 °C. IR (KBr) per cm: 1583,
1
1508, 1452 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.83
–2.86 (m, 4H, (CH2)2), 3.44–3.47 (m, 4H, (CH2)2), 3.80 (s,
2H, CH2), 7.22–8.64 (m, 8H, Ar-H), 8.88 (s, 1H, N=CH).
MS m/z: 379 (M + 1). Anal. Calcd. for C20H19ClN6: C,
63.40; H, 5.05; N, 22.18. Found: C, 63.48; H, 5.09; N,
26.22.
6-(4-(4-bromobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5j)
Yield: 79%; m.p. 182–183 °C. IR (KBr) per cm: 1585,
1
6-(4-(3-chlorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5e)
1512, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.64
–2.65 (m, 4H, (CH2)2), 3.33–3.36 (m, 4H, (CH2)2), 3.51 (s,
2H, CH2), 7.16–8.53 (m, 8H, Ar-H), 8.77 (s, 1H, N=CH).
MS m/z: 423 (M + 1). Anal. Calcd. for C20H19BrN6: C,
56.75; H, 4.52; N, 19.85. Found: C, 56.81; H, 4.62; N,
19.83.
Yield: 81%; m.p. 158–160 °C. IR (KBr) per cm: 1582,
1
1508, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.64
–2.65 (m, 4H, (CH2)2), 3.33–3.36 (m, 4H, (CH2)2), 3.52 (s,
2H, CH2), 7.16–8.49 (m, 8H, Ar-H), 8.75 (s, 1H, N=CH).
MS m/z: 379 (M + 1). Anal. Calcd. for C20H19ClN6: C,
63.40; H, 5.05; N, 22.18. Found: C, 63.48; H, 5.09; N,
26.22.
6-(4-benzylpiperazin-1-yl)-[1,2,4]triazolo[3,4-a]
phthalazine (5k)
Yield: 82%; m.p. 130–132 °C. IR (KBr) per cm: 1585,
1
6-(4-(4-chlorobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5f)
1508, 1450 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.60
–2.67 (m, 4H, (CH2)2), 3.34–3.39 (m, 4H, (CH2)2), 3.57 (s,
2H, CH2), 7.16–8.53 (m, 9H, Ar-H), 8.77 (s, 1H, N=CH).
MS m/z: 345 (M + 1). Anal. Calcd. for C20H20N6: C,
69.75; H, 5.85; N, 24.40. Found: C, 69.78; H, 5.89; N,
24.46.
Yield: 79%; m.p. 186–188 °C. IR (KBr) per cm: 1585,
1
1508, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.63
–2.64 (m, 4H, (CH2)2), 3.32–3.33 (m, 4H, (CH2)2), 3.52 (s,
2H, CH2), 7.19–8.53 (m, 8H, Ar-H), 8.77 (s, 1H, N=CH).
MS m/z: 379 (M + 1). Anal. Calcd. for C20H19ClN6: C,
63.40; H, 5.05; N, 22.18. Found: C, 63.46; H, 5.10; N,
26.21.
6-(4-(4-methylbenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5l)
Yield: 86%; m.p. 178–180 °C. IR (KBr) per cm: 1585,
1
6-(4-(2,4-dichlorobenzyl)piperazin-1-yl)-[1,2,4]
triazolo[3,4-a]phthalazine (5g)
1512, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.26
(s, 3H, CH3), 2.64–2.66 (m, 4H, (CH2)2), 3.31–3.33 (m,
4H, (CH2)2), 3.52 (s, 2H, CH2), 7.06–8.50 (m, 8H, Ar-H),
8.75 (s, 1H, N=CH). MS m/z: 359 (M + 1). Anal. Calcd. for
C21H22N6: C, 70.37; H, 6.19; N, 23.45. Found: C, 70.38;
H, 6.22; N, 23.46.
Yield: 83%; m.p. 212–213 °C. IR (KBr) per cm: 1586,
1
1508, 1456 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.63
–2.64 (m, 4H, (CH2)2), 3.26–3.28 (m, 4H, (CH2)2), 3.56 (s,
2H, CH2), 7.08–8.48 (m, 7H, Ar-H), 8.69 (s, 1H, N=CH).
MS m/z: 413 (M + 1). Anal. Calcd. for C20H18Cl2N6: C,
58.12; H, 4.39; N, 20.33. Found: C, 58.16; H, 5.43; N,
20.36.
6-(4-(4-methoxybenzyl)piperazin-1-yl)-[1,2,4]
triazolo[3,4-a]phthalazine (5m)
Yield: 83%; m.p. 180–182 °C. IR (KBr) per cm: 1612,
1
6-(4-(2-bromobenzyl)piperazin-1-yl)-[1,2,4]triazolo
[3,4-a]phthalazine (5h)
1512, 1454 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.65
–2.66 (m, 4H, (CH2)2), 3.34–3.52 (m, 4H, (CH2)2), 3.72 (s,
2H, CH2), 3.75 (s, 3H, CH3), 7.00–8.55 (m, 8H, Ar-H),
8.78 (s, 1H, N=CH). MS m/z: 375 (M + 1). Anal. Calcd. for
Yield: 87%; m.p. 178–180 °C. IR (KBr) per cm: 1585,
1
1508, 1450 (C=N). H-NMR (CDCl3, 300 MHz, ppm): 2.62
Chem Biol Drug Des 2013
3

Wu et al.
C21H22N6O: C, 67.36; H, 5.92; N, 22.44. Found: C,
67.39; H, 5.99; N, 22.46.
6-(4-(4-fluorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8c)
Yield: 93%; m.p. 136–138 °C. IR (KBr) per cm: 1604,
1508 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.63–2.64
(m, 4H, (CH2)2), 3.33–3.35 (m, 4H, (CH2)2), 3.61 (s, 2H,
CH2), 6.96–8.46 (m, 8H, Ar-H). MS m/z: 364 (M + 1). Anal.
Calcd. for C19H18FN7: C, 62.80; H, 4.99; N, 26.98. Found:
C, 62.85; H, 5.03; N, 26.96.
Synthesis of 1-chloro-4-hydrazinylphthalazine (6)
To a solution of hydrazine hydrate (1.2 mmol) in THF
(10 mL) was added dropwise a solution of compound 3
(1 mmol) in THF (25 mL) at room temperature. The mixture
was stirred at 60 °C for 1 h. Then half of the solvent was
removed under reduced pressure, and the solution was
poured into petroleum ether. The precipitate was filtered,
washed with petroleum ether, and kept below 0 °C. The
compounds obtained were pure enough for the following
step.
6-(4-(2-chlorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8d)
Yield: 90%; m.p. 158–160 °C. IR (KBr) per cm: 1589,
1502 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.73–2.75
(m, 4H, (CH2)2), 3.49–3.50 (m, 4H, (CH2)2), 3.66 (s, 2H,
CH2), 7.07–8.52 (m, 8H, Ar-H). MS m/z: 380 (M + 1). Anal.
Calcd. for C19H18ClN7: C, 60.08; H, 4.78; N, 25.81.
Found: C, 60.10; H, 4.82; N, 26.79.
Synthesis of 6-chlorotetrazolo[5,1-a]phthalazine (7)
To a solution of compound 6 (0.8 mmol) in 30% H2SO4,
10% NaNO2 solution was added dropwise below 5 °C.
The mixture was stirred and cooled below 5 °C for 1 h.
The precipitate was filtered, washed with petroleum ether
to afford compound 7 in 85% yield.
6-(4-(3-chlorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8e)
Yield: 91%; m.p. 152–153 °C. IR (KBr) per cm: 1589,
1500 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.78–2.81
(m, 4H, (CH2)2), 3.60–3.62 (m, 4H, (CH2)2), 3.63 (s, 2H,
CH2), 7.20–8.65 (m, 8H, Ar-H). MS m/z: 380 (M + 1). Anal.
Calcd. for C19H18ClN7: C, 60.08; H, 4.78; N, 25.81.
Found: C, 60.13; H, 4.85; N, 26.77.
General procedure for the synthesis of compounds
8a–m
A mixture of 7 (1.0 mmol), monosubstituted piperazine
(2.0 mmol), and anhydrous potassium carbonate in ace-
tone was heated at reflux with stirring for 12 h. The solvent
was then evaporated under reduced pressure, and the
resulting residue dissolved in dichloromethane. The dichlo-
romethane solution was then washed sequentially with
water and brine, dried over MgSO4, and distilled to dry-
ness under reduced pressure. The resulting residue was
purified by silica gel column chromatography with dichlo-
romethane and methanol (60:1). The yield, melting point,
and spectral data of each compound are given below.
6-(4-(4-chlorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8f)
Yield: 94%; m.p. 159–161 °C. IR (KBr) per cm: 1586,
1500 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.66–2.67
(m, 4H, (CH2)2), 3.49–3.50 (m, 4H, (CH2)2), 3.52 (s, 2H,
CH2), 7.14–8.54 (m, 8H, Ar-H). MS m/z: 380 (M + 1). Anal.
Calcd. for C19H18ClN7: C, 60.08; H, 4.78; N, 25.81.
Found: C, 60.15; H, 4.86; N, 26.76.
6-(4-(2-fluorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8a)
6-(4-(2,4-dichlorobenzyl)piperazin-1-yl)tetrazolo
[5,1-a]phthalazine (8g)
Yield: 92%; m.p. 150–152 °C. IR (KBr) per cm: 1600,
1508 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 1H-NMR
(CDCl3, 300 MHz, ppm): 2.84–2.85 (m, 4H, (CH2)2), 3.61–
3.63 (m, 4H, (CH2)2), 3.76 (s, 2H, CH2), 7.05–8.66 (m, 8H,
Ar-H). MS m/z: 364 (M + 1). Anal. Calcd. for C19H18FN7:
C, 62.80; H, 4.99; N, 26.98. Found: C, 62.89; H, 5.03; N,
26.92.
Yield: 93%; m.p. 186–188 °C. IR (KBr) per cm: 1588,
1502 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.72–2.75
(m, 4H, (CH2)2), 3.52–3.60 (m, 4H, (CH2)2), 3.64 (s, 2H,
CH2), 7.14–8.56 (m, 7H, Ar-H). MS m/z: 414 (M + 1). Anal.
Calcd. for C19H17Cl2N7: C, 55.08; H, 4.14; N, 23.67.
Found: C, 55.10; H, 4.22; N, 23.60.
6-(4-(3-fluorobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8b)
6-(4-(2-bromobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8h)
Yield: 90%; m.p. 146–148 °C. IR (KBr) per cm: 1599,
1508 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.67–2.70
(m, 4H, (CH2)2), 3.50–3.53 (m, 4H, (CH2)2), 3.56 (s, 2H,
CH2), 7.01–8.57 (m, 8H, Ar-H). MS m/z: 364 (M + 1). Anal.
Calcd. for C19H18FN7: C, 62.80; H, 4.99; N, 26.98. Found:
C, 62.88; H, 5.01; N, 26.93.
Yield: 91%; m.p. 210–212 °C. IR (KBr) per cm: 1580,
1489 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.73–2.75
(m, 4H, (CH2)2), 3.51–3.55 (m, 4H, (CH2)2), 3.60 (s, 2H,
CH2), 7.19–8.07 (m, 8H, Ar-H). MS m/z: 424 (M + 1). Anal.
Calcd. for C19H18BrN7: C, 53.78; H, 4.28; N, 23.11.
Found: C, 53.80; H, 4.32; N, 23.08.
4
Chem Biol Drug Des 2013

Synthesis and Biological Evaluation of [1,2,4]Triazolo[3,4-a]phthalazine and Tetrazolo[5,1-a]phthalazine
6-(4-(3-bromobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
the compounds synthesized. Milrinone (Shuzhou Unite
Pharmaceutical Co., Shuzhou, China) and 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 Æ 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 imme-
diately with N-2-hydroxyethyl piperazine-N-2-ethanesulfon-
ic acid (HEPES) buffer solution by means of a peristaltic
pump (1.25 mL/min) at 34 °C. The composition of the buf-
fer 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,
phthalazine (8i)
Yield: 90%; m.p. 216–218 °C. IR (KBr) per cm: 1583,
1490 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.76–2.79
(m, 4H, (CH2)2), 3.53–3.54 (m, 4H, (CH2)2), 3.62 (s, 2H,
CH2), 7.21–8.09 (m, 8H, Ar-H). MS m/z: 424 (M + 1). Anal.
Calcd. for C19H18BrN7: C, 53.78; H, 4.28; N, 23.11.
Found: C, 53.82; H, 4.36; N, 23.07.
6-(4-(4-bromobenzyl)piperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8j)
Yield: 92%; m.p. 213–215 °C. IR (KBr) per cm: 1581,
1489 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.79–2.80
(m, 4H, (CH2)2), 3.50–3.53 (m, 4H, (CH2)2), 3.64 (s, 2H,
CH2), 7.28–8.17 (m, 8H, Ar-H). MS m/z: 424 (M + 1). Anal.
Calcd. for C19H18BrN7: C, 53.78; H, 4.28; N, 23.11.
Found: C, 53.81; H, 4.30; N, 23.10.
6-(4-benzylpiperazin-1-yl)tetrazolo[5,1-a]
phthalazine (8k)
Table 1: Positive inotropic activity of the compounds tested
Yield: 91%; m.p. 138–140 °C. IR (KBr) per cm: 1586,
1486 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.66–2.67
(m, 4H, (CH2)2), 3.34–3.39 (m, 4H, (CH2)2), 3.59 (s, 2H,
CH2), 7.19–8.503 (m, 9H, Ar-H). MS m/z: 346 (M + 1).
Anal. Calcd. for C19H19N7: C, 66.07; H, 5.54; N, 28.39.
Found: C, 66.10; H, 5.59; N, 28.40.
N
R
N
R
N
N
N
N
N
N
N
N
N
5a-m
8a-m
N
N
Compound
R
Increased stroke volumea (%)
6-(4-(4-methylbenzyl)piperazin-1-yl)tetrazolo[5,1-
a]phthalazine (8l)
5a
5b
5c
5d
5e
5f
2-F
3-F
4-F
3.67 Æ 0.07
0.75 Æ 0.04
0.67 Æ 0.03
1.69 Æ 0.05
1.01 Æ 0.05
4.33 Æ 0.04
4.12 Æ 0.03
Yield: 90%; m.p. 170–172 °C. IR (KBr) per cm: 1581,
1489 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.29 (s,
3H, CH3), 2.67–2.70 (m, 4H, (CH2)2), 3.50–3.53 (m, 4H,
(CH2)2), 3.55 (s, 2H, CH2), 7.08–8.62 (m, 8H, Ar-H). MS
m/z: 360 (M + 1). Anal. Calcd. for C20H21N7: C, 66.83; H,
5.89; N, 27.28. Found: C, 66.88; H, 5.92; N, 27.26.
2-Cl
3-Cl
4-Cl
2,4-Cl2
2-Br
3-Br
4-Br
H
4-CH3
4-OCH3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2,4-Cl2
2-Br
3-Br
4-Br
H
5g
5h
5i
b
—
0.96 Æ 0.02
1.08 Æ 0.02
—
5j
6-(4-(4-methoxybenzyl)piperazin-1-yl)tetrazolo[5,1-
a]phthalazine (8m)
5k
5l
—
Yield: 92%; m.p. 174–176 °C. IR (KBr) per cm: 1608,
1496 (C=N). 1H-NMR (CDCl3, 300 MHz, ppm): 2.68–2.71
(m, 4H, (CH2)2), 3.40–3.45 (m, 4H, (CH2)2), 3.75 (s, 2H,
CH2), 3.78 (s, 3H, CH3), 7.06–8.56 (m, 8H, Ar-H). MS m/z:
376 (M + 1). Anal. Calcd. for C20H21N7O: C, 63.98; H,
5.64; N, 26.12. Found: C, 63.99; H, 5.70; N, 26.10.
5m
8a
8b
8c
8d
8e
8f
9.92 Æ 0.09
0.62 Æ 0.04
0.74 Æ 0.04
3.74 Æ 0.09
0.81 Æ 0.03
1.01 Æ 0.05
3.14 Æ 0.06
2.27 Æ 0.04
—
8g
8h
8i
Evaluation of positive inotropic effects in vitro
The method of measuring left atrium stroke volume was
described previously (12,13). The features of CHF are car-
diac dilatation, poor contractility of cardiac muscle,
decreased ejection fraction, and depression of left ventric-
ular pressure maximum alleosis. Therefore, macroscopic
measurement of the variance in left atrium stroke volume
can be used to estimate the positive inotropic effects of
—
8j
2.63 Æ 0.04
8k
8l
—
4-CH3
4-OCH3
—
8m
Milrinone
12.02 Æ 0.20
2.46 Æ 0.04
aThe concentration for the test sample is 3 9 10À5 M.
bNone or negative stroke volume increase.
Chem Biol Drug Des 2013
5

Wu et al.
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 dias-
tole. 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 inter-
vals, 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 9 10À5 M.
Samples were dissolved in DMSO and diluted with the HE-
PES buffer to 0.1% DMSO. 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. To
assess differences, repeated measurements were com-
pared by means of an ANOVA test followed by the Bonfer-
roni’s multiple-comparison test. Statistical significance was
defined as p < 0.05, and the data are presented as
means Æ SE.
in the presence of potassium carbonate afforded com-
pounds 5a–m (17). Compound 6 was prepared by the
reaction of compound 3 with hydrazine hydrate in ethanol
and was subsequently reacted with sodium nitrite in 30%
hydrochloric acid to afford compound 7 (18). Finally, com-
pounds 8a-m were obtained in high yield from the reac-
tions of 7 with a variety of different monosubstituted
piperazines in refluxing acetone in the presence of potas-
sium carbonate.
Biological results and discussion
As shown in Table 1,19 of the 26 compounds tested dis-
played inotropic effects against the isolated rabbit heart
preparations, with eight of the compounds exhibiting more
potent effects than milrinone (2.46 Æ 0.07% at
3 9 10À5 M). Compound 8m in particular was the most
potent
with
an
increased
stroke
volume
of
12.02 Æ 0.20%. Furthermore, compound 8m was found
to be 4.9-fold more potent than milrinone.
For the [1,2,4]triazolo[3,4-a]phthalazine series of derivatives
bearing substituted benzylpiperazine moieties (5a–m), the
presence of different substituents on the phenyl ring of the
benzyl group at the 4-position of the piperazine ring was
found to exert a significant influence on the observed ino-
tropic activity. For the different halogen substituted deriva-
tives (5a–j), an activity order of Cl > F > Br was observed.
Although the position of the substituents on the phenyl
ring was also found to influence the activity, no clear pat-
tern could be found for the structure–activity relationship.
For the halo substituted derivatives, an activity order of
o > m > p was observed for the fluorinated compounds,
2,4-Cl > p > o > m for the chlorinated compounds, and
p > m > o for the brominated compounds. For the deriva-
tives containing electron-donating substituents, such as
CH3 and OCH3, the methoxy-substituted derivative 5m
Results and Discussion
Chemistry
Compounds 5a–m were synthesized according to the syn-
thetic route depicted in Scheme 1. Isoindoline-1,3-dione
(1) was reacted with hydrazine hydrate in ethanol to afford
2,3-dihydrophthalazine-1,4-dione (2), which was subse-
quently reacted with refluxing phosphorus oxychloride
(POCl3) to give 1,4-dichlorophthalazine (3) (14,15). Com-
pound 4 was synthesized from compound 3 by reaction
with formylhydrazine in n-butyl alcohol (16). The nucleo-
philic aromatic substitution reactions of 4 with a variety of
different monosubstituted piperazines in acetone refluxing
N
R
Cl
N
Cl
N
c
d
N
N
N
N
b
4
N
5a-m
Cl
3
N
O
O
N
O
N
a
NH
NH
N
O
e
O
N
R
1
Cl
N
Cl
N
2
N
N
N
N
d
f
R= a: 2-F
b: 3-F
f: 4-Cl
g: 2,4-Cl
h: 2-Br
i : 3-Br
j: 4-Br
k: H
l : 4-CH3
N
N
8a-m
2
N
N
H2NHN
c: 4-F
d: 2-Cl
e: 3-Cl
N
m: 4-OCH3
N
6
7
N
Scheme 1: Synthetic scheme for the synthesis of compound 5a–m and 8a–m. Reagents and conditions: (A) NH2NH2ÁH2O/ethanol, reflux
12 h; (B) POCl3/TEA, reflux 4 h; (C) NH2NHCOH/N-BuOH, reflux 8 h; (D) monosubstituted piperazines, K2CO3, acetone, reflux, 12–48 h.
(E) NH2NH2ÁH2O/ethanol, reflux 4 h; (F) NaNO2/HCl.
6
Chem Biol Drug Des 2013

Synthesis and Biological Evaluation of [1,2,4]Triazolo[3,4-a]phthalazine and Tetrazolo[5,1-a]phthalazine
(9.92 Æ 0.09%) presented the most favorable activity in
of the bromine substituent did not have a clear impact on
the observed activity, and only one of the compounds
(para-substituted 8j) was found to exhibit any activity. In a
similar trend to that observed in the [1,2,4]triazolo[3,4-a]
phthalazine derivative 5a–m, the corresponding para-
methyl-substituted and para-methyl-unsubstituted com-
pounds 8l and 8k showed no activity, whereas the meth-
oxy-substituted derivative 8m (9.92 Æ 0.09%) presented
the most favorable activity of the series.
this series, although the para-methyl-substituted and
para-methyl-unsubstituted derivatives 5l and 5k showed
no activity.
In the tetrazolo[5,1-a]phthalazine series of derivatives 8a–
m, the fluorinated and chlorinated compounds showed
better activity than the brominated compounds. The posi-
tion of the halogen substituent (F, Cl) on the phenyl ring
was found to exert a significant influence on the observed
activity, and an activity order of p > m > o was found for
the fluorinated compounds, whereas an activity order of
p > 2,4-Cl2 > m > o was observed for the chlorinated
compounds. For the brominated compounds, the position
The dynamics of the test compounds in perfused beating
rabbit atria were also evaluated in the current study, reveal-
ing that compounds 5m and 8m exhibited more desirable
atrial dynamic profiles than milrinone (Figure 3A,C). The
A
B
D
C
Figure 3: Effects of milrinone and compounds 5a, 5f, 5g, 5m, 8c, 8f, 8j, and 8m on stroke volume in beating rabbit atria (1.5 Hz).
Values are means Æ SE. ***p < 0.001 versus control.
A
B
Figure 4: Concentration-response curves of compounds 5m, 8m and milrinone on stroke volume in beating rabbit artia (1.5 Hz). Values
are means Æ SE. ***p < 0.001 versus control.
Chem Biol Drug Des 2013
7

Wu et al.
Table 2: Changes of heart rate caused by compounds in isolated
rabbit heart preparations
Acknowledgment
This work was supported by the National Science Founda-
tion of China (81160381).
Compound
Mean Æ SEa
Mean Æ SEb
5a
103.97 Æ 0.04
116.73 Æ 0.07
120.86 Æ 0.12
101.67 Æ 0.06
99.52 Æ 0.09
113.36 Æ 0.18
115.63 Æ 0.07
109.26 Æ 0.12
106.36 Æ 0.02
103.89 Æ 0.05
116.36 Æ 0.09
121.84 Æ 0.08
102.68 Æ 0.09
90.01 Æ 0.26c
113.86 Æ 0.16
115.39 Æ 0.10
120.25 Æ 0.08
106.38 Æ 0.03
5f
5g
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compounds 5m and 8m exhibited promising cardiovascu-
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none. In particular, compound 8m was found to be 4.9-
fold more active than milrinone. These results suggest that
the further development of these compounds may be of
interest and, with this in mind, further biological tests
including in vivo evaluation, coronary vasodilation analysis,
and studies into the possible mechanism of action are cur-
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8
Chem Biol Drug Des 2013

Synthesis and Biological Evaluation of [1,2,4]Triazolo[3,4-a]phthalazine and Tetrazolo[5,1-a]phthalazine
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Chem Biol Drug Des 2013
9