Synthesis and evaluation of the anticonvulsant activities of 2,3-dihydrophthalazine-1,4-dione derivatives

Article abstract of DOI:10.1007/s00044-017-1896-8

A series of 2,3-dihydrophthalazine-1,4-dione derivatives with triazole (6a–y) and other heterocyclic substituents (8a–b, 9a–b, 10a–b, 12a–b, and 14a–b) was synthesized and their structures characterized by using IR, ¹H-NMR, ¹³C-NMR, and MS. Their anticonvulsant activities were evaluated using the maximal electroshock test, and their neurotoxicity was evaluated using the rotarod neurotoxicity test (Tox). The results showed that 5-(3-(trifluoromethyl)benzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6e) had the most potent anticonvulsant activity with an ED₅₀ value of 6.8 mg/kg and protective index (PI = TD₅₀/ED₅₀) value of 11.5 (i.p.); its anticonvulsant activity was found to be stronger than that of the lead compound and the standard drug carbamazepine. In particular, compound 6e protected against the seizures induced by pentylenetetrazole and bicuculline.

Full text of DOI:10.1007/s00044-017-1896-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1896-8
ORIGINAL RESEARCH
Synthesis and evaluation of the anticonvulsant activities of
2,3-dihydrophthalazine-1,4-dione derivatives
Xiang-Jun Liu1 Hong-Jian Zhang1 Zhe-Shan Quan1
●
●
Received: 16 January 2017 / Accepted: 7 April 2017
© Springer Science+Business Media New York 2017
Abstract A series of 2,3-dihydrophthalazine-1,4-dione
derivatives with triazole (6a–y) and other heterocyclic
substituents (8a–b, 9a–b, 10a–b, 12a–b, and 14a–b) was
synthesized and their structures characterized by using IR,
1H-NMR, 13C-NMR, and MS. Their anticonvulsant activ-
ities were evaluated using the maximal electroshock test,
and their neurotoxicity was evaluated using the rotarod
neurotoxicity test (Tox). The results showed that 5-(3-(tri-
fluoromethyl)benzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6
(5H)-one (6e) had the most potent anticonvulsant activity
with an ED50 value of 6.8 mg/kg and protective index (PI =
TD50/ED50) value of 11.5 (i.p.); its anticonvulsant activity
was found to be stronger than that of the lead compound
and the standard drug carbamazepine. In particular, com-
pound 6e protected against the seizures induced by penty-
lenetetrazole and bicuculline.
Introduction
Epilepsy is a common disease that affects more than 60
million people worldwide, as reported in epidemiological
studies (Löscher 1998). Current anticonvulsants drugs can
only control about 60–70% of seizures in people, and these
drugs are associated with both notable adverse side effects,
such as drowsiness, ataxia, gastrointestinal disturbance,
hepatotoxicity, and megaloblastic anemia (Leppik 1994;
Perucca 1996), as well as other potentially life-threatening
conditions (Al-Soud et al. 2003; Kanner 2014; Fiest et al.
2014). Thus, it is a challenge for the field of pharmaceutical
chemistry to discover and develop new, safer, and more
effective drugs for epilepsy treatment.
We previously described the synthesis and antic-
onvulsant activity of various compounds containing tria-
zole; the majority showed potent activity as assessed using
the maximal electroshock (MES) test and several chemical
models, such as the pentylenetetrazole-induced and
isoniazid-induced seizure tests (Fig. 1) (Xie et al. 2005; Guo
et al. 2009; Zhang et al. 2010; Deng et al. 2010).
●
Keywords 2,3-Dihydrophthalazine-1,4-dione derivatives
Anticonvulsant Maximal electroshock Pentylenetetrazole
Bicuculline
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●
●
Evaluation of the currently used antiepileptic drugs
shows that the major characteristics vital to newly synthe-
sized compounds are the inclusion of a hydrophobic site
and H-bond donors/acceptors. With respect to the com-
pounds mentioned above, the hydrophobic site requirement
can clearly be fulfilled by the phenyl group and its sub-
stituents, while the H-bond acceptor requirement can be
fulfilled by triazole.
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1896-8) contains supplementary material,
which is available to authorized users.
Previously, we found that 6-(substituted-alkoxy)-[1,2,4]
triazolo[3,4-a]phthalazine (V) compounds had potent
anticonvulsant activities. The most promising of these, 6-
* Zhe-Shan Quan
zsquan@ybu.edu.cn
heptyloxy-[1,2,4]triazolo[3,4-a]phthalazine, showed
median effective dose of 11.0 mg⁄kg and had a protective
a
1
Key Laboratory of Natural Resources and Functional Molecules of
the Changbai Mountain, Affiliated Ministry of Education, College
index (PI) value of 8.0 (Zhang et al. 2009).
of Pharmacy, Yanbian University, Yanji, Jilin 133002, China

Med Chem Res
Fig. 1 Structures of some
compounds in containing
triazole and design of target
compounds
In this study, we attempted to introduce an amide bond
between the 5-position and 6-position of the lead compound
V, resulting in the creation of two H-bond acceptors, a
triazole and amide, in the same structure. The target com-
pounds, 5-(substituted)-[1,2,4]triazolo[3,4-a]phthalazin-6
(5H)-one (6a–y), were designed and synthesized, and their
anticonvulsant activity was evaluated in a preliminary
manner. Because compounds 6e and 6t showed a greater
protective effect against MES-induced seizures, other het-
erocyclic substituents (8a–b, 9a–b, 10a–b, 12a–b, and
14a–b) were obtained using bioisosterism, similarity theo-
rem, and other principles and methods (Fig. 1). After the
preliminary evaluation of the target compounds 8a–b, 9a–b,
10a–b, 12a–b, and 14a–b, compound 6e was selected for
quantitative evaluation of its anticonvulsant activity. Sei-
zures were chemically induced to investigate the com-
pound’s possible mechanism of action.
intermediate compound 5 via a triethyl orthoformate stirring
reaction with compound 4 at 80 °C (Wang et al. 2014).
Compounds 6a–y, 7, 12a–b, and 14a–b were obtained via
alkylation (Yamaguchi et al. 1993). The intermediate 3 was
subjected to reaction with the appropriate haloalkane in
acetonitrile to yield compound 7 (Yamaguchi et al. 1993).
Compound 7 was subjected to reaction with 1,2,4-triazole,
pyrazole, and imidazole to yield compounds 8a–b, 9a–b,
and 10a–b, respectively (Stappert et al. 2015; Yamaguchi
et al. 1993; Kassem et al. 1989). The intermediate 4 was
subjected to reaction with acetylacetone and acetic anhy-
dride to yield compound 11 and compound 13, respectively
(Habib et al. 2011; Dunham and Miya 1957). (Scheme 1)
The structures of the synthesized compounds were con-
1
firmed via IR, H-NMR, 13C-NMR, and mass spectra.
Anticonvulsant activity
Results and discussion
Chemistry
The experiments were carried out on adult Kun Ming mice
(18–22 g). They were maintained on a 12-h light/dark cycle
in a temperature-controlled (23 2 °C) laboratory. Food
and water were available ad libitum. The tested compounds
and reference drug were dissolved in dimethyl sulfoxide
(DMSO), and injected intraperitoneally at a standard
volume of 0.1 ml/20 g body weight. The tested compounds
and reference drug were dissolved in carboxy methylated
cellulose–Na, and given orally (p.o.) at a standard volume
of 0.2 ml/20 g body weight. The methods were carried out
in accordance with the Administration Rule of Laboratory
The starting materials, 2,3-dihydrophthalazine-1,4-dione
(compound 1) and POCl3, were refluxed for 3 h to obtain
compound 2 (Xue et al. 2014). Compound 2 was stirred in
glacial acetic acid at 100 °C for 2 h to afford compound 3
(Monsieurs et al. 2007). Compound 4 was obtained when
compound 3 reacted with pure hydrazine hydrate at 80 °C
for 24 h (Brizzi et al. 1986). It was easy to obtain the

Med Chem Res
Scheme 1 Synthesis of
compounds 6a–y, 8a–b, 9a–b,
10a–b, 12a–b, and 14a–b. a
Phosphorus oxychloride,
105 °C, 3 h; b CH3COOH,
100 °C, 2 h; c Hydrazine
hydrate, 100 °C, 24 h; d Triethyl
orthoformate, absolute ethanol,
80 °C, 3 h; e RX, NaH, DMF,
100 °C, 3 h; f RX, K2CO3,
DMF, 80 °C, 3 h; g 1,2,4-
Triazole sodium, DMF, 100 °C,
6 h; h Pyrazole, K2CO3, DMF,
100 °C, 3 h; i Imidazole,
K2CO3, DMF, 100 °C, 6 h; j
(CH3CO)2CH2, acetonitrile,
82 °C, 6 h; k RX, K2CO3, DMF,
80 °C, 3 h; l Acetic anhydride,
100 °C, 3 h; m RX, K2CO3,
DMF, 80 °C, 3 h
Animals of China. All experimental protocols were
approved by the Medical Ethics Committee of China.
The MES test is an experimental model that aids in the
identification of clinical candidates that prevent the spread of
tonic-clonic seizures and partial convulsions (Clark et al.
1984). The neurotoxicity of the active compounds was
established using the rotarod test. The anticonvulsant activ-
ities of 6a–y are listed in Table 1. Compounds 6e, 6n, 6o, 6p,
6t, and 6x showed protection in 3/3 at 100 mg/kg; only
compound 6e showed complete protection of the tested mice
at 30 mg/kg. From pharmacological data, we found that the
length of the alkyl chain of 2,3-dihydrophthalazine-1,4-dione
derivatives has a significant impact on anticonvulsant activity
and neurotoxicity. From compound 6a to 6i, as the length of
the alkyl chain increased, the anticonvulsant activity first
increased and then decreased, peaking at a length of seven
carbon atoms. The key structure of compounds 6i–y was an
aromatic ring. The anticonvulsant activity of compounds with
different substituents on the benzene ring was as follows: m-
CF3 > m-Cl, p-CH3 > o, p-Cl ≥ o, p-Br ≥ o, p-CF3 ≥ 2,6-Cl2,
2,6-Cl2 ≥ p-F > o, m-F. The compound with a strong electron-
withdrawing group (–CF3) showed stronger activity than the
other derivatives with different halogen substituents did.
Additionally, the m-position was found to be the best position
to enhance anticonvulsant activity among the –CF3-sub-
stituted derivatives.
The anticonvulsant activities of compounds 8a–b, 9a–b,
10a–b, 12a–b, and 14a–b are shown in Table 2. As only
compound 14b showed both weaker anticonvulsant activities
at 100 mg/kg and no anticonvulsant activity at 30 mg/kg, the
results indicate that the triazole group was the heterocyclic
substituent responsible for anticonvulsant activities.
In accordance with the results of the preliminary
screening, compound 6e was subjected to further testing and
quantification of anticonvulsant activity (as indicated by
ED50) and neurotoxicity (as indicated by TD50) in mice.
Results of the quantitative testing for the selected com-
pounds, along with the data on the standard drug carba-
mazepine, are presented in Table 3. 5-Heptyl-[1,2,4]triazolo
[3,4-a] phthalazin-6(5H)-one (6e) was determined to have
an ED50 value of 6.8 mg/kg and a TD50 value of 77.9 mg/
kg, resulting in a PI of 11.5, which is superior to that of the

Med Chem Res
Table 1 Preliminary anticonvulsant activity of compounds 6a–y
administered intraperitoneally (i.p.) to mice
Table 2 Preliminary anticonvulsant activity of compounds 8a–b,
9a–b, 10a–b, 12a–b, 14a–b administered intraperitoneally (i.p.) to
mice
Compounds
R
MESa (mg/kg)
Compounds
R
MESa (mg/kg)
100
30
10
100
30
c
6a
6b
6c
6d
6e
6f
n-C3H7
0/3b
1/3
2/3
2/3
3/3
–
–
–
c
8a
n-C7H15
0/3b
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
1/3
–
n-C4H9
–
–
8b
–CH2C6H4(m-CF3)
n-C7H15
–
n-C5H11
0/3
1/3
3/3
1/3
1/3
–
–
9a
–
n-C6H13
0/3
1/3
0/3
0/3
–
9b
–CH2C6H4(m-CF3)
n-C7H15
–
n-C7H15
10a
10b
12a
12b
14a
14b
a
–
n-C8H17
–CH2C6H4(m-CF3)
n-C7H15
–
6g
6h
6i
n-C9H19
–
–
n-C10H21
0/3
0/3
0/3
1/3
1/3
2/3
3/3
3/3
3/3
2/3
2/3
2/3
3/3
2/3
2/3
2/3
3/3
0/3
–CH2C6H4(m-CF3)
n-C7H15
–
n-C12H25
–
–
–
6j
–CH2C6H5
–
–
–CH2C6H4(m-CF3)
0/3
6k
6l
–CH2C6H4 (o-F)
–CH2C6H4 (m-F)
–CH2C6H4 (p-F)
–CH2C6H4 (o-Cl)
–CH2C6H4 (m-Cl)
–CH2C6H4 (p-Cl)
–CH2C6H4 (o-Br)
–CH2C6H4 (p-Br)
–CH2C6H4 (o-CF3)
–CH2C6H4 (m-CF3)
–CH2C6H4 (p-CF3)
–CH2C6H3 (2,4-Cl2)
–CH2C6H3 (2,6-Cl2)
–CH2C6H4 (p-CH3)
–CH2C6H4 (p-CN)
–
–
–
–
Maximal electroshock: doses of 30 and 100 mg/kg were admini-
strated intraperitoneally in mice. The animals were examined 0.5 h
after administration
6m
6n
6o
6p
6q
6r
6s
6t
0/3
0/3
1/3
0/3
0/3
0/3
0/3
2/3
0/3
0/3
0/3
1/3
–
–
–
b
n1/n2, number of animals protected/number of animals tested
0/3
–
c
Not tested
–
Table 3 Phase-II quantitative anticonvulsant evaluation in Mice
–
PIc
a
b
Compound
ED50 (MES)
TD50 (NT)
–
6e (i.p.)
6e (p.o.)
6.8 (6.55–7.05)d
77.9 (49.2–68.2)
11.5
0/3
–
6u
6v
6w
6x
6y
a
24.89 (19.05–32.52) 593.3 (553.3–636.3) 23.84
–
Carbamazepine 11.8 (8.5–16.4)
(i.p.)
76.1 (55.8–103.7)
6.45
–
Carbamazepine 27.3 (19.7–37.9)
(p.o.)
328.6 (229.9–469.7) 12.0
0/3
–
a
ED50: median effective dose affording anticonvulsant protection in
50% of animals, the dose is measured in mg/kg
Maximal electroshock: doses of 30 and 100 mg/kg were admini-
strated intraperitoneally in mice. The animals were examined 0.5 h
after administration
b
TD50 : median toxic dose eliciting minimal neurological toxicity in
b
50% of animals, the dose is measured in mg/kg
n1/n2, number of animals protected/number of animals tested
c
c
PI: protective index (TD50/ED50
)
Not tested
d
95% confidence intervals given in parentheses
standard drug carbamazepine and the lead compound, 6-
heptyloxy-[1,2,4]triazolo[3,4-a]phthalazine (Zhang et al.
2009).
To evaluate its oral bioavailability, we investigated the
oral time of peak effect (TPE) of compound 6e. The time-
course of compound 6e in the MES test is shown in Fig. 2;
we found the TPE of compound 6e to be 2.0 h after
administration. We then evaluated the oral anticonvulsant
activity of compound 6e, with the results of Phase-II testing
shown in Table 3. 5-Heptyl-[1,2,4]triazolo[3,4-a]phthala-
zin-6(5H)-one (6e) was found to have an ED50 value of
24.89 mg/kg and a TD50 of 593.3 mg/kg, resulting in a PI of
23.84. Thus, we concluded that compound 6e was safer
Fig. 2 Time-course of compound 6e in the MES test (40 mg/kg, p.o.)
The subcutaneous pentylenetetrazole (sc-PTZ) test eval-
uates the clinical efficacy of compounds in the prevention of
absence seizures by determining whether they elevate the
seizure threshold (Okada et al. 1989). In the sc-PTZ model,
than the anticonvulsant drug carbamazepine (ED50
=
27.3 mg/kg, TD50 = 328.6 mg/kg, PI = 12.0) as per MES
and TOX testing under oral conditions.

Med Chem Res
Table 4 Effects of compound
6e on chemical-induced seizures
in mice (i.p.)
Chemical
substances
Compound Doses
(mg/kg)
Test Time Clonic seizures Tonic seizures Lethality
(h)
(%)
(%)
(%)
PTZ
DMSO
0.5
0.5
0.5
0.5
0.5
0.5
100
30
40
0
40
10
0
6e
50
50
CBZ
DMSO
6e
100
100
10
0
BIC
40
0
40
20
20
50
50
CBZ
90
0
carbamazepine inhibits clonic seizures, tonic seizures, and
death at rates of 0, 100, and 100%, respectively (Table 4).
Compound 6e inhibited clonic seizures, tonic seizures, and
lethality at rates of 70, 100, and 90%, respectively. From
these data, we can see that compound 6e can better inhibit
the clonic seizures induced by PTZ than carbamazepine.
PTZ has been reported to produce seizures by inhibiting γ-
aminobutyric acid (GABA) neurotransmission (Olsen 1981;
Gale 1992). GABA is the main inhibitory neurotransmitter
of the brain, and is widely implicated in epilepsy. Inhibition
of GABAergic neurotransmission or activity has been
shown to promote and facilitate seizures (Badawy et al.
1990), while GABAergic enhancement is known to inhibit
or attenuate seizures. The ability of compound 6e to inhibit
PTZ-induced clonic seizures might be related to the
enhancement of GABAergic neurotransmission or activity.
In the bicuculline (BIC)-induced seizure model, carba-
mazepine inhibits clonic seizures, tonic seizures, and leth-
ality at rates of 10, 100, and 80%, respectively (Table 4).
Compound 6e inhibited clonic seizures, tonic seizures, and
lethality at rates of 90, 100, and 80%, respectively. Hence,
we can conclude that compound 6e can inhibit BIC-induced
seizures with a lethality rate lower than that of the control
drug, carbamazepine. BIC is a mild sensitive competitive
antagonist of the GABAA receptor, and produces convul-
sions through its antagonism of the GABAA receptor
(Macdonald and Barker 1978). As compound 6e inhibited
the seizures induced by BIC, we hypothesize that it exerts
its anticonvulsant activity through GABAA-mediated
mechanisms.
the standard drug carbamazepine and the lead compound 6-
heptyloxy-[1,2,4]triazolo[3,4-a]phthalazine.
Experimental
Chemistry
The starting material and major chemicals were purchased
from Sigma-Aldrich Corporation. Melting points were
determined in open capillary-tubes and were uncorrected.
The progress of the reactions was monitored by thin layer
chromatography and purified using silica gel column chro-
matography. Infrared spectra were recorded (in KBr) using
a FTIR1730 spectrometer (PerkineElmer, Waltham, MA,
USA). 1H-NMR and 13C-NMR spectra were made to
identify at 300 MHz and 75 MHz by an AV-300 spectro-
meter (Bruker, Switzerland). All spectra were obtained in
CDCl3 or DMSO-d6. All chemical shifts were given in parts
per million relative to tetramethylsilane. Mass spectra were
measured using an HP1100 LC spectrometer (Agilent
Technologies, USA).
Synthesis of 1,4-dichloro-1,2-dihydrophthalazine (2)
Compound 1 (5.0 g, 30.8 mmol) was dissolved in phos-
phorus oxychloride (50 ml) and stirred for 2 h at 105 °C.
After the reaction was completed, the mixture was poured
into ice water (100 ml). The precipitate that separated was
collected via filtration, washed with water, and dried to give
4-chlorophthalazin-1(2H)-one (compound 2) as a white
solid.
Conclusion
Thirty-five 2,3-dihydrophthalazine-1,4-dione derivatives
were synthesized and evaluated for anticonvulsant activ-
ities. Compound 6e was found to be the most potent, with
an ED50 value of 6.8 mg/kg and PI value of 11.5. It also
protected against seizures induced by pentylenetetrazole
and BIC. In addition, compound 6e showed an ED50 value
of 24.89 mg/kg and PI > 23.84 p.o. indicating that it pos-
sesses better anticonvulsant activity and lower toxicity than
Synthesis of 4-chlorophthalazin-1(2H)-one (3)
Compound 2 (5.0 g, 25.0 mmol) was dissolved in glacial
acetic acid (50 ml) and stirred at 100 °C for 1 h. After the
reaction was completed, the mixture was cooled to room
temperature, filtered, and washed, and compound 3 was
obtained as a white powder.

Med Chem Res
Synthesis of 4-hydrazinylphthalazin-1(2H)-one (4)
8.84 (s, 1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ:
13.47, 18.71, 30.14, 67.83, 118.58, 122.40, 123.65, 124.83,
130.70, 133.89, 139.48, 141.35. ESI-HRMS calcd for
C13H15N4O+ ([M + H]+): 243.1240; found: 243.1235.
A mixture of compound 3 (5.0 g, 27.8 mmol), ethanol (50
ml), and hydrazine monohydrate (20 ml) was stirred at 70 °
C for 24 h. The reaction mixture was subjected to eva-
poration under reduced pressure, before washing with water
to yield compound 4 as a grass-green powder.
5-Pentyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6c)
Yield: 49.6%, m.p.: 161–163 °C. IR (KBr) cm−1: 1657
(C=O), 1130. 1H-NMR (CDCl3, 300 MHz) δ: 0.98 (t, 3H, J
= 6.0 Hz, –CH3), 1.45–1.54 (m, 4H, –(CH2)2–), 1.90–2.00
(m, 2H, –CH2–), 4.49 (t, 2H, J = 6.0 Hz, –CH2–), 7.79 (t,
H, J = 6.0 Hz, Ar–H), 7.93 (t, 1H, J = 6.0 Hz, Ar–H), 8.20
(d, 1H, J = 9.0 Hz, Ar–H), 8.61 (d, 1H, J = 9.0 Hz, Ar–H),
8.84 (s, 1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ:
13.67, 21.75, 27.66, 27.73, 68.12, 118.63, 122.44, 123.68,
124.86, 130.75, 133.93, 139.51, 141.39, 157.80. ESI-
HRMS calcd for C14H17N4O+ ([M + H]+): 257.1397;
found: 257.1394.
Synthesis of [1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (5)
Compound 4 (5.0 g, 28.0 mmol) was dissolved in absolute
ethanol (20 ml) and stirred at room temperature. A total
volume of 4.9 ml triethyl orthoformate (4.3 g, 29.0 mmol)
was added dropwise, and the mixture was stirred at 80 °C
for 3 h. After the reaction was completed, the solvent was
evaporated under reduced pressure and filtered off. The
residue was washed with petroleum ether, and dried to yield
compound 5 as a yellowish powder.
5-Hexyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6d)
Yield: 51.3%, m.p.: 201–203 °C. IR (KBr) cm−1: 1657
(C=O), 3114 (C–H). H-NMR (CDCl3, 300 MHz) δ: 0.95
Synthesis of 5-substituted-[1,2,4]triazolo[3,4-a]phthalazin-
6(5H)-one (6a–y)
1
(t, 3H, J = 6.0 Hz, –CH3), 1.39–1.43 (m, 4H, –(CH2)2–),
1.51–1.61 (m, 2H, –CH2–), 1.90–1.99 (m, 2H, –CH2–),
4.50 (t, 2H, J = 6.0 Hz, –CH2–), 7.79 (t, 1H, J = 9.0 Hz,
Ar–H), 7.93 (t, 1H, J = 9.0 Hz, Ar–H), 8.20 (d, 1H, J = 9.0
Hz, Ar–H), 8.62 (d, 1H, J = 9.0 Hz, Ar–H), 8.84 (s, 1H,
Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 13.69,
21.91, 25.11, 28.03, 30.88, 68.16, 118.70, 122.50, 123.73,
124.92, 130.81, 134.00, 139.55, 141.43, 157.86. ESI-
HRMS calcd for C15H19N4O+ ([M + H]+): 271.1553;
found: 271.1561.
A mixture of compound 5 (0.5 g, 2.7 mmol) and NaH (0.2
g, 8.1 mmol) was dissolved in N,N-dimethylformamide (20
ml). The mixture was stirred and refluxed for 30 min. The
reaction was cooled to room temperature, and alkyl bromide
or benzyl chloride was added dropwise to the mixture as
appropriate before refluxing for 3 h. After the reaction was
completed, the solvent was removed under reduced pres-
sure. The residue was washed with water (10 ml × 3), fil-
tered off, and dried and purified using silica gel column
chromatography (CH2Cl2/CH3OH, 60:1) to yield com-
pounds 6a–y, a white powder.
5-Heptyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6e)
Yield: 54.2%, m.p.: 181–183 °C. IR (KBr) cm−1: 1657
(C=O), 1133. 1H-NMR (CDCl3, 300 MHz) δ: 0.92 (t, 3H, J
= 6.0 Hz, –CH3), 1.34–1.43 (m, 6H, –(CH2)3–), 1.50–1.57
(m, 2H, –CH2–), 1.89–1.99 (m, 2H, –CH2–), 4.49 (t, 2H, J
= 6.0 Hz, –CH2–), 7.78 (t, 1H, J = 6.0 Hz, Ar–H), 7.92 (t,
1H, J = 6.0 Hz, Ar–H), 8.19 (d, 1H, J = 9.0 Hz, Ar–H),
8.61 (d, 1H, J = 9.0Hz, Ar–H), 8.84 (s, 1H, Triazole-H).
13C-NMR (DMSO-d6, 75 MHz) δ: 13.89, 22.07, 25.46,
28.02, 28.46, 31.23, 67.98, 118.36, 122.39, 123.42, 124.74,
130.77, 133.97, 139.61, 141.23, 157.59. ESI-HRMS calcd
for C16H21N4O+ ([M + H]+): 285.1710; found: 285.1701.
5-Propyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6a)
Yield: 47.5%, m.p.: 192–194 °C. IR (KBr) cm−1: 1640
1
(C=O), 1136. H-NMR (DMSO-d6, 300 MHz) δ: 1.07 (t,
3H, J = 6.0 Hz, –CH3), 1.84–1.95 (m, 2H, –CH2–), 4.42 (t,
2H, J = 7.5 Hz, –CH2–), 7.90 (t, H, J = 7.5 Hz, Ar–H), 8.04
(t, 1H, J = 7.5 Hz, Ar–H), 8.19 (d, 1H, J = 9.0 Hz, Ar–H),
8.46 (d, 1H, J = 9.0 Hz, Ar–H), 9.38 (s, 1H, Triazole-H).
13C-NMR (CDCl3, 75 MHz) δ: 10.57, 22.00, 69.85, 119.27,
123.23, 124.13, 125.13, 130.55, 133.59, 139.29, 142.29,
158.44. ESI-HRMS calcd for C12H13N4O+ ([M + H]+):
229.1084; found: 229.1076.
5-Octyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6f)
Yield: 48.9%, m.p.: 163–165 °C. IR (KBr) cm−1: 1662
(C=O), 1121. 1H-NMR (CDCl3, 300 MHz) δ: 0.91 (t, 3H, J
= 6.0Hz, –CH3), 1.32–1.40 (m, 8H, –(CH2)4–), 1.50–1.57
(m, 2H, –CH2–), 1.89–1.99 (m, 2H, –CH2–), 4.49 (t, 2H, J
= 6.0 Hz, –CH2–), 7.79 (t, 1H, J = 9.0 Hz, Ar–H), 7.92 (t,
1H, J = 6.0 Hz, Ar–H), 8.20 (d, 1H, J = 9.0 Hz, Ar–H),
8.61 (d, 1H, J = 9.0 Hz, Ar–H), 8.83 (s, 1H, Triazole-H).
5-Butyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6b)
Yield: 52.1%, m.p.: 143–145 °C. IR (KBr) cm−1: 1654
(C=O), 1130. 1H-NMR (CDCl3, 300 MHz) δ: 1.05 (t, 3H, J
= 6.0 Hz, –CH3), 1.53–1.66 (m, 2H, –CH2–), 1.88–1.98 (m,
2H, –CH2–), 4.50 (t, 2H, J = 6.0 Hz, –CH2–), 7.79 (t, H, J
= 9.0 Hz, Ar–H), 7.93 (t, 1H, J = 9.0 Hz, Ar–H), 8.20 (d,
1H, J = 9.0 Hz, Ar–H), 8.61 (d, 1H, J = 9.0 Hz, Ar–H),

Med Chem Res
13C-NMR (DMSO-d6, 75 MHz) δ: 13.70, 21.95, 25.46,
28.05, 28.51, 28.62, 31.15, 68.12, 118.62, 122.45, 123.69,
124.84, 130.71, 133.93, 139.48, 141.38, 157.79. ESI-
HRMS calcd for C17H23N4O+ ([M + H]+): 299.1866;
found: 299.1858.
6.0 Hz, Ar–H), 9.17 (s, 1H, Triazole-H). 13C-NMR
(DMSO-d6, 75 MHz) δ: 69.82, 118.88, 122.97, 123.97,
125.46, 128.64, 128.83, 129.03, 131.45, 134.64, 136.02,
140.18, 141.85, 157.97. ESI-HRMS calcd for C16H13N4O+
([M + H]+): 277.1084; found: 277.1077.
5-Nonyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6g)
Yield: 44.2%, m.p.: 220–222 °C. IR (KBr) cm−1: 1600
(C=O), 1134. 1H-NMR (CDCl3, 300 MHz) δ: 0.90 (t, 3H, J
= 6.0 Hz, –CH3), 1.24–1.42 (m, 8H, –(CH2)4–), 1.53–1.60
(m, 2H, –CH2–), 1.90–1.99 (m, 2H, –CH2–), 4.49 (t, 2H, J
= 6.0 Hz, –CH2–), 7.79 (t, 1H, J = 6.0 Hz, Ar–H), 7.93 (t,
1H, J = 6.0 Hz, Ar–H), 8.20 (d, 1H, J = 9.0 Hz, Ar–H),
8.61 (d, 1H, J = 9.0 Hz, Ar–H), 8.83 (s, 1H, Triazole-H).
13C-NMR (DMSO-d6, 75 MHz) δ: 13.69, 21.96, 25.45,
28.04, 28.53, 28.66, 28.83, 31.19, 68.11, 118.59, 122.43,
123.68, 124.80, 130.67, 133.90, 139.46, 141.35, 157.77.
ESI-HRMS calcd for C18H25N4O+ ([M + H]+): 313.2023;
found: 213.2020.
5-(2-fluorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6k) Yield: 59.2%, m.p.: 213–215 °C. IR (KBr) cm−1
:
1674 (C=O), 1124. 1H-NMR (DMSO-d6, 300 MHz) δ: 5.61
(s, 2H, –CH2–), 7.28–7.36 (m, 2H, Ar–H), 7.47–7.53 (m,
1H, Ar–H), 7.75 (t, 1H, J = 6.0 Hz, Ar–H), 7.88 (t, 1H, J =
9.0 Hz, Ar–H), 8.05 (t, 1H, J = 9.0 Hz, Ar–H), 8.16 (d, 1H,
J = 9.0 Hz, Ar–H), 8.48 (d, 1H, J = 9.0 Hz, Ar–H), 9.45 (s,
1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 66.88,
118.44, 122.58, 123.81, 124.99, 127.45, 129.45, 130.25,
130.42, 130.94, 132.91, 133.09, 134.22, 139.62, 141.49,
157.46. ESI-HRMS calcd for C16H12FN4O+ ([M + H]+):
295.0990; found: 295.0992.
5-(3-Fluorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
5-Decyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6h)
Yield: 51.6%, m.p.: 196–198 °C. IR (KBr) cm−1: 1654
(C=O), 1122 (C–H). H-NMR (CDCl3, 300 MHz) δ: 0.90
one (6l) Yield: 55.8%, m.p.: 253–254 °C. IR (KBr) cm−1
:
1
1660 (C=O), 1122. H-NMR (CDCl3, 300 MHz) δ: 5.61(s,
2H, –CH2–), 7.57–7.62 (m, 1H, Ar–H), 7.69 (d, 1H, J =
6.0 Hz, Ar–H), 7.73–7.83 (m, 3H, Ar–H), 7.96 (t, 1H, J =
9.0 Hz, Ar–H), 8.21(d, 1H, J = 9.0 Hz, Ar–H), 8.64 (d, 1H,
J = 9.0 Hz, Ar–H), 8.87 (s, 1H, Triazole-H). 13C-NMR
(CDCl3, 75 MHz) δ: 69.04, 118.81, 122.12, 123.33, 124.19,
124.97, 125.06, 125.49, 129.33, 130.75, 131.50, 133.94,
136.27, 139.20, 142.23, 157.89. ESI-HRMS calcd for
C16H12FN4O+ ([M + H]+): 295.0990; found: 295.0986.
1
(t, 3H, J = 6.0 Hz, –CH3), 1.22–1.44 (m, 12H, –(CH2)6–),
1.50–1.60 (m, 2H, –CH2–), 1.90–1.99 (m, 2H, –CH2–),
4.49 (t, 2H, J = 6.0 Hz, –CH2–), 7.79 (t, 1H, J = 9.0 Hz,
Ar–H), 7.93 (t, 1H, J = 9.0 Hz, Ar–H), 8.19 (d, 1H, J = 9.0
Hz, Ar–H), 8.61 (d, 1H, J = 9.0 Hz, Ar–H), 8.84 (s, 1H,
Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 13.70,
21.95, 25.45, 28.05, 28.51, 28.64, 28.85, 28.87, 31.21,
68.14, 118.07, 122.48, 123.73, 124.88, 130.74, 133.97,
139.50, 141.40, 157.84. ESI-HRMS calcd for C19H27N4O+
([M + H]+): 327.2179; found: 327.2175.
5-(4-Fluorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6m) Yield: 50.1%, m.p.: 238–240 °C. IR (KBr)
1
cm−1: 1665 (C=O), 1121. H-NMR (CDCl3, 300 MHz) δ:
5-Dodecyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6i)
Yield: 54.2%, m.p.: 177–179 °C. IR (KBr) cm−1: 1654
(C=O), 1107. 1H-NMR (CDCl3, 300 MHz) δ: 0.90 (t, 3H, J
= 6.0 Hz, –CH3), 1.29–1.44 (m, 16H, –(CH2)8–), 1.53–1.57
(m, 2H, –CH2–), 1.90–1.98 (m, 2H, –CH2–), 4.49 (t, 2H, J
= 6.0 Hz, –CH2–), 7.79 (t, 1H, J = 9.0 Hz, Ar–H), 7.93 (t,
1H, J = 9.0 Hz, Ar–H), 8.20 (d, 1H, J = 9.0 Hz, Ar–H), 8.61
(d, 1H, J = 9.0 Hz, Ar–H), 8.84 (s, 1H, Triazole-H). 13C-
NMR (DMSO-d6, 75 MHz) δ: 13.68, 21.96, 25.45, 28.05,
28.59, 28.65, 28.77, 28.89, 28.92, 28.94, 31.22, 68.11,
118.50, 122.45, 123.71, 124.69, 130.53, 133.80, 139.39,
141.37, 141.37, 157.69. ESI-HRMS calcd for C21H31N4O+
([M + H]+): 355.2492; found: 355.2486.
5.51 (s, 2H, –CH2–), 7.08–7.17 (m, 2H, Ar–H), 7.51–7.56
(m, 2H, Ar–H), 7.77 (t, 1H, J = 9.0 Hz, Ar–H), 7.93 (t, 1H,
J = 9.0 Hz, Ar–H), 8.19 (d, 1H, J = 8.14 Hz, Ar–H), 8.61
(d, 1H, J = 9.0 Hz, Ar–H), 8.86 (s, 1H, Triazole-H). 13C-
NMR (CDCl3, 75 MHz) δ: 69.23, 115.82, 116.60, 118.92,
123.19, 124.05, 125.09, 129.16, 130.40, 131.12, 133.79,
134.59, 139.21, 157.97. ESI-HRMS calcd for
C16H12FN4O+ ([M + H]+): 295.0990; found: 295.0982
5-(2-Chlorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6n) Yield: 55.5 %, m.p.: 205–207 °C. IR (KBr)
1
cm−1: 1668 (C=O), 1136. H-NMR (CDCl3, 300 MHz) δ:
5.65 (s, 2H, –CH2–), 7.36–7.40 (m, 2 H, Ar–H), 7.47–7.50
(m, 1H, Ar–H), 7.61–7.64 (m, 1H, Ar–H), 7.79 (t, 1H, J =
9.0 Hz, Ar–H), 7.94 (t, 1H, J = 9.0 Hz, Ar–H), 8.23 (d, 1H,
J = 6.0 Hz, Ar–H), 8.63 (d, 1H, J = 6.0 Hz, Ar–H), 8.87 (s,
1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 66.68,
118.44, 122.58, 1233.81, 124.99, 127.45, 129.49, 130.25,
130.42, 130.94, 132.91, 133.09, 134.22, 139.62, 141.49,
5-Benzyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-one (6j)
Yield: 57.3%, m.p.: 244–246 °C. IR (KBr) cm-1: 1671
1
(C=O), 1122. H-NMR (DMSO-d6, 300 MHz) δ: 5.67 (s,
2H, –CH2–), 7.33–7.43 (m, 5H, Ar–H), 7.87 (t, 1H, J = 9.0
Hz, Ar–H), 8.01 (t, 1H, J = 9.0 Hz, Ar–H), 8.36 (d, 2H, J =

Med Chem Res
157.46. ESI-HRMS calcd for C16H12ClN4O+ ([M + H]+):
311.0694; found: 331.0691.
5-(2-(Trifluoromethyl)benzyl)-[1,2,4]triazolo[3,4-a]phthala-
zin-6(5H)-one (6s) Yield: 55.4%, m.p.: 222–224 °C. IR
(KBr) cm−1: 1658 (C=O), 1139. 1H-NMR (DMSO-d6, 300
MHz) δ: 5.66 (s, 2H, –CH2–), 7.80–7.88 (m, 4H, Ar–H),
7.90 (t, 1H, J = 9.0 Hz, Ar–H), 8.06 (t, 1H, J = 9.0 Hz,
Ar–H), 8.23 (d, 1H, J = 9.0 Hz, Ar–H), 8.48 (d, 1H, J =
9.0 Hz, Ar–H), 9.40 (s, 1H, Triazole-H). 13C-NMR (CDCl3,
75 MHz) δ: 52.03, 124.94, 125.58, 126.96, 127.72, 128.79,
129.26, 129.70, 130.11, 132.77, 133.47, 133.80, 136.75,
144.08, 152.93, 158.90. ESI-HRMS calcd for
C17H12F3N4O+ ([M + H]+): 345.0958; found: 345.0952.
5-(3-Chlorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6o) Yield: 60.2%, m.p.: 267–269 °C. IR (KBr) cm−1
:
1
1680 (C=O), 1143. H-NMR (CDCl3, 300 MHz) δ: 5.53(s,
2H, –CH2–), 7.39–7.44 (m, 3H, Ar–H), 7.55 (s, 1H, Ar–H),
7.81 (t, 1H, J = 9.0 Hz, Ar–H), 7.96 (t, 1H, J = 9.0 Hz,
Ar–H), 8.23 (d, 1H, J = 6.0 Hz, Ar–H), 8.64 (d, 1H, J =
6.0 Hz, Ar–H), 8.87 (s, 1H, Triazole-H). 13C-NMR
(DMSO-d6, 75 MHz) δ: 68.51, 118.45, 122.57, 123.61,
125.12, 126.74, 127.88, 128.30, 130.51, 131.08, 133.26,
134.30, 138.13, 139.72, 141.48, 157.50. ESI-HRMS calcd
for C16H12ClN4O+ ([M + H]+): 311.0694; found:
311.0684.
5-(3-(Trifluoromethyl)benzyl)-[1,2,4]triazolo[3,4-a]phthala-
zin-6(5H)-one (6t) Yield: 46.8%, m.p.: 276–278 °C. IR
(KBr) cm−1: 1671 (C=O), 1141. 1H-NMR (DMSO-d6, 300
MHz) δ: 5.65 (s, 2H, –CH2–), 7.68–7.74 (m, 1H, Ar–H),
7.79 (d, 1H, J = 6.0 Hz, Ar–H), 7.91–7.99 (m, 2H, Ar–H),
8.06 (t, 1 H, J = 6.0 Hz, Ar–H), 8.20 (d, 1H, J = 9.0 Hz,
Ar–H), 8.35 (t, 1H, J = 6.0 Hz, Ar–H), 8.49 (d, 1H, J = 9.0
Hz, Ar–H), 9.44 (s, 1H, Triazole-H). 13C-NMR (CDCl3, 75
MHz) δ: 51.26, 125.65, 125.97, 126.07, 126.51, 126.97,
127.43, 128.44, 128.80, 133.48, 134.98, 137.55, 146.03,
153.47, 158.95, 159.57. ESI-HRMS calcd for
C17H12F3N4O+ ([M + H]+): 345.0958; found: 345.0949.
5-(4-Chlorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6p) Yield: 47.4%, m.p.: 250–252 °C. IR (KBr) cm−1
:
1665 (C=O), 1120. 1H-NMR (DMSO-d6, 300 MHz) δ: 5.55
(s, 2H, –CH2–), 7.52 (d, 2H, J = 6.0 Hz, Ar–H), 7.65 (d,
2H, J = 6.0 Hz, Ar–H), 7.90 (t, 1H, J = 9.0 Hz, Ar–H), 8.05
(t, 1H, J = 9.0 Hz, Ar–H), 8.20 (d, 1H, J = 6.0 Hz, Ar–H),
8.48 (d, 1H, J = 6.0 Hz, Ar–H), 9.42 (s, 1H, Triazole-H).
13C-NMR (CDCl3, 75 MHz) δ: 73.78, 123.68, 127.77,
128.80, 129.98, 133.57, 134.55, 135.59, 138.69, 138.71,
138.99, 144.03, 146.91, 162.65. ESI-HRMS calcd for
C16H12ClN4O+ ([M + H]+): 311.0694; found: 311.0686.
5-(4-(Trifluoromethyl)benzyl)-[1,2,4]triazolo[3,4-a]phthala-
zin-6(5H)-one (6u) Yield: 55.7%, m.p.: 260–262 °C. IR
(KBr) cm−1: 1660 (C=O), 1112. 1H-NMR (DMSO-d6, 300
MHz) δ: 5.71 (s, 2H, –CH2–), 7.66 (t, 1H, J = 9.0 Hz,
Ar–H), 7.81 (t, 1H, J = 9.0 Hz, Ar–H), 7.85–7.99 (m, 3H,
Ar–H), 8.06 (t, 1H, J = 6.0 Hz, Ar–H), 8.17 (d, 1H, J = 9.0
Hz, Ar–H), 8.48 (d, 1H, J = 9.0 Hz, Ar–H), 9.45 (s, 1H,
Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 66.47,
109.19, 118.69, 123.04, 124.05, 125.44, 126.70, 127.68,
129.70, 131.31, 133.51, 134.81, 140.26, 141.86, 157.70.
ESI-HRMS calcd for C17H12F3N4O+ ([M + H]+):
345.0958; found: 345.0947.
5-(2-Bromobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6q) Yield: 50.2%, m.p.: 267–269 °C. IR (KBr) cm−1
:
1660 (C=O), 1135. 1H-NMR (DMSO-d6, 300 MHz) δ: 5.57
(s, 2H, –CH2–), 7.37 (t, 1H, J = 6.0 Hz, Ar–H), 7.49 (t, 1H,
J = 6.0 Hz, Ar–H), 7.73 (d, 1H, J = 6.0 Hz, Ar–H), 7.79 (d,
1H, J = 6.0 Hz, Ar–H), 7.89 (t, 1H, J = 9.0 Hz, Ar–H), 8.04
(t, 1H, J = 9.0 Hz, Ar–H), 8.19 (d, 1H, J = 6.0 Hz, Ar–H),
8.46 (d, 1H, J = 6.0 Hz, Ar–H), 9.46 (s, 1H, Triazole-H).
13C-NMR (DMSO-d6, 75 MHz) δ: 69.36, 118.75, 123.06,
123.39, 124.04, 125.51, 128.53, 130.89, 131.01, 131.56,
133.19, 134.74, 135.03, 140.27, 141.94, 157.76. ESI-
HRMS calcd for C16H12BrN4O+ ([M + H]+): 355.0189;
found: 355.0179.
5-(2,4-Dichlorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6
(5H)-one (6v) Yield: 43.8%, m.p.: 288–290 °C. IR (KBr)
cm−1: 1674 (C=O), 1135. 1H-NMR (DMSO-d6, 300 MHz)
δ: 5.60 (s, 2 H, –CH2–), 7.52–7.55 (m, 1H, Ar–H), 7.76 (s,
1H, Ar–H), 7.82 (d, 1H, J = 9.0 Hz, Ar–H), 7.90 (t, 1H, J
= 9.0 Hz, Ar–H), 8.06 (t, 1H, J = 9.0 Hz, Ar–H), 8.20 (d,
1H, J = 9.0 Hz, Ar–H), 8.47 (d, 1H, J = 9.0 Hz, Ar–H),
9.45 (s, 1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ:
66.68, 118.72, 123.08, 124.06, 125.50, 128.14, 129.49,
131.56, 132.09, 132.68, 134.20, 134.46, 134.86, 140.25,
141.89, 157.73. ESI-HRMS calcd for C16H11Cl2N4O+ ([M
+ H]+): 345.0304; found: 345.0299.
5-(4-Bromobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6r) Yield: 57.0%, m.p.: 291–293 °C. IR (KBr) cm−1
:
1668 (C=O), 1109. 1H-NMR (DMSO-d6, 300 MHz) δ: 5.53
(s, 2H, –CH2–), 6.58 (d, 1H, J = 6.0 Hz, Ar–H), 6.65 (d,
1H, J = 6.0 Hz, Ar–H), 7.89 (t, 1H, J = 9.0 Hz, Ar–H), 8.05
(t, 1H, J = 9.0 Hz, Ar–H), 8.18 (d, 1H, J = 9.0 Hz, Ar–H),
8.47 (d, 1H, J = 9.0 Hz, Ar–H), 9.41 (s, 1H, Triazole-H).
13C-NMR (CDCl3, 75 MHz) δ: 73.78, 123.66, 127.17,
127.74, 128.78, 130.01, 134.88, 135.65, 136.54, 138.77,
139.27, 144.09, 146.88, 162.62. ESI-HRMS calcd for
C16H12BrN4O+ ([M + H]+): 355.0189; found: 355.0183.
5-(2,6-Dichlorobenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6
(5H)-one (6w) Yield: 52.6%, m.p.: 244–246 °C. IR (KBr)

Med Chem Res
cm−1: 1662 (C=O), 1124. 1H-NMR (DMSO-d6, 300 MHz)
δ: 5.73 (s, 2H, –CH2–), 7.53 (t, 1H, J = 9.0 Hz, Ar–H), 7.62
(d, 2H, J = 9.0Hz, Ar–H), 7.84(t, 1H, J = 6.0 Hz, Ar–H),
8.03 (m, 2H, Ar–H), 8.48 (d, 1H, J = 9.0 Hz, Ar–H), 9.46
(s, 1H, Triazole-H). 13C-NMR (DMSO-d6, 75 MHz) δ:
65.18, 118.54, 123.12, 124.09, 125.39, 129.38,
131.00,131.57, 132.55, 134.88, 136.76, 140.25, 141.91,
157.86. ESI-HRMS calcd for C16H11Cl2N4O+ ([M + H]+):
345.0304; found: 345.0296.
triazole (0.13 g, 1.88 mmol) was then added, and the mix-
ture was stirred at 100 °C for 6 h. After the reaction was
completed, the solvent was evaporated under reduced
pressure. The residue was washed with water, filtered off,
and dried to yield compounds 8a–b, a solid.
2-Heptyl-4-(1H-1,2,4-triazol-1-yl)phthalazin-1(2H)-one
(8a) Yield: 40.2%, m.p.: 87–89 °C. IR (KBr) cm−1: 1674
(C=O), 1131. 1H-NMR (CDCl3, 300 MHz) δ: 0.84 (t, 3H, J
= 6.0 Hz, –CH3), 1.24–1.32 (m, 8H, –(CH2)4–), 1.74–1.83
(m, 2H, –CH2–), 4.16 (t, 2 H, J = 6.0 Hz, –CH2–),
7.85–7.88 (m, 1H, Ar–H), 7.97–8.02 (m, 2H, Ar–H),
8.37–8.40 (m, 1H, Ar–H), 8.44 (s, 1H, Triazole-1H), 9.12
(s, 1H, Triazole-1H). 13C-NMR (CDCl3, 75 MHz) δ: 14.35,
22.48, 26.41, 28.28, 28.75, 31.58, 125.27, 125.72, 127.25,
128.40, 133.53, 134.51, 136.82, 146.02, 153.39, 158.58.
ESI-HRMS calcd for C17H22N5O+ ([M + H]+): 312.1819;
found: 312.1810.
5-(4-Methylbenzyl)-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (6x) Yield: 54.8%, m.p.: 230–232 °C. IR (KBr) cm−1
:
1660 (C=O), 1130. 1H-NMR (CDCl3, 300 MHz) δ: 2.42 (s,
3H, –CH3), 5.51 (s, 2H, –CH2–), 7.27 (d, 2H, J = 6.0 Hz,
Ar–H), 7.45 (d, 2H, J = 6.0 Hz, Ar–H), 7.77(t, 1H, J = 9.0
Hz, Ar–H), 7.93 (t, 1H, J = 9.0 Hz, Ar–H), 8.21 (d, 1H, J =
9.0 Hz, Ar–H), 8.62 (d, 1H, J = 9.0 Hz, Ar–H), 8.88 (s, 1H,
Triazole-H). 13C-NMR (CDCl3, 75 MHz) δ: 21.27, 69.95,
119.17, 123.26, 124.19, 125.25, 128.51, 129.41, 130.59,
132.25, 133.73, 138.63, 139.27, 142.31, 158.21. ESI-
HRMS calcd for C17H15N4O+ ([M + H]+): 291.1240;
found: 291.1234.
4-(1H-1,2,4-triazol-1-yl)-2-(3-(trifluoromethyl)benzyl)phtha-
lazin-1(2H)-one (8b) Yield: 47.6%, m.p.: 101–103 °C. IR
(KBr) cm−1: 1685 (C=O), 1129. 1H-NMR (CDCl3, 300
MHz) δ: 5.57 (s, 2H, –CH2–), 7.36 (d, 1H, J = 6.0 Hz,
Ar–H), 7.52 (t, 1H, J = 6.0 Hz, Ar–H), 7.61 (t, 1H, J = 6.0
Hz, Ar–H), 7.81 (d, 1H, J = 9.0 Hz, Ar–H), 7.95–7.98 (m,
1H, Ar–H), 8.03–8.06 (m, 2H, Ar–H), 8.40–8.43 (m, 1H,
Ar–H), 8.44 (s, 1H, Triazole-1H), 9.05 (s, 1H, Triazole-1H).
13C-NMR (CDCl3, 75 MHz) δ: 51.26, 125.65, 125.97,
126.07, 126.51, 126.97, 127.43, 128.44, 128.80, 133.48,
133.83, 134.98, 137.55, 137.73, 146.03, 153.47, 158.95,
159.57. ESI-HRMS calcd for C18H13F3N5O+ ([M + H]+):
372.1067; found: 372.1058.
4-((6-Oxo-[1,2,4]triazolo[3,4-a]phthalazin-5(6H)-yl)methyl)
benzonitrile (6y) Yield: 52.3%, m.p.: 270–272 °C. IR
(KBr) cm−1: 1682 (C=O), 1189. 1H-NMR (DMSO-d6, 300
MHz) δ: 5.66 (s, 2H, –CH2–), 7.82 (d, 2H, J = 9.0 Hz,
Ar–H), 7.90–7.95 (m, 3H, Ar–H), 8.26(t, 1 H, J = 9.0 Hz,
Ar–H), 8.49 (d, 1H, J = 9.0 Hz, Ar–H), 9.42 (s, 1H, Tria-
zole-H). 13C-NMR (DMSO-d6, 75 MHz) δ: 53.99, 125.57,
125.90, 127.34, 128.49, 128.99, 130.43, 132.82, 133.71,
134.74, 135.91, 137.36, 146.10, 153.44, 158.66. ESI-HRMS
calcd for C17H12N5O+ ([M + H]+): 302.1036; found:
302.1033.
Synthesis of 2-substituted-4-(1H-pyrazol-1-yl)phthalazin-1
(2H)-one (9a–b)
Synthesis of 2-substituted-4-chlorophthalazin-1(2H)-one
(7a–b)
A mixture of compound 7 (0.5 g, 1.8 mmol) and K2CO3
(0.5 g, 3.6 mmol) was dissolved in N,N-dimethylformamide
(30 ml) and stirred at room temperature for 30 min. 1H-
pyrazole (0.13 g, 1.9 mmol) was then added, and the mix-
ture was stirred at 100 °C for 6 h. After the reaction was
completed, the solvent was evaporated under reduced
pressure. The residue was washed with water, filtered off,
and dried to yield compounds 9a–b, a solid.
A mixture of compound 3 (1.0 g, 5.6 mmol) and NaH (0.26 g,
11.2 mmol) was dissolved in N,N-dimethylformamide (30
ml). The mixture was stirred and refluxed for 30 min. Alkyl
bromide or benzyl chloride was added as appropriate, and
the mixture was stirred at 80°C for 3 h. After the reaction
was completed, the solvent was removed under reduced
pressure. The residue was washed with water, filtered off,
and dried to yield compounds 7a–b as a white powder.
2-Heptyl-4-(1H-pyrazol-1-yl)phthalazin-1(2H)-one (9a)
Yield: 50.4%, m.p.: 54–56 °C. IR (KBr) cm−1: 1660
1
Synthesis of 2-substituted-4-(1H-1,2,4-triazol-1-yl)
(C=O), 1135. H-NMR (CDCl3, 300 MHz) δ: 0.75 (s, 3H,
phthalazin-1(2H)-one (8a–b)
–CH3), 1.17–1.25 (m, 8H, –(CH2)4–), 1.76–1.77 (m, 2H,
–CH2–), 4.12–4.13 (m, 2H, –CH2–), 6.39 (m, 1H, Pyrazole-
H), 7.65–7.71 (m, 3H, Ar–H), 7.97–7.99 (m, 1H, Ar–H),
8.27–8.35 (m, 2H, Pyrazole-2H). 13C-NMR (CDCl3, 75
MHz) δ: 13.99, 22.48, 26.54, 28.37, 28.85, 31.61, 51.03,
A mixture of compound 7 (0.5 g, 1.8 mmol) and K2CO3
(0.5 g, 3.6 mmol) was dissolved in N,N-dimethylformamide
(30 ml) and stirred at room temperature for 30 min. 1,2,4-

Med Chem Res
106.94, 125.56, 126.16, 127.08, 128.83, 130.35, 131.92,
133.02, 138.97, 141.92, 158.68. ESI-HRMS calcd for
C18H23N4O+ ([M + H]+): 311.1866; found: 311.1858. MS:
m/z 311.13 (M + H).
142.34, 158.97. ESI-HRMS calcd for C19H14F3N4O+ ([M
+ H]+): 371.1114; found: 371.1107.
Synthesis of 4-(3,5-dimethyl-1H-pyrazol-1-yl)phthalazin-1
(2H)-one (11)
4-(1H-pyrazol-1-yl)-2-(3-(trifluoromethyl)benzyl)phthala-
zin-1(2H)-one (9b) Yield: 46.7%, m.p.: 128–130 °C. IR
Compound 4 (2.0 g, 11.4 mmol) was dissolved in acetoni-
trile (30 ml). Acetylacetone (1.2 g, 12.0 mmol) was added,
and the mixture was stirred at 82 °C for 6 h. The solvent was
evaporated under reduced pressure, and then dried to yield
compound 11.
1
(KBr) cm−1: 1668 (C=O), 1125. H-NMR (CDCl3, 300
MHz) δ: 5.47 (s, 2H, –CH2–), 6.55 (s, 1H, Pyrazole-H),
7.48 (t, 1H, J = 6.0 Hz, Ar–H), 7.59 (m, t, 1H, J = 6.0 Hz,
Ar–H), 7.80 (d, 1H, J = 9.0 Hz, Ar–H), 7.79–7.88(m, 4H,
Ar–H), 8.03–8.04 (m, 1H, Ar–H), 8.45–8.54 (m, 2H, Pyr-
azole-H). 13C-NMR (CDCl3, 75 MHz) δ: 54.15, 101.28,
124.84, 124.89, 125.59, 125.64, 125.87, 126.66, 127.49,
128.97, 129.18, 130.51, 132.22, 132.44, 133.60, 137.27,
139.77, 142.34, 158.97. ESI-HRMS calcd for
C19H14F3N4O+ ([M + H]+): 371.1114; found: 371.1119.
Synthesis of 2-substituted-4-(3,5-dimethyl-1H-pyrazol-1-yl)
phthalazin-1(2H)-one (12a–b)
A mixture of compound 11 (1.0 g, 4.2 mmol) and K2CO3
(0.58 g, 4.2 mmol) was dissolved in N,N-dimethylforma-
mide (30 ml) and stirred at room temperature for 30 min.
Alkyl bromide or benzyl chloride was added as appropriate,
and the mixture was stirred at 80 °C for 3 h. The reaction
mixture was evaporated under reduced pressure, washed
with water, and dried and purified using silica gel column
chromatography (CH2Cl2/CH3OH, 80:1) to yield com-
pounds 12a–b, a white powder.
Synthesis of 2-substituted-4-(1H-imidazol-1-yl)phthalazin-1
(2H)-one (10)
A mixture of compound 7 (0.5 g, 1.8 mmol) and K2CO3
(0.5 g, 3.6 mmol) was dissolved in N,N-dimethylformamide
(30 ml) and stirred at room temperature for 30 min. 1H-
imidazole (0.13 g, 1.9 mmol) was added, and the mixture
stirred at 100 °C for 6 h. After the reaction was completed,
the solvent was evaporated under reduced pressure. The
residue was washed with water, filtered off, and then dried
to yield compound 10, a solid.
4-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-heptylphthalazin-1
(2H)-one (12a) Yield: 37.5%, m.p.: 232–234 °C. IR
(KBr) cm−1: 1654 (C=O), 1121. H-NMR (CDCl3, 300
1
MHz) δ: 0.87 (t, 3H, J = 6.0 Hz, –CH3), 1.28–1.37 (m, 8H,
–(CH2)4–), 1.82–1.92 (m, 2H, –CH2–), 2.30 (s, 3H, –CH3),
2.35 (s, 3H, –CH3), 4.26 (t, 2H, J = 9.0 Hz, Ar–H), 6.09 (s,
1H, pyrazole-H), 7.63–7.66 (m, 1H, Ar–H), 7.79–7.82 (m,
2H, Ar–H), 8.48–8.51 (m, 1H, Ar–H). 13C-NMR (CDCl3,
75 MHz) δ: 11.64, 13.68, 14.05, 22.56, 26.65, 28.57, 28.97,
31.70, 51.29, 106.84, 125.53, 127.18, 127.69, 128.84,
132.10, 133.36, 139.14, 141.95, 150.42, 159.11. ESI-
HRMS calcd for C20H27N4O+ ([M + H]+): 339.2179;
found: 339.2168.
2-Heptyl-4-(1H-imidazol-1-yl)phthalazin-1(2H)-one (10a)
Yield: 39.7%, m.p.: 67–69 °C. IR (KBr) cm−1: 1651 (C=O),
1135. H-NMR (CDCl3, 300 MHz) δ: 0.87 (s, 3H, –CH3),
1
1.28–1.37 (m, 8H, –(CH2)4–), 1.87–1.88 (m, 2H, –CH2–),
4.24 (t, 2H, J = 6.0 Hz, –CH2–), 7.31–7.32 (m, 1H, Ar–H),
7.37 (s, 1H, Imidazole-H), 7.60–7.62 (m, 1H, Imidazole-H),
7.86–7.93 (m, 3H, Ar–H), 8.53–8.55 (m, 1H, Imidazole-H).
13C-NMR (CDCl3, 75 MHz) δ: 11.04, 22.55, 26.61, 28.42,
28.90, 31.68, 51.37, 119.90, 123.63, 126.25, 127.89,
128.79, 130.05, 132.61, 133.72, 136.97, 137.36, 158.73.
ESI-HRMS calcd for C18H23N4O+ ([M + H]+): 311.1866;
found: 311.1853.
4-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-(3-(trifluoromethyl)
benzyl)phthalazin-1(2H)-one (12b) Yield: 32.0%, m.p.:
1
207–209 °C. IR (KBr) cm−1: 1674 (C=O), 1118. H-NMR
4-(1H-imidazol-1-yl)-2-(3-(trifluoromethyl)benzyl)phthala-
zin-1(2H)-one (10b) Yield: 41.8%, mp: 180–182 °C. IR
(CDCl3, 300 MHz) δ: 2.17 (s, 3H, –CH3), 2.35 (s, 3H,
–CH3), 5.48 (s, 2H, –CH2–), 6.08 (s, 1H, pyrazole-H), 7.47
(t, 1H, J = 9.0 Hz, Ar–H), 7.58 (t, 1H, J = 9.0 Hz, Ar–H),
7.68–7.75 (m, 3H, Ar–H), 7.78–7.84 (m, 2 H, Ar–H),
8.48–8.52 (m, 1H, Ar–H). 13C-NMR (CDCl3, 75 MHz) δ:
11.49, 13.67, 54.10, 107.08, 124.79, 124.84, 125.49,
125.54, 126.08, 127.34, 127.73, 128.83, 129.17, 132.33,
132.42, 133.72, 137.41, 139.75, 142.75, 142.08, 150.63,
159.07. ESI-HRMS calcd for C21H18F3N4O+ ([M + H]+):
399.1427; found: 399.1418.
1
(KBr) cm−1: 1646 (C=O), 1126. H-NMR (CDCl3, 300
MHz) δ: 5.45 (s, 2H, –CH2–), 7.31–7.35 (m, 2H, Imidazole-
H), 7.48 (t, 1H, J = 9.0 Hz, Ar–H), 7.57–7.65 (m, 2H,
Ar–H), 7.71 (d, 1H, J = 6.0 Hz, Ar–H), 7.75 (s, 1H, Ar–H),
7.86–7.90 (m, 3H, Ar–H), 8.54–8.57(m, 1H, Imidazole-H).
13C-NMR (CDCl3, 75 MHz) δ: 54.15, 101.28, 124.84,
124.89, 125.59, 125.64, 125.87, 126.66, 127.49, 128.97,
129.18, 130.51, 132.22, 132.44, 133.60, 137.27, 139.77,

Med Chem Res
Synthesis of 3-methyl-[1,2,4] triazolo[3,4-a]phthalazin-6
(5H)-one (13)
induced seizures was defined as the abolition of the hind leg
and tonic maximal extension component of the seizure
(Krall et al. 1978; Porter et al. 1984).
A mixture of compound 4 (2.0 g, 11.4 mmol) in acetic
anhydride (30 ml) was stirred and heated at 100 °C for 3 h.
After the reaction was completed, the solvent was removed
under reduced pressure. The residue was washed with
water, and dried to give compound 13, a white solid.
Neurotoxicity (NT) screening
The neurotoxicity of the compounds was measured in mice
by the rotarod test. The mice were trained to stay on an
accelerating rotarod of diameter 3.2 cm that rotates at 10 r.p.
m. Trained animals were given an intraperitoneal injection
of the test compounds. Neurotoxicity was indicated by the
inability of the animal to maintain equilibrium on the rod
for at least 1 min in each of the trials.
Synthesis of 5-substituted-3-methyl-[1,2,4]triazolo[3,4-a]
phthalazin-6(5H)-one (14a-b)
A mixture of compound 13 (1.0 g, 5.0 mmol) and K2CO3
(0.69 g, 5.0 mmol) was dissolved in N,N-dimethylforma-
mide (30 ml) and stirred at room temperature for 30 min.
Alkyl bromide or benzyl chloride was added as appropriate,
and the mixture stirred at 80 °C for 3 h. The reaction mix-
ture was evaporated under reduced pressure, washed with
water, and dried and purified using silica gel column
chromatography (CH2Cl2/CH3OH, 50:1) to yield com-
pounds 14a–b, a white powder.
sc-PTZ induced seizures test
At 0.5 h after the administration of the test compounds,
85 mg/kg PTZ dissolved in saline was administered sc. The
animals (10 mice in one group) were placed in individual
cages and observed for 0.5 h. The numbers of clonic sei-
zures (rang from exaggerated twitches of the limbs to vio-
lent shaking or vibrating of the stiffened extremities) and
tonic seizures (the extremities pull towards the body or
rigidly push away from it, usually maximal extension of the
hind leg, as well as the numbers of deaths were noted
(Zomkowski et al. 2006; Elliott et al. 1986).
5-Heptyl-3-methyl-[1,2,4]triazolo[3,4-a]phthalazin-6(5H)-
one (14a) Yield: 42.2%, m.p.: 258–260 °C. IR (KBr)
1
cm−1: 1634 (C=O), 1134. H-NMR (CDCl3, 300 MHz) δ:
0.92 (s, 3H, –CH3), 1.35–1.55 (m, 8H, –(CH2)4–),
1.92–1.96 (m, 2H, –CH2–), 2.74 (s, 3H, –CH3), 4.51 (t, 2H,
J = 9.0 Hz, –CH2–), 7.74 (t, 1H, J = 9.0 Hz, Ar–H), 7.85 (t,
1H, J = 9.0 Hz, Ar–H), 8.16 (d, 1H, J = 9.0 Hz, Ar–H),
8.56 (d, 1H, J = 9.0 Hz, Ar–H). 13C-NMR (CDCl3, 75
MHz) δ: 9.79, 14.08, 22.61, 26.07, 28.60, 29.01, 31.75,
68.20, 118.74, 123.04, 124.57, 125.01, 130.18, 133.40,
142.43, 147.59, 157.88. ESI-HRMS calcd for C17H23N4O+
([M + H]+): 299.1866; found: 299.1858.
Bicuculline-induced seizures test
At 0.5 h after the administration of compounds, the animals
(10 mice in one group) were given a subcutaneous dose of
2.7 mg/kg for Bicuculline (within 15–45 min after pre-
paration due to instability). Individual mice were then
placed in isolation cages and observed for at least 0.5 h for
the presence or absence of clonic seizures, and tonic sei-
zures, and lethality was also recorded (Shank et al. 1994).
3-Methyl-5-(3-(trifluoromethyl)benzyl)-[1,2,4]triazolo[3,4-
a]phthalazin-6(5H)-one (14b) Yield: 49.9%, m.p.:
1
240–242 °C. IR (KBr) cm−1: 1625 (C=O), 1123. H-NMR
Acknowledgements This work was supported by the National
(CDCl3, 300 MHz) δ: 2.76 (s, 3H, –CH3), 5.63 (s, 2H,
–CH2–), 7.59 (t, 1H, J = 9.0 Hz, Ar–H), 7.69 (t, 1H, J =
9.0 Hz, Ar–H), 7.74–7.79 (m, 2H, Ar–H), 7.86–7.94 (m,
2H, Ar–H), 8.20 (d, 1H, J = 9.0 Hz, Ar–H), 8.59 (d, 1H, J
= 9.0 Hz, Ar–H). 13C-NMR (CDCl3, 75 MHz) δ: 9.90,
50.86, 118.45, 123.32, 125.07, 125.35, 125.40, 125.61,
129.41, 130.53, 131.74, 133.94, 136.57, 142.53, 147.72,
157.45. ESI-HRMS calcd for C18H14F3N4O+ ([M + H]+):
359.1114; found: 359.1111.
Natural Science Foundation of China (No. 81360468 and 21662036)
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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