Design, synthesis of 8-alkoxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-ones with anticonvulsant activity

Article abstract of DOI:10.1016/j.ejmech.2008.09.003

A new series of 8-alkoxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-one derivatives were synthesized. Their anticonvulsant activities were evaluated by the maximal electroshock (MES) test, and their neurotoxicities were evaluated by the rotarod neurotox

Full text of DOI:10.1016/j.ejmech.2008.09.003

European Journal of Medicinal Chemistry 44 (2009) 1265–1270
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis of 8-alkoxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-ones
with anticonvulsant activity
,
,b,
*
Xian-Yu Sun a b, Lei Zhang a, Cheng-Xi Wei a, Hu-Ri Piao a, Zhe-Shan Quan a
a Key Laboratory of Organism Functional Factors of the Changbai Mountain, Yanbian University, Ministry of Education, Yanji, Jilin 133002, China
b College of Pharmacy, Yanbian University, Yanji, Jilin 133002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 April 2008
Received in revised form 16 July 2008
Accepted 1 September 2008
Available online 16 September 2008
A new series of 8-alkoxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-one derivatives were synthesized.
Their anticonvulsant activities were evaluated by the maximal electroshock (MES) test, and their
neurotoxicities were evaluated by the rotarod neurotoxicity test. The results showed that 8-heptyloxy-
5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-one 5t was the most potent with median effective dose
(ED50) value of 11.4 mg/kg, median toxicity dose (TD50) of 114.1 mg/kg, providing a protective index
(PI ¼ TD50/ED50) value of 10.0, which is much greater than the PI of the prototype drug carbamazepine
(PI ¼ 6.4). To explain the possible mechanism of anticonvulsant activity, the compound 5t was tested in
chemically induced seizures.
Keywords:
1,2,4-Triazino[4,3-a]quinolin-1-one
Triazone
Anticonvulsant
MES
Ó 2008 Elsevier Masson SAS. All rights reserved.
Pentylenetetrazole
Isoniazid
Thiosemicarbazide
3-Mercaptopropionic acid
Strychnine
1. Introduction
7-benzyloxyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline(compound
II), which showed ED50 values of 17.3 mg/kg and 24 mg/kg in the MES
Epilepsy,
a
ubiquitous disease characterized by recurrent
and the sc PTZ tests, respectively [7].
seizures, inflicts more than 60 million people worldwide according
to epidemiological studies [1]. For epilepsy treatment, nearly 95% of
clinically available drugs were approved before 1985 and they could
provide satisfactory seizure control for 60–70% of patients. These
drugs, however, also cause notable adverse side effects such as
drowsiness, ataxia, gastrointestinal disturbance, hepatotoxicity and
megaloblastic anemia [2–4], and even life threatening conditions
[5]. Research to find more effective and safer antiepileptic drugs is,
therefore, imperative and challenging in medicinal chemistry.
In our previous work [6], a series of derivatives of 6-alkoxy-3,4-
dihydro-2(1H)-quinoline were first found to have anticonvulsant
activities, among which 6-benzyloxy-3,4-dihydro-2(1H)-quinoline
(compound I) showed the strongest activity with an ED50 value of
29.6 mg/kg in the MES test Fig. 1. Introduction of triazole ring to
Aimed at exploring effective compounds with better anticon-
vulsant activity and lower neurotoxicity, compounds 5a–5v, 6a
and 6s were designed and synthesized, which substituted by
triazone the triazole in compound II. The hypothesis was that
triazone compound may have higher affinity to the receptor due to
the carbonyl group, and thus may increase the anticonvulsant
activity. As shown in the preliminary work [8], the introduction of
alkoxy to the 7th position of 1,2,4-triazole quinoline remarkably
increased their anticonvulsant activity. So, we designed and
synthesized a series of 8-alkoxy-5,6-dihydro-[1,2,4]triazino[4,3-
a]quinolin-1-one derivatives, to discuss the influence of the
8-alkoxy to anticonvulsant activity; the structure–activity rela-
tionship was also discussed in this paper. Their structures were
characterized using IR, 1H NMR, MS, and elemental analysis
techniques. In additional, their anticonvulsant activity was eval-
uated using MES test and reported for the first time. Their
neurotoxicity was evaluated using the rotarod test in mice. For
explaining the possible mechanism of action, the compound 5t
was tested in pentylenetetrazole (PTZ), isoniazid, thio-
semicarbazide, 3-mercaptopropionic acid (3-MP) and strychnine
induced test. The anti-MES activity and the neurotoxicity of the
the first and second positions of this compound
I caused
a remarkable increase in the anticonvulsant activity, as seen in
* Corresponding author. College of Pharmacy, Yanbian University, No. 121 JuZi
Street, Yanji City, Jilin Province 133000, China. Tel.: þ86 433 2660606; fax: þ86 433
2660568.
E-mail address: zsquan@ybu.edu.cn (Z.-S. Quan).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.003

1266
X.-Y. Sun et al. / European Journal of Medicinal Chemistry 44 (2009) 1265–1270
filtered and washed with petroleum ether, and then kept below
0 ꢀC. The compounds obtained were pure enough for the next step.
The structures of compounds 4a–4v may change gradually at room
temperature in about 10 h, and the molecular weight indicated the
disubstitution of hydrazine with compounds 3a–3v, as shown in
Scheme 2. But this change could be avoided by keeping compounds
4a–4v below 0 ꢀC.
O
O
N
N
N
N
H
O
compound I
compound II
Compounds 4a–4v reacted further with glyoxylic acid mono-
hydrate in ethanol at room temperature, then evaporated the solvent
under reduced pressure, the residue continued to react in refluxing
toluene for about 2 h to obtain target compounds 5a–5v. Compounds
4a and 4s reacted further with pyruvic acid in ethanol at room
temperature, then evaporated the solvent under reduced pressure,
the residue continued to react in refluxing acetic acid for about 2 h to
obtain target compounds 8-(benzyloxy)-2-methyl-5,6-dihydro-
[1,2,4]triazino[4,3-a]quinolin-1-one 6a and 8-hexyloxy-2-methyl-
5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-one 6s, respectively [9].
Fig. 1. Previous active quinolines reported by Quan et al. I: 6-(benzyloxy)-3,4-dihy-
droquinolin-2(1H)-one; II: 7-benzyloxyl-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline.
marketed agent carbamazepine were evaluated in our laboratory
under the same conditions for the purpose of comparison.
2. Results and discussions
2.1. Chemistry
2.2. Pharmacology
Compounds were prepared according to Scheme 1. The starting
material 6-hydroxy-3,4-dihydro-2(1H)-quinolone reacted with an
appropriate amount of alkyl halide in a solution of sodium
hydroxide in DMF and yielded 6-alkoxy-3,4-dihydroquinolin-
2(1H)-one 2a–2v. 6-Alkoxy-3,4-dihydroquinolin-2(1H)-thiones
3a–3d and 3j–3v were prepared by the reaction of compounds 2a–
2d and 2j–2v with phosphorous pentasulfide in acetonitrile in the
presence of triethylamine. Since compounds 2e–2i, with Cl atom
substituted at 8-alkoxy group, did not dissolve in acetonitrile,
toluene was used as solvent and compounds 2e–2i and phospho-
rous pentasulfide reacted in toluene with stirring and refluxing,
which produced derivatives 3e–3i with a moderate yield.
Compounds 3a–3v reacted further with hydrazine hydrate in
THF to afford 1-(6-alkoxy-3,4-dihydroquinolin-2(1H)-ylidene)hy-
drazines 4a–4v. Briefly, to a solution of hydrazine hydrate in THF,
a solution of compounds 3a–3v in THF was added dropwise at room
temperature, and the mixture was stirred at 60 ꢀC for 1 h. Then, half
of the solvent was removed under reduced pressure, and the
mixture was poured into petroleum ether. The precipitate was
The results of pharmacology test of all synthesized compounds
and reference drug are shown in Table 1. As shown in Table 1, most of
the compounds showed remarkable anticonvulsant activity. But 1-
methyl derivatives 6a and 6s did not exhibit anticonvulsant activityat
dosages up to 100 mg/kg. The result illustrated that 1-methyl may
block the combination of 1,2,4-triazinone ring and receptor.
Analyzing the activities of 14 phenyl-substituted derivatives 5a–
5n, the following structure–activity relationship was obtained.
Compared the activities of these halogen atom substituted deriva-
tives; it was found that the Cl atom gave more contribution to the
anticonvulsant activity than the Br, F atom on the whole level. And
the position of halogen atom on the phenyl ring greatly influenced
the anticonvulsant activity, the activity order is o > m > p. The o-Cl
derivative 5g exhibited the most potent activity with ED50 value of
13.7 mg/kg and TD50 value of 105.6 mg/kg, providing a PI value of
7.7. Although the o-Br derivative 5j exhibited weaker activity than
5g, with ED50 value of 13.7 mg/kg, it possessed lower neurotoxicity
and a safer PI value was gained. The derivatives 5h and 5i, con-
taining double Cl atom, did not exhibited better anticonvulsant
activity than derivatives with single Cl atom. The compounds 5h
and 5i, which were 2,4-Cl2 and 2,6-Cl2 substituted-benzyloxyl at
the eighth position, respectively, had corresponding ED50 values in
the MES test of 26.4 mg/kg and 20.5 mg/kg. Compared the influ-
ence of electric-donor group to anticonvulsant activity, their
contribution order is p-CH3 > 3,4-(OCH3)2– > p-OCH3. Three elec-
tric-donor derivatives 5l–5n showed weaker anticonvulsant
activity ranging from 27.4 mg/kg to 56.8 mg/kg than non-
substituted derivative 5a with ED50 value of 22.8 mg/kg.
The length of the alkyl chain appeared to have a direct impact
on anticonvulsant activity of the 8-alkoxy-5,6-dihydro-[1,2,4]tri-
azino[4,3-a]quinolin-1-one derivatives. From compounds 5m–5v,
as alkyl chain length increased, ED50 gradually increased with the
compound 5t (with the n-heptyl substituted group) being the
most active. The trend reversed, however, when the alkyl chain
had more than seven carbon atoms. Compound 5t, with ED50 value
of 11.4 mg/kg, was comparable to reference agent carbamazepine
in anti-MES activity, but it possessed weaker neurotoxicity with
TD50 value of 114.1 mg/kg, so a safer PI value of 10.00 was
RO
HO
P2S5
RX
NaOH
N
H
2a-2v
O
N
H
O
1
RO
RO
NH2NH2·H2O
N
H
NNH2
N
H
S
3a-3v
4a-4v
RO
R1COCOOH
N
O
N
N
RO
RO
H
N
N
N
H
N
R1
N
H
NNH2
5a-5v, 6a, 6s
OR
Scheme 1. The synthesis route of target compounds 5a–5v, 6a and 6s.
Scheme 2. Structural change of compounds 4a–4v.

X.-Y. Sun et al. / European Journal of Medicinal Chemistry 44 (2009) 1265–1270
1267
Table 1
Quantitative anticonvulsant activity in MES test in mice (test drug administered ip)
O
R
N
N
N
O
R1
Comp.
R
R1
ED50 (mg/kg)
TD50c (mg/kg)
PI
b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
6a
6s
–C6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–CH3
–CH3
–
22.8 (15.3–28.4)a
38.0 (27.9–51.8)
31.7 (23.3–43.2)
23.7 (17.1–32.9)
32.9 (24.1–44.8)
22.8 (16.7–31.1)
13.7 (10.1–18.7)
26.4 (19.6–34.7)
20.5 (16.5–25.4)
16.4 (12.0–22.3)
64.2 (47.1–87.5)
56.8 (40.9–78.8)
27.4 (20.1–37.4)
45.6 (33.5–62.1)
39.4 (28.9–53.6)
32.9 (24.1–44.8)
22.7 (16.4–31.5)
21.9 (16.1–29.8)
19.7 (14.5–26.8)
11.4 (8.4–15.5)
12.7 (9.3–17.3)
54.8 (40.2–74.7)
>100
–
–
–
–
–
–
–
–
–
–
7.7
–
–
8.0
–
–
–
–
–
–
–
–
–C6H4(p-F)
–C6H4(m-F)
–C6H4(o-F)
–C6H4(p-Cl)
–C6H4(m-Cl)
–C6H4(o-Cl)
–C6H3(2,4-Cl2)
–C6H3(2,6-Cl2)
–C6H4(o-Br)
–C6H4(p-Br)
–C6H4(p-OCH3)
–C6H4(p-CH3)
–C6H3(3,4-(OCH3)2)
n-C2H5
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C12H25
–C6H5
–
–
105.6 (79.3–140.5)
–
–
132.0 (99.2–175.7)
–
–
–
–
–
–
–
–
82.2 (60.3–112.0)
114.1 (83.7–155.5)
98.6 (72.4–134.4)
4.2
10.0
7.76
–
–
–
–
–
6.4
n-C6H13
–
>100
11.8 (8.5–16.4)
Carbamazepine
76.1(55.8–103.7)
a
The 95% confidence limits.
Not tested.
b
c
Taking consideration of their anti-MES activity, only compounds with an ED50 value lower than 20 mg/kg in the MES test have been selected to neurotoxicity test.
obtained. Compound 5t, could be considered as a potentially
useful and safe therapeutic. And the anticonvulsant activity
decreased obviously when alkyl chain number lengthened to 12,
even showed effective anticonvulsant activity at dosages up to
54.8 mg/kg.
GABAergic neurotransmission is known to inhibit or attenuate
seizures. The findings of the present study tend to suggest that the
derivatives in this study might have inhibited or attenuated pen-
tylenetetrazole-induced and isoniazid-induced seizures in mice by
enhancing GABAergic neurotransmission.
As a result of preliminary test, compound 5t exhibited the
strongest anti-MES activity and the lowest neurotoxicity, it was
then selected for further investigations against seizures induced by
pentylenetetrazole, 3-MP, thiosemicarbazide, isoniazid and
strychnine to prove its anticonvulsant activity and speculate about
the possible mechanism of anticonvulsant action. As shown in
Table 2, compound 5t was effective against the seizures induce by
pentylenetetrazole, isoniazid, 3-mercaptopropionic acid and thio-
semicarbazide, and the potency of compound 5t was stronger than
carbamazepine in these chemically induced seizures.
Pentylenetetrazole and isoniazid have been reported to produce
seizures by inhibiting gamma-aminobutyric acid (GABA) neuro-
transmission [10]. GABA is the main inhibitory neurotransmitter
substance in the brain, and is widely implicated in epilepsy. Inhi-
bition of GABAergic neurotransmission or activity has been shown
to promote and facilitate seizures, while enhancement of
3-Mercaptopropionic acid and thiosemicarbazide were seen as
the competitive inhibitors of GABA synthesis; enzyme glutamate
decarboxylase (GAD) inhibited the synthesis of GABA to decrease
the GABA level in the brain [11]. The moderate antagonism of 3-
mercaptopropionic acid-induced and thiosemicarbazide-induced
seizures suggests that this series of compounds might activate GAD
or inhibit (GABA)-
a-oxoglutarate aminotransferase (GABA-T) in the
brain.
As shown in Table 2, compound 5t failed to protect animals from
seizure induced by strychnine. It is known that strychnine directly
antagonizes the inhibitory spinal reflexes of glycine [12], so the result
suggested that compounds could not influence glycine system.
In conclusion, in the present study, we found that 8-heptyloxy-
5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-one (5t) possessed
comparable anticonvulsant activity to marketed drug carbamaze-
pine, with ED50 value of 11.4 mg/kg, but a safer PI value of 10.0 was
Table 2
Anticonvulsant activity of compound 5t in chemically induced seizures tests
Comp.
sc PTZ
ip Isoniazid
ip Thiosemicarbazide
sc 3-MP
ip Strychnine
a
5t
11.3 (8.7–14.5)
11.4 (8.4–15.5)
19.7 (14.5–26.8)
22.8 (16.7–31.1)
15.8 (11.6–21.5)
19.7 (14.5–26.8)
11.8 (8.5–16.4)
13.7 (10.0–18.7)
–
–
Carbamazepine
a
Compound failed to control the seizure induced by strychnine.

1268
X.-Y. Sun et al. / European Journal of Medicinal Chemistry 44 (2009) 1265–1270
obtained. Especially, compound 5t produced significant antagonism
activity against seizures induced by pentylenetetrazole, 3-mercap-
topropionic acid, thiosemicarbazide and isoniazid, suggested that
the compound 5t might have effects on GABAergic neurotransmis-
sion and activate glutamate decarboxylase (GAD) or inhibit (GABA)-
pressure, the acid intermediate continued to react in refluxing acetic
acid for about 2 h to obtain targets 6a and 6s, respectively.
3.2.1. 8-Benzyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5a)
a-oxoglutarate aminotransferase (GABA-T) in the brain.
M.p. 167–169 ꢀC, yield ¼ 58.6%. 1H NMR (CDCl3, 300 MHz)
d
3.02–3.25 (m, 4H, H-5, H-6), 5.10 (s, 2H, –OCH2–), 6.94–7.43 (m,
8H, Ar-H), 8.44 (s, 1H, H-2). IR (KBr) cmꢁ1: 1702 (C]O). MS m/z 304
(M þ 1). Anal. Calcd. for C18H15N3O2: C 70.81, H 4.95, N 13.76.
Found: C 70.92, H 4.97, N 13.73.
3. Experimental procedures
3.1. Chemistry
3.2.2. 8-(4-Fluorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5b)
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) on an
FTIR1730. 1H NMR spectra were measured on an AV-300 (Bruker,
Switzerland), and all chemical shifts were given in parts per million
relative to tetramethylsilane. Mass spectra were measured on an
HP1100LC (Agilent Technologies, USA). Elemental analyses were
performed on a 204Q CHN (Perkin–Elmer, USA). Microanalyses of C,
N, and H were performed using a Heraeus CHN Rapid Analyzer. The
major chemicals were purchased from Aldrich Chemical Corpora-
tion. All other chemicals were of analytical grade.
M.p. 126–128 ꢀC, yield ¼ 63.7%. 1H NMR (CDCl3, 300 MHz)
d
3.01–3.23 (m, 4H, H-5, H-6), 5.06 (s, 2H, –OCH2–), 6.93–7.44 (m,
7H, Ar-H), 8.57 (s, 1H, H-2). IR (KBr) cmꢁ1: 1698 (C]O). MS m/z 324
(M þ 1). Anal. Calcd. for C18H14FN3O2: C 66.87, H 4.36, N 13.00.
Found: C 66.90, H 4.37, N 13.02.
3.2.3. 8-(3-Fluorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5c)
M.p. 121–123 ꢀC, yield ¼ 60.3%. 1H NMR (CDCl3, 300 MHz)
d
3.01–3.24 (m, 4H, H-5, H-6), 5.10 (s, 2H, –OCH2–), 6.92–7.39 (m,
3.1.1. 6-Alkoxy-3,4-dihydro-2(1H)-quinolones (2a–2v)
7H, Ar-H), 8.58 (s, 1H, H-2). IR (KBr) cmꢁ1: 1704 (C]O). MS m/z 324
(M þ 1). Anal. Calcd. for C18H14FN3O2: C 66.87, H 4.36, N 13.00.
Found: C 66.91, H 4.37, N 13.01.
The starting compound 1 (10 mmol) and appropriate alkyl
halide (10 mmol) were added to a solution of sodium hydroxide in
DMF with stirring and refluxing for 3 h. The reaction mixture was
cooled and then poured into ice water. The white precipitate was
collected through filtration and dried in a vacuum to produce the
crude products 2a–2v with a moderate yield and sufficient purity
for the next stage.
3.2.4. 8-(2-Fluorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5d)
M.p. 200–202 ꢀC, yield ¼ 65.9%. 1H NMR (CDCl3, 300 MHz)
d
3.02–3.24 (m, 4H, H-5, H-6), 5.17 (s, 2H, –OCH2–), 6.96–7.53 (m,
7H, Ar-H), 8.59 (s, 1H, H-2). IR (KBr) cmꢁ1: 1701 (C]O). MS m/z 324
(M þ 1). Anal. Calcd. for C18H14FN3O2: C 66.87, H 4.36, N 13.00.
Found: C 66.91, H 4.37, N 13.02.
3.1.2. 6-Alkoxy-3,4-dihydro-1H-quinoline-2-thiones (3a–3v)
To a stirring mixture of acetonitrile and triethylamine in a three-
necked round-bottomed flask in an ice bath, P2S5 (1.2 equiv), divided
into multiple portions, was added one portion at a time after the
previous portion had completely dissolved. Then, compounds 2a–2d
and 2j–2v were added and the solution was refluxed for 3 h under
nitrogen. After cooling, the light yellow precipitate was collected
through filtration and dried in a vacuum to produce the crude
products 3a–3d and 3j–3v; compounds 2e–2i and phosphorous
pentasulfide reacted in toluene with stirring and refluxing for about
2 h. The solvent was evaporated under reduced pressure; the residue
was washed with water to produce a light yellow solid 3e–3i.
3.2.5. 8-(4-Chlorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5e)
M.p. 179–181 ꢀC, yield ¼ 59.6%. 1H NMR (CDCl3, 300 MHz)
d
3.01–3.25 (m, 4H, H-5, H-6), 5.07 (s, 2H, –OCH2–), 6.92–7.38 (m,
7H, Ar-H), 8.60 (s, 1H, H-2). IR (KBr) cmꢁ1: 1693 (C]O). MS m/z 340
(M þ 1), 342 (M þ 3). Anal. Calcd. for C18H14ClN3O2: C 63.63, H 4.15,
N 12.37. Found: C 63.71, H 4.16, N 12.32.
3.2.6. 8-(3-Chlorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5f)
3.1.3. 6-Alkoxy-3,4-dihydro-2-hydrazine-1H-quinolines (4a–4v)
A solution of compounds 3a–3v (5 mmol) in 30 mL THF was
added dropwise to a solution of hydrazine hydrate (25 mmol) in
20 mL THF at room temperature, then the mixture was stirred and
heated at 60 ꢀC for 1 h. After the reaction, half of the solvent was
evaporated under reduced pressure, then the solution was poured
to petroleum ether, the yellow precipitate was filtered and then
kept at 0 ꢀC for the next step.
M.p. 114–116 ꢀC, yield ¼ 56.3%. 1H NMR (CDCl3, 300 MHz)
d
3.01–3.24 (m, 4H, H-5, H-6), 5.08 (s, 2H, –OCH2–), 6.92–7.45 (m,
7H, Ar-H), 8.58 (s, 1H, H-2). IR (KBr) cmꢁ1: 1696 (C]O). MS m/z 340
(M þ 1), 342 (M þ 3). Anal. Calcd. for C18H14ClN3O2: C 63.63, H 4.15,
N 12.37. Found: C 63.72, H 4.16, N 12.31.
3.2.7. 8-(2-Chlorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5g)
M.p. 185–187 ꢀC, yield ¼ 60.6%. 1H NMR (CDCl3, 300 MHz)
3.2. General procedure for the synthesis of 6-alkoxy-
[1,2,4]triazolo[3,4-a]phthalazine (5a–5v, 6a and 6s)
d 3.03–3.25 (m, 4H, H-5, H-6), 5.20 (s, 2H, –OCH2–), 6.96–7.56 (m,
7H, Ar-H), 8.61 (s, 1H, H-2). IR (KBr) cmꢁ1: 1693 (C]O). MS m/z 340
(M þ 1), 342 (M þ 3). Anal. Calcd. for C18H14ClN3O2: C 63.63, H 4.15,
N 12.37. Found: C 63.72, H 4.16, N 12.32.
A solution of compounds 4a–4v (4 mmol) and glyoxylic acid
monohydrate (4 mmol) in 30 mL of ethanol was stirred for 2 h at
room temperature. The mixture was evaporated under reduced
pressure. The acid intermediate was then reacted in refluxing toluene
for 2–3 h. The solution was evaporated to dryness, and the oily
residue was filtered on a silica gel chromatographic column
(ethyl acetate:methanol, 15:1) to give a white or light yellow solid.
Compounds 4a and 4s (4 mmol) reacted with pyruvic acid in ethanol
at room temperature, then evaporated the solvent under reduced
3.2.8. 8-(2,4-Dichlorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5h)
M.p. 170–172 ꢀC, yield ¼ 62.5%. 1H NMR (CDCl3, 300 MHz)
d
3.02–3.24 (m, 4H, H-5, H-6), 5.15 (s, 2H, –OCH2–), 6.94–7.50 (m,
6H, Ar-H), 8.58 (s, 1H, H-2). IR (KBr) cmꢁ1: 1689 (C]O). MS m/z 374
(M þ 1), 376 (M þ 3). Anal. Calcd. for C18H13Cl2N3O2: C 57.77, H 3.50,
N 11.23. Found: C 57.82, H 2.51, N 11.16.

X.-Y. Sun et al. / European Journal of Medicinal Chemistry 44 (2009) 1265–1270
1269
3.2.9. 8-(2,6-Dichlorobenzyloxy)-5,6-dihydro-[1,2,4]triazino
3.2.17. 8-Butyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
[4,3-a]quinolin-1-one (5i)
one (5q)
M.p. 206–208 ꢀC, yield ¼ 64.3%.1H NMR (CDCl3, 300 MHz)
M.p. 100–102 ꢀC, yield ¼ 51.1%. 1H NMR (CDCl3, 300 MHz)
d
0.99
d
3.03–3.25 (m, 4H, H-5, H-6), 5.31 (s, 2H, –OCH2–), 7.01–7.41 (m,
(t, 3H, J ¼ 7.4 Hz, –CH3), 1.45–1.84 (m, 4H, –CH2–CH2–), 3.00–3.22
(m, 4H, H-5, H-6), 3.99 (t, 2H, J ¼ 9.5 Hz, –OCH2–), 6.85–7.32 (m, 3H,
Ar-H), 8.57 (s, 1H, H-2). IR (KBr) cmꢁ1: 1696 (C]O). MS m/z 272
(M þ 1). Anal. Calcd. for C15H17N3O2: C 66.40, H 6.32, N 15.49.
Found: C 66.45, H 6.34, N 15.43.
6H, Ar-H), 8.59 (s, 1H, H-2). IR (KBr) cmꢁ1: 1687 (C]O). MS m/z 374
(M þ 1), 376 (M þ 3). Anal. Calcd. for C18H13Cl2N3O2: C 57.77, H 3.50,
N 11.23. Found: C 57.81, H 2.51, N 11.13.
3.2.10. 8-(2-Brominebenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5j)
3.2.18. 8-Pentyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5r)
M.p. 158–160 ꢀC, yield ¼ 50.8%.1H NMR (CDCl3, 300 MHz)
d
3.02–3.24 (m, 4H, H-5, H-6), 5.17 (s, 2H, –OCH2–), 6.95–7.64 (m,
M.p. 115–117 ꢀC, yield ¼ 56.8%. 1H NMR (CDCl3, 300 MHz)
d 0.94
7H, Ar-H), 8.57 (s, 1H, H-2). IR (KBr) cmꢁ1: 1690 (C]O). MS m/z 384
(M þ 1). Anal. Calcd. for C18H14BrN3O2: C 56.27, H 3.67, N 10.94.
Found: C 56.33, H 3.68, N 10.91.
(t, 3H, J ¼ 7.1 Hz, –CH3), 1.34–1.85 (m, 6H, –(CH2)3–), 2.99–3.22 (m,
4H, H-5, H-6), 3.97 (t, 2H, J ¼ 6.5 Hz, –OCH2–), 6.84–7.32 (m, 3H, Ar-
H), 8.56 (s, 1H, H-2). IR (KBr) cmꢁ1: 1689 (C]O). MS m/z 286
(M þ 1). Anal. Calcd. for C16H19N3O2: C 67.35, H 6.71, N 14.73. Found:
C 67.45, H 6.74, N 14.62.
3.2.11. 8-(4-Brominebenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5k)
M.p. 207–209 ꢀC, yield ¼ 54.4%.1H NMR (CDCl3, 300 MHz)
3.2.19. 8-Hexyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5s)
d
3.01–3.24 (m, 4H, H-5, H-6), 5.05 (s, 2H, –OCH2–), 6.91–7.55 (m,
7H, Ar-H), 8.60 (s, 1H, H-2). IR (KBr) cmꢁ1: 1695 (C]O). MS m/z 384
(M þ 1). Anal. Calcd. for C18H14BrN3O2: C 56.27, H 3.67, N 10.94.
Found: C 56.34, H 3.68, N 10.90.
M.p. 98–100 ꢀC, yield ¼ 51.8%. 1H NMR (CDCl3, 300 MHz)
d 0.92
(t, 3H, J ¼ 7.0 Hz, –CH3), 1.35–1.83 (m, 8H, –(CH2)4–), 3.01–3.24 (m,
2H, H-5, H-6), 3.98 (t, 2H, J ¼ 6.5 Hz, –OCH2–), 6.86–7.33 (m, 3H, Ar-
H), 8.60 (s, 1H, H-2). IR (KBr) cmꢁ1: 1694 (C]O). MS m/z 300
(M þ 1). Anal. Calcd. for C17H21N3O2: C 68.20, H 7.07, N 14.04.
Found: C 68.29, H 7.10, N 13.94.
3.2.12. 8-(4-Methoxybenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5l)
M.p. 163–165 ꢀC, yield ¼ 58.8%.1H NMR (CDCl3, 300 MHz)
d
3.01–3.24 (m, 4H, H-5, H-6), 3.84 (s, 3H, –OCH3), 5.02 (s, 2H,
3.2.20. 8-Heptyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5t)
–OCH2–), 6.93–7.38 (m, 7H, Ar-H), 8.59 (s, 1H, H-2). IR (KBr) cmꢁ1
:
1697 (C]O). MS m/z 336 (M þ 1). Anal. Calcd. for C19H17N3O3: C
M.p. 94–96 ꢀC, yield ¼ 63.2%. 1H NMR (CDCl3, 300 MHz)
d 0.88–
68.05, H 5.11, N 12.53. Found: C 58.14, H 5.12, N 12.49.
1.88 (m, 13H, –(CH2)5CH3), 3.00–3.23 (m, 2H, H-5, H-6), 3.98 (t, 2H,
J ¼ 6.5 Hz, –OCH2–), 6.86–7.33 (m, 3H, Ar-H), 8.58 (s, 1H, H-2). IR
(KBr) cmꢁ1: 1688 (C]O). MS m/z 314 (M þ 1). Anal. Calcd. for
C18H23N3O2: C 68.98, H 7.40, N 13.41. Found: C 67.04, H 7.38, N
13.36.
3.2.13. 8-(4-Methylbenzyloxy)-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (5m)
M.p. 143–145 ꢀC, yield ¼ 53.9%. 1H NMR (CDCl3, 300 MHz)
d
2.38 (s, 3H, –CH3) 2.88–3.22 (m, 4H, H-5, H-6), 3.84 (s, 3H,
–CH3), 5.30 (s, 2H, –OCH2–), 6.92–7.34 (m, 7H, Ar-H), 8.56 (s, 1H,
H-2). IR (KBr) cmꢁ1: 1694 (C]O). MS m/z 320 (M þ 1). Anal.
Calcd. for C19H17N3O2: C 71.47, H 5.37, N 13.16. Found: C 71.52, H
5.39, N 13.09.
3.2.21. 8-Octyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5u)
M.p. 63–65 ꢀC, yield ¼ 53.7%. 1H NMR (CDCl3, 300 MHz)
d 0.88–
1.83 (m, 15H, –(CH2)6CH3), 3.00–3.23 (m, 2H, H-5, H-6), 3.98 (t, 2H,
J ¼ 6.5 Hz, –OCH2–), 6.85–7.33 (m, 3H, Ar-H), 8.59 (s, 1H, H-2). IR
(KBr) cmꢁ1: 1692 (C]O). MS m/z 328 (M þ 1). Anal. Calcd. for
C19H25N3O2: C 69.70, H 7.70, N 12.83. Found: C 69.79, H 7.72, N 12.76.
3.2.14. 8-(3,4-Dimethoxybenzyloxy)-5,6-dihydro-
[1,2,4]triazino[4,3-a]quinolin-1-one (5n)
M.p. 168–170 ꢀC, yield ¼ 51.7%. 1H NMR (CDCl3, 300 MHz)
d
3.01–3.23 (m, 4H, H-5, H-6), 3.91 (s, 6H, –OCH3), 5.02 (s, 2H,
3.2.22. 8-Dodecyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5v)
–OCH2–), 6.88–7.34 (m, 6H, Ar-H), 8.58 (s, 1H, H-2). IR (KBr) cmꢁ1
:
1689 (C]O). MS m/z 366 (M þ 1). Anal. Calcd. for C20H19N3O4: C
M.p. 62–64 ꢀC, yield ¼ 63.1%. 1H NMR (CDCl3, 300 MHz)
d 0.87–
65.74, H 5.24, N 11.50. Found: C 65.81, H 5.25, N 11.42.
1.82 (m, 23H, –(CH2)10CH3), 3.00–3.22 (m, 2H, H-5, H-6), 3.97 (t, 2H,
J ¼ 6.5 Hz, –OCH2–), 6.86–7.32 (m, 3H, Ar-H), 8.57 (s, 1H, H-2). IR
(KBr) cmꢁ1: 1690 (C]O). MS m/z 384 (M þ 1). Anal. Calcd. for
C23H33N3O2: C 72.03, H 8.67, N 10.96. Found: C 72.14, H 8.69, N
10.84.
3.2.15. 8-Ethoxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5o)
M.p. 165–167 ꢀC, yield ¼ 65.5%. 1H NMR (CDCl3, 300 MHz)
d
1.43 (t, 3H, J ¼ 7.0 Hz, –CH3), 2.99–3.21 (m, 4H, H-5, H-6), 4.02–
4.08 (q, 2H, J ¼ 7.0 Hz, –OCH2–), 6.84–7.32 (m, 3H, Ar-H), 8.56 (s,
1H, H-2). IR (KBr) cmꢁ1: 1698 (C]O). MS m/z 244 (M þ 1). Anal.
Calcd. for C13H13N3O2: C 64.19, H 5.39, N 17.27. Found: C 64.23, H
5.38, N 17.21.
3.2.23. 2-Methyl-8-benzyloxy-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (6a)
M.p. 156–158 ꢀC, yield ¼ 58.6%. 1H NMR (CDCl3, 300 MHz)
d 2.55
(s, 3H, –CH3), 3.01–3.25 (m, 4H, H-5, H-6), 5.08 (s, 2H, –OCH2–),
6.94–7.44 (m, 8H, Ar-H). IR (KBr) cmꢁ1: 1794 (C]O). MS m/z 318
(M þ 1). Anal. Calcd. for C19H17N3O2: C 71.46, H 5.37, N 13.16. Found:
C 71.53, H 5.40, N 13.06.
3.2.16. 8-Propyloxy-5,6-dihydro-[1,2,4]triazino[4,3-a]quinolin-1-
one (5p)
M.p. 123–125 ꢀC, yield ¼ 59.3%. 1H NMR (CDCl3, 300 MHz)
d 1.03
(t, 3H, J ¼ 7.0 Hz, –CH3), 1.78–1.87 (m, 2H, –CH2–), 3.00–3.22 (m, 4H,
H-5, H-6), 3.94 (t, 2H, J ¼ 6.5 Hz, –OCH2–), 6.85–7.32 (m, 3H, Ar-H),
8.57 (s, 1H, H-2). IR (KBr) cmꢁ1: 1695 (C]O). MS m/z 258 (M þ 1).
Anal. Calcd. for C14H15N3O2: C 65.35, H 5.88, N 16.33. Found: C
65.40, H 5.87, N 16.29.
3.2.24. 2-Methyl-8-hexyloxy-5,6-dihydro-[1,2,4]triazino
[4,3-a]quinolin-1-one (6s)
M.p. 71–73 ꢀC, yield ¼ 58.9%. 1H NMR (CDCl3, 300 MHz)
d 0.93 (t,
3H, J ¼ 7.0 Hz, –CH3), 1.31–1.80 (m, 8H, –(CH2)4–), 2.57 (s, 3H, –CH3),
3.01–3.24 (m, 2H, H-5, H-6), 3.99 (t, 2H, J ¼ 6.5 Hz, –OCH2–), 6.896–

1270
X.-Y. Sun et al. / European Journal of Medicinal Chemistry 44 (2009) 1265–1270
7.34 (m, 3H, Ar-H). IR (KBr) cmꢁ1: 1693 (C]O). MS m/z 314 (M þ 1).
Anal. Calcd. for C18H23N3O2: C 68.98, H 7.40, N 13.41. Found: C 69.04,
H 7.42, N 13.33.
3.3.5. 3-MP-induced seizures [17]
30 min after the administration of the compounds at various
doses, 40 mg/kg 3-MP in saline solution was injected sc. This dose
was then administered to animals 1 h after different treatments.
The doses were calculated which prevented 50% of the treated
animals from tonic convulsions (ED50).
3.3. Pharmacology
The MES test and rotarod test were carried out by the standard
described in the Antiepileptic Drug Development Program (ADD) of
the National Institutes of Health following previously described
testing procedures (USA) [13,14]. All compounds, which were dis-
solved in polyethyleneglycol-400, were evaluated for anticonvul-
sant activities with C57B/6 mice in the 18–25 g weight range.
Groups of 10 mice were given a range of intraperitoneal doses of the
test drug until at least three points were established in the range of
10–90% seizure protection or minimal observed neurotoxicity. From
the plots of these data, the respective ED50 and TD50 values, 95%
confidence intervals, slopes of the regression line and the standard
error of the slopes were calculated by means of a computer
program written by the National Institute of Neurological Disorders
and Stroke.
3.3.6. Strychnine-induced seizures [18]
30 min after the administration of the compounds, the animals
were given a subcutaneous dose of strychnine chlorhydrate in
saline (1.2 mg/mL, 1 mL/kg), a dose at which 100% of the naı¨ve
animals showed convulsive reactions. The mice were placed in
individual cages and observed for 30 min.
3.3.7. Rotarod test [19]
30 min after the administration of the compounds, the animals
were tested on a 1-in. diameter knurled plastic rod rotating at
6 rpm for 1 min. Neurotoxicity was indicated by the inability of an
animal to maintain equilibrium in each of three trials.
Acknowledgment
3.3.1. MES test
Seizures were elicited with a 60-Hz alternating current of 50 mA
intensity in mice. The current was applied via corneal electrodes for
0.2 s. Protection against the spread of MES-induced seizures was
defined as the abolition of the hind leg and tonic maximal exten-
sion component of the seizure. 30 min after the administration of
the compounds, the activities were evaluated in MES test.
This work was supported by the National Natural Science
Foundation of China (No. 30460151 and No. 30760290) and
Important Item Foundation of Ministry of Education PR China (No.
20070422029).
References
3.3.2. PTZ-induced seizure [13,14]
30 min after the administration of the synthesized compounds
at various doses, the animals were given a subcutaneous dose of
pentylenetetrazole (85 mg/kg), a dose at which 100% of the naı¨ve
animals showed convulsive reactions. The dose which prevented
50% of the treated animals from tonic convulsions (ED50) was then
calculated.
[1] W. Loscher, Eur. J. Pharmacol. 342 (1998) 1.
[2] I.E. Leppik, Epilepsia 35 (S4) (1994) 29.
[3] E. Perucca, Br. J. Clin. Pharmacol. 42 (1996) 531.
[4] Z. Lin, P.K. Kadaba, Med. Res. Rev. 17 (1997) 537.
[5] Y.A. Al-Soud, N.A. Al-Masoudi, Ael-R. Ferwanah, Bioorg. Med. Chem. 11 (2003)
1701.
[6] Z.S. Quan, J.M. Wang, J.R. Rho, K.C. Kwak, H.C. Kang, C.S. Jun, K.Y. Chai, Bull.
Korean Chem. Soc. 26 (2005) 1757.
[7] L.J. Cui, Z.F. Xie, H.R. Piao, G. Li, K.Y. Chai, Z.S. Quan, Biol. Pharm. Bull. 28 (2005)
1216.
[8] Z.F. Xie, K.Y. Chai, H.R. Piao, K.C. Kwak, Z.S. Quan, Bioorg. Med. Chem. Lett. 15
(2005) 4803.
[9] D.S. Federico, P. Giampaolo, T. Sabrina, M.M. Anna, L.M. Concettina, N. Ettore,
G. Giovanni, L. Antonio, T. Letizia, M. Claudia, J. Med. Chem. 44 (2001) 316.
[10] R. Okada, N. Negishi, H. Nagaya, Brain Res. 480 (1989) 383.
[11] W. Loscher, Biochem. Pharmacol. 28 (1979) 1397.
[12] U. Sayin, S. Cengiz, T. Altug, Pharm. Res. 28 (1993) 325.
[13] R.J. Krall, J.K. Penry, B.G. White, H.J. Kupferberg, Epilepsia 19 (1978) 409.
[14] R.J. Poter, J.J. Cereghino, G.D. Gladding, B.J. Hessie, H.J. Kupferberg, B. Scoville,
Cleve. Clin. Q. 51 (1984) 293.
[15] R. Bernasconi, M. Klein, P. Martin, P. Christen, T. Hafner, C. Portet, M. Schmutz,
J. Neural. Transm 72 (1988) 213.
[16] W. Loscher, D. Schmidt, Epilepsy Res. 2 (1988) 145.
[17] A. Arnoldi, A. Bonsignori, P. Melloni, L. Merlini, M.L. Quadri, A.C. Rossi,
M. Valsecchi, J. Med. Chem. 33 (1990) 2865.
[18] M. Geurts, J.H. Poupaert, G.K. Scriba, D.M. Lambert, J. Med. Chem. 41 (1998) 24.
[19] X.Y. Sun, Y.Z. Jin, F.N. Li, G. Li, K.Y. Chai, Z.S. Quan, Arch. Pharm. Res. 29 (2006)
1080.
3.3.3. Isoniazid-induced seizure [15]
30 min after the administration of the compounds at various
doses, the animals were given an ip dose of isoniazid (250 mg/kg),
a dose at which 100% of the na¨ıve animals showed convulsive
reactions. The mice were placed in individual cages and observed
for 1 h. The dose which prevented 50% of the treated animals from
tonic convulsions (ED50) was calculated.
3.3.4. Thiosemicarbazide-induced seizure [16]
30 min after the administration of the compounds at various
doses, the animals were given an ip dose of thiosemicarbazide
(50 mg/kg), a dose at which 100% of the animals showed convulsive
reaction. This dose was then administered to animals 1 h after
different treatments. The doses were calculated which prevented
50% of the treated animals from tonic convulsions (ED50).