Synthesis and anti-inflammatory activity evaluation of some novel 6-alkoxy(phenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.07.049

Starting from phthalic anhydride, several new 6-alkoxy(phenoxy)-[1,2,4] triazolo[3,4-a]phthalazine-3-amine derivatives were synthesized as potent anti-inflammatory agent. The study showed that the compounds 6h (6-(2-chlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine) and 6s (6-(4-aminophenoxy)-[1,2,4] triazolo[3,4-a]phthalazine-3-amine) exhibited the highest anti-inflammatory activity (81% and 83% inhibition, respectively, at 0.5 h after i.p. administration) which were slightly more potent than the reference drug Ibuprofen (61%). Furthermore, the peak activity of 6h and 6s was observed at the 3 h after p.o. administration, and they exhibited stronger anti-inflammatory activity than Ibuprofen at the dose of 50 mg/kg at the peak time.

Full text of DOI:10.1016/j.ejmech.2010.07.049

European Journal of Medicinal Chemistry 45 (2010) 4807e4812
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-inflammatory activity evaluation of some novel 6-alkoxy
(phenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine derivatives
,
,
,
a,
*
Xian-Yu Sun a c, Chuan Hu a, Xian-Qing Deng a, Cheng-Xi Wei a b, Zhi-Gang Sun b , Zhe-Shan Quan
*
a College of Pharmacy, Yanbian University, No. 1829, JuZi Street, Yanji, Jilin 133000, China
b Institute of Neurosurgery, Inner Mongolia University for Nationalities, No. 1742, Holin River Street, Tongliao, Inner Mongolia Autonomous Region 028007, China
c College of Animal Technique, Bayi Agriculture University, Daqing, Heilongjiang 163319, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from phthalic anhydride, several new 6-alkoxy(phenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-
3-amine derivatives were synthesized as potent anti-inflammatory agent. The study showed that the
compounds 6h (6-(2-chlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine) and 6s (6-(4-amino-
phenoxy)-[1,2,4] triazolo[3,4-a]phthalazine-3-amine) exhibited the highest anti-inflammatory activity
(81% and 83% inhibition, respectively, at 0.5 h after i.p. administration) which were slightly more potent
than the reference drug Ibuprofen (61%). Furthermore, the peak activity of 6h and 6s was observed at the
3 h after p.o. administration, and they exhibited stronger anti-inflammatory activity than Ibuprofen at
the dose of 50 mg/kg at the peak time.
Received 21 May 2010
Received in revised form
24 July 2010
Accepted 28 July 2010
Available online 6 August 2010
Keywords:
3-Amine triazole
Phthalazine
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
[1,2,4]Triazolo[3,4-a]phthalazine-3-amine
Anti-inflammatory
1. Introduction
[3,4-a]phthalazine-3-amine derivatives. Quinoline ring and
phthalazine ring could be regarded as generalized Bioisosterism
Non-steroidal anti-inflammatory drugs (NSAIDs) are useful
tools in the treatment of acute and chronic inflammation [1], pain
[2], and fever [3]. However, long-term clinical usage of NSAIDs is
associated with significant side effects of gastrointestinal lesions,
bleeding, and nephrotoxicity [4,5]. Therefore the discovery of new
and safer anti-inflammatory drugs represents a challenging goal for
such a research area [6]. As resistance to anti-inflammatory drugs is
widespread, there is an increasing need for identification of novel
structure leads that may be of use in designing new, potent and less
toxic anti-inflammatory agents.
[13,14]. Bioisosterism, can be defined as the property by which
the substituents, or groups with similar physical or chemical
properties, impart similar biological properties to a chemical
compound is a useful strategy for both medicinal chemistry and
rational design of new drugs. Herein, we report the synthesis of
6-alkoxy(phenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine
derivatives and evaluation of their experimental anti-inflamma-
tory activity.
2. Chemistry
In the previous studies [7e12], the pharmacological activities
of several 4,5-dihydro[1,2,4]triazolo[4,3-a]quinolin compounds
have been evaluated, among these compounds, 7-alkoxy-4,5-
dihydro[1,2,4]triazolo[4,3-a]quinolin-1-amine (Fig. 1. I) was
identified to possess anti-inflammatory activity. Analysed the
structure of compound I, 3-amine triazole was assumed to be the
main pharmacological moiety playing anti-inflammatory activity.
So, on the basic of compound I, made the quinoline ring
substituted with phthalazine ring and retained 3-amine triazole
moiety, designed a series of 6-alkoxy(phenoxy)-[1,2,4]triazolo
Compounds were prepared according to Scheme 1. On the
basis of the previous studies carried out in our laboratory, we
designed and prepared 6-alkoxy(phenoxy)-[1,2,4]triazolo[3,4-a]
phthalazine-3-amine derivatives (6aeu). The target compounds
6aeu were synthesized according to Scheme 1. The starting
material phthalic anhydride reacted with hydrazine hydrate in
ethanol to yield 2,3-dihydrophthalazine-1,4-dione (compound
2), which reacted further with the refluxing phosphorus oxy-
chloride (POCl3) to yield 1,4-dichlorophthalazine (compound 3)
[15]. Compound 3 reacted further with hydrazine hydrate in THF
to produce compound 4 (1-hydrazine-4-chrorophthalazin) [16].
* Corresponding authors. Tel./fax: þ86 433 2660606.
E-mail addresses: sxianyu@sohu.com (X.-Y. Sun), sunzhigang101@yahoo.cn
(Z.-G. Sun), zsquan@ybu.edu.cn (Z.-S. Quan).
Then, 6-chlorom-[1,2,4]triazolo[3,4-a]phthalazine-3-amine
5
was prepared by cyclising compound 4 with cyanogene bromide
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.049

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X.-Y. Sun et al. / European Journal of Medicinal Chemistry 45 (2010) 4807e4812
4. Results and discussion
R
O
O
In the primary screening, all the synthesized compounds was
tested in the xylene-induced ear edema test in mice. These
compounds were evaluated for anti-inflammatory activity through
monitoring their ability to inhibit xylene-induced ear edema, and
made comparison with vehicle (DMSO) and Ibuprofen. As shown in
Table 1, most of the tested compounds exhibited anti-inflammatory
activity at a dose of 100 mg/kg administered intraperitoneally (i.p.),
especially, the compounds 6h (6-(2-chlorophenoxy)-[1,2,4]triazolo
[3,4-a]phthalazine-3-amine) and 6s (6-(4-aminophenoxy)-[1,2,4]
triazolo[3,4-a]phthalazine-3-amine) showed the highest anti-
inflammatory inhibition rate, 81% and 83%, respectively.
N
N
R
NH2
N
N
N
N
N
H2N
I
title compound
Fig. 1. Structure of compound I and title compound.
Among these alkyloxy substituted compounds, compounds 6d,
6f and 6g possessed similar anti-inflammatory activity with
Ibuprofen, but compounds 6a and 6b did not exhibited anti-
inflammatory activity compared with the vehicle group, suggesting
that appropriate length of the alkyl chain at C-6 position, or
appropriate lipophilic property, was essential to the anti-inflam-
matory activity of these compounds [19,20]. All the fourteen
aryl-substituted derivatives 6heu exhibited anti-inflammatory
in the presence of Na2CO3 [17]. Finally, compound 5 reacted with
appropriate alkanol and substituted phenol to produce the
target compounds, 6-alkoxy(phenoxy)-[1,2,4]triazolo[3,4-a]
phthalazine-3-amine derivatives (6aeu). The compounds
synthesized were characterized by IR, 1H NMR, MS, and
elemental analysis.
3. Pharmacology
inhibition to a certain extent (38.4%e83.0%). Among them,
compounds 6i, 6j, 6o, 6p, 6t and 6u exhibited similar anti-inflam-
matory activity with Ibuprofen, the compounds 6h and 6s were
slightly more potent than the reference drug Ibuprofen. Compar-
ison of the halogen substituted derivatives indicated that different
halogen atoms contributed to the anti-inflammatory activity in the
order of Cl > F ¼ Br; but bichloride 6k did not showed strong anti-
inflammatory activity. Furthermore, the position of substituted
group on the phenyl ring greatly influenced the anti-inflammatory
activity with an activity order of o > m > p, compared with the non-
substituted phenyl derivative 6u, compounds containing electron
donor group on the phenyl ring did not appeared to be more potent
in the anti-inflammatory activity, only one derivative 6s (p-amino
substituted) showed increased activity.
All the synthesized compounds were evaluated the anti-
inflammatory activity via the method of the xylene-induced ear
edema in Kunming mice, 20e25 g body weight, 10 animals per
group purchased from the Laboratory of Animal Research, College
of Pharmacy, Yanbian University [18]. Firstly, the test compounds
were evaluated their intraperitoneal (i.p.) administration anti-
inflammatory activities at
a dose of 100 mg/kg. Then, the
outstanding compounds (6h, 6s) were administered orally (p.o.) to
mice and quantified their anti-inflammatory activities at different
intervals (1 h, 2 h, 3 h, 4 h, 5 h and 24 h) under dose of 200 mg/kg.
The peak activity of 6h and 6s was observed at the 3 h after p.o.
administration. At last, the anti-inflammatory activities of the two
compounds were investigated at different p.o. doses (50, 100,
200 mg/kg) at the peak time and made comparison with Ibuprofen.
Based on the results of primary screening, two outstanding
derivatives 6h and 6s were chosen to be evaluated in the further
O
Cl
O
NH2NH2 .H2O
POCl3
NH2NH2 .H2O
THF
NH
NH
N
N
O
O
O
2
Cl
3
1
R
N
Cl
Cl
O
BrCN, NaCO3
N
N
ROH
N
N
N
NH2
NH2
N
NHNH2
N
N
N
4
5
6a-6u
R:
a: -CH3
b: -C2H5
h: -C6H4(o-Cl)
i: -C6H4(m-Cl)
j: -C6H4(p-Cl)
k:- C6H3(2,4-Cl2)
l: -C6H4(p-F)
o: -C6H4(o-CH3)
p: -C6H4(m-CH3)
q: -C6H4(p-CH3)
r: -C6H4(o-NH2)
s: -C6H4(p-NH2)
t: -C6H4(m-NH2)
u: -C6H5
c: i-C3H7
d: n-C4H9
e: n-C6H13
f: n-C7H15
g: n-C8H17
m: -C6H4(p-Br)
n: -C6H4(p-OCH3)
Scheme 1. Synthesis of compounds 6aeu.

X.-Y. Sun et al. / European Journal of Medicinal Chemistry 45 (2010) 4807e4812
4809
Table 1
Table 3
Anti-inflammatory activity of compounds 6aeu administrated i.p.
Anti-inflammatory activity of compounds 6h and 6s administered orally at different
doses.
Comp.
R
Dose
(mg/kg) of mice
Number Edema mean
Inhibition
rate (%)
ꢂ S.D. (mg)
Time (h)
Dose (mg/kg)
Inhibition (%)
DMSO
Ibuprfen
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
e
e
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
13.36 ꢂ 2.84
e
6h
6s
Ibuprofen
e
100
100
100
100
100
100
100
100
100
100
100
100
100
100
5.19 ꢂ 1.67* 61.2
3
3
3
200
100
50
75.05
46.70
30.80*
84.35*
37.86
22.81*
64.58
26.93
e
n-CH3
n-C2H5
i-C3H7
n-C4H9
n-C6H13
n-C7H15
n-C8H17
-C6H4(o-Cl)
-C6H4(p-Cl)
-C6H4(m-Cl)
-C6H3(2,4-Cl2)
-C6H4(p-F)
-C6H4(p-Br)
9.09 ꢂ 2.63
e
e
10.30 ꢂ 2.66
8.10 ꢂ 5.57* 39.4
4.33 ꢂ 2.35* 67.6
*: p < 0.01 compared with Ibuprofen at the corresponding dose.
e: no anti-inflammatory activity.
8.09 ꢂ 2.27
39.5
4.59 ꢂ 2.52* 65.7
5.62 ꢂ 4.43* 58.0
2.51 ꢂ 2.15* 81.2
4.16 ꢂ 2.86* 68.8
3.34 ꢂ 2.12* 75.0
7.96 ꢂ 3.90* 40.4
6.84 ꢂ 3.04* 48.8
6.90 ꢂ 3.36* 48.4
7.48 ꢂ 1.92* 44.0
4.24 ꢂ 2.92* 68.3
4.28 ꢂ 2.92* 68.0
6.31 ꢂ 3.03* 52.7
8.23 ꢂ 3.05* 38.4
2.28 ꢂ 0.82* 83.0
4.28 ꢂ 1.69* 68.0
6.03 ꢂ 3.18* 55.0
5. Conclusion
A new series of anti-inflammatory compounds, 6-alkoxy(phe-
noxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amines, were synthe-
sized and their anti-inflammatory activities were evaluated by an in
vivo test. Two compounds 6h (6-(2-chlorophenoxy)-[1,2,4]triazolo
[3,4-a]phthalazine-3-amine) and 6s (6-(4-aminophenoxy)-[1,2,4]
triazolo[3,4-a]phthalazine-3-amine) showed promising anti-
inflammatory activity (81% and 83% inhibition, respectively, at 0.5 h
after i.p. administration) which were slightly more potent than the
reference drug Ibuprofen (61%).
-C6H4(p-OCH3) 100
-C6H4(o-CH3)
-C6H4(m-CH3)
-C6H4(p-CH3)
-C6H4(o-NH2)
-C6H4(p-NH2)
-C6H4(m-NH2)
-C6H5
100
100
100
100
100
100
100
6t
6u
*p < 0.01 compared with vehicle group.
6. Experimental protocols
screening where the dose was 200 mg/kg by oral administration,
but multiple intervals (1 h, 2 h, 3 h, 4 h, 5 h and 24 h) for xylene
application were assessed. The results were shown in Table 2. As
the interval lengthened, the anti-inflammatory activity of
compounds 6h and 6s first increased and then declined; the
peak activity was observed at the 3 h interval. Both compound
6h and 6s exhibited higher anti-inflammatory inhibition rate
from 1 to 5 h in the test. Notably, compound 6h possessed more
potential activity than Ibuprofen at the 4 h interval, and showed
no difference with Ibuprofen at the other time interval.
Comparing 6s and Ibuprofen, compound 6s showed stronger
anti-inflammatory activity than Ibuprofen at the 2e4 h interval,
and showed similar activity level as the reference drug at other
time points.
In succession, the ear inflammation inhibition rate of
compounds 6h, 6s and reference drug Ibuprofen at lower doses
(100 mg/kg and 50 mg/kg) administered 3 h before xylene appli-
cation were evaluated and compared (Table 3). Both the two
compounds showed similar effects as Ibuprofen at the dose of
100 mg/kg; but they exhibited stronger anti-inflammatory activity
than Ibuprofen at the dose of 50 mg/kg, at which dose Ibuprofen
did not showed any anti-inflammatory activity after 3 h adminis-
tration p.o. in the test. A conclusion of compounds 6h, 6s possessing
a slightly long-term anti-inflammatory action in animal test could
be indicated by the present result.
6.1. Chemistry
Melting points were determined in open capillary tubes and
were uncorrected. 1H NMR spectra’s were measured on an AV-300
(Bruker, Fällanden, Switzerland), and all chemical shifts were
reported in parts per million relative to tetramethylsilane. Mass
spectra were measured on an HP1100LC (Agilent Technologies,
Santa Clara, USA). Elemental analyses were performed on a 204Q
CHN (PerkinElmer, Fremont, USA). Major chemicals were
purchased from Aldrich Chemical Corporation (Shanghai, China).
All other chemicals were of analytical grade.
6.1.1. Synthesis of 2,3-dihydrophthalazine-1,4-dione (2)
The starting compound phthalic anhydride 1 (50 g, 337.8 mmol)
was dissolved in ethanol (500 mL) in 30 min, then to this mixture
was added hydrazine hydrate (20.3 g, 405.4 mmol) dropwise under
ice-bath. The reaction mixture was stirred at room temperature
(r.t.) for 1 h then made cool in the ice-bath. The white solid was
collected through filtration and dried in a vacuum to give the
compound 2,3-dihydrophthalazine-1,4-dione 43.0 g. M.p.
181e183 ꢀC, yield ¼ 78.5%. 1H NMR (DMSO, 300 MHz)
d 7.59e7.63
(m, 2H, H-6, H-7), 8.14 (d, 2H, J ¼ 7.9 Hz, H-5, H-8), 9.04 (s, 2H,
eCOeNHeNHeCOe).
6.1.2. Synthesis of 1,4-dichlorophthalazine (3)
Compound 2 (8.5 g, 52.5 mmol) was dissolved in phosphorus
oxychloride (45 mL) and stirred under reflux for 4 h. Then the
solvent was removed under vacuum. The residue was dissolved in
dichloromethane (200 mL) and stirred rapidly, and the solution was
neutralized by the addition of solid and aqueous sodium hydrogen
carbonate (cautiously). When effervescence had ceased, the organic
layer was separated and the aqueous layer was extracted with
dichloromethane (2 ꢁ 200 mL). The combined organic layers were
dried over MgSO4, filtered, and evaporated; the residue was puri-
fied by silica gel column chromatography with ethyl acetate:
petroleum ether (1:8) and gained 8.5 g. M.p. 192e194 ꢀC,
Table 2
Anti-inflammatory activity of compounds 6h and 6s administered orally at different
times before xylene application.
Time (h)
Dose (mg/kg)
Inhibition (%)
6h
6s
Ibuprofen
1
2
3
4
5
24
200
200
200
200
200
200
52.70
59.05
75.05
64.11*
57.17
8.24
60.46
29.63
38.58
64.58
36.33
23.39
13.18
81.29**
84.35**
80.58**
55.87
yield ¼ 81.2%. 1H NMR (CDCl3, 300 MHz)
5, H-8), 7.90e7.94 (m, 2H, H-6, H-7).
d
7.59 (d, J ¼ 7.9 Hz, 2H, H-
18.92
*p < 0.05, **p < 0.01 compared with Ibuprofen at the corresponding time.

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X.-Y. Sun et al. / European Journal of Medicinal Chemistry 45 (2010) 4807e4812
6.1.3. Synthesis of 1-hydrazine-4-chrorophthalazin (4)
10), 8.45 (d, 1H, J ¼ 7.9 Hz, H-7). 13C NMR (DMSO, 300 MHz)
d 22.05,
A solution of compound 3 (5 g, 25.1 mmol) in THF (60 mL) was
added dropwise to a solution of hydrazine hydrate (6.28 g,
125.6 mmol) in THF (10 mL) at room temperature. The mixture was
stirred and heated 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 and washed with
petroleum ether, and then kept below 0 ꢀC. The compounds
obtained were pure enough for the following step.
71.26, 118.34, 121.99, 125.08, 125.51, 130.09, 134.16, 138.30, 151,17,
155,71. MS m/z 244 (Mþ). Anal. Calcd. for C12H13N5O: C 59.25, H
5.39, N 28.79. Found: C 59.40, H 5.64, N 29.07.
6.1.5.4. 6-Butoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6d).
M.p. 160e162 ꢀC, yield ¼ 78%. IR (KBr) cmꢃ1: 3414, 3073, 1644,
1240, 1047. 1H NMR (CDCl3, 300 MHz)
d
1.05 (t, 3H, J ¼ 7.4 Hz,
eCH3), 1.52e1.65 (m, 2H, eCH2e), 1.87e1.96 (m, 2H, eCH2e), 4.48
(t, 2H, J ¼ 6.5 Hz, -OCH2-), 4.91 (s, 2H, eNH2), 7.68 (t, 1H, J ¼ 7.7 Hz,
H-8), 7.84 (t, 1H, J ¼ 7.2 Hz, H-9) 8.12 (d, 1H, J ¼ 8.0 Hz, H-10), 8.45
(d, 1H, J ¼ 7.9 Hz, H-7). MS m/z 258 (Mþ). Anal. Calcd. for
C13H15N5O: C 60.69, H 5.88, N 27.22. Found: C 60.82, H 5.98, N 27.45.
6.1.4. Synthesis of 6-chloro-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (5)
In
a three-neck round-bottomed flask with thermometer,
compound 4 (2.2 g, 11.3 mmol) was dissolved in dioxane (60 mL),
and the solution was treated with Na2CO3 (1.2 g, 11.3 mmol) in H2O
(20 mL). Then cyanogene bromide (1.3 g, 12.5 mmol) in dioxane
(20 mL) was added dropwise to the mixture under ice-bath and
kept the reaction temperature below 10 ꢀC, after stirring for 2 h, the
solvent was removed under reduced pressure. The residue was
purified by silica gel chromatography (dichloromethane-methanol
10:1). M.p. 218e220 ꢀC, yield ¼ 30%. 1H NMR (DMSO, 300 MHz)
6.1.5.5. 6-(Hexyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6e).
M.p.130e132 ꢀC, yield ¼ 76%. IR (KBr) cmꢃ1: 3425, 3072,1642,1241,
1047. 1H NMR (CDCl3, 300 MHz)
d
0.95 (t, 3H, J ¼ 6.5 Hz, eCH3),
1.34e1.41 (m, 4H, e(CH2)2e), 1.49e1.59 (m, 2H, eCH2e), 1.87e1.96
(m, 2H, eCH2e), 4.46 (t, 2H, J ¼ 6.5 Hz, eOCH2e), 5.40 (s, 2H,
eNH2), 7.68 (t, 1H, J ¼ 7.5 Hz, H-8), 7.83 (t, 1H, J ¼ 7.4 Hz, H-9) 8.11
(d, 1H, J ¼ 8.1 Hz, H-10), 8.43 (d, 1H, J ¼ 7.8 Hz, H-7). MS m/z 286
(Mþ). Anal. Calcd. for C15H19N5O: C 63.14, H 6.71, N 25.54. Found: C
63.40, H 6.90, N 25.20.
d
6.67 (s, 2H, eNH2), 7.86 (t, 1H, J ¼ 7.7 Hz, H-8), 8.03 (t, 1H,
J ¼ 7.6 Hz, H-9) 8.18 (d, 1H, J ¼ 8.0 Hz, H-10), 8.34 (d, 1H, J ¼ 7.9 Hz,
H-7).
6.1.5.6. 6-(Heptyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6f).
M.p. 114e116 ꢀC, yield ¼ 72%. IR (KBr) cmꢃ1: 3412, 3062, 1634, 1240,
6.1.5. General procedure for synthesis of 6-alkoxy(phenoxy)-[1,2,4]
triazolo[3,4-a]phthalazine-3-amine derivatives (6aeu)
1052. 1H NMR (CDCl3, 300 MHz)
d
0.92 (t, 3H, J ¼ 6.5 Hz, eCH3),
Compound 5 (0.4 g, 1.83 mmol), NaOH (80 mg, 2.01 mmol), and
alkanol (15 mL) were reacted together by stirring and refluxing for
approximately 5 h. After the alkanol was removed under reduced
pressure, the solid residue was purified by silica gel chromatog-
raphy (dichloromethane:methanol, 10:1) to obtain compounds
6aeg.
1.34e1.43 (m, 6H, e(CH2)3e), 1.50e1.55 (m, 2H, eCH2e), 1.89e1.98
(m, 2H, eCH2e), 4.48 (t, 2H, J ¼ 6.5 Hz, eOCH2e), 5.02 (s, 2H,
eNH2), 7.69 (t, 1H, J ¼ 7.5 Hz, H-8), 7.84 (t, 1H, J ¼ 7.7 Hz, H-9) 8.13
(d, 1H, J ¼ 8.1 Hz, H-10), 8.45 (d, 1H, J ¼ 7.8 Hz, H-7). MS m/z 300
(Mþ). Anal. Calcd. for C16H21N5O: C 64.19, H 7.07, N 23.39. Found: C
64.31, H 7.12, N 23.42.
A mixture of appropriate substituted phenol (2.01 mmol), NaOH
(80 mg, 2.01 mmol) in DMF (20 mL) was stirred at 50 ꢀC for 15 min,
then to this mixture was added compound 5 (0.4 g, 1.83 mmol), the
mixture was stirred at 120 ꢀC for approximately 5 h. After the DMF
was removed under reduced pressure, the solid residue was puri-
fied by silica gel chromatography (dichloromethane:methanol,
10:1) to obtain compounds 6heu. The yield, melting point data of
each compound were given below.
6.1.5.7. 6-(Octyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6g).
M.p. 98e100 ꢀC, yield ¼ 70%. IR (KBr) cmꢃ1: 3416, 3064, 1651, 1246,
1056. 1H NMR (CDCl3, 300 MHz)
d
0.90 (t, 3H, J ¼ 6.9 Hz, eCH3),
1.32e1.54 (m, 10H, e(CH2)5e), 1.88e1.95 (m, 2H, eCH2e), 4.48 (t,
2H, J ¼ 6.5 Hz, eOCH2e), 5.09 (s, 2H, eNH2), 7.68 (t, 1H, J ¼ 7.7 Hz,
H-8), 7.83 (t, 1H, J ¼ 7.7 Hz, H-9) 8.12 (d, 1H, J ¼ 7.8 Hz, H-10), 8.46
(d, 1H, J ¼ 8.1 Hz, H-7). MS m/z 314 (Mþ). Anal. Calcd. for
C17H23N5O: C 65.15, H 7.40, N 22.35. Found: C 65.30, H 7.58, N 22.52.
6.1.5.1. 6-Methoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6a).
M.p. 198e200 ꢀC, yield ¼ 70%. IR (KBr) cmꢃ1: 3422, 3059, 1640,
6.1.5.8. 6-(2-Chlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine
1239, 1045. 1H NMR (DMSO, 300 MHz)
d
4.14 (s, 3H, eOCH3), 6.24 (s,
(6h). M.p. 278e280 ꢀC, yield ¼ 85%. IR (KBr) cmꢃ1: 3418, 3080,
2H, eNH2), 7.74 (t, 1H, J ¼ 7.7 Hz, H-8), 7.90 (t, 1H, J ¼ 7.6 Hz, H-9)
8.07 (d, 1H, J ¼ 8.0 Hz, H-10), 8.26 (d, 1H, J ¼ 7.7 Hz, H-7). MS m/z
216 (Mþ). Anal. Calcd. for C10H9N5O: C 55.81, H 4.22, N 32.54.
Found: C 55.56, H 4.48, N 32.78.
1630, 1230, 1056. 1H NMR (DMSO, 300 MHz)
d 6.03 (s, 2H, eNH2),
7.38e7.68 (m, 4H, Ar-H), 7.88 (t, 1H, J ¼ 7.7 Hz, H-8), 8.03 (t, 1H,
J ¼ 7.6 Hz, H-9) 8.30 (d, 1H, J ¼ 8.0 Hz, H-10), 8.35 (d, 1H, J ¼ 7.9 Hz,
H-7). MS m/z 312 (Mþ), 314 (Mþ3). Anal. Calcd. for C15H10ClN5O: C
57.79, H 3.23, N 22.47. Found: C 57.91, H 3.34, N 22.78.
6.1.5.2. 6-Ethoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6b).
M.p.196e198 ꢀC, yield ¼ 73%. IR (KBr) cmꢃ1: 3408, 3069,1641,1230,
6.1.5.9. 6-(4-Chlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine
1048. 1H NMR (DMSO, 300 MHz)
d
1.48 (t, 3H, J ¼ 7.0 Hz, eOCH3),
(6i). M.p. 204e206 ꢀC, yield ¼ 87%. IR (KBr) cmꢃ1: 3407, 3083, 1635
4.55(q, 2H, J ¼ 7.0 Hz, eOCH2e), 6.28 (s, 2H, eNH2), 7.76 (t, 1H,
J ¼ 7.6 Hz, H-8), 7.93 (t, 1H, J ¼ 7.4 Hz, H-9) 8.07 (d, 1H, J ¼ 8.0 Hz, H-
1227, 1052. 1H NMR (DMSO, 300 MHz)
d 6.38 (s, 2H, eNH2), 7.63 (m,
4H, Ar-H), 7.83 (t, 1H, J ¼ 7.7 Hz, H-8), 8.00 (t, 1H, J ¼ 7.6 Hz, H-9)
10), 8.25 (d, 1H, J ¼ 7.9 Hz, H-7). 13C NMR (DMSO, 300 MHz)
d 22.05,
8.25 (d, 1H, J ¼ 8.0 Hz, H-10), 8.33 (d, 1H, J ¼ 7.9 Hz, H-7). 13C NMR
71.25, 118.34, 121.99, 125.08, 125.51, 130.10, 134.18, 138.29, 151,17,
155,71. MS m/z 230 (Mþ). Anal. Calcd. for C11H11N5O: C 57.63, H
4.84, N 30.55. Found: C 57.49, H 5.10, N 30.81.
(DMSO, 300 MHz) d 91.23, 115.97, 116.44, 116.98, 122.50, 123.62,
123.99, 125.76, 126.34, 130.81, 134.63, 138.06, 151.28, 158.39, 175.57.
MS m/z 312 (Mþ), 314 (Mþ3). Anal. Calcd. for C15H10ClN5O: C 57.79,
H 3.23, N 22.47. Found: C 57.92, H 3.38, N 22.62.
6.1.5.3. 6-Isopropoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6c).
M.p.176e178 ꢀC, yield ¼ 80%. IR (KBr) cmꢃ1: 3407, 3066,1633,1234,
6.1.5.10. 6-(3-Chlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
1051. 1H NMR (CDCl3, 300 MHz)
d
1.51 (d, 6H, J ¼ 6.1 Hz, eCH
amine (6j). M.p. 247e249 ꢀC, yield ¼ 90%. IR (KBr) cmꢃ1: 3409,
(CH3)2), 5.02 (s, 2H, eNH2), 5.38e5.46 (m, 1H, eOCHe), 7.67 (t, 1H,
J ¼ 7.7 Hz, H-8), 7.83 (t, 1H, J ¼ 7.6 Hz, H-9) 8.12 (d, 1H, J ¼ 8.0 Hz, H-
3078, 1627, 1241, 1054. 1H NMR (DMSO, 300 MHz)
d 6.13 (s, 2H,
eNH2), 7.37e7.62 (m, 4H, Ar-H), 7.83 (t, 1H, J ¼ 7.7 Hz, H-8), 8.01

X.-Y. Sun et al. / European Journal of Medicinal Chemistry 45 (2010) 4807e4812
4811
(t, 1H, J ¼ 7.6 Hz, H-9) 8.23 (d, 1H, J ¼ 8.0 Hz, H-10), 8.33 (d, 1H,
J ¼ 7.9 Hz, H-7). MS m/z 312 (Mþ), 314 (M þ 3). Anal. Calcd. for
C15H10ClN5O: C 57.79, H 3.23, N 22.47. Found: C 57.70, H 3.40, N
22.22.
H-10), 8.33 (d,1H, J ¼ 7.8 Hz, H-7). MS m/z 292 (Mþ). Anal. Calcd. for
16H13N5O: C 65.97, H 4.50, N 24.04. Found: C 65.73, H 4.21, N 24.10.
C
6.1.5.18. 6-(2-Aminophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6r). M.p. 300e302 ꢀC, yield ¼ 81%. IR (KBr) cmꢃ1: 3413,
6.1.5.11. 6-(2,4-Dichlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-
3074, 1634, 1248, 1049. 1H NMR (DMSO, 300 MHz)
d 5.13 (s, 2H,
3-amine (6k). M.p. 232e234 ꢀC, yield ¼ 82%. IR (KBr) cmꢃ1: 3407,
Ar-NH2), 5.86 (s, 2H, eNH2), 6.58e7.18 (m, 4H, Ar-H), 7.83 (t, 1H,
J ¼ 7.5 Hz, H-8), 7.99 (t, 1H, J ¼ 7.5 Hz, H-9) 8.26 (d, 1H, J ¼ 8.0 Hz,
H-10), 8.32 (d, 1H, J ¼ 7.9 Hz, H-7). MS m/z 293 (Mþ). Anal. Calcd.
for C15H12N6O: C 61.64, H 4.14, N 28.75. Found: C 61.69, H 4.01, N
28.60.
3075, 1631, 1233, 1049. 1H NMR (DMSO, 300 MHz)
d 6.13 (s, 2H,
eNH2), 7.53e7.84 (m, 3H, Ar-H), 7.86 (t, 1H, J ¼ 7.7 Hz, H-8), 8.05 (t,
1H, J ¼ 7.6 Hz, H-9) 8.30 (d, 1H, J ¼ 7.9 Hz, H-10), 8.34 (d, 1H,
J ¼ 7.9 Hz, H-7). MS m/z 346 (Mþ), 348 (Mþ3). Anal. Calcd. for
C15H9Cl2N5O: C 52.04, H 2.62, N 20.23. Found: C 52.20, H 2.78, N
20.54.
6.1.5.19. 6-(4-Aminophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6s). M.p. 248e250 ꢀC, yield ¼ 78%. IR (KBr) cmꢃ1: 3411,
6.1.5.12. 6-(4-Fluorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
3083, 1633, 1232, 1047. 1H NMR (DMSO, 300 MHz)
d 5.10 (s, 2H,
amine (6l). M.p. 238e240 ꢀC, yield ¼ 81%. IR (KBr) cmꢃ1: 3420,
Ar-NH2), 5.96 (s, 2H, eNH2), 6.61e7.11 (m, 4H, Ar-H), 7.82 (t, 1H,
J ¼ 7.6 Hz, H-8), 7.99 (t, 1H, J ¼ 7.5 Hz, H-9) 8.25 (d, 1H, J ¼ 8.0 Hz,
H-10), 8.31 (d, 1H, J ¼ 7.8 Hz, H-7). MS m/z 293 (Mþ). Anal. Calcd.
for C15H12N6O: C 61.64, H 4.14, N 28.75. Found: C 61.78, H 4.01, N
28.52.
3068, 1620, 1240, 1056. 1H NMR (DMSO, 300 MHz)
d 6.06 (s, 2H,
eNH2), 7.29e7.55 (m, 4H, Ar-H), 7.84 (t, 1H, J ¼ 7.7 Hz, H-8), 8.00 (t,
1H, J ¼ 7.6 Hz, H-9) 8.27 (d, 1H, J ¼ 8.0 Hz, H-10), 8.33 (d, 1H,
J ¼ 7.9 Hz, H-7). 13C NMR (DMSO, 300 MHz)
d 90.87, 116.61, 116.92,
117.96, 122.17, 123.39, 123.51, 125.44, 125.74, 130.40, 134.81, 138.65,
151.10, 158.23, 179.40. MS m/z 296 (Mþ). Anal. Calcd. for
C15H10FN5O: C 61.02, H 3.41, N 23.72. Found: C 60.89, H 3.27, N
23.79.
6.1.5.20. 6-(3-Aminophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6t). M.p. 266e268 ꢀC, yield ¼ 84%. IR (KBr) cmꢃ1: 3415,
3076, 1630, 1235, 1054. 1H NMR (DMSO, 300 MHz)
d 5.31 (s, 2H,
Ar-NH2), 6.12 (s, 2H, eNH2), 6.45e7.10 (m, 4H, Ar-H), 7.83 (t, 1H,
J ¼ 7.7 Hz, H-8), 8.01 (t, 1H, J ¼ 7.6 Hz, H-9) 8.18 (d, 1H, J ¼ 8.0 Hz,
H-10), 8.34 (d, 1H, J ¼ 7.9 Hz, H-7). MS m/z 293 (Mþ). Anal. Calcd.
for C15H12N6O: C 61.64, H 4.14, N 28.75. Found: C 61.79, H 4.32, N
28.92.
6.1.5.13. 6-(4-Bromophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6m). M.p. 218e220 ꢀC, yield ¼ 75%. IR (KBr) cmꢃ1: 3408,
3083, 1622, 1255, 1058. 1H NMR (DMSO, 300 MHz)
d 6.11 (s, 2H,
eNH2), 7.45e7.68 (m, 4H, Ar-H), 7.83 (t, 1H, J ¼ 7.7 Hz, H-8), 8.00 (t,
1H, J ¼ 7.6 Hz, H-9) 8.25 (d, 1H, J ¼ 8.0 Hz, H-10), 8.32 (d, 1H,
J ¼ 7.9 Hz, H-7). MS m/z 355 (Mþ). Anal. Calcd. for C15H10BrN5O: C
50.58, H 2.83, N 19.66. Found: C 50.30, H 3.12, N 19.49.
6.1.5.21. 6-Phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (6u).
M.p. 212e214 ꢀC, yield ¼ 73%. IR (KBr) cmꢃ1: 3410, 3067,1630,1244,
1049. 1H NMR (DMSO, 300 MHz)
d 6.05 (s, 2H, eNH2), 7.28e7.52 (m,
6.1.5.14. 6-(4-Methoxyphenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-
5H, Ar-H), 7.84 (t, 1H, J ¼ 7.7 Hz, H-8), 8.01 (t, 1H, J ¼ 7.6 Hz, H-9)
8.26 (d, 1H, J ¼ 8.1 Hz, H-10), 8.34 (d, 1H, J ¼ 7.9 Hz, H-7). MS m/z
278 (Mþ). Anal. Calcd. for C15H11N5O: C 64.97, H 4.00, N 25.26.
Found: C 64.81, H 3.85, N 25.50.
amine (6n). M.p. 224e226 ꢀC, yield ¼ 82%. IR (KBr) cmꢃ1: 3398,
3078, 1635, 1254, 1046. 1H NMR (DMSO, 300 MHz)
d 3.80 (s, 3H,
eOCH3), 6.00 (s, 2H, eNH2), 7.00e7.41 (m, 4H, Ar-H), 7.83 (t, 1H,
J ¼ 7.3 Hz, H-8), 8.00 (t, 1H, J ¼ 7.2 Hz, H-9) 8.27 (d, 1H, J ¼ 7.9 Hz, H-
10), 8.32 (d, 1H, J ¼ 7.8 Hz, H-7). 13C NMR (DMSO, 300 MHz)
d
69.41,
6.2. Pharmacology
115.07, 118.09, 122.15, 122.53, 125.38, 125.77, 129.02, 130.26, 130.40,
134.36,134.72,138.67,146.55,15.06,157.08. MS m/z 3.08 (Mþ). Anal.
Calcd. for C16H13N5O2: C 62.53, H 4.26, N 22.79. Found: C 62.78, H
4.29, N 22.91.
The anti-inflammatory activity was evaluated by an in vivo
inhibition assay by monitoring xylene-induced ear edema in
mice [18]. In the primary screening, all tested compounds were
freshly prepared (dissolved with DMSO) prior to administered
i.p. at a dose of 100 mg/kg to mice and at a concentration of
0.05 mL/20 g of mice weight. Control mice received the vehicle
only (DMSO, 0.05 mL/20 g of mice weight). 0.5 h after adminis-
tration i.p., animals were used in the xylene-induced ear edema
test. In the latter evaluation, tested compounds (6h, 6s and
Ibuprofen) were homogenized with 0.5% sodium carboxymeth-
ylcellulose (CMC-Na) and administered orally to mice at
a concentration of 0.2 mL/10 g mice weight. Control mice
received the vehicle only (0.5% CMC-Na, 0.2 mL/10 g). At a spec-
6.1.5.15. 6-(2-Methylphenoxyl)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6o). M.p. 265e267 ꢀC, yield ¼ 85%. IR (KBr) cmꢃ1: 3413,
3068, 1623, 1239, 1052. 1H NMR (DMSO, 300 MHz)
d 2.24 (s, 3H,
eCH3), 5.93 (s, 2H, eNH2), 7.23e7.39 (m, 4H, Ar-H), 7.85 (t, 1H,
J ¼ 7.7 Hz, H-8), 8.02 (t, 1H, J ¼ 7.6 Hz, H-9) 8.33 (t, 2H, H-7, H-10).
MS m/z 292 (Mþ). Anal. Calcd. for C16H13N5O: C 65.97, H 4.50, N
24.04. Found: C 65.70, H 4.75, N 24.23.
6.1.5.16. 6-(4-Methylphenoxyl)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6p). M.p. 166e168 ꢀC, yield ¼ 80%. IR (KBr) cmꢃ1: 3409,
ified later time, 20 mL xylene was applied to the surface of the
3070, 1640, 1247, 1054. 1H NMR (DMSO, 300 MHz)
d
2.35 (s, 3H,
right ear of each mouse by a micropipette. Mice were sacrificed
30 min later and a cylindrical plug (7 mm diameter) was excised
from each of the treated and untreated ears. Edema was quan-
tified by the difference in weight between the two plugs. The
anti-inflammatory activity was expressed as percent edema
reduction as compared to the CMC-Na administered control
group. The NSAID drug Ibuprofen was tested in parallel as an
activity reference. Edema values, expressed as mean ꢂ standard
deviation, were compared statistically using one-way-ANOVA
followed by Dunnet’s test. A level of p < 0.05 was adopted as the
test of significance.
eCH3), 6.01 (s, 2H, eNH2), 7.26e7.35 (m, 4H, Ar-H), 7.82 (t, 1H,
J ¼ 7.7 Hz, H-8), 7.99 (t, 1H, J ¼ 7.6 Hz, H-9) 8.24 (d, 1H, J ¼ 8.0 Hz, H-
10), 8.32 (d, 1H, J ¼ 7.9 Hz, H-7). MS m/z 292 (Mþ). Anal. Calcd. for
C16H13N5O: C 65.97, H 4.50, N 24.04. Found: C 66.12, H 4.23, N 24.09.
6.1.5.17. 6-(3-Methylphenoxyl)-[1,2,4]triazolo[3,4-a]phthalazine-3-
amine (6q). M.p. 250e252 ꢀC, yield ¼ 86%. IR (KBr) cmꢃ1: 3421,
3056, 1637, 1258, 1048. 1H NMR (DMSO, 300 MHz)
d 2.36 (s, 3H,
eCH3), 6.06 (s, 2H, eNH2), 7.10e7.39 (m, 4H, Ar-H), 7.83 (t, 1H,
J ¼ 8.1 Hz, H-8), 8.01 (t, 1H, J ¼ 7.5 Hz, H-9) 8.22 (d, 1H, J ¼ 8.0 Hz,

4812
X.-Y. Sun et al. / European Journal of Medicinal Chemistry 45 (2010) 4807e4812
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This work was supported by the National Natural Science
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Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2010.07.049.
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