Synthesis and anti-inflammatory activity evaluation of a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.02.008

The transcription factor nuclear factor-κB (NF-κB) controls many physiological processes including inflammation, immunity, and apoptosis. In this study, a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives were synthesized as potent anti-inflammatory agents, which acted on tumor necrosis factor (TNF-α) as inhibitors of NF-κB activation. We showed that compounds 6h (6-(2,4-dichlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide) and 6i (6-(3-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide) showed more prominent anti-inflammatory activity than other compounds, with similar activities as the reference drug dihydrotanshinone; compound 6i showed the lowest cellular toxicity among the tested compounds. In vivo evaluation of the anti-inflammatory activity showed that compound 6i exhibited excellent anti-inflammatory activity with 58.19% inhibition at 50?mg/kg intraperitoneal (i.p.), with equal efficacy as the positive control indomethacin (100?mg/kg i.p.; 59.21% inhibition).

Full text of DOI:10.1016/j.bmcl.2016.02.008

Accepted Manuscript
Synthesis and anti-inflammatory activity evaluation of a novel series of 6-phe-
noxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives
Da-Chuan Liu, Guo-Hua Gong, Cheng-Xi Wei, Xue-Jun Jin, Zhe-Shan Quan
PII:
S0960-894X(16)30111-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.008
BMCL 23559
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 November 2015
1 February 2016
4 February 2016
Please cite this article as: Liu, D-C., Gong, G-H., Wei, C-X., Jin, X-J., Quan, Z-S., Synthesis and anti-inflammatory
activity evaluation of a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.008
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Synthesis and anti-inflammatory activity evaluation of a novel
series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide
derivatives
Da-Chuan Liu a, Guo-Hua Gong b, c, Cheng-Xi Wei b, Xue-Jun Jin a, *, Zhe-Shan
Quan a, *
a Key Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian
University, Yanji, Jilin, 133002, China.
b Medicinal Chemistry and Pharmacology Institute, Inner Mongolia University for the
Nationalities, Tongliao, Inner Mongolia, 208002, China.
c Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000,
Inner Mongolia, P.R. China.
*Corresponding author Email: zsquan@ybu.edu.cn (Z,-S. Quan);
xjjin@ybu.edu.cn(X,-J. Jin).

Abstract:
The transcription factor nuclear factor-κB (NF-κB) controls many physiological
processes including inflammation, immunity, and apoptosis. In this study, a novel
series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives were
synthesized as potent anti-inflammatory agents, which acted on tumor necrosis factor
(TNF-α) as inhibitors of NF-κB activation. We showed that compounds 6h
(6-(2,4-dichlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide) and 6i
(6-(3-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide)
showed
more
prominent anti-inflammatory activity than other compounds, with similar activities as
the reference drug dihydrotanshinone; compound 6i showed the lowest cellular
toxicity among the tested compounds. In vivo evaluation of the anti-inflammatory
activity showed that compound 6i exhibited excellent anti-inflammatory activity with
58.19% inhibition at 50 mg/kg intraperitoneal (i.p.), with equal efficacy as the
positive control indomethacin (100 mg/kg i.p; 59.21% inhibition).
Keywords: synthesis, 3-carboxamide triazole, phthalazine, anti-inflammatory,
nuclear factor-κB, TNF-α, MTT
Inflammation is a common pathophysiological phenomenon that is involved in
many diseases and is the most primitive protective response of the body to noxious
stimuli.1 There are many possible causes of inflammation, but the basic pathological
changes are quite similar, such as tissue and cell degeneration, partial response for
microvascular leakage of blood components, necrosis, hyperplasia, and repair; the
clinical symptoms also show a similar trend and are characterized by redness,
swelling, fever, pain, and dysfunction.2-4 Non-steroidal anti-inflammatory drugs
(NSAIDs) showed promising effect in the treatment of acute and chronic
inflammation,5 pain,6 and fever,7 through inhibition of cyclooxygenase (COX).
However, their clinical usage is associated with undesirable and numerous side effects
such as nephrotoxicity, gastrointestinal lesions, and bleeding;8, 9 meanwhile, the
resistance of body to anti-inflammatory drugs is widespread.10 Therefore, it is
particularly important to find and research new targets of prevention and treatment to
inflammation with less adverse effects. Nuclear factor-kappaB (NF-κB) is a key factor

in the immune response triggered by a wide variety of molecules including
inflammatory cytokines with higher specificity. This transcription factor represents a
new target for the development of anti-inflammatory molecules.11 Multiple studies
also have demonstrated the central role of NF-κB in the regulation of many genes
which involved in immunity and inflammation.12-14 Therefore, the inhibitors of NF-κB
function could modulate the inflammatory processes and thus to achieve the purposes
of anti-inflammatory with less adverse effects.
Phthalazine derivatives were reported to possess anticonvulsant,15 cardiotonic,16
vasorelaxant,17 antimicrobial,18 antifungal,19 anticancer,20 and anti-inflammatory21
applications, and our previous study demonstrated their anti-inflammatory activity.22
As a leading compound, we continued to modify on it. Various derivatives of
1,2,4-triazole have been reported to possess anti-inflammatory activity.23-27 Some
studies have shown that many amide compounds have anti-inflammatory activity.28, 29
Therefore, we designed a series of phthalazine derivatives with an amide group at the
first position of the 1,2,4-triazole ring to examine if this could yield compounds with
better activity. Followed the design concept above, we synthesized a series of
6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives and evaluated
their anti-inflammatory activity. However, little attention has been paid to cytokines
tumor necrosis factor (TNF-α) which play an important role in the inflammatory
process and immune responses, even though the anti-inflammatory and
immunomodulatory activities of the factors have been mentioned.30,31 Therefore, we
performed luciferase reporter assay (examination of the inhibitory effect of the
synthesized compounds on the pro-inflammatory mediator TNF-α) to evaluate their
experimental anti-inflammatory activities. Then, the most active compound was
selected to evaluate its anti-inflammatory activity in vivo. We believed that these
screening methods could help us discover potential anti-inflammatory agents.
Compounds were synthesized according to Scheme 1. On the basis of the
previous studies carried out in our laboratory, we designed and synthesized
6-phenoxy-[1,2,4] triazolo[3,4-a]phthalazine-3-carboxamide derivatives (6a-6k). The
starting material 2,3-dihydrophthalazine-1,4-dione (compound 1) was reacted with

refluxing phosphorus oxychloride (POCl3) to yield 1,4-dichlorophthalazine
(compound 2).32 Compound 2 was further reacted with hydrazine hydrate in THF to
yield 1-hydrazine-4-chrorophthalazin (compound 3).33 Then, compound 3 was added
to diethyl oxalate and refluxed to yield ethyl 6-chloro-[1,2,4]triazolo[3,4-a]
phthalazine-3-carboxylate (compound 4). Then, compound 5 was reacted with the
appropriate substituted phenols to yield 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3
-carboxylate derivatives (5a-5k). Finally, the target compounds, 6-phenoxy-
[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives (6a-6k), were obtained
by reacting compounds 5a-5k with concentrated ammonia stirred at room
temperature.34
O
Cl
Cl
Cl
CO2C2H5
CO2C2H5
POCl3
NH
NH
NH2NH2
N
N
N
N
O
HN NH2
1
3
2
R
R
Cl
OH
O
O
R
N
N
NH3
N
N
N
N
O
O
O
K2CO3
O
NH2
N
O
N
N
N
N
N
6a-6k
4
5a-5k
R:
6a: -C6H4(o-F)
6e: -C6H4(p-Cl)
6b: -C6H4(m-F)
6f: -C6H4(p-Br)
6j: -C6H4(p-CH3)
6c: -C6H4(p-F)
6d: -C6H4(m-Cl)
6g: -C6H3(2, 4-2Cl) 6h: -C6H4(o-CH3)
6k: -C6H4(p-OCH3)
6i: -C6H4(m-CH3)
Reagents and conditions: (a) POCl3, reflux, 4 h, yield 83.5%; (b) NH2NH2·H2O, ethyl
acetate, 25-60 ℃, yield 75.6%; (c) diethyl oxalate, reflux, 1h, yield 52.6%; (d)
substituted phenol, K2CO3, ethyl acetate, 70℃, 4h, H2O, 25℃, 0.5h, yield 51.2-61.3%;
(e) NH3 (28% wt. in H2O), 45℃, 1h, yield 45.9-59.8%.
Scheme 1: The synthesis of title compounds 6a-6k.

All the target compounds were screened for their anti-inflammatory activity
using the luciferase reporter assay. NF-κB-dependent luciferase activity was measured
using the Dual Luciferase Reporter Assay system. Cytotoxicity was assessed by the
MTT assay. MTT assay indicates cell viability in response to different compounds at
various concentration, and the data reflected cytotoxicity of the compounds under
evaluation. Then, compound (6i) was evaluated for its anti-inflammatory activity in
vivo by the method of xylene-induced ear edema in Kunming mice (18-22 g body
weight), purchased from the Laboratory of Animal Research, College of Pharmacy,
Yanbian University, then we divided them into 3 groups: the drug treated group
(compound 6i), positive control group (Indomethacin) and normal control group, each
group had 8 mice. The anti-inflammatory activity of compound 6i was evaluated by
intraperitoneal (i.p.) administration at the dose of 50 mg/kg. Then, compound 6i (50
mg/kg) was administered orally (p.o.) to mice and its activities were evaluated at
different intervals (1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). The peak activity of 6i was
observed at 4 h after p.o. administration and compared with that of indomethacin (50
mg/kg, positive control).
It is known that NF-κB regulates several hundreds of genes, including those
involved in immunity, inflammation, anti-apoptosis, cell proliferation, tumorigenesis,
and the negative feedback of the NF-κB signal. NF-κB regulates the transcription of
various inflammatory cytokines, including TNF-α. Therefore, pharmacological
inhibition of NF-κB could be a valuable strategy to modulate the inflammatory
processes. Therefore, in this study, we demonstrated the anti-inflammatory activity of
the compounds (6a-6k) by examining their ability to inhibit TNF-α-induced
NF-κB-dependent reporter gene expression.
To investigate the effect of the compounds on the expression of NF-κB induced
by TNF-α, we performed NF-κB reporter assay. After cells were transiently
transfected with the NF-κB-regulated luciferase reporter vector, the cells were further
incubated with TNF-α in the presence of various concentrations of the compounds.
Dihydrotanshinone is a component of the traditional Chinese medicinal plant Salvia
miltiorrhiza Bunge. It has multiple therapeutic activities and is used to treat

vasculocardiac disease, hepatitis, inflammation, and cancer.35, 36 Previous studies have
demonstrated the anti-inflammatory activity of dihydrotanshinone and proved that the
activity was reacted through inhibits TNF-α-induced NF-κB activation.37, 38 So it was
used as positive control in this study.
We found that all the eleven compounds 6a-6k exhibited anti-inflammatory
activity to a certain extent (Fig. 1, compared with the TNF-α-induced group). Among
the halogen-substituted derivatives, 6-(2,4-dichlorophenoxy)-[1,2,4]triazolo[3,4-a]
phthalazine-3-carboxamide (compound 6g) showed better activity than the others and
its activity was similar to the normal group (untreated with TNF-α). However, we
found that the position of substituted group on the phenyl ring barely influenced the
anti-inflammatory activity of these compounds and compound 6g exhibited more
cellular toxicity. Then, we focused our attention on the compounds containing
electron donor group on the phenyl ring. Notable, 6-(3-tolyloxy)-[1,2,4]triazolo[3,4-a]
phthalazine-3-carboxamide (compound 6i) showed better activity than the normal
group and had comparable anti-inflammatory activity with dihydrotanshinone. Taken
together, 6i was found to have the highest anti-inflammatory activity among the
derivatives in this series.

Fig. 1: HeLa cells were transiently transfected with a NF-κB-dependent reporter gene
for 48 h and then pretreated for 8 h with the 100 μM of the compounds 6a-6k
followed by stimulation for 8 h with TNF-α (10 ng/ml), and the luciferase activity was
determined as described in “6.2. Pharmacology”. Data represented as mean ± standard
deviation of three independent experiments. **P＜0.01, ***P＜0.001, significantly
different when compared with TNF-α-stimulated normal cells.
Based on the results of primary screening, two most active derivatives 6g and 6i
were chosen for evaluation in the further screening. We found that TNF-α-induced
NF-κB reporter activity was substantially suppressed by compounds 6g and 6i in a
dose-dependent manner (Fig. 2). Both of them showed strong anti-inflammatory
activities at 100 μM, but when the dose was reduced, their activity markedly
diminished. Nevertheless, they also had some anti-inflammatory activities at 30 μM,
and compound 6i, and not 6g, exhibited anti-inflammatory activity even at 10 μM.

Fig. 2: HeLa cells were transiently transfected with a NF-κB-dependent reporter gene
for 48 h and then pretreated for 8 h with the indicated concentrations of compounds 6i
and 6g followed by stimulation for 8 h with TNF-α (10 ng/ml), and the luciferase
activity was determined as described in “6.2. Pharmacology”. Data represented as
mean ± standard deviation of three independent experiments. **P＜0.01, ***P＜
0.001, significantly different when compared with TNF-α-stimulated normal cells.
To evaluate the effect of compounds 6g and 6i on viability of HeLa cells, the
MTT assay was performed. The cytotoxicity of both compounds decreased at
decreasing dosage. Compound 6g showed higher cytotoxicity, but compound 6i
exhibited slight cellular toxicity on HeLa cells at 100 μM (Fig. 3). Generally,
compound 6i showed reliable anti-inflammatory potential and lower toxicity than 6g.

Fig. 3: HeLa cells were treated with the indicated concentrations of the compounds 6i
and 6g. After 24 h incubation, cell viability was determined by MTT assays. Data
represented as mean ± standard deviation of three independent experiments.
Based on the above results, the anti-inflammatory activity of compound 6i
further evaluated in vivo. In this experiment, we used indomethacin as the control
positive drug. Indomethacin is a widely used non-steroidal anti-inflammatory drugs
(NSAIDs) with anti-inflammatory, analgesic and antipyretic activity. Compound 6i
showed significant anti-inflammatory activity with 58.19% inhibition at 50 mg/kg
(0.5 h after i.p.) in the Kunming mice. The results are shown in Table 1. Apparent
statistical significance was observed between the test group and the control group. It
is worth mentioning that compound 6i and the control positive drug indomethacin (at
a dosage of 100 mg/kg) showed equivalent anti-inflammatory activities. To determine
the oral time to peak effect (TPE) of compound 6i, we conducted a time-course test by

administering oral medications (p.o.; Table 2). As the interval lengthened, the
anti-inflammatory activity of compound 6i first increased and then declined at the
dosage of 50 mg/kg; the peak activity was observed at the 4 h interval with 38.8%
inhibition, which was lower than that observed with the positive control drug.
Table 1. Anti-flammatory activites of compound 6i and the positive control
administrated i.p.
Number
Edema mean
±SEM.(mg)
3.70±0.73*
3.61±0.49*
8.85±1.00
Inhibition
rate(%)
58.19
59.21
-
Comp.
Dose (mg/kg)
of mice
50
100
-
8
8
8
6i
Indomethacin
DMSO
*p＜0.05 compared with vehicle control group.
Table 2. anti-inflammatory activity of compound 6i administrered orally at different
times before xylene application.
Inhibition
Time (h)
Indomethacin (Dose 100 mg/kg)
6i (Dose 50 mg/kg)
1
2
3
4
5
6
5.63
8.92
19.05
22.49
22.73
38.85*
22.44
8.19
27.67*
63.39*
53.46*
29.65
*p＜0.05 compared with vehicle control group at the corresponding time.
In conclusion, a new series of anti-inflammatory 6-phenoxy-[1,2,4]triazolo[3,4-a]
phthalazine-3-carboxamide derivatives were synthesized and their anti-inflammatory
activities were evaluated by the luciferase reporter assay. Two compounds 6g and 6i

showed promising anti-inflammatory activity, which was much better than the
TNF-α-induced group and slightly more potent than the normal control group. It is
worth mentioning that compound 6i had comparable activity with the reference drug
dihydrotanshinone, and showed lower cellular toxicity than compound 6g, indicating
that 6i is most likely safer than 6g. Then, compound 6i was selected for
anti-inflammatory evaluation in vivo. The activity screening indicated that compound
6i at 50 mg/kg showed 58.19% inhibition compared with the control positive drug at
the dose of 100 mg/kg in mice at 0.5 h after i.p.. It means that compound 6i
could achieve a similar anti-inflammatory activity with a lower dosage than
Indomethacin, the widely used NSAIDs. We then reported that intraperitoneal
administration of the drug was superior to oral one (only with 38.8 % inhibition by
p.o.). We inferred that the anti-inflammatory activity of compound 6i might be
affected by the first-pass effect.
Conflict of interest: We declare that we have no conflict of interest with respect
to this study.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(No. 81160382) and National Science and Technology Major Project of China (No.
2012ZX09103-101-044).
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Synthesis and anti-inflammatory activity evaluation of a novel series
of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide
derivatives
Da-Chuan Liu a, Guo-Hua Gong b, c, Cheng-Xi Wei b, Xue-Jun Jin a, *, Zhe-Shan Quan a, *
A new series of anti-inflammatory 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3
-carboxamide derivatives were synthesized and their anti-inflammatory activities were
evaluated
by
the
luciferase
reporter
assay.
6-(3-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine -3-carboxamide (compound 6i) had
comparable activity with the reference drug dihydrotanshinone with low cellular
toxicity. Then, compound 6i was selected for anti-inflammatory evaluation in vivo.
The activity screening indicated that compound 6i at 50 mg/kg showed 58.19%
inhibition at 0.5 h after i.p. and 38.8 % inhibition by p.o..
R
O
O
O
NH
NH
N
N
N
N
O
O
O
NH2
NH2
N
N
N
N
R: m-CH3
Target compound