Design, synthesis and biological evaluation of novel anti-cytokine 1,2,4-triazine derivatives

Article abstract of DOI:10.1016/j.bmc.2013.08.009

A series of 16 novel 1,2,4-triazine derivatives bearing hydrazone moiety (7a-7p) have been designed, synthesized and evaluated for their activity to inhibit IL-1β and TNF-α production. All compounds are reported for the first time. The chemical structures of all compounds were confirmed by spectroscopic methods and elemental analyzes. Most of the synthesized compounds were proved to have potent anti-cytokine activity and low toxicity on PBMC and MCF-7 cell lines. Compounds 7f, 7k, 7l and 7j presented simultaneously good levels of inhibition of both cytokines. Moreover, compound 7l exhibited good anti-inflammatory effect in carrageenan-induced rat paw edema. The results of Western blotting demonstrated that the anti-cytokine potential of compound 7l is mainly mediated through the inhibition of p38 MAPK signaling pathway. Molecular docking was performed to position compound 7l into p38α binding site in order to explore the potential target. The information of this work might be helpful for the design and synthesis of novel scaffold toward the development of new therapeutic agent to fight against inflammatory diseases.

Full text of DOI:10.1016/j.bmc.2013.08.009

Accepted Manuscript
Design, synthesis and biological evaluation of novel anti-cytokine 1,2,4-triazine
derivatives
Mehdi Khoshneviszadeh, Mohammad H. Ghahremani, Alireza Foroumadi,
Ramin Miri, Omidreza Firuzi, Armin Madadkar-Sobhani, Najmeh Edraki,
Maliheh Parsa, Abbas Shafiee
PII:
S0968-0896(13)00691-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.009
BMC 11025
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 May 2013
5 August 2013
5 August 2013
Please cite this article as: Khoshneviszadeh, M., Ghahremani, M.H., Foroumadi, A., Miri, R., Firuzi, O., Madadkar-
Sobhani, A., Edraki, N., Parsa, M., Shafiee, A., Design, synthesis and biological evaluation of novel anti-cytokine
1,2,4-triazine derivatives, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.08.009
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Design, synthesis and biological evaluation of novel anti-cytokine 1,2,4-triazine derivatives
Mehdi Khoshneviszadeha,b, Mohammad H. Ghahremanic,d, Alireza Foroumadia,e, Ramin Mirib,
Omidreza Firuzib, Armin Madadkar-Sobhanif, Najmeh Edrakia,b, Maliheh Parsac, Abbas
Shafieea,*
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran.
b
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran.
cDepartment of Toxicology-Pharmacology, School of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran.
dToxicology & poisoning Research Center, Tehran university of Medical Sciences, Tehran,
Iran.
eNeuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran.
f Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
* Corresponding author: AbbasShafiee, Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran14176, Iran.
Tel.: +98 2166406757; Fax: +98 21 66461178, E-mail: ashafiee@ams.ac.ir
1

Abstract
A series of 16 novel 1,2,4-triazine derivatives bearing hydrazone moiety (7a-7p) have been
designed, synthesized and evaluated for their activity to inhibit IL-1β and TNF-α production. All
compounds are reported for the first time. The chemical structures of all compounds were
confirmed by spectroscopic methods and elemental analyzes. Most of the synthesized
compounds were proved to have potent anti-cytokine activity and low toxicity on PBMC and
MCF-7 cell lines. Compounds 7f, 7k, 7l and 7j presented simultaneously good levels of
inhibition of both cytokines. Moreover, compound 7l exhibited good anti-inflammatory effect in
carrageenan-induced rat paw edema. The results of western blotting demonstrated that the anti-
cytokine potential of compound 7l is mainly mediated through the inhibition of p38 MAPK
signaling pathway. Molecular docking was performed to position compound 7l into p38α binding
site in order to explore the potential target. The information of this work might be helpful for the
design and synthesis of novel scaffold toward the development of new therapeutic agent to fight
against inflammatory diseases.
Keywords: IL-1β, TNF-α, p38 MAPK, 1,2,4-triazine, anti-inflammatory activity
2

1. Introduction
Inflammation is a natural, beneficial and spontaneous biological response to noxious stimuli,
such as pathogens, chemicals, thermal and mechanical injuries. It is a complex defensive process
that may vary from a localized to a generalized response characterized by the accumulation of
local fluids and leukocytes with the objective of eliminating the harmful stimulus. The
inflammation is produced and regulated by the interaction of a variety of inflammatory mediators
such as histamine, 5-hydroxytryptamine, various chemotactic factors such as bradykinin,
leukotrienes, prostaglandins and pro- inflammatory cytokines (IL-1β, TNF-α, etc).1-5
The pro- inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1-beta (IL-
1β) are small molecular weight proteins secreted mainly by the immune cells. They act as
important mediators involved in the pathophysiology of several chronic inflammatory diseases,
including rheumatoid arthritis (RA), inflammatory bowel disease, septic shock, and
osteoporosis.6, 7
The main roles of TNF-α and IL-1β in chronic inflammatory diseases are demonstrated by
several studies in animal models as well as therapeutic experience in humans. Inhibition of TNF-
α in animal models of RA has been shown to suppress joint swelling, while neutralization of IL-
1β prevents cartilage damage and reduces the inflammatory cell recruitment.8 According to these
findings, a number of biologic agents which specifically inhibit TNF-α or IL-1β have been
approved for the treatment of inflammatory disorders such as rheumatoid arthritis, psoriasis and
inflammatory bowel disease. While these biological agents (Enbrel®, Remicade®,Humira® and
Kineret®) have changed the treatment paradigm and have significantly improved the quality of
life of RA patients, they have disadvantages of most of biologic agents, such as high cost, lack of
oral efficacy, short half-life, potential immune reactions and inconvenient dosing regimens.
9Therefore, these facts suggest that small molecules representing both inhibitions of IL-1β and
TNF-α would display a highly attractive alternative for the treatment of inflammatory disease.
Simply, inhibition of the effects of TNF-α and IL-1β by non-peptidic, small molecules is
currently being investigated through several different strategies.6, 10
Activation of the mitogen-activated protein (MAP) kinase p38 is a key step in the signaling
pathway leading to the production of pro-inflammatory cytokines such as TNF-α and IL-1β at
the levels of transcription and translation.11, 12
3

p38 MAP kinase, a 38 kDa protein, is a serine/threonine kinase activated in response to pro-
inflammatory cytokine including TNF-α, transforming growth factor-β (TGF-β), osmotic shock,
endotoxin (bacterial lipopolysaccharide, LPS) and UV light.13, 14Thep38 kinase consists of four
isoforms (α, β, δ and γ), which have a large degree of sequence identity, but different tissue
distributions. The p38α kinase is believed to be the isoform primarily responsible for regulation
of inflammation; therefore compounds that selectively inhibit p38α and β relative to δ and γ
produce anti-inflammatory effects. Like other kinase inhibitors, first generation p38 MAPK
16
inhibitors like the pyridinyl-imidazole (SB203580) (Fig. 1)15are fully competitive with ATP.
The co-crystal structure of p38 MAPK with SB203580 showed that these inhibitors partially
occupy the ATP binding site, preventing further phosphorylation of p38 MAPK. The most
important interactions observed between p38 MAPK and SB203580 in the co-crystal structure
are (Fig. 1): (i) a hydrogen bond between the N3 of the imidazole core and a hydrogen atom of
the –NH2 group of Lys53 (ii) The pyridine ring nitrogen of the inhibitor forms a hydrogen bond
to the backbone NH of Met109 in the linker region connecting the small and the large lobe of the
kinase something, which is also observed for the ATP adenosine ring; and (iii) a π-stacking
interaction between the phenyl ring of the methyl sulfinylphenyl moiety and the phenyl ring of
Tyr35. Additionally, in close proximity to this interaction site, a hydrophobic pocket more
spacious in p38 than in other Ser/Thr kinases accommodates the 4-fluorophenyl ring of
SB203580 and located behind and orthogonally to the ATP binding site.17, 18
Figure 1
In our continuing effort towards the identification of anti-inflammatory agents, 19-22 in the present
study, a series of novel anti-inflammatory compounds possessing 5,6-diaryl-1,2,4-triazine are
designed based on ‘analogue-based’ design strategy.
1,2,4-Triazine nucleus is a prominent structural core system present in numerous biologically
active compounds, possessing a wide range of biological activities. Many investigations revealed
the anticancer potential of triazine derivatives.23Triazine derivatives have been also introduced as
antitumor, 24, 25 anti-inflammatory, 26 radical scavenger,27β-sheet breaker,28and antifungal29agents.
The purpose of this study is to investigate the benefits of such hybridization on the anticipated
biological activities, and to determine whether this will lead to improved biological activity of
4

synthesized compounds. Finally, we have employed in vitro assays to evaluate anti-cytokine
activity of designed compounds and determined the mechanism of action by using western blot
analysis.
2. Result and discussion
2.1. Analogue-based pharmacophore design
The novel 1,2,4-triazine derivatives based pharmacophore for p38 MAPK inhibitors was
elucidated by analogue-based design that uses two selective p38 MAPK inhibitors (pamapimod
(5) and R1487 (6))30and IL-1β production inhibitors 3 that were introduced by Matsuda et al.31,
32( Fig.2). Some evidences suggested that the hydrazone moiety ofphenylhydrazone derivative
(1) possesses a pharmacophoric character for the inhibition of COX. Moreover; hydrazone type
containing compounds such as compound 2 (Fig. 2) were described as dual COX/5-LOX
inhibitors.33Mantlo et al. showed that amino pyridine derivatives (4) and amino pyridazine
derivatives were potent anti–cytokine agents.34In addition, various scaffolds have been identified
as p38-MAPK inhibitors.35This implies the flexibility of p38-MAPK active site to accommodate
different chemical scaffolds. Therefore, the main focus of this study was the design of anti-
cytokine agents that exert anti-inflammatory activity through various mechanisms. To this end,
we first considered the key fragment of selected lead compounds of relevant anti-cytokine
scaffold (IL1-β production inhibitor, p38-MAPK inhibitor and COX and LOX inhibitor) for
design of multi-target scaffold. Finally, the new anti-cytokine scaffold was designed through the
hybridization method which is common in the medicinal chemistry.
In attempt to build a complete pharmacophore, herein, we selected 1,2,4-triazine as central core
because the nitrogens (N1 or N2) of this six member heterocycle form the key hydrogen bond
with Met 109 of p38 active site and the adjacent nitrogen facilities this interaction. The ‘diaryl’
system, which is common in 3 was considered as the side arm attachment to central 1,2,4-triazine
core. Further, attachment of the amine moiety into the position 3 of central core could be
involved in additional hydrogen bond with Met 109 or other amino acids of active site as it was
established in pamapimod (5) and R1487 (6). Finally, in order to improve anti-cytokine and anti-
inflammatory activity of these designed compounds, this amine side chain was further replaced
by hydrazone moiety.
5

Figure 2
2.2. Chemistry
The reactions were carried out by environmentally friendly conditions (‘green chemistry’)
through elimination of hazardous substances to human health and the environment. The synthetic
pathway for the preparation of 1,2,4-triazine derivatives (7a-7p) is outlined in Scheme 1. We
used Thiamine hydrochloride (Vitamin B1) instead of highly toxic cyanide ion to prepare 2-
hydroxy-1,2-bis(4-methoxyphenyl)ethanone (2). Compound (2) oxidized in the presence of nitric
acid to get corresponding benzyl (3)36. 5,6-Diaryl-1,2,4-triazine-3-thiol (4) was prepared by
cyclization of compound (3) with thiosemicarbazide as reported previously.37, 38 The compound
(5) was synthesized by the reaction of compound (4) with methyl iodide in basic ethanol. 1-(5, 6-
bis (4-methoxyphenyl)-1,2,4-triazin-3-yl)hydrazine (6) was obtained by the reaction of 5,6-bis(4-
methoxyphenyl)-3-(methylthio)-1,2,4-triazine (5) with excess amount of hydrazine hydrate in
ethanol at reflux condition. Finally, target compounds (7a-7p) were prepared in 70-90% yield
using the general synthetic method previously described.39Accordingly, Hydrazone (7a-7p) were
obtained by coupling (6) with equimolar amounts of the appropriate commercially available
1
carboxaldehydes in warm ethanol. The synthetic yields, melting points, H NMR, 13C NMR and
EI-MS analysis of unpublished compounds are described in the Section 4.
Scheme 1
2.3 Biological activity
2.3.1. In vitro assay for TNF-α and IL-1β inhibition
The TNF-α and IL-1β have important role in inflammation. Furthermore, p38 MAPK is known to
regulate signaling cascades coupled to the TNF-α and IL-1β receptors. Therefore, the p38 pathway
mediates TNF-α and IL-1β function as well as their production. Thus, regulation of TNF-α and
IL-1β via inhibition of p38 MAP kinase is considered an attractive target for anti-inflammatory
therapy.40
Lipopolysaccharide (LPS) is an important structural component of the outer membrane of gram-
negative bacteria, and it is a well-studied immunostimulator that induces a systemic
6

inflammation response, 41and especially, the expression of proinflammatory cytokines, such as
TNF-α and IL-1β.
The synthesized compounds were evaluated for their inhibitory abilities against LPS induced
TNF-α and IL-1β release in PBMC (Peripheral Blood Mononuclear Cells). The cells were pre-
incubated with 10 μM compounds and DMSO as a control for 30 min and then incubated with 1
μg/ml LPS for 6 h at 37 Co. The amount of TNF-α and IL-1β in media was detected through
enzyme-linked immunosorbant assay (ELISA). A well-known inhibitor of the inflammatory
pathway key intermediate p38 MAP kinase was used as a reference compound (SB202190;
Sigma Chemical Co., St. Louis, MO). The results of the anti-inflammatory assay of synthesized
compounds are summarized in Table 1.
SB202190 reduced LPS-mediated TNF-α and IL-1β expression at 87.2% and 93.4%,
respectively. Among these 16 compounds, the majority of the tested compounds inhibited LPS-
induced TNF-α and IL-1β expression to various degrees. All Compounds showed considerable
inhibitory ability against LPS-induced TNF- α expression.
Table 1
Compounds 7a, 7d, 7f, 7g, 7k and 7l showed inhibition of TNF- α production in a range of 50-
72%. The most potent 1,2,4-triazine derivatives on inhibiting IL-1β production, compounds 7l,
7g and 7j, demonstrated the inhibitory percent equal to 89.8, 82.3 and 86.9 at 10µM respectively.
7l, namely 2,3-dimethoxy-substituted compound, showed the strongest inhibitory effect on LPS-
induced TNF-α and IL-1β release among the tested synthesized compounds and its inhibitory
potential reached 71.8% and 89.8% at 10µM respectively. Considering the data of Table 1, the
following structure activity relationship may be developed:
-
The introduction of strong electron-withdrawing groups such as nitro or fluoro on the
aromatic ring led to a reduced inhibitory potential as deduced from compound 7h because
this kind of electron-attracting substituent reduced capability of N-H to form hydrogen
bond with Met 109or other aminoacidsin active site of p38 MAPK protein.
The presence of electron-donating or electron-withdrawing substituent on position 4 of
aromatic ring slightly diminished anti-cytokine activity of synthesized compounds as can
be seen in compounds7i, 7m and 7o.
-
-
Five membered heterocyclic rings similar to substituted phenyl showed considerable
inhibition of cytokine production (compounds 7d and 7k).
7

2.3.2. Western blotting
In order to investigate the p38 inhibitory activity as a possible mechanism of anti-
cytokine potential of synthesized derivatives, we further evaluated the effect of potent anti-
cytokine agent of 1,2,4-triazine scaffold 7l on the p38 signaling pathway in a cell-based assay
using MCF-7 breast cancer cells. Protein levels of phospho-p38 and phospho-ATF-2 were
determined by western blot analysis. We have previously examined the effect of etoposide
42
treatment on MCF-7 cells in a dose and time-dependent manner. Etoposide-induced activation
of p38 signaling, as shown for the p38 phosphorylation target activating transcription factor 2
(ATF-2), was inhibited at doses 20μM (Fig3).
In this study, our synthesized compounds are designed as inhibitors of p38 kinase which lead to
blocking the phosphorylation (activation) of downstream targets. However, compound 7l shows
no effect on phospho-p38 levels that was facilitated by MKK. The p38 kinas activity of our
synthesized compound is presented by decreasing in phosphorylation of ATF-2 (the downstream
target of p38) following drug treatments. In the other words, specific inhibition effect of this
series on p38 activity has been clarified by lack of effect on MKK (upstream to p38) and
blocking of its downstream targets like ATF-2. The results confirmed that the anti-cytokine
activity by 7l may be mediated by the inhibition of active p38 (or phospho-p38) MAPK.
Figure 3
2.3.3. Reduction of carrageenan-induced inflammatory condition in rats
The most active compound 7l was selected amongst all derivatives for the carrageenan
test. The effect of the tested compound and indomethacin, as a reference, was measured before
and 1, 2, 3 and 4h after carrageenan injection. Percent edema inhibition was calculated with
regard to saline as control group (Table 2). The selected compound showed a reasonable
inhibition of edema size in comparison with indomethacin. The result showed a 68% paw edema
reduction after 4h administration of compound 7l.
Table 2
8

2.3.4 Molecular docking study
In order to obtain better understanding on the mechanism and potency of the synthesized
compounds and guide further structure–activity relationships (SAR) studies, the molecular
docking was performed by placing inhibitor 7l into binding site of p38, the same binding site of
pamapimod. All docking runs were applied by docking program Autodock 4.2.43
The binding models of compound 7l with p38 (PDB code: 3FLY) were depicted in Fig.4. The
ATP competitive inhibitor 7l binds in the ATP binding site located in the cleft between the N-
and the C-terminal domains of p38α MAP kinase. Three important hydrogen bond interactions
could be seen between inhibitor and active site residues:
-
Key Hydrogen bond interaction between the nitrogen atom of 1, 2, 4-triazine heterocycle
core of 7l and the backbone NH of Met109. (distance : 2.83 Å)
-
-
Hydrogen bond between NH of 7l and carbonyl of His107 (distance: 1.99 Å)
Oxygen atom of methoxy group exhibited hydrogen bond with NH of Lys53 (distance:
3.22 Å, angle: about 127°)
These interactions are analogous to the H-bonding interactions typically seen between the
adenine of ATP and a kinase. The 2,3-dimethoxyphenyl group occupied a lipophilic pocket
located past the gate keeper residue, Thr-106. In order to evaluate the precision of resulted
docking orientation, we also examined the docking process with other software, which also
confirmed our results.
Figure 4
2.3.5 Cytotoxicity test
The inhibitory activity of the compounds is sometimes a result of their toxic effect and
consequently might cause an erroneous conclusion, the cytotoxicity of all the synthesized 1,2,4-
triazine derivatives were evaluated using LPS-activated peripheral blood mononuclear cells
(PBMC) and MCF-7. Potential compound toxicity at examined concentrations (10 and 20 µM)
was assessed by MTT assay. Values are reported as percent of cell viability (the viability percent
of more than 90% was considered as acceptable viability). The data showed that most of the
synthesized compounds have a quite low cytotoxicity.
9

3. Conclusion
In conclusion, a series of novel 1,2,4-triazinederivatives containing hydrazone moiety
have been designed, synthesized and their biological activities were evaluated as potent anti-
cytokine activity. The results showed promising activity of most of the compounds as inhibitor
of both cytokines with very low cytotoxicity. Among them, compound 7l presented the highest
level of inhibition of TNF-α, being also one of the major inhibitors of IL-1β. The results of
western blotting demonstrated that compound 7l blocks etoposide-induced activation of p38
MAPK signaling pathway in MCF-7 cells.
A computer model of the interaction of compound 7l with the p38α MAPK kinase in the ATP
binding pocket was proposed. The model involved a favorable H-bond network with the
conserved hydrogen bonds to Met109 and adjacent His107 in complete pharmacophoric
equivalency to the known ATP binding mode. Furthermore, aryl moiety occupies the kinase’s
hydrophobic region I and methoxy group on aryl moiety involving third hydrogen bond with
Lys53.Finally, compound 7l showed good anti-inflammatory activity in carrageenan-induced
inflammation model.
The information of this work might be helpful for the design and synthesis of a leading
compound7l toward the development of new therapeutic agent to fight against inflammatory
diseases.
4. Experimental protocols
4.1. Chemistry
The starting materials, chemical reagents and solvents used in this study were obtained from
commercial sources. NMR-spectra were recorded on Brucker 500 spectrometer or Varian unity
400 spectrometer and chemical shifts (δ) are reported in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard using CDCl3 or DMSO-d6 as solvent. The mass
spectra were run on an Agilent 6410. The IR spectra were obtained on a FT-IR Perkin-Elmer
Precisely system spectrophotometer (potassium bromide disks). Melting points were determined
on a Kofler hot stage apparatus and are uncorrected. Elemental analyses were carried out on a
CHN-O-rapid elemental analyzer (Heraeus, Hanau, Germany) for C, H and N, and the results are
within ± 0.4% of the theoretical values.
10

Merck silica gel 60 F254 plates were used for analytical TLC. Preparative thin-layer
chromatography was done with prepared glass-backed plates (20 20 cm2, 500 µ) using silica
gel (MerckKieselgel 60 HF254, Art. 7739).
General procedures:
The 2-hydroxy-1,2-bis(4-methoxyphenyl)ethanone (2) and 1,2-bis(4-methoxyphenyl)ethane-1,2-
dione (3) were prepared according to the previously described procedure.36
4.1.1. Synthesis of 5,6-bis(4-methoxyphenyl)-1,2,4-triazine-3-thiol (4)
The 1,2-bis(4-methoxyphenyl)ethane-1,2-dione (10 g, 37 mmol) was added to 60 ml. of acetic
o
acid and the mixture was heated to about 100 C with stirring. Thiosemicarbazide (6.84 g, 75.04
mmol) was added and the mixture was refluxed for about two hours. The mixture was cooled and
the solid product was collected by filtration. The solid was washed with cold acetic acid and
water. The product was air dried to yield 10.84 g. of 5,6-bis(4-methoxyphenyl)-1,2,4-triazine-3-
o
thiol;mp: 237-239 C.36, 43.Anal.Calcd for C17H15N3O2S: C 62.75, H 4.65, N 12.91, found: C
62.61, H 4.59, N 12.66.
4.1.2. 5,6-Bis(4-methoxyphenyl)-3-(methylthio)-1,2,4-triazine (5)
The sodium hydroxide (0.8 g, 20 mmol) was dissolved in 60 ml of ethanol by warming. The
basic solution was cooled to room temperature and 5,6-bis(4-methoxyphenyl)-3-(methylthio)-
1,2,4-triazine (6.7 g, 20 mmol) was added and stirred for 15 min. Methyl iodide (6.7g, 47 mmol)
was added dropwise to the reaction mixture and the mixture immediately became a slurry. Thirty
milliliters of ethanol was added to the reaction mixture and stirring was continued for about 4
hours at room temperature. To the mixture 15 ml of water was added and the yellow precipitate
was filtered, washed with ethanol and subsequently dried under reduced pressure (yield:
91%)44;mp: 157-160◦C, 1H NMR (CDCl3, 500 MHz): δH= 7.96 (d, J=8.7 Hz , 2H, ArH), 7.58 (d,
J=8.7 Hz, 2H, ArH), 6.91 (d, J=8.5 Hz, 2H, ArH), 6.85 (d, J=8.5 Hz, 2H, ArH), 3.85 (s, 6H,
2×O-CH3), 2.74 ppm (s, 3H, S-CH3).Anal. Calcd for C18H17N3O2S: C 63.70, H 5.05, N 12.38,
found: C 63.51, H 4.89, N 12.68.
4.1.3 3-Hydrazinyl-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (6)
11

A solution of NH2NH2.H2O (3 ml) in 30 ml. ethanol was treated with 5,6-bis(4-methoxyphenyl)-
3-(methylthio)-1,2,4-triazine (4g, 11.8 mmol). The reaction mixture was stirred under reflux for
12 h, and allowed to cool to room temperature. The precipitated product was filtered off, washed
with cold ethanol, dried and recrystallized from ethanol to give dark yellow crystalline 3-
hydrazinyl-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (yield 83%); mp: 185-188◦C,1H NMR
(DMSO-d6, 500 MHz): δH= 8.75 (s, 1H, N-H), 7.45 (d, J=8.6 Hz , 2H, ArH), 7.32-7.28 ( m, 2H,
ArH), 6.94-6.91(m, 4H, ArH), 4.44 (brs, 2H, NH), 3.78 (s, 3H, O-CH3), 3.77 ppm ( s, 3H, O-
CH3); IR (KBr): ν = 3248, 2936, 1607, 1519, 1174, 830 cm-1; MS (EI) m/z (%): 323 (M+, 100),
252 (15), 238 (91), 223 (73), 195 (18), 180 (12).Anal. Calcd for C17H17N5O2: C 63.15, H 5.30, N
21.66, found: C 62.81, H 5.41, N 21.38.
4.1.4. General method for synthesis of 3-(2-arylidenehydrazinyl)-5,6-bis(4-methoxyphenyl)-
1,2,4-triazine (7a-7p)
To a well-stirred warm (70-75◦C) solution of 3-hydrazinyl-5,6-bis(4-methoxyphenyl)-1,2,4-
triazine (1 mmol) in 10 ml ethanol was added equivalent amount of appropriate aldehyde (1
mmol) and treated with a few drops of glacial acetic acid. The reaction mixture was stirred for 2
h. The completion of the reaction was monitored by TLC. After completion, the precipitate was
filtered off, washed with cold ethanol and recrystallized from suitable solvent to yield the
desirable pure products 7a-p.
3-(2-Benzylidenehydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7a)
Compound 7a was prepared by the described procedure using 106 mg (1 mmol) of benzaldehyde
1
(yeild 83%): mp: 245-247◦C; H NMR (DMSO-d6, 500 MHz): δH= 10.29 (s, 1H, NH), 8.24 (s,
1H, N=CH), 7.81 (d, J= 7.1 Hz, 2H, ArH), 7.66 (d, J= 8.8 Hz, 2H, ArH), 7.50 (d, J= 8.7 Hz, 2H,
ArH), 7.43-7.38 (m, 3H, ArH), 6.93 (d, J= 8.7 Hz, 2H, ArH), 6.88 (d, J= 8.7 Hz, 2H, ArH), 3.86
ppm (s, 6H, 2×O-CH3); IR (KBr): ν = 3440, 3211, 1605, 1571, 831 cm-1; MS (EI) m/z (%): 411.2
(M+, 29), 334 (20), 308 (14), 238 (100), 223 (49), 195 (12), 152 (11). Anal.Calcd for
C24H21N5O2: C 70.06, H 5.14, N 17.02, found: C 70.21, H 4.86, N16.95.
5,6-Bis(4-methoxyphenyl)-3-(2-(4-nitrobenzylidene)hydrazinyl)-1,2,4-triazine (7b)
12

The titled compound was synthesized by the described procedure from 4-nitrobenzaldehyde (151
mg, 1 mmol) as yellow solid (yield 88%): mp: 265-267◦C; 1H NMR (DMSO-d6, 500 MHz): δH=
12.23 (s, 1H, NH), 8.36 (s, 1H, N=CH), 8.31 (d, J= 8.6 Hz, 2H, ArH), 7.99 (d, J = 8.0 Hz, 2H,
ArH), 7.52 (d, J= 8.3 Hz, 2H, ArH), 7.39 (d, J= 8.6 Hz, 2H, ArH), 6.98-6.96 (m, 4H, ArH), 3.79
ppm (s, 6H, 2×O-CH3); IR (KBr): ν = 3421, 3205, 1607, 1585, 838 cm-1; MS (EI) m/z (%): 456
(M+, 21), 308 (16), 238 (100), 223 (25), 195 (11).Anal. Calcd for C24H20N6O4: C 63.15, H 4.42,
N 18.41, found: C 62.78, H 4.12, N 18.51.
3-(2-(3,4-Dimethoxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7c)
The yellow solid product 7c was prepared in similar described manner using 3,4-
1
dimethoxybenzaldehyde (166 mg , 1mmol): (yield 82%), mp: 250-253◦C; H NMR (DMSO-d6,
500 MHz): δH=11.70 (s, 1H, NH), 8.19 (s, 1H, N=CH), 7.49 (d, J=8.6 Hz, 2H, ArH), 7.37-7.35
(m, 3H, ArH), 7.22 (d, J=8.2 Hz, 1H, ArH), 7.03 (d , J=8.2Hz, 1H, ArH), 6.96-6.94 (m, 4H,
ArH), 3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.78ppm (s, 6H, 2×O-CH3); 13C NMR (DMSO-
d6, 125MHz): δC=160.91, 159.31, 158.19, 155.29, 150.30, 150.07, 148.95, 143.62, 142.85,
131.14, 130.15, 128.74, 127.96, 127.59,120.64, 113.80, 113.71, 111.54, 108.32, 55.83,55.50,
55.3, 55.27, 55.10ppm; IR (KBr): ν = 3444, 3216, 1609, 1508, 836 cm-1; MS (EI) m/z (%): 456
(M+, 16), 308 (28), 238 (100), 223 (35), 195 (13), 152 (10). Anal. Calcd for C26H25N5O4: C
66.23, H 5.34, N 14.85, found: C 66.34, H 5.21, N 14.65.
5,6-Bis(4-methoxyphenyl)-3-(2-(thiophen-2-ylmethylene)hydrazinyl)-1,2,4-triazine (7d)
The yellow solid product 7d was prepared from thiophene-2-carbaldehyde (112 mg, 1 mmol) in
1
similar manner described in general procedure(yield: 91%): mp: 250-253◦C; H NMR (DMSO-
d6, 500 MHz): δH= 11.79 (s, 1H, NH), 8.45 (s, 1H, N=CH), 7.61 (s, 1H, ArH), 7.44-7.46 (m, 2H,
ArH), 7.39-7.35 (m, 3H, ArH), 7.12 (brs, 1H, ArH), 6.96-6.95 (m, 4H, ArH), 3.78 ppm (s, 6H,
2×O-CH3); 13C NMR (DMSO-d6, 125MHz): δC= 160.94, 159.34, 158.03, 155.37, 151.23,
150.33, 139.65, 138.66, 131.13, 130.17, 129.28, 128.67, 127.90, 127.76, 55.27, 55.10 ppm. IR
(KBr): ν = 3350, 3220, 1608, 1514, 1360, 831 cm-1; MS (EI) m/z (%): 417 (M+, 61), 308 (10),
283 (10), 252 (11), 238 (100), 223 (57), 195 (20), 180 (13), 152 (23), 39 (16). Anal. Calcd for
C22H19N5O4S: C 66.23, H 4.59, N 16.78, found: C 65.94, H 4.74, N 16.94.
13

3-(2-(4-Hydroxy-3-methoxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-
triazine (7e)
Compound 7e was obtained according to the described general procedure using 4-hydroxy-3-
methoxybenzaldehyde (152 mg, 1 mmol) as yellow solid (yield 88%): mp: 245-248◦C; 1H NMR
(DMSO-d6, 500 MHz): δH= 11.62 (s, 1H, NH), 9.46 (s, 1H, N=CH), 8.15 (s, 1H,ArOH), 7.49 (d,
J= 8.5 Hz, 2H, ArH), 7.36 (d, J= 8.5 Hz 2H, ArH), 7.31 (s, 1H, ArH), 7.11 (d, J= 8.1 Hz, 1H,
ArH), 6.96-6.94 (m, 4H, ArH), 6.84 (d, J=8.1 Hz, 1H, ArH), 3.84 (s, 3H, O-CH3), 3.78ppm (s,
13
6H, 2×O-CH3); C NMR (DMSO-d6, 125 MHz): δC= 161.48, 159.29, 158.22,155.28, 149.99,
148.27, 147.91, 144.08, 131.12,130.93, 130.72, 130.14, 129.16, 128.78, 128.01, 126.29, 120.87,
115.51, 113.79, 113.70, 113.57, 109.18, 55.50, 55.25, 55.09 ppm; IR (KBr):ν = 3485, 3225,
1607, 1508, 1257, 843 cm-1; MS (EI) m/z (%): 417 (M+, 61), 323 (10), 308 (37), 252 (14), 238
(100), 223 (53), 195 (14), 152 (12). Anal. Calcd for C25H23N5O4: C 65.63, H 5.07, N 15.31,
found: C 65.93, H 5.36, N 15.51.
3-(2-(2,4-Dichlorobenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7f)
The titled compound 7f was synthesized according to the general procedure using 2,4-
1
dichlorobenzaldehyde (175 mg, 1mmol) as pale yellow solid (yield 78%): mp: 248-251◦C; H
NMR (DMSO-d6, 500 MHz): δH= 12.14 (s, 1H, NH), 8.61 (s, 1H, N=CH), 8.06 (d, J=8.6 Hz,
1H, ArH), 7.72 (d, J=2.0 Hz, 1H, ArH), 7.53 (dd, J =2.0 Hz, J =8.5 Hz, 1H, ArH), 7.50 (d ,
J=8.7 Hz, 2H, ArH), 7.38 (d, J =8.7 Hz, 2H, ArH), 6.97-6.95 (m, 4H, ArH), 3.79 ppm
(s,6H,2×O-CH3); IR (KBr): ν = 3444, 3183, 1607, 1511,1369,1252, 835 cm-1; MS (EI) m/z (%):
480 (M+, 10), 484 (M+4, 2), 482 (M+2, 5), 334 (16), 308 (15), 238 (100), 223 (45), 195 (13), 152
(10). Anal. Calcd for C24H19Cl2N5O2: C 60.01, H 3.99, N 14.58, found: C 59.84, H 4.16, N
14.41.
5,6-Bis(4-methoxyphenyl)-3-(2-[(1H-indol-3-yl)methylene]hydrazinyl)-1,2,4-triazine(7g)
The titled compound 7g was synthesized according to the general described procedure using 1H-
indole-3-carbaldehyde (145 mg, 1 mmol) as yellow solid (yield: 74%): mp: 258-262◦C; 1H NMR
(DMSO-d6, 500 MHz): δH= 11.48 (s, 1H, NH), 8.51 (d, J = 7.2 Hz, 1H, ArH), 8.45 (s, 1H,
N=CH), 7.77 (brs, 1H, NH), 7.56 (d, J = 7.7 Hz, 2H, ArH), 7.44 (d, J = 7.7Hz, 2H, ArH), 7.39
(d, J =8.4 Hz, 2H, ArH), 7.21-7.18 (m, 2H, ArH), 6.99-6.96 (m, 4H, ArH), 3.80ppm (s, 6H, 2×O-
14

CH3); 13C NMR (DMSO-d6, 125MHz): δC=160.95, 159.24,158.44, 155.01, 149.39, 140.71,
136.98, 131.82, 131.12, 130.13, 129.01, 128.92, 128.26, 124.44, 122.50, 122.32, 120.23, 113.83,
113.74, 112.35, 111.68, 55.32, 55.12 ppm; IR (KBr):ν = 3414, 3010, 1609, 1510,1249,1065, 839
cm-1; MS (EI) m/z (%): 450 (M+, 4), 308 (14), 238 (100), 223 (47), 195 (15), 142 (18), 116 (16).
Anal. Calcd for C26H22N6O2: C 69.32, H 4.92, N 18.66, found: C 68.97, H 4.76, N 18.34.
3-(2-(2-Chloro-6-nitrobenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7h)
The yellow solid product 7h was prepared from 2-chloro-6-nitrobenzaldehyde (185 mg, 1 mmol)
1
according to the general procedure (yield: 71%): mp: 258-262◦C; H NMR (DMSO-d6, 500
MHz): δH= 12.33 (s, 1H, NH), 8.68 (s, 1H, N=CH), 8.14-8.13 (m, 2H, ArH), 7.70 (dd, J=2.45 Hz
, J= 8.8 Hz, 1H, ArH), 7.51 (d,J = 8.8 Hz, 2H, ArH), 7.39 (d, J=8.7 Hz, 2H, ArH), 6.98-6.96 (m,
4H, ArH), 3.79 ppm (s, 6H, 2×O-CH3); IR (KBr):ν = 3424, 3121, 1602, 1519, 1125, 831 cm-1;
MS (EI) m/z (%): 490 (M+, 21), 492 (M+2, 6), 334 (16), 308 (15), 238 (100), 223 (45), 195 (13),
152 (10). Anal. Calcd for C24H19ClN6O4: C 58.72, H 3.90, N 17.12, found: C 58.97, H 3.72, N
17.16.
3-(2-(1,3-Dihydroxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7i)
The titled compound 7i was synthesized according to the general procedure from 2,4-
1
dihydroxybenzaldehyde(138 mg, 1 mmol) as yellow solid (yield: 86%): mp: 262-266◦C; H
NMR (DMSO-d6, 500 MHz): δH= 11.93 (s, 1H, NH), 11.71 (s, 1H, ArOH), 9.89 (s, 1H, ArOH)
8.31 (s, 1H, N=CH), 7.77 (s, 1H, ArH), 7.50 (d, J=8.7 Hz , 2H, ArH), 7.38 (d, J=8.7 Hz, 2H,
ArH), 7.25 (d, J= 8.4 Hz, 1H, ArH), 6.97-6.95 (m, 4H, ArH), 6.37-6.35 (dd, J= 2.2 Hz , J= 8.3
Hz, 1H, ArH), 3.79 ppm(s,6H,2×O-CH3); IR (KBr):ν = 3352, 2837, 1608, 1517, 1253, 834 cm-1;
MS (m/z, %): 443 (M+, 22), 308 (11), 238 (100), 195 (11). Anal. Calcd for C24H21N5O4: C 65.01,
H 4.77, N 15.79, found: C 64.91, H 4.83, N 16.01.
3-(2-(Benzo[d][1,3]dioxol-5-ylmethylene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-
triazine(7j)
The titled compound 7j was synthesized by the described general procedure using 1,3-
1
benzodioxole-5-carbaldehyde (piperonal)(150 mg, 1mmol)(yield 88%): mp: 268-272◦C; H
NMR (DMSO-d6, 400 MHz): δH= 11.62 (s, 1H, NH), 8.16 (s, 1H, N=CH), 7.49 (d, J= 7.6 Hz,
15

2H, ArH), 7.40-7.34 (m, 2H, ArH), 7.31 (s, 1H, ArH), 7.14 (d, J= 7.6 Hz, 2H, ArH), 6.98-6.94
(m, 4H, ArH), 6.08 (s, 2H, ArH), 3.78 ppm (s, 6H, 2×O-CH3); IR (KBr): ν = 3429, 2962, 1606,
1515, 1175, 836 cm-1; MS (EI) m/z (%): 455 (M+, 18), 334 (10), 308 (16), 238 (100), 223 (38),
195 (11). Anal. Calcd for C25H21N5O4: C 65.93, H 4.65, N 15.38, found: C 65.71, H 5.01, N
15.65.
3-(2-(Furan-2-ylmethylene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7k)
The yellow solid product 7k was prepared from furan-2-carbaldehyde (96 mg, 1 mmol)
1
according to the general procedure (yield: 71%): mp: 258-262◦C; H NMR (DMSO-d6, 500
MHz): δH= 11.79 (s, 1H, NH), 8.15 (s, 1H, N=CH), 7.82 (s, 1H, ArH), 7.47 (d, J=8.6 Hz, 2H,
ArH), 7.36 (d, J=8.6 Hz, 2H, ArH), 6.96-6.94 (m, 4H, ArH), 6.85 (d, J=3.2, 1H, ArH), 6.63 (brs,
13
1H, ArH), 3.78 ppm (s, 6H, 2×O-CH3); C NMR (DMSO-d6, 125MHz): δC= 161.57, 161.42,
159.99, 158.72, 156.17, 151.82, 150.92, 150.33, 145.73, 144.97, 134.10, 131.66, 130.71, 129.16,
128.44, 116.15, 114.30, 114.22, 112.57, 55.77, 55.61ppm; IR (KBr):ν = 3443, 2839, 1608, 1254,
834 cm-1; MS (EI) m/z (%): 401 (M+, 66), 372 (23), 308 (15), 267 (47), 252 (14), 238 (100), 223
(89), 195 (22), 152 (21), 135 (37). Anal. Calcd for C22H19N5O3: C 65.83, H 4.77, N 17.45, found:
C 65.51, H 5.03, N 17.12.
3-(2-(2,3-Dimethoxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7l)
Compound 7l was synthesized according to the general procedure from 2,3-
1
dimethoxybenzaldehyde (166 mg, 1 mmol) as yellow solid (yield: 88%): mp: 219-222◦C; H
NMR (DMSO-d6, 500 MHz): δH= 11.82 (s, 1H, NH), 8.56 (s, 1H, N=CH), 7.52-7148 (m, 2H,
ArH), 7.37 (d, J= 8.4 Hz, 2H, ArH), 7.15-7.09 (m, 3H, ArH), 6.97-6.95 (m, 4H, ArH), 3.84 (s,
6H, 2×O-CH3), 3.78 ppm (s, 6H, 2×O-CH3); IR (KBr):ν = 3444, 3211, 1607, 1587, 1255, 831
cm-1; MS (EI) m/z (%): 471 (M+, 16), 334 (10), 308 (24), 238 (100), 223 (46), 195 (14).
Anal.Calcd for C26H25N5O4: C 66.23, H 5.34, N 14.85, found: C 65.98, H 5.71, N 15.02.
3-(2-(4-Methoxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7m)
The titled compound 7m was synthesized according to the general procedure from 4-
methoxybenzaldehyde(136 mg, 1 mmol) as yellow solid (yield: 91%): mp: 254-256◦C; 1H NMR
(DMSO-d6, 500 MHz): δH= 11.72 (s, 1H, NH), 8.21 (s, 1H, N=CH), 7.67 (d, J= 8.0 Hz, 2H,
16

ArH), 7.49 (d, J= 8.0 Hz,.2H, ArH),7.36 (d, J=8.0Hz, 2H, ArH), 7.02-6.94 (m, 6H, ArH), 3.80
(s, 3H, O-CH3), 3.78 ppm (s, 6H, 2×O-CH3); IR (KBr):ν = 3444, 3215, 1608, 1577, 1248, 834
cm-1; MS (EI) m/z (%): 441 (M+, 14), 308 (31), 238 (100), 223 (49), 195 (12), 152 (10). Anal.
Calcd for C25H23N5O3: C 68.01, H 5.25, N 15.86, found: C 68.28, H 4.93, N 15.67.
3-(2-(1,2-Dihydroxybenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7n)
The yellow solid product 7n was prepared from 2,3-dihydroxybenzaldehyde (138 mg, 1 mmol)
1
according to the general procedure(yield: 85%): mp: 268-271◦C; H NMR (DMSO-d6, 500
MHz): δH= 12.13 (s, 1H, NH), 11.41 (s, 1H, Ar-OH), 9.15 (s, 1H, Ar-OH), 8.93 (s, 1H, N=CH),
7.51 (d,J=8.5 Hz, 2H, ArH), 7.38 (d, J=8.6 Hz, 2H, ArH), 6.97-6.95 (m, 4H, ArH), 6.92
(d,J=7.7, 1H, ArH), 6.84 (d, J=7.7 Hz, 1H, ArH), 6.75-6.72 (m, 1H, ArH), 3.78 ppm(s, 6H, 2×O-
13
CH3); C NMR (DMSO-d6, 125MHz): δC= 161.04, 159.39, 157.76, 155.51, 150.66, 145.61,
145.51, 145.21, 131.17, 130.25, 128.62, 127.84, 119.92, 119.02, 116.75, 113.82, 55.29, 55.10
ppm; IR (KBr):ν = 3428, 3207, 2837, 1606, 1515, 1252, 836 cm-1; MS (m/z, %): 443 (M+, 54),
334 (11), 309 (10), 238 (100), 223 (53), 195 (16), 152 (17). Anal. Calcd for C24H21N5O4: C
65.00, H 4.77, N 15.79, found: C 65.61, H 5.12, N 15.03.
3-(2-(4-Fluorobenzylidene)hydrazinyl)-5,6-bis(4-methoxyphenyl)-1,2,4-triazine (7o)
The titled compound 7o was synthesized by the general procedure using 4-fluorobenzaldehyde
1
(124 mg, 1mmol)(yield 72%): mp: 257-259◦C; H NMR (DMSO-d6, 500 MHz): δH= 11.64 (s,
1H, NH), 8.25 (s, 1H, N=CH), 8.06-8.02 (m, 2H, ArH), 7.49 (d, J= 8.5Hz, 2H, ArH), 7.35 (d, J=
8.5 Hz, 2H, ArH), 7.30-7.26 (m, 2H, ArH ), 6.96-6.93 (m, 4H, ArH), 3.78 ppm(s,6H,2×O-
CH3);13C NMR (DMSO-d6, 125 MHz): δC= 163.39, 161.45, 160.99, 159.39, 158.21, 155.45,
150.64, 141.87, 137.48, 131.19, 130.86, 130.80, 130.21, 128.63, 127.87, 122.86, 116.03, 115.86,
113.83, 112.42, 112.24, 55.27, 55.11 ppm; IR (KBr):ν = 3444, 3223, 2913, 1607, 1589, 1514,
1256, 836 cm-1; MS (EI) m/z (%):429 (M+, 36), 334 (29), 308 (10), 238 (100), 223 (53), 195
(12), 152 (11). Anal. Calcd for C24H20FN5O2: C 67.12, H 4.69, N 16.31, found: C 66.85, H 4.81,
N 15.98.
5,6-Bis(4-methoxyphenyl)-3-(2-(pyridin-4-ylmethylene)hydrazinyl)-1,2,4-triazine (7p)
17

The yellow solid product 7p was prepared from pyridine-4-carbaldehyde (107 mg, 1 mmol)
1
according to the general procedure(yield: 81%): mp: 255-258◦C; H NMR (DMSO-d6, 500
MHz): δH= 12.17 (s, 1H, NH), 8.63 (d, J= 4.4 Hz, 2H, ArH), 8.23 (s, 1H, N=CH), 7.67 (d, J=
5.7Hz, 2H, ArH), 7.52 (d, J= 8.8 Hz, 2H, ArH), 7.39 (d, J=8.6 Hz, 2H, ArH), 6.98-6.96 (m, 4H,
ArH), 3.79 ppm(s, 6H, 2×O-CH3); IR (KBr):ν = 3434, 3146, 1609, 1569, 1507, 1237, 841 cm-1;
MS (EI) m/z (%): 412 (M+, 32), 334 (16), 308 (12), 238 (100), 223 (25), 195 (10).Anal.Calcd for
C23H20N6O2: C 66.98, H 4.89, N 20.38, found: C 66.75, H 5.71, N 19.93.
4.2. Biological assays
4.2.1. Reagents
RPMI 1640 media, FBS (Fetal Bovine Serum), trypsin, penicillin and streptomycin were
purchased from Biosera (Austria). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide], Etoposide, SB202190 and Ficoll-hypaque were purchased from Sigma (USA).Western
blott detection kit and polyvinyl difluoride (PVDF) membrane were from Roche (Germany).
Antibodies to phospho-p38 and phospho-ATF-2 were from cell signaling company (U.S.A) and
β-actin antibody was purchased from Santa Cruz Biotech (USA). All other reagents were from
Merck (Germany).
4.2.2. In vitro assay for TNF-α and IL-1β inhibition
Human PBMCs (peripheral blood mononuclear cells) were isolated from whole blood collected
from healthy donors. Blood was diluted into RPMI 1640 containing 2.5 mM EDTA, and Ficoll-
Hypaque (sigma) was overlaid with the mixture and centrifuged at 1500 rpm for 30 min. the
buffy coat was collected, and cells were washed three times and resuspended at a concentration
of 1× 106 cells/mL in assay medium (RPMI medium containing 10% fetal bovine serum). Cells
were then seeded (100μl/mL) into 96-well tissue culture treated plates. Test compounds were
added to appropriate wells and incubated with cells for 30 min. Cells were then stimulated by the
addition of bacterialLPS (Sigma), with a final concentration of 1μg/ml, and incubated for 6h at
37 oC, 5% CO2. Following incubation, the culture medium was harvested (100 μl) and stored at -
o
70 C. TNF-α and IL-1β concentration in the medium was quantified using an enzyme-linked
immunosorbent assay (ELISA) kit following the manufacturer’s instructions (assay designs). The
18

absorbance in each well was measured with a microplate reader at 450 nm and corrected at 570
nm. Concentrations of cytokines released were obtained and the percentage of inhibition of
cytokines production was calculated as compared to control conditions.
4.2.3. Cell cultures and drug treatment
MCF-7 cells were obtained from EACC. Cells were cultured in RPMI 1640 media with 10%
fetal bovine serum, penicillin (100 IU/m1), and streptomycin (100 mg/ml) at 37 °C in a 5% CO2
humid atmosphere. Cells were treated with 10 and 20 μM of compound 7l and SB202190 for 30
min, then in order to induce p38 MAPK pathway, cells treated with 100μMetoposide and
incubated for 3h.
4.2.3. Western blotting
42
These assays were performed according to the protocols described previously. Briefly, after
incubation period, cells were lysed with cell lysis buffer (Tris 62.5 mM, pH 6.8, dithiothreitol 50
mM, sodiumdodecylsulphate (SDS) 2%, Glycerol 10% and Bromophenol Blue 0.25% (w/v).
Equal amount of protein samples were subjected to 12% SDS–PAGE; and then transferred to
PVDF membranes. Membranes were blocked with 1% casein and then incubated with anti-
phospho-p38 (1:1000) or anti-phospho-ATF-2 (1:1000) antibodies at 4 °C, overnight. Anti-β-
actin antibody (1:5000) was used as internal control. The blots were then treated with HRP-
conjugated secondary antibody (1:10000) for 1 h at room temperature. Protein bands were
detected using a chemiluminescence kit on X-ray film.
4.2.4. In-vivo Anti-inflammatory activity
Rat paw edema was performed as described by Winter et al.45 The anti-inflammatory activity
was determined in-vivo using the carrageenan induced rat paw edema test. Edema was induced in
the right hind paw of all rats by subcutaneous injection of 0.1mL of 1% (W/V) carrageenan
lambda (a pro-inflammatory agent; prepared in 0.9% NaCl) subcutaneously in the sub-plantar
region of right hind paw, 1 h after administration of the intra-peritoneal (IP) administration of the
test substances.46 The paw thickness was measured from the ventral to the dorsal surfaces using a
dial caliper immediately before and after injection of 1% carrageenan at different time intervals
(1, 2, 3, 4 h). Changes in paw volume were recorded at the above time intervals with reference to
19

the initial volume before administration of the inflammatory agent. The anti-inflammatory
activity was expressed similar to the inhibition percentage of edema when compared with the
control group and was calculated using the following formula:
%Inhibition = (1 - Tt / Tc) × 100
In this formula, Tt and Tc are defined as the thickness variation of test group and control group,
respectively.
4.3. Molecular docking
Autodock 4.2 and Auto Dock Tools 1.5.4 (ADT) were used for docking study of 1,2,4-triazine
containing hydrazine moiety derivatives.47
Preparation of the protein: The X-ray crystal structure of p38α in complex with 6-(2,4-
difluorophenoxy)-8-methyl-2-[(1-methylethyl)amino]pyrido[2,3-d]pyrimidin-7(8H)-one (PDB
ID: 3FLY) was retrieved from the Protein Data Bank (http://www.rcsb.org). Water molecules
and cognate ligand were removed from the receptor. All hydrogens were properly added to the
receptor PDB and non-polar hydrogens were merged into related carbon atoms of the receptor
using ADT and Kollman charges were also assigned.
Preparation of ligands: The three-dimensional structures of the ligands were constructed using
Chem3D Ultra 8.0 software to obtain standard 3D structures. The constructed compounds were
energetically minimized (100 steepest descent steps using MM+ force field with a gradient
convergence value of 0.1 kcal/mol) using HyperChem software. The Gastiger charges (empirical
atomic partial charges) and torsional degrees of freedom were assigned on the generated PDB
files by ADT 1.5.4.
Calculation of Grid map: The grid maps were calculated with AutoGrid (part of the AutoDock
package). The created three-dimensional grids were 40 * 40 * 40 (x, y, z) with a grid spacing of
0.375 Å and the cubic grids were centered on the binding site of native ligand.
Preparation of docking parameters and running the automated docking simulation:
Automated docking studies were carried out using Lamarckian Genetic Algorithm (LGA) to
model the interaction between p38α and ligands. For LGA method; 2500,000 energy evaluation,
population size of 100, a gene mutation rate of 0.02; a crossover rate of 0.8 and number of 250
independent docking runs were used for each case. Cluster analysis was performed on the
docked results using a root mean square (RMS) tolerance of 2.0 Å.
20

Acknowledgment
The authors acknowledge the financial support of Tehran University of Medical Sciences, Vice
Chancellor of Research, Iran National Elite Foundation (INEF) and Iran National Science
Foundation (INSF). This study was part of the PhD thesis project of Mehdi Khoshneviszadeh.
21

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24

Table1: Results summarized for both % of cytokines inhibition are presented as the mean ± SEM
from three different experiments (n =3).
Compounds
7a
% Inhibition at 10 μM
TNF-α
Ar
IL1-β
53.13± 9.05
71.52±2.33
37.56±3.09
15.52±8.81
39.86±6.82
7b
7c
NO2
44.97±24.31
OMe
OMe
57.45±10.27
46.07±23.52
81.79±5.96
84.28±4.56
7d
7e
S
OMe
OH
69.96±13.94
86.92±12.48
7f
Cl
Cl

53.27±2.64
82.29±10.17
7g
H
N
47.91±12.51
32.66±13.77
2.81±13.27
51.75±6.24
7h
7i
O2N
Cl
HO
OH
52.20±15.36
71.12±16.22
71.84±11.21
86.91±9.57
84.05±16.24
89.78±6.08
7j
7k
7l
O
O
O
OMe
MeO
7m
7n
OMe
OH
48.49±3.95
46.67±2.11
35.94±11.34
78.20±10.72
HO

34.48±10.15
41.80±21.23
27.92±3.94
7o
7p
F
60.81±12.32
N
---------
88.19±4.11
94.45±2.73
SB-202190

Table 2.Anti-inflammatory activity of the compound 7l against carrageenan-induced paw
edema in rat.
%Oedema inhibition relative tocontrol
Compound
Dose(mg/Kg)
at 1h
at 2h
at 3h
at 4h
Indomethacine
10
38.8±8.04 45.32±6.31 64.24±11.21
75.61±4.91
50
19.24±4.06 28.38±6.16 48.22±12.31
68.21±5.11
7l

MeO
O
O
O
OH
CHO
N
N
N
c
a
b
MeO
SH
1
4
MeO
OMe
3
MeO
2
OMe
MeO
MeO
MeO
MeO
N
N
N
N
N
N
f
N
N
d
e
N
Ar
N
H
NH2
N
NH
S
MeO
7a-7p
5
6
MeO
MeO
Scheme 1.Synthesis pathway for the preparation of triazine derivatives. Reagents and conditions: (a)
thiamine hydrochloride, NaOH, EtOH, reflux, 3h (b) HNO3, 60 oC (c) thiosemicarbazide, CH3COOH,
reflux, 3 h (d) CH3I, NaOH, EtOH, 2h, rt (e) NH2NH2.H2O, EtOH, reflux, 12h (f) ArCHO, EtOH, 1-10 h,
rt