Design, green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents*

Article abstract of DOI:10.3109/14756366.2015.1060480

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were syn

Full text of DOI:10.3109/14756366.2015.1060480

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Design, green synthesis and pharmacological
evaluation of novel 5,6-diaryl-1,2,4-triazines
bearing 3-morpholinoethylamine moiety as potential
antithrombotic agents
a
a
a
a
Riyaj S. Tamboli , Rajani Giridhar , Hardik P. Gandhi , Ashish M. Kanhed , Hemant M.
b
a
Mande & Mange Ram Yadav
a
Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The Maharaja
Sayajirao University of Baroda, Vadodara, Gujarat, India and
b
Department of Chemistry, Faculty of Science, Sayajiganj, The Maharaja Sayajirao
University of Baroda, Vadodara, Gujarat, India
Published online: 02 Jul 2015.
To cite this article: Riyaj S. Tamboli, Rajani Giridhar, Hardik P. Gandhi, Ashish M. Kanhed, Hemant M. Mande & Mange
Ram Yadav (2015): Design, green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-
morpholinoethylamine moiety as potential antithrombotic agents, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1060480
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1060480
!
RESEARCH ARTICLE
Design, green synthesis and pharmacological evaluation of novel
,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety
as potential antithrombotic agents*
5
1
1
1
1
2
Riyaj S. Tamboli , Rajani Giridhar y, Hardik P. Gandhi , Ashish M. Kanhed , Hemant M. Mande , and
1
Mange Ram Yadav
1
Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat,
2
India and Department of Chemistry, Faculty of Science, Sayajiganj, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India
Abstract
Keywords
The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines
(
1
,2,4-Triazines, antithrombotic agents,
antiplatelet agents, aspirin, green
synthesis, molecular docking
3a–3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet
activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by
environment benign route and their structures were unambiguously confirmed by spectral
data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out
of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six
of them were found to be prominent in ex vivo assays and one compound (3d) was found
to have the most promising antithrombotic profile in vivo. Moreover, compound (3d)
demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most
promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of
molecular docking studies and the results were correlated with the biological activity.
History
Received 24 February 2015
Revised 31 May 2015
Accepted 2 June 2015
Published online 1 July 2015
Introduction
(TXA ) in platelets but also prostaglandin I (PGI ) in vascular
2 2 2
7
endothelium . Consequently, it induces stomach ulcers, limiting
its clinical use. Development of newer COX inhibitors devoid of
side effects of the currently available antiplatelet agents is a major
goal in thromboembolic research.
Syntheses of small molecules possessing vicinal diaryl scaffold
as a privileged substructure have received wide attention in the
8,9
field of medicinal chemistry . Molecules incorporating this
Ischemic stroke is the third-leading cause of morbidity and
1
mortality in western countries . Platelets play a vital role in both
thrombosis and hemostasis by maintaining the integrity of the
circulatory system and respond rapidly at vascular injury. The
activation of platelets leads to a series of responses that impart a
2
critical role in thrombosis . This activation generally occurs when
they are brought in contact with agents, such as collagen,
thrombin and adenosine diphosphate (ADP). Currently, acetyl-
salicylic acid (ASA, aspirin) and thienopyridines, such as
clopidrogel and ticlopidine, are widely used as antiplatelet
aggregating agents (Figure 1). Nearly half of the global population
is resistant to antiplatelet drugs resulting in increased risk of
recurrent myocardial infarction and stroke or cardiac related
scaffold have demonstrated biological activity of varying nature,
11–13
10
especially as COX inhibitors and antiplatelet agents
.
Some of the diarylthiazoles were reported to be potent
14
antiplatelet agents with vasodilatory action . This study helped
to conclude that guanidine moiety present in these structures plays
a major role in vasodilatation. L-Arginine (S-2-amino-5-guanidi-
nopentanoic acid) is a well-known essential amino acid which acts
as a vasodilator through nitric oxide-dependant pathway (NO
3
,4
deaths . Though dual antiplatelet therapy using a combination of
aspirin and a thienopyridine drug has been implemented during
the primary stage, atherothrombotic disease remains as one of the
15
16
donor) . Moreover, agamatine (4-aminobutylguanidine) and its
derivatives like GYKI-14766 (D-methyl-phenylalanyl-prolyl-argi-
5
major causes of morbidity and mortality . The most widely used
antiplatelet drug aspirin inhibits cyclooxygenase (COX) irrevers-
17
nal) were accessed as experimental medicines in anticoagulant
6
ibly . Aspirin prevents not only the synthesis of thromboxane A
and antithrombotic therapy. Timegadine, a trisubstituted guan-
19
2
18
idine derivative , and some other acyclic guanidines possess
potent vasodilatory actions.
*This article is dedicated to fond memories of Late Prof. (Mrs.) Rajani
Giridhar.
yDeceased
Drugs containing morpholinoethyl and morpholinoethylamino
groupings have gained noteworthy attention in cardiovascular
2
0–24
20
drug research
. Drugs like moricizine
possess potent
antiarrhythmic properties while some antipsychotics like moclo-
Address for correspondence: Prof. Mange Ram Yadav, Pharmacy
Department, Faculty of Technology and Engineering, Kalabhavan, The
Maharaja Sayajirao University of Baroda, Vadodara 390 001, Gujarat,
India. Tel: +91 265 2434187. Fax: +91 265 2418927. E-mail:
mryadav11@yahoo.co.in
2
1
22
bemide and aphobazole are specially used in patients with
cardiovascular diseases. Some of the molecules like sulfonated
morpholinoethyl derivatives
2
3
and morpholinoethylamino

2
R. S. Tamboli et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
ꢀ
1
O
OH
O
OMe
N
IR (in cm ) spectra in KBr pellets were taken on a Bruker
1
13
ALPHA-T instrument. H NMR and C NMR spectra were
recorded in CDCl3 on a Bruker Avance II spectrometer
(400 MHz), using tetramethylsilane (TMS) as an internal stand-
ard. Chemical shift is reported in parts per million (ꢀ in ppm)
where s, brs, t and m are designated for singlet, broad singlet,
triplet and multiplet, respectively. Mass spectra were carried out
on Thermoscientific DSQ-II mass spectrometer equipped with an
electron spray ionization (ESI) or an electron impact ionization
O
Me
N
S
O
Cl
Ticlopidine
S
Cl
Clopidogrel
Aspirin
Figure 1. Currently used oral antiplatelet drugs.
(EI) interface. Flash column chromatography was performed on
Combiflash R 200 (Teledyne Esco) using flash grade silica gel
F
(
230–400 mesh). Elemental analyses were done on
a
Thermoscientific Flash-2000 CHN analyzer. Single crystals for
compounds (3l and 3m) for X-ray crystallography were obtained
from mixture of MeOH:CHCl (30:70) and analyzed on Agilent
3
single crystal X-ray model having Xcalibur, Eos, Gemini
diffractometer operated at RT. Thin-layer chromatography
(
TLC) was performed on precoated Silica gel Merck plates.
Compounds were visualized by illuminating with UV light
254 nm) or exposure to iodine vapors. All chemicals and solvents
(
Figure 2. Designing hypothesis for the new compounds by fusing three were of reagent grade and were purified and dried using standard
pharmacophores in a single molecule for antiplatelet and vasodilatory methods. All the animal experiments were carried out as per the
action without ulcerogenesis.
guidelines of Institutional Animal Ethics Committee of Pharmacy
Department, The Maharaja Sayajirao University of Baroda,
Gujarat, India (IAEC Reg. No. 404/01/a/CPCSEA).
2
4
derivatives of benzothiazoles were patented, respectively, for
their potent antiplatelet and antiulcer properties.
Green synthesis of 5,6-diaryl-N-(2-morpholinoethyl-1,2,4-
triazin-3-amines (3a–3q)
1
pharmacological activities, such as antiplatelet
,2,4-Triazine scaffold has been associated with diversified
14,25
26
, anticancer ,
2
7
thromboxane synthetase inhibition , anti-inflammatory and General procedure: 5,6-Diaryl-3-methylthio-1,2,4-triazines were
28
2
antimalarial . Further, the diaryl system attached to 1,2,4-triazine synthesized as per the green chemical procedure previously
9
3
8
heterocycle has been reported to be a potent anticytokine, reported by our group . A mixture of the corresponding
mediating through the inhibition of p38 MAPK signaling 3-methylthiotriazine (1.0 mmol) and 4-(2-aminoethyl)morpho-
3
0
ꢁ
pathway . Recent studies on 5,6-diaryl triazines showed neuro- line (4.0 mmol) was stirred at 100–110 C. The progress of the
pharmacological applications of this moiety, such as adenosine reaction was monitored by TLC using a mixture of 10%
A2A antagonism, in Parkinson’s disease , anti-neuroinflamma- methanol in chloroform as eluent. After completion, the
3
1
3
2
33
tory activity in Alzheimer’s disease and as neuroprotectants . reaction mixture was added to crushed ice. The precipitated
More recently, potent neuroprotective action of some 1,2,4- product was filtered, washed thoroughly with water and dried.
triazines by activating Wnt/b-catenin signaling pathway has been The products showed single spots on TLC. However, for
3
4
demonstrated by our research group . Some patents have also characterization purposes the compounds were further recrys-
disclosed the topical anti-inflammatory and antithrombotic tallized in methanol.
3
5
3
activity of diaryl triazines .
5
Based on the previous findings, it was thought logical to
combine the diaryl 1,2,4-triazine scaffold with an amino group at
position 3, providing a cyclic guanidine moiety. Such a system
could prove beneficial in thromboembolic disorders due to its
antiplatelet as well as vasodilatory actions (Figure 2). The well-
documented antiplatelet activity associated with the 1,2,4-triazine
N-(2-Morpholinoethyl)-5,6-diphenyl-1,2,4-triazin-3-amine (3a)
ꢁ
Yield 48%, yellow crystals from MeOH, mp: 150–152 C; FT-IR
ꢀ1
(
(
KBr, cm ): 3223, 1580, 1522, 1114; ESI-MS (m/z) [%]: 361.40
+
+
1
M) [31.66], 362.40 (M + 1) [100]; H NMR (CDCl ): ꢀ 7.20–
3
7
.41 (m, 10H, Ar–H), 5.97 (brs, 1H, –NH), 3.58–3.68 (m, 6H,
1
4,15,25
–NH–CH and 2ꢂ CH –O), 2.61 (t, J ¼ 5.9 Hz, 2H, –NH–CH –
pharmacophore
motivated us to synthesize 1,2,4-triazines
2
2
2
1
3
CH ), 2.46 (t, J ¼ 4.0 Hz, 4H, 2ꢂ –N–CH –CH –O); C NMR
with concomitantly substituted vicinal diaryl group along with a
flexible 3-morpholinoethylamino side chain. As a part of our
ongoing research program, aiming at the discovery of novel diaryl
heterocycle-based antiplatelet agents, we have recently reported
potent antiplatelet effect of compounds containing a vicinal diaryl
fragment as a key structural element attached to 1,4-diazepine and
2
2
2
(
CDCl ): ꢀ 37.65, 53.44, 57.16, 66.99, 128.33, 129.22, 129.56,
3
130.24, 136.37, 136.45, 149.67, 156.79, 160.29; Anal. Calcd. for
C H N O; C, 69.78%; H, 6.41%; N, 19.38%. Found: C, 69.98%;
H, 6.23%; N, 19.19%.
21 23
5
-aminopyrimidine as the heterocyclic scaffolds36,37. This paper 5,6-Bis(4-chlorophenyl)-N-(2-morpholinoethyl)-1,2,4-triazin-3-
2
amine (3b)
describes the synthesis of diaryl-1,2,4-triazines with biologically
favourable morpholinoethylamino group at 3 position and their
in vitro, ex vivo and in vivo pharmacological evaluation as
potential antiplatelet/antithrombotic leads.
ꢁ
Yield 60%, yellow crystals from MeOH, mp: 160–162 C; FT-IR
ꢀ1
(
(
KBr, cm ): 3223, 1595, 1522, 831; ESI-MS (m/z) [%]: 429.40
+
M ) [100], 431.50 (M + 1) [87.27], 432.70 (M + 2) [21.81]; H
+
+
1
NMR (CDCl ): ꢀ 7.21–7.37 (m, 8H, Ar–H), 6.03 (brs, 1H, –NH),
3
Materials and methods
3.60–3.68 (m, 6H, –NH–CH2 and 2ꢂ –CH –O), 2.61
2
(
t, J ¼ 5.9 Hz, 2H, –NH–CH –CH ), 2.46 (t, J ¼ 4.1 Hz, 4H,
2
2
General
2ꢂ
–N–CH –CH –O); Anal. Calcd. for C H Cl N O;
2 2 21 21 2 5
Melting points were determined in capillaries using Veego C, 58.61%; H, 4.92%; N, 16.27%. Found: C, 58.87%; H, 4.68%;
programmable melting point apparatus and are uncorrected. N, 16.08%.

DOI: 10.3109/14756366.2015.1060480
Novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine
5-(4-Bromophenyl)-6-(4-chlorophenyl)-N-(2-morpholinoethyl)-
3
N-(2-Morpholinoethyl)-5,6-di(p-tolyl)-1,2,4-triazin-3-amine (3c)
1
,2,4-triazin-3-amine (3h)
ꢁ
Yield 65%, light yellow crystals from MeOH, mp: 159–161 C;
1
ꢀ
ꢁ
FT-IR (KBr, cm ): 3221, 1595, 1523, 1115; ESI-MS (m/z) [%]: Yield 53%, light yellow crystals from MeOH, mp: 152–154 C;
ꢀ1
90.50 (M + 1) [100]; H NMR (CDCl ): ꢀ 7.03–7.32 (m, 8H, IR (KBr, cm ) 3441, 1601, 1114, 830; EI-MS (m/z) [%]:
3
+
1
3
+
+
+
Ar–H), 5.91 (brs, 1H, –NH), 3.60–3.67 (m, 6H, –NH–CH and 2ꢂ 474.08 (M) [35], 476.11 (M + 2) [18], 478.00 (M + 4) [4];
2
1
–
CH –O), 2.59 (t, J ¼ 5.9 Hz, 2H, –NH–CH –CH ), 2.45
H NMR (CDCl ): ꢀ (ppm) 7.26–7.48 (m, 8H, Ar–H), 6.10 (brs,
3
2
2
2
(
t, J ¼ 4.0 Hz, 4H, 2ꢂ –N–CH –CH –O), 2.27 (s, 6H, 2ꢂ 1H, N–H), 3.56–3.74 (m, 6H, 2ꢂ CH –O and HN–CH ),
3 3 2 2
2
2
2
2
1
3
Ar–CH ); C NMR (CDCl ): ꢀ 21.30, 21.44, 37.63, 53.43, 2.67 (t, J ¼ 5.8 Hz, 2H, HN–CH –CH –N), 2.53 (t, J ¼ 4.0 Hz,
5
1
7.20, 66.96, 129.01, 129.27, 129.50, 133.67, 138.06, 140.50, 4H, 2ꢂ N–CH –CH O); Anal. Calcd. for C H BrClN O:
23 27 5
2
2
21 21
5
49.62, 156.58, 160.22; Anal. Calcd. for C H N O; C, 70.92%; C, 53.12%; H, 4.46%; N, 14.75%; Found: C, 53.38%; H, 4.26%;
N, 14.43%.
H, 6.99%; N, 17.98%. Found: C, 70.72%; H, 6.67%; N, 17.69%.
5-(4-Chlorophenyl)-N-(2-morpholinoethyl)-6-(p-tolyl)-1,2,4-tria-
zin-3-amine (3d)
6
1
-(4-Bromophenyl)-5-(4-chlorophenyl)-N-(2-morpholinoethyl)-
,2,4-triazin-3-amine (3i)
ꢁ
Yield 53%, light yellow crystals from MeOH, mp: 158–160 C;
ꢀ
ꢁ
Yield 51%, yellow crystals from MeOH, mp: 161–163 C; IR
ꢀ1
1
FT-IR (KBr, cm ): 3225, 1595, 1521, 1399, 1118, 830; ESI-MS
(
KBr, cm ) 3440, 1602, 1114, 830; EI-MS (m/z) [%]: 473.83
+
M) [72], 478.64 (M + 4) [57]; H NMR (CDCl ): ꢀ (ppm)
+
m/z) [%]: 409.70 (M) [100], 411.60 (M + 2) [53.84]; H NMR
+
1
(
+
1
(
3
(
CDCl ): ꢀ 7.05–7.31 (m, 8H, Ar–H), 5.94 (brs, 1H, –NH), 3.62–
3
7
.26–7.46 (m, 8H, Ar–H), 6.11 (brs, 1H, N–H), 3.57–3.73 (m, 6H,
ꢂ CH –O and HN–CH ), 2.67 (t, J ¼ 6.0 Hz, 2H, HN–CH –
3
.67 (m, 6H, –NH–CH and 2ꢂ –CH –O), 2.61 (t, J ¼ 5.9 Hz, 2H,
2 2 2 2
2
2
2
2
2
2
–
O), 2.29 (s, 3H, Ar–CH ); Anal. Calcd. for C H ClN O; C,
NH–CH –CH –N), 2.45 (t, J ¼ 4.1 Hz, 4H, 2ꢂ –N–CH –CH –
CH –N), 2.52 (t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O); Anal.
2
2
2
3
22 24
5
Calcd. for C H BrClN O: C, 53.12%; H, 4.46%; N, 14.75%;
5
2
1
21
67.55%; H, 6.16%; N, 13.70%. Found: C, 67.28%; H, 6.39%;
N, 13.50%.
Found: C, 53.33%; H, 4.19%; N, 14.50%.
6
1
-(4-Chlorophenyl)-5-(4-fluorophenyl)-N-(2-morpholinoethyl)-
,2,4-triazin-3-amine (3j)
6-(4-Chlorophenyl)-N-(2-morpholinoethyl)-5-(p-tolyl)-1,2,4-tria-
zin-3-amine (3e)
ꢁ
ꢁ
Yield 49%, light yellow crystals from MeOH, mp: 149–151 C; IR
Yield 22%, yellow crystals from MeOH, mp: 150–152 C; FT-IR
ꢀ1
ꢀ
1
KBr, cm ): 3226, 1594, 1522, 1400, 1116, 821; EI-MS (m/z)
(KBr, cm ) 3448, 1598, 1115, 839; EI-MS (m/z) [%]: 413.54
1
(M) [100], 415.54 (M + 2) [30]; H NMR (CDCl ): ꢀ (ppm)
3
(
[
(
+
+
+
%]: 409.60 (M ) [100], 411.70 (M + 2) [54.21]; H NMR
+
1
7
.00–7.50 (m, 8H, Ar–H), 6.11 (brs, 1H, N–H), 3.70-3.75 (m, 6H,
ꢂ CH –O and HN–CH ), 2.68 (t, J ¼ 5.9 Hz, 2H, HN–CH –
CDCl ): ꢀ 7.05–7.38 (m, 8H, Ar–H), 5.93 (brs, 1H, –NH), 3.62–
3
2
3
.68 (m, 6H, –NH–CH and 2ꢂ –CH –O), 2.62 (t, J ¼ 5.9 Hz, 2H,
2
2
2
2
2
1
3
CH –N), 2.53 (t, J ¼ 4.3 Hz, 4H, 2ꢂ N–CH –CH O); C NMR
–
O), 2.29 (s, 3H, Ar–CH ); C NMR (CDCl ): ꢀ 21.35, 37.65,
NH–CH –CH –N), 2.46 (t, J ¼ 4.0 Hz, 4H, 2ꢂ –N–CH –CH –
2
2
2
2
2
2
2
1
3
(CDCl ): ꢀ 37.59, 53.39, 57.05, 66.90, 115.56, 115.68, 128.71,
3
3
3
1
1
1
30.38, 130.89, 131.65, 134.65, 136.67, 148.36, 155.43, 162.77,
65.27; Anal. Calcd. for C H ClFN O: C, 60.94%; H, 5.11%; N,
6.92%; Found: C, 60.63%; H, 5.33%; N, 16.55%.
5
1
3.46, 57.15, 67.01, 128.65, 129.05, 129.26, 130.99, 133.19,
35.05, 136.52, 138.48, 149.51, 155.56, 160.34; Anal. Calcd.
21 21
5
for C H ClN O; C, 67.55%; H, 6.16%; N, 13.70%. Found: C,
2
2
24
5
6
7.28%; H, 6.33%; N, 13.45%.
5-(4-Chlorophenyl)-6-(4-fluorophenyl)-N-(2-morpholinoethyl)-
1,2,4-triazin-3-amine (3k)
5-(4-Fluorophenyl)-N-(2-morpholinoethyl)-6-phenyl-1,2,4-tria-
zin-3-amine (3f)
ꢁ
Yield 52%, yellow crystals from MeOH, mp: 143–145 C; IR
ꢀ1
ꢁ
Yield 48%, Light yellow crystals from MeOH, mp: 128–130 C; (KBr, cm ) 3420, 1603, 1116, 841; EI-MS (m/z) [%]: 413.37
ꢀ
1
IR (KBr, cm ) 3424, 1602, 703; EI-MS (m/z) [%]: 379.14 (M)
[
+
+
+
1
(M) [100], 415.28 (M + 2) [30]; H NMR (CDCl ): ꢀ (ppm)
3
1
58]; H NMR (CDCl ): d (ppm) 6.97–7.58 (m, 9H, Ar–H), 6.05 7.00–7.48 (m, 8H, Ar–H), 6.09 (brs, 1H, N–H), 3.68–3.73 (m, 6H,
3
(
brs, 1H, N–H), 3.55–3.92 (m, 6H, 2ꢂ CH –O and HN–CH ), 2ꢂ CH –O and HN–CH ), 2.68 (t, J ¼ 5.2 Hz, 2H, HN–CH –
2
2
2
2
2
1
3
2
4
5
1
.68 (t, J ¼ 5.5 Hz, 2H, HN–CH –CH –N), 2.54 (t, J ¼ 4.0 Hz, CH –N), 2.53 (t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O); C NMR
2
2
2
2
2
1
3
H, 2ꢂ N–CH –CH O); C NMR (CDCl ): ꢀ 37.64, 53.46, (CDCl ): ꢀ 37.58, 53.40, 57.03, 66.94, 115.58, 115.79, 128.71,
2 2 3
3
7.14, 67.00, 115.55, 115.66, 128.50, 129.20, 129.51, 130.38, 130.38, 130.97, 131.65, 132.16, 134.73, 136.70, 148.31, 162.79,
31.05, 131.86, 136.31, 156.70, 161.63, 164.10; Anal. Calcd. for 165.30; Anal. Calcd. for C H ClFN O: C, 60.94%; H, 5.11%; N,
2
1
21
5
C H FN O: C, 66.47%; H, 5.84%; N, 18.46%; Found: C, 16.92%; Found: C, 60.68%; H, 5.43%; N, 16.58%.
2
1
22
5
6
6.65%; H, 5.49%; N, 18.23%.
5-(4-Chlorophenyl)-N-(2-morpholinoethyl)-6-phenyl-1,2,4-tria-
zin-3-amine (3l)
6-(4-Fluorophenyl)-N-(2-morpholinoethyl)-5-phenyl-1,2,4-tria-
zin-3-amine (3g)
ꢁ
Yield 56%, light yellow crystals from MeOH, mp: 163–165 C;
ꢀ1
ꢁ
Yield 46%, yellow crystals from MeOH, mp: 121–123 C; IR IR (KBr, cm ) 3440, 1584, 1119, 705; EI-MS (m/z) [%]:
ꢀ
1
+
+
1
(
(
KBr, cm ) 3434, 1602, 1186, 701; EI-MS (m/z) [%]: 379.28 395.41 (M) [4], 397.27 (M + 2) [1]; H NMR (CDCl ):
+
M) [62]; H NMR (CDCl ): ꢀ (ppm) 6.96–7.56 (m, 9H, Ar–H), ꢀ (ppm) 7.26-7.47 (m, 9H, Ar–H), 6.08 (brs, 1H, N–H),
3
1
3
6
.09 (brs, 1H, N–H), 3.70–3.74 (m, 6H, 2ꢂ CH –O and 3.55-3.73 (m, 6H, 2ꢂ CH
2
–O and HN–CH
–N), 2.53 (t, J ¼ 4.0 Hz, 4H,
): ꢀ 37.60, 53.40, 57.07,
37.61, 53.41, 57.10, 66.99, 115.40, 115.61, 128.45, 129.16, 66.96, 128.49, 128.56, 129.44, 130.39, 130.43, 134.41, 134.83,
30.33, 130.92, 131.35, 131.73, 136.27, 156.70, 161.63, 164.10; 136.16, 148.53, 156.70, 162.79; Anal. Calcd. for C21 22ClN O:
2
), 2.68
2
HN–CH ), 2.67 (t, J ¼ 5.9 Hz, 2H, HN–CH –CH –N), 2.53 (t, J ¼ 5.2 Hz, 2H, HN–CH
–CH
2
2
2
2
2
1
3
13
(
ꢀ
1
t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O); C NMR (CDCl ): 2ꢂ N–CH
–CH O); C NMR (CDCl
2 2 3
2
2
3
H
5
Anal. Calcd. for C H FN O: C, 66.47%; H, 5.84%; N, 18.46%; C, 63.71%; H, 5.60%; N, 17.69%; Found: C, 63.36%; H, 5.86%;
1
2
22
5
Found: C, 66.73%; H, 5.62%; N, 18.28%.
N, 17.38%.

4
R. S. Tamboli et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
6-(4-Chlorophenyl)-N-(2-morpholinoethyl)-5-phenyl-1,2,4-tria-
zin-3-amine (3m)
Pharmacological (antiplatelet/antithrombotic) activity
In vitro platelet aggregation assay
ꢁ
Yield 54%, yellow crystals from MeOH, mp: 129–131 C; IR
ꢀ
1
KBr, cm ) 3441, 1596, 1115, 702; EI-MS (m/z) [%]: 395.01
The newly synthesized compounds were studied for their in vitro
platelet aggregation inhibitory activity on whole human blood as
37
previously reported by us .
(
(
+
M) [90], 397.41 (M + 2) [40]; H NMR (CDCl ): ꢀ (ppm)
+
1
3
7
2
.27–7.51 (m, 9H, Ar–H), 6.07 (brs, 1H, N–H), 3.71–3.76 (m, 6H,
ꢂ CH –O and HN–CH ), 2.67 (t, J ¼ 5.8 Hz, 2H, HN–CH –
2
2
2
Ex vivo antiplatelet study
CH –N), 2.53 (t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O); Anal.
2
2
2
Calcd. for C H ClN O: C, 63.71%; H, 5.60%; N, 17.69%; The suspensions of the test compounds were triturated with
2
1
22
5
Found: C, 63.46%; H, 5.86%; N, 17.53%.
minimum quantity of gum acacia (0.9% w/v). Test compounds
were orally administered (10 mg/kg) and the first blood sample
was withdrawn after 2 h via the retro-orbital puncture. All samples
were collected into vaccue tubes containing 1/10 volume of
heparin (100 IU/ml) and kept at room temperature until further
N-(2-Morpholinoethyl)-6-(4-nitrophenyl)-5-(p-tolyl)-1,2,4-tria-
zin-3-amine (3n)
ꢁ
Yield 66%, yellow crystals from MeOH, mp: 150–152 C; IR experimentation, for a period ranging from 5 min to 1 h. Diluted
ꢀ
+ 1
1
(
(
KBr, cm ) 3432, 1585, 1516, 1115; EI-MS (m/z) [%]: 420.26 whole blood (490 ml) with isotonic saline (500 ml) was transferred
M) [14]; H NMR (CDCl ): ꢀ (ppm) 7.13–8.19 (m, 8H, Ar–H), to an aggregometer cuvette placed in the thermostated (37 C)
ꢁ
3
6
.22 (brs, 1H, N–H), 3.71–3.76 (m, 6H, 2ꢂ CH –O and cuvette holder of the whole-blood aggregometer (Model 592,
2
HN–CH ), 2.69 (t, J ¼ 5.3 Hz, 2H, HN–CH –CH –N), 2.55 Chrono-Log Corp., Hawertown, PA). Siliconized stirring bar and
2
2
2
(
t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O), 2.38 (s, 3H, Ar–CH ); electrode were dipped inside and the system was allowed to come
2
2
3
1
3
C NMR (CDCl ): ꢀ 21.38, 37.58, 53.39, 57.01, 66.88, 123.53, to equilibrium. Ten microliters of ADP (10 mM, Chrono-Log
3
1
1
29.02, 129.68, 130.62, 132.73, 138.86, 141.37, 143.15, Corp.) were added and the change in impedance, which
47.43, 153.54, 160.23; Anal. Calcd. for C H N O : reflected platelet aggregation around the electrodes, is recorded
2
2 24 6 3
C, 62.84%; H, 5.75%; N, 19.99%; Found: C, 62.46%; H, 5.98%; until the maximum extent of aggregation was reached. Each
N, 19.68%.
blood sample was tested thrice and the mean was calculated for
each group.
Maximal reading (mean ± SEM) of platelet aggregation in
whole blood induced by 10 mM ADP was determined without the
test sample (Blank, i.e. Maximum Aggregation) and a given
concentration of each test sample. The percentage inhibition was
calculated considering the blank reading as 100% by the formula
as given below:
N-(2-Morpholinoethyl)-5-(4-nitrophenyl)-6-(p-tolyl)-1,2,4-tria-
zin-3-amine (3o)
ꢁ
Yield 68%, yellow crystals from MeOH, mp: 141–143 C; IR
ꢀ
1
KBr, cm ) 3440, 1586, 1516, 1115; EI-MS (m/z) [%]: 420.52
(
(
+
M) [18]; H NMR (CDCl ): ꢀ (ppm) 7.13–8.17 (m, 8H, Ar–H),
1
3
6
.22 (brs, 1H, N–H), 3.73–3.75 (m, 6H, 2ꢂ CH –O and
2 2 2
2
Blank ꢀ Sample
Percentage Inhibition ¼
ꢂ 100
ð1Þ
HN–CH ), 2.69 (t, J ¼ 5.7 Hz, 2H, HN–CH –CH –N), 2.54
Sample
(
t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O), 2.38 (s, 3H, Ar–CH );
2
2
3
Anal. Calcd. for C H N O : C, 62.84%; H, 5.75%; N, 19.99%;
22 24 6 3
Found: C, 62.59%; H, 5.98%; N, 19.73%.
In vivo antithrombotic study
Ferric chloride induced thrombosis
5-(3-Chlorophenyl)-N-(2-morpholinoethyl)-6-(p-tolyl)-1,2,4-tria-
zin-3-amine (3p)
Rats weighing between 250 and 300 g were anaesthetized with
ketamine (100 mg/kg) and a polyethylene catheter (PE-205) was
ꢁ
Yield 55%, light yellow crystals from MeOH, mp: 142–144 C; inserted into the trachea via tracheotomy to facilitate breathing.
ꢀ
1
IR (KBr, cm ) 3450, 1596, 1117, 696; EI-MS (m/z) [%]: Catheters were also placed in the femoral artery for blood samples
09.56 (M) [22], 411.25 (M + 2) [16]; H NMR (CDCl3): and measurement of arterial blood pressure and in the jugular vein
+
+
1
4
ꢀ
(ppm) 7.12–7.55 (m, 8H, Ar–H), 6.06 (brs, 1H, N–H), 3.68–3.74 for administration of test samples. The right carotid artery was
(
m, 6H, 2ꢂ CH –O and HN–CH ), 2.67 (t, J ¼ 5.7 Hz, 2H, isolated and a small piece of Parafilm ‘‘M’’ was placed under the
2
2
HN–CH –CH –N), 2.53 (t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O), vessel to isolate it from surrounding tissues throughout the
2 2 2 2
2
.36 (s, 3H, Ar–CH ); Anal. Calcd. for C H ClN O: experiment. The test sample was administered by an intravenous
3 22 24 5
C, 64.46%; H, 5.90%; N, 17.09%; Found: C, 64.78%; H, 5.68%; injection at a defined time prior to initiation of thrombus
N, 16.82%.
formation. Thrombus formation was induced by the application
of filter paper (2 ꢂ 5 mm), saturated with FeCl solution, to the
3
carotid artery. Concentration of FeCl solution that caused
3
6-(3-Chlorophenyl)-N-(2-morpholinoethyl)-5-(p-tolyl)-1,2,4-tria-
zin-3-amine (3q)
consistent thrombus formation was determined under our experi-
mental conditions and it was found out to be 42.30% w/v. The
paper was allowed to remain on the vessel for 10 min before
removal. The temperature probe (SS6L, BIOPAC Inc., Goleta,
CA) was placed distal to the filter paper piece towards the
cephalic end to measure any changes in temperature during the
experiment. The experiment was continued for 60 min after the
induction of thrombosis. At that time, the thrombus was removed
ꢁ
Yield 58%, yellow crystals from MeOH, mp: 138–140 C; IR
ꢀ
1
KBr, cm ) 3447, 1595, 1117, 697; EI-MS (m/z) [%]: 409.02
(
(
+
+
M) [24], 411.37 (M + 2) [8]; H NMR (CDCl ): ꢀ (ppm) 7.12–
1
3
7
.55 (m, 8H, Ar–H), 6.09 (brs, 1H, N–H), 3.70–3.74 (m, 6H, 2ꢂ
CH –O and HN–CH ), 2.67 (t, J ¼ 5.6 Hz, 2H, HN–CH –CH –
2
2
2
2
N), 2.53 (t, J ¼ 4.0 Hz, 4H, 2ꢂ N–CH –CH O), 2.36 (s, 3H,
2
2
3
9
1
Ar–CH ); C NMR (CDCl ): ꢀ 21.45, 37.60, 53.41, 57.11, 66.95,
3
and weighed . The percentage inhibition of thrombosis was
calculated using following formula:
3
3
1
1
27.40, 128.28, 129.07, 129.17, 129.39, 129.48, 133.03, 134.31,
38.46, 140.94, 148.27, 156.74, 160.34; Anal. Calcd. for
A ꢀ A1
Percentage Inhibition of Thrombosis ¼
ꢂ 100
ð2Þ
C H ClN O: C, 64.46%; H, 5.90%; N, 17.09%; Found: C,
2
2
24
5
A
6
4.83%; H, 5.68%; N, 16.88%.

DOI: 10.3109/14756366.2015.1060480
Novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine
5
where A ¼ thrombus weight of control and A ¼ thrombus weight complex, 10 docking experiments were performed using
1
after treatment with the test sample or standard.
Lamarckian genetic algorithm to determine the binding energy
docked energy), and by using the obtained binding energy the Ki
(
AV shunt model for thrombosis
value was determined. The maximum number of energy evalu-
ations of 25 million was applied for each docking experiment.
Rats were anaesthetized and fixed in supine position on a
ꢁ
temperature-controlled (37 C) heating plate to maintain body
temperature. The left carotid artery and the right jugular vein were
Results and discussion
catheterized with short polyethylene catheters. The catheters were Synthesis
filled with isotonic saline solution and clamped. The two ends of
The diaryltriazine derivatives presented in this paper were
prepared according to the green synthetic route described in
Scheme 1. The starting materials, 1,2-diketones (1a–1j), were
the catheters were connected with a 2 cm glass capillary with an
internal diameter of 1 mm. At a defined time after administration
of the test sample (3d), the clamps which were occluding the
AV-shunt were opened. If the blood started flowing, then the
temperature would rise from room temperature to body tempera-
ture. In contrast, decrease in temperature indicated the formation
of an occluding thrombus. The temperature was measured
44
prepared as per our previous reported method . All the reactions
were carried out by environment friendly protocols developed in
our laboratory without the use of volatile organic compounds
(VOCs). Green chemistry proposes optimized synthetic meth-
odologies for high product yields and generation of byproducts
and/or waste material that offer little or no harm to environment.
The changing paradigm to use alternative reaction media rather
than volatile organic solvents emphasizes on the use of ionic
liquids and solvent-free solid-phase syntheses. As per our interest
continuously (as mentioned in the procedure for FeCl model)
3
4
over 30 min after opening of the shunt .
0
Bleeding time
The rats were given per oral dose of 10 mg/kg bw of test sample in developing green synthetic methodologies for biologically
3
4,38
and anesthetized after 2 h. Then they were fixed in supine position active small molecules
ꢁ
, first step of the synthesis was carried
on a temperature-controlled (37 C) heating-table. Catheterization out in ionic liquid and the latter one in solvent-less condition. The
+
ꢀ
of a carotid artery (for measurement of blood pressure) and a ionic liquid 1,3-dibutylimidazolium bromide [Bbim Br ]:DMSO
jugular vein was performed. After a defined latency period, the (1:10) promoted the one pot reaction of the corresponding
tail of the rat was transected with a razor blade at a distance of diketones (1a–1j) with methyl iodide and thiosemicarbazide
4 mm from the tip of the tail. Immediately after transection, the offering 3-methylthio-diaryl-1,2,4-triazines (2a–2q) at 70 C in a
3
tail was immersed into a bath filled with isotonic saline solution at very short period of time . The regioisomers formed were
ꢁ
8
ꢁ
37 C. The amount of time elapsed from tail transection to separated using flash chromatography using 5% ethyl acetate in n-
cessation of bleeding was measured up to 30 min and was hexane as eluent. The separated isomers were unambiguously
4
assigned as the bleeding time .
1
confirmed by their spectral data and one of them (2f) by single
3
8
crystal X-ray structure (CCDC 969264) . In the IR spectra, the
Gastric ulceration
1,2-diketones showed absorption bands for C¼O in the range
ꢀ
1
650–1670 cm . The cyclized products (2a–2q) were confirmed
1
Female Wistar rats (150–170 g) were fasted for 48 h having access
to drinking water ad libitum. Test compounds and standard drug
aspirin were orally administered to groups of five rats 5 h before
autopsy. The stomachs were macroscopically inspected and the
number of ulcers is noted and the severity recorded with the
following scores: 0 ¼ no ulcer; 1 ¼ superficial ulcers; 2 ¼ deep
by the appearance of C¼N stretching in the IR spectrum at 1590–
ꢀ1
1
610 cm
and absence of carbonyl stretching of diketones.
Demethylsulfurization and instantaneous amination of these
compounds occurred due to sufficient basicity of morpholi-
noethylamine without the use of any added base/solvent/catalyst.
The resulting compounds (2a–2q) when treated with excess of
1
4
ulcers; 3 ¼ perforation .
ꢁ
neat 4-(2-aminoethyl)morpholine (4 eq.) at 100–110 C for 2–4 h
afforded 3-morpholinoethylamine derivatives of 1,2,4-triazine
Molecular docking studies
(
3a–3q) as the desired products.
Compounds (3a–3q) showed characteristic bands at 1610–
42,43
The docking studies were performed by using AutoDock4
validate the docking study, the co-crystallized ligand within the 1590 cm
. To
ꢀ1
for C¼N stretching, asymmetric and symmetric
ꢀ
1
D structure of COX-1 and COX-2 (PDB Code: 2OYE and stretching N–H bands at around 3500 and 3350 cm , respectively
6
of the enzymes. The docking study of the most active ligand (3d) proton was observed at around ꢀ 6.0. The four morpholinyl
3
1
COX, respectively) was re-docked into the respective active sites in the IR spectra. In the H NMR spectra, a broad singlet of N–H
on active sites of the target enzymes COX-1 and COX-2 was protons of the two CH groups directly attached to oxygen and the
2
performed to get further insights into the possible interactions two CH protons of the side chain attached to NH appeared as
2
between the ligand and the target enzyme. For the ligand–enzyme multiplet (mixed signal of a broad triplet and a sharp triplet) at ꢀ
X
X
X
X/Y
O
O
N
N
N
N
SMe
N
N
a
N
b
N
O
N
N
SMe
N
H
Y
Y
Y
Y/X
1a-1j
2a-2q
3a-3q
Scheme 1. Green synthesis of 5,6-diaryl-N-morpholinoethyl-1,2,4-triazin-3-amines (3a–3q). Reagents and conditions: (a) thiosemicarbazide, methyl
+
iodide, ionic liquid [Bbim Br ]: DMSO (1:10), 70 C, 10–110 min; (b) 4-(2-aminoethyl)morpholine (4 eq.), neat, 100–110 C, 2–4 h.
ꢀ
ꢁ
ꢁ

6
3
R. S. Tamboli et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
.5–4.0. The CH protons of side chain attached to the tertiary
2
From the in vitro evaluation of the test compounds (3a–3q), 10
nitrogen of the ring always appeared as a distinct triplet at ꢀ 2.6– compounds were found to have prominent activity. Compounds
.7. The remaining four morpholinyl CH2 protons directly (3a, 3c, 3d, 3f, 3g, 3j, 3k, 3l, 3p and 3q) produced more than 60%
2
attached to the ring nitrogen appeared as a triplet (occasionally inhibition while compounds (3e, 3h, 3i, 3m, 3n and 3o) were
broad) at ꢀ 2.4–2.6. The aromatic protons appeared as multiplet at found to be comparable in activity with aspirin. Out of these
1
7.0–8.0. In the C NMR spectra, the ꢀ values of the carbons are compounds, 3d was the most potent, being twice as potent
3
ꢀ
in accordance with the structures of the synthesized compounds. as aspirin.
The single crystal X-ray structures were also affirmed for two
of the compounds 3l and 3m (Figure S1 in Supplementary
Information, CCDC 1028877 and 1028686, respectively). The
crystal structure data of these positional isomers confirmed their
respective structures. All the final compounds (3a–3q) were
characterized by spectral and elemental analysis.
Ex vivo antiplatelet aggregation study
Almost all of the compounds showed good to moderate inhibition
of platelet aggregation in the in vitro study. Hence, all the
compounds were screened further for their ex vivo platelet
aggregation inhibition. The whole blood aggregation method was
used for this purpose in which blood was withdrawn from
animals, 2 h after pretreatment of the group with test samples by
oral dosing, along with the control group and the aspirin-treated
group (Table 2).
From the ex vivo study of the tested compounds (3a–3q), six
compounds (3a, 3d, 3e, 3i, 3l and 3n) were found with noticeable
activity. Out of these five compounds, 3d again was found to be
the most potent (53.19% inhibition), which was almost double the
activity of aspirin. Compounds (3a, 3e, 3i, 3l and 3n) produced
comparable inhibition of platelet aggregation to aspirin. Even
though these compounds were found to be less potent in
comparison to another standard drug clopidogrel which acted
through a different mechanism of action (P2Y12 inhibition), their
potential vasodilatory effect could give them an additional
advantage.
Antiplatelet/antithrombotic activity
In vitro platelet aggregation assay
The newly synthesized compounds were studied for their in vitro
platelet aggregation inhibitory activity. Our earlier study has
shown that this type of compounds does not exhibit any
cytotoxicity rather show protective role in Malondialdehyde
(MDA), catalase and intracellular reactive oxygen species (ROS)
generation assays . The whole human blood aggregation study
3
4
3
7
was done as per our previously reported procedure . Each
reading was taken in triplicate for the test samples, using control
and a standard drug aspirin for comparative reading each time.
The percent inhibition of platelet aggregation was determined for
each test sample on comparison with the control and the standard
aspirin. The inhibitory values were calculated accordingly as
shown in Table 1.
In vivo antithrombotic study
Ferric chloride induced thrombosis
Table 1. Structures and in vitro antiplatelet aggregation activity data of
the synthesized 3-substituted 5,6-diaryl-1,2,4-triazines (3a–3q).
All the synthesized compounds were evaluated for their
antithrombotic activity using FeCl -induced thrombosis model,
X
3
3
9
as reported earlier . From Table 2, it could be seen that almost all
the compounds (3a–3q) inhibited the thrombus formation as
evidenced from their respective thrombus weight after treatment
at a dose of 10 mg/kg bw p.o. Compounds (3b, 3c, 3d, 3j and 3q)
decreased the thrombus weight significantly as compared to the
standard drug and the control. Two compounds (3c and 3d)
showed noteworthy efficacy in this model. The time to occlusion
N
N
O
N
N
N
H
Y
3a-3q
(TTO) was also measured as a parameter indicating obstruction to
.
blood flow after formation of clot/thrombus. Increase in TTO was
considered as a preventive measure for prolongation of thrombus
formation. Compounds (3d, 3g, 3j, 3k, 3p and 3q) significantly
increased TTO as compared to the standard drug and the control
%
Inhibition of platelet
aggregation
(in vitro, 10 mg/ml)
Compd.
no.
X
Y
Mol. Form.
(Table 2).
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
H
H
C
21
C
21
C
23
C
22
C
22
C
21
C
21
C
21
C
21
C
21
C
21
C
21
C
21
C
22
C
22
C
22
C
22
–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
23
N
5
O
63.95
34.79
71.25
75.00
48.02
68.88
66.93
39.51
41.93
68.72
70.16
58.79
36.29
49.68
46.77
58.87
62.50
34.00
4-Cl
4-CH
4-CH
4-Cl
H
4-Cl
4-CH
4-Cl
4-CH
4-F
2 5
21Cl N O
N O
27 5
24ClN
24ClN
22FN
22FN
21BrClN
21BrClN
21ClFN
21ClFN
22ClN
22ClN
Arterio-venous (AV) shunt thrombosis
3
3
3
3
5
O
O
Further, to ensure antithrombotic efficacy of the compound (3d),
4
0
5
it was evaluated by previously known AV shunt model . No
temperature fall was observed up to 20 min which showed the
non-occlusive response indicating the potential antithrombotic
action of compound (3d).
5
O
O
4-F
H
5
4-Cl
4-Br
4-Cl
4-F
4-Br
4-Cl
4-F
4-Cl
4-Cl
H
5
O
O
5
5
O
O
5
Bleeding time
H
5
O
O
4-Cl
4-CH
4-NO
4-CH
3-Cl
–
5
Bleeding time was used as a parameter to evaluate the hemor-
rhagic properties of antithrombotic drugs. Hence, tail bleeding
3
4-NO
2
24
N
N
6
O
3
O
3
4
1
2
4-CH
3-Cl
4-CH
–
3
24
6
method was used at a dose of 10 mg/kg of body weight for the
test samples. Compounds (3d, 3i, 3g, 3j and 3k) showed increased
but significantly lower bleeding time than standard drugs
clopidogrel and aspirin. The optimum increase in bleeding time
3
24ClN
24ClN
5
O
O
3
5
Aspirin
The bold values suggest the noteworthy/comparable activity of respective by these compounds could be of great advantage since that would
compounds.
overcome the fatal bleeding complications (Table 2).

DOI: 10.3109/14756366.2015.1060480
Novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine
7
Table 2. Antithrombotic activity (ex vivo and in vivo) data of the synthesized 3-substituted 5,6-diaryl-1,2,4-triazines
3a–3q) at a dose of 10 mg/kg bw p.o.
(
Comp.
no.
% Inhibition of platelet
aggregation (ex vivo)
Thrombus wt.
(mg)
% Inhibition
of thrombosis
Time to occlusion
(min)
Bleeding time
(min)
Control
–
6.60
4.80
4.20
4.00
4.00
5.00
4.90
4.30
4.90
4.85
4.20
4.25
4.75
4.50
4.25
4.25
4.00
4.10
3.00
3.00
–
15.0
12.5
18.0
21.0
430
12.0
12.5
27.0
20.5
21.5
29.0
29.5
19.0
23.5
21.0
21.5
27.0
27.5
430
430
18.0
17.0
16.0
19.5
25.0
17.5
24.0
25.5
18.0
20.5
24.5
25.0
20.5
19.5
16.5
16.5
430
430
430
430
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
37.65
32.53
19.87
53.19
44.47
22.37
19.11
23.82
36.47
19.94
17.64
45.29
26.17
36.42
35.58
17.64
18.58
84.03
47.80
27.27
36.36
39.39
39.39
24.24
25.75
34.84
25.75
26.51
36.36
35.60
28.03
31.81
35.60
35.60
39.39
37.87
54.54
54.54
Clopidogrel*
Aspiriny
The bold values suggest the noteworthy/comparable activity of respective compounds.
Clopidogrel used at a dose of 25 mg/kg body weight.
yAspirin used at a dose of 50 mg/kg body weight.
*
Figure 3. Effect of compound (3d) and aspirin on gastric mucosa. (a) Control with no gastric lesions; (b) Compound (3d) showing mild gastric lesions
10 mg/kg); (c) Compound (3d) showing moderate gastric lesions (100 mg/kg); (d) Aspirin showing severe gastric lesions (100 mg/kg).
(
Gastric ulceration
100 mg/kg dose the ulceration was much less than the standard
drug aspirin which caused severe ulceration at the same dose
1
4
The gastric ulceration model was selected to ascertain the
undesired side effects, if any, of the test sample (3d). The results
suggested that there was no gastric ulceration in the control
(Figure 3a) while a mild ulceration was observed after 10 mg/kg
dose (Figure 3b) of the test compound (3d), whereas moderate The effect of various groups on the basic 5,6-diaryl-1,2,4-triazine
ulceration was observed at 100 mg/kg dose (Figure 3c). Still, at scaffold was studied with respect to antiplatelet activity of the
(100 mg/kg) (Figure 3d).
Structure–activity relationships

8
R. S. Tamboli et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 4. Docking simulations of compound (3d) with active sites of (a) COX-1 (PDB: 2OYE) and (b) COX-2 (PDB: 6COX), respectively.
synthesized compounds (Table 1). Among the compounds in the on the 5-aryl ring favor antiplatelet activity. While compound (3d)
series, compound (3d) was found to be the most potent inhibitor showed the most prominent activity, its positional isomer (3e)
of in vitro platelet aggregation. This suggests that in the present with p-methyl substituent on the 5-aryl ring and p-Cl substituent
series, p-methyl substituent on the 6-aryl ring and p-Cl substituent on the 6-aryl ring showed almost half the activity of 3d. p-Halo

DOI: 10.3109/14756366.2015.1060480
Novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine
9
substituents on the 5-aryl ring showed better activity in almost all further investigation as a new potential drug candidate for
of the compounds except for compound (3b). Halo substitution at thromboembolic diseases.
these positions increases the lipophilic character of the molecule.
This could be one of the reasons for higher in vitro activity of p- Molecular docking studies
halo substituted compounds at the 5-aryl ring. Compounds
Molecular docking studies of the most active compound (3d) with
without halogens on both of the aryl rings showed overall
COX-1 and COX-2 enzymes were performed to understand the
decreased activity (3n and 3o). Compounds with m-Cl substituent
possible nature of its binding interactions within the active sites
on the 5-aryl ring showed lower activity than the p-Cl substituted
derivatives (compound 3p versus compound 3d). The compound
with p-Br substituent on the 5-aryl ring (3h) showed a drastic
reduction in the activity whereas p-F substituent on this position
retained the activity (3f and 3j). Surprisingly, a compound with p-
Cl substituent on both the aryl rings (3b) has shown diminished
activity while p-methyl substituents on at both the aryl rings
retained the same level of activity (3c).
45
45
(
PDB Code: 2OYE and 6COX , respectively).
The most active molecule (3d), when docked into the COX-1
active site, comfortably entered the active site and showed good
interactions with binding energy of ꢀ10.59 kcal/mole. The p-tolyl
group showed hydrophobic interactions with Phe381, Leu384,
Tyr385 and Trp387, whereas, the p-chlorophenyl ring depicted
hydrophobic interactions with Tyr355, Val349 and Ile523. The in
silico binding affinity (K ) value for the ligand–receptor complex
i
In case of ex vivo activity of the synthesized compounds, five of
the compounds (3b, 3d, 3e, 3i and 3l) showed better activity than
the rest. Compound (3d) again emerged as the most potent
compound with 53.19% inhibition of platelet aggregation ex vivo.
In case of bleeding time, compound 3b with p-Cl substituents
on both of the aryl rings showed the least bleeding time which was
unexpectedly low. Compound (3d) showed the maximum
bleeding time of 25 min in the series, in contrast to the reference
drugs aspirin and clopidogrel which had still higher bleeding
times (430 min). The increased bleeding time after treatment by
the current drugs is considered to be the main drawback, which
causes fatal bleeding and loss of preventive mechanism of
hemostasis. Hence, compounds with optimum bleeding time, such
as 3d, would be beneficial for antiplatelet/antithrombotic therapy.
Thrombus formation is one of the major causes of the
thromboembolic diseases. To evaluate the antithrombotic efficacy
was obtained as 17.16 nM. However, when we studied its
interactions with the active site of COX-2 enzyme, the p-tolyl
ring oriented itself near the secondary pocket (Leu352, Arg513)
of the active site of the receptor. The in silico binding energy for
this complex was observed to be ꢀ8.23 kcal/mole and the binding
affinity value was calculated to be 927.04 nM.
In brief, we compared the binding interactions of the most
active molecule (3d) with COX-1 and COX-2 enzymes (Figure 4).
The in silico interactions of compound (3d) with COX-1 and COX-
2 showed that the compound is having more affinity and selectivity
towards COX-1 while moderate activity towards COX-2. These
results were in accord with the observations of the previous studies
7,46
of nonselective COX inhibition as antiplatelet agents
.
Conclusions
of the tested compounds, FeCl -induced thrombosis model was
3
A novel series of 5,6-diaryl-1,2,4-trizines has been reported with
several electron withdrawing as well as donating functions
attached to the vicinal diaryl rings. In vitro, ex vivo and in vivo
evaluations indicated that some of the compounds showed
moderate to potent antiplatelet/antithrombotic activities which
could be helpful for the development of new antiplatelet drugs.
The most promising compound (3d) of the series seems to possess
a potentially better therapeutic profile.
utilized. FeCl at an optimum concentration of 42.30% w/v causes
3
clot/thrombus formation in vein/artery and the weight of the
thrombus formed was measured carefully after isolation. The
weight of the thrombus formed was used as one of the parameters
to find the preventive role of the synthesized compounds on
thrombosis. It was observed that almost all the compounds
inhibited thrombus formation to variable extents. Compounds (3c
and 3p) were quite active but compound (3d) again was found to
be the most potent in this test. Percent inhibition of thrombosis
was also calculated using the thrombus weight. The estimated
Declaration of interest
potency of 3d observed in this model was further confirmed by We thank All India Council for Technical Education (AICTE), New
evaluating it using AV shunt model.
Delhi, India for National Doctoral Fellowship to RST [File No. 1–10/RID/
NDF/PG/36/2009–2010]. The authors also thank the DST-PURSE Single
Crystal X-Ray Diffraction Facility at the Faculty of Science, The
Maharaja Sayajirao University of Baroda, Vadodara for single crystal
X-ray crystallographic studies.
Eventually, TTO was measured for all the test compounds.
Almost one-third of the compounds significantly inhibited the
occlusion for more than 20 min. Compounds (3d, 3g, 3i, 3k, 3m,
3
hypothesized vasodilatory action of the tested compounds might
p and 3q) showed better profile in this parameter. The previously
The authors declare that they have no conflicts of interest to disclose.
1
4–18
be responsible for this phenomenon
. Compound (3d) showed
TTO of more than 30 min which was equal to the standard drugs
clopidogrel. Compound (3e) was found to be the least active
amongst all the compounds and was comparable to the
unsubstituted diaryl compound (3a).
As several COX inhibiting drugs show moderate to severe
gastric ulcerations, the most potent compound (3d) was evaluated
for its propensity to cause gastric ulceration. It was found that it
did not show any undesirable side effect at its normal dose of
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Supplementary material available online
Supplementary Figure S1