Biphenyl-3-oxo-1,2,4-triazine linked piperazine derivatives as potential cholinesterase inhibitors with anti-oxidant property to improve the learning and memory

Article abstract of DOI:10.1016/j.bioorg.2018.12.017

A series of novel piperazine tethered biphenyl-3-oxo-1,2,4-triazine derivatives were designed, and synthesized. Amongst the synthesized analogs, compound 6g showed significant non-competitive inhibitory potential against acetylcholinesterase (AChE, IC₅₀; 0.2 ± 0.01 μM) compared to standard donepezil (AChE, IC₅₀: 0.1 ± 0.002 μM). Compound 6g also exhibited significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE (22.22 ± 1.11%) and showed good CNS permeability in PAMPA-BBB assay (P_{e (exp)}, 6.93 ± 0.46). The in vivo behavioral studies of compound 6g indicated significant improvement in cognitive dysfunctions against scopolamine-induced amnesia mouse models. Further, ex vivo studies showed a significant AChE inhibition and reversal of the scopolamine-induced oxidative stress by compound 6g. Moreover, molecular docking and dynamics simulations of compound 6g showed a consensual binding affinity and active site interactions with the PAS and active catalytic site (CAS) residues of AChE.

Full text of DOI:10.1016/j.bioorg.2018.12.017

Accepted Manuscript
Biphenyl-3-oxo-1,2,4-triazine linked piperazine derivatives as potential choli-
nesterase inhibitors with anti-oxidant property to improve the learning and
memory
Prabhash Nath Tripathi, Pavan Srivastava, Piyoosh Sharma, Manish Kumar
Tripathi, Ankit Seth, Avanish Tripathi, Sachchida Nand Rai, Surya Pratap
Singh, Sushant K. Shrivastava
PII:
S0045-2068(18)31004-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.017
YBIOO 2681
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 September 2018
3 December 2018
12 December 2018
Please cite this article as: P. Nath Tripathi, P. Srivastava, P. Sharma, M. Kumar Tripathi, A. Seth, A. Tripathi, S.
Nand Rai, S. Pratap Singh, S.K. Shrivastava, Biphenyl-3-oxo-1,2,4-triazine linked piperazine derivatives as
potential cholinesterase inhibitors with anti-oxidant property to improve the learning and memory, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.017
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Biphenyl-3-oxo-1,2,4-triazine linked piperazine derivatives as potential
cholinesterase inhibitors with anti-oxidant property to improve the
learning and memory
Prabhash Nath Tripathia, Pavan Srivastavaa, Piyoosh Sharmaa, Manish Kumar Tripathia,
Ankit Setha, Avanish Tripathia, Sachchida Nand Raib, Surya Pratap Singhb, Sushant K.
Shrivastavaa*
aPharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical
Engineering & Technology, Indian Institute of Technology (Banaras Hindu University),
Varanasi – 221 005 (India)
bDepartment of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi
221005, India
*Address correspondence to:
Dr. Sushant Kumar Shrivastava
Professor
Department of Pharmaceutical Engineering & Technology,
Indian Institute of Technology (BHU), Varanasi- 221 005, India
Email: skshrivastava.phe@itbhu.ac.in
Phone: +91-542-6702725
1

ABSTRACT
A series of novel piperazine tethered biphenyl-3-oxo-1,2,4-triazine derivatives were
designed, and synthesized. Amongst the synthesized analogs, compound 6g showed
significant non-competitive inhibitory potential against acetylcholinesterase (AChE, IC50; 0.2
± 0.01 μM) compared to standard donepezil (AChE, IC50: 0.1 ± 0.002 μM). Compound 6g
also exhibited significant displacement of propidium iodide from the peripheral anionic site
(PAS) of AChE (22.22 ± 1.11%) and showed good CNS permeability in PAMPA-BBB assay
(Pe(exp), 6.93 ± 0.46). The in vivo behavioral studies of compound 6g indicated significant
improvement in cognitive dysfunctions against scopolamine-induced amnesia mouse models.
Further, ex vivo studies showed a significant AChE inhibition and reversal of the
scopolamine-induced oxidative stress by compound 6g. Moreover, molecular docking and
dynamics simulations of compound 6g showed a consensual binding affinity and active site
interactions with the PAS and active catalytic site (CAS) residues of AChE.
Keywords: Acetylcholinesterase; butyrylcholinesterase; 1,2,4-triazine; piperazine; PAMPA-
BBB; cognitive dysfunctions; Alzheimer’s disease; donepezil.
2

1. INTRODUCTION
Cognition is a combination of awareness, attention, acquaintance, memory, decision making,
and judgments [1]. Cognitive impairment is a pathological condition associated with many
neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD),
depression, and schizophrenia [2]. The neurotransmitters like acetylcholine (ACh), dopamine,
serotonin, glutamate, etc. are responsible for regulating the cognitive symptoms and
functionalities. Amongst them, ACh plays a pivotal role in cognitive functionalities as its
deficiency in the brain causes memory disorder [3]. ACh is metabolized by
acetylcholinesterase (AChE) into choline and acetic acid [4, 5]. Hence, the augmentation of
ACh level in the brain could be achieved by targeting the AChE through the AChE inhibitors
(AChEIs) [6-8]. In the current scenario, there are only three FDA approved AChEIs
(donepezil, rivastigmine, and galantamine) available which provide symptomatic cure rather
than modifying the disease progression [9]. Therefore, the discovery and development of new
drugs for the treatment of AD is required to increase the lifespan of human beings. In the past
decade, AChEIs (metrifonate and tesofensine) were identified as promising molecules to treat
dementia but withdrawn from the clinical trials due to their toxicity and poor CNS stability
[10-12]. Hence, there is an unmet necessity to discover the novel neurotherapeutic agents to
diminish the AChE activity and modifying the disease progression.
The oxidative stress is a detrimental factor of memory dysfunctions caused by an imbalance
of antioxidant enzymes and overproduction of reactive oxygen species (ROS) such as
•
superoxide anion, hydroxyl radical (OH•), and hydrogen peroxide radicals (H2O2 ) [13-16].
These anions and free radicals oxidize various biomolecules and support the progression of
the disease. ROS are reported to cause the memory impairment and synaptic plasticity [17,
18]. Therefore, AChE inhibitor with promising antioxidant activity could prove itself as a
good therapeutic candidate to treat dementia.
3

1.1. Designing Considerations
In this study, the classical bivalent bioisosteric strategy was adopted to design the novel
hybrid molecules [19]. The triazine nucleus being an attractive moiety was used owing to its
binding ability and better inhibition characteristics against several enzymes [20]. Compounds
containing 1,2,4-triazine nucleus showed a wide array of applications including the potential
AChE inhibitory activities [21-26]. The studies revealed that vicinal biaryl-1,2,4-triazines (1)
modulate the cholinesterase (ChE) activity through intrinsic antioxidant property [27],
whereas 3-Thioalkyl-5,6-biaryl-1,2,4-triazine derivatives (2) suppressed the ROS generation
and oxidative stress-induced cell death [28]. The 1,2,4-triazine group also displayed a
significant role in anchoring the 5,6-biaryl group into the peripheral anionic site (PAS) of
AChE through hydrophobic interactions [29]. Additionally, the molecule containing 5,6-
biaryl-3-oxo-1,2,4-triazine group were reported to devoid of gastric or cardiac toxicity [30,
31] Therefore, the 5,6-biaryl-3-oxo-1,2,4-triazine was chosen as a core scaffold and tethered
with other pharmacophoric functionalities.
Donepezil (3) is an FDA approved drug used for the treatment of dementia having benzyl
piperidine nucleus, which is oriented towards the catalytic active site (CAS) of AChE [32-
34]. The piperazine moiety was chosen in place of piperidine ring owing to the bioisosteric
nature. Though piperazine ring possesses lower AChE affinity [35], but several compounds
comprising this nucleus have been reported for nootropic effect with substantial
neuroprotective activity [36-38].
Therefore, molecular hybrids consisted of the 5,6-biphenyl-3-oxo-1,2,4-triazine nucleus and
substituted piperazines were connected using the different lengths of alkyl linkers and
evaluated for their potential ChE inhibition and antioxidant properties (Fig. 1). In the present
work, biphenyl-3-oxo-1,2,4-triazine linked piperazine analogs were synthesized and
evaluated for ChE inhibition, propidium iodide displacement, BBB permeability, and
4

neuroblastoma cell line assay. The propitious compound was further subjected to in vivo
behavioral studies by scopolamine-induced mouse models. The ex vivo studies and
biochemical estimation of the oxidative stress biomarkers were also affirmed their AChE
inhibitory and antioxidant potential, respectively. Finally, the interactions and binding modes
of the compound 6g on active site of AChE were assessed by in silico molecular docking and
dynamic simulations studies.
Fig. 1. The designed compounds.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The target compounds (5a-g, 6a-g, 7a-g, and 8a-g) were synthesized according to Scheme 1.
Initially, 5,6-Biphenyl-3-oxo-1,2,4-triazine (4) was synthesized by refluxing 1,2-
diphenylethandione with semicarbazide hydrochloride in the presence of glacial acetic acid
[31]. Compound 4 was further reacted with different bromochloroalkanes in the presence of
potassium hydroxide and N, N-dimethylformamide (DMF) to afford the key intermediates (5,
6, 7 and 8). In 1H NMR spectra, the disappearance of (-NH) proton peak of compound 4, and
5

the appearances of triplet signal of two protons of (N-CH2) between 4.53-4.29 ppm (J = 7.0
Hz) and multiplates of ten protons of biphenyl group at 7.51-7.28 ppm. 13C NMR spectral
13
assignment was based on the distinctive carbon peaks of all the synthesized derivatives. C
NMR spectra of compounds (5, 6, 7, and 8) displayed the characteristic signals of -N-CH2
and -CH2-Cl at 53.0-49.8 ppm, and 44.7-41.0 ppm, respectively. The signal of carbonyl
(triazinone,>C=O) of 1,2,4-triazine-3-oxo nucleus appeared at 166.5-166.2 ppm. The target
compounds (5a-g, 6a-g, 7a-g, and 8a-g) were synthesized by refluxing the intermediates (5,
6, 7 and 8) with various piperazines in the presence of potassium carbonate, potassium
iodide, and acetonitrile. The synthesized compounds were preliminarily identified by positive
Dragendroff test on TLC (Thin Layer Chromatography) [39, 40] and further purified by
1
column chromatography to afford the pure compounds (60-84% yield). H NMR spectra of
compounds (5a-g, 6a-g, 7a-g, and 8a-g) showed the presence of methylene protons (CH2-N)
and absence of single -NH proton of piperazine moieties. The compounds having 2-pyridyl
(5d, 6d, 7d, and 8d) moiety exhibited the signals in the range of 8.31-6.60 ppm. The
compounds (5e, 6e, 7e, and 8e) bearing 4-methoxy substituent showed a diagnostic singlet
peak of three protons between 3.73-3.62 ppm. The compounds (5f, 6f, 7f, and 8f) having
biphenyl methyl (-CH) group exhibited a distinctive singlet peak of one proton approximately
at ~4.12 ppm. Compounds (5g, 6g, 7g, and 8g) displayed the characteristic signals of two
13
protons (N-CH2-phenyl) between 3.45-3.19 ppm. In the C NMR spectra, the compounds
(5e, 6e, 7e, and 8e) having 4-methoxyphenyl piperazine displayed the signals of methoxy (-
OCH3), and methoxy attached carbon between 57.7-55.6, 153.7-152.9 ppm, respectively. The
synthesized derivatives (5f, 6f, 7f, and 8f) with biphenyl methyl (-CH) group exhibited a
diagnostics signals at around ~76.3 ppm. The distinctive piperazine-CH2-phenyl signals of
the compounds (5g, 6g, 7g, and 8g) appeared at 63.2-62.9 ppm. The purity of all the
6

synthesized compounds was determined by elemental analyses, and their results were found
within ± 0.4% range of the theoretical values.
Scheme 1. Reagents and conditions: (I) semicarbazide hydrochloride, glacial acetic acid,
reflux; (II) potassium hydroxide, Br(CH2)nCl (n = 2, 3, 4, and 5), DMF, room temperature, 1
h; (III) substituted piperazines, anhydrous potassium carbonate, potassium iodide,
acetonitrile, reflux, 13 h.
2.2. Pharmacology
2.2.1. In vitro cholinesterase inhibition assay
All the synthesized compounds (5a-g, 6a-g, 7a-g, and 8a-g) and standard donepezil were
screened for ChE inhibition activity using Ellman’s method (Table 1).
7

Table 1. The results of ChE inhibition assay, selectivity index, and enzyme kinetics study.
AChE
Types of
Inhibition
(AChE)
aSI
ratio
Comp.
Code
AChE
BChE
n
Ar
Ki (µM) ± SD
IC50 (µM) ± SD IC50 (µM) ± SD
2
2
2
2
2
2
2
3
3
3
3
-C6H5
4-NO2C6H5-
4-FC6H5-
-C5H4N
1.2 ± 0.08*
0.6 ± 0.04*
0.9 ± 0.06*
1.8 ± 0.11*
0.6 ± 0.04*
0.5 ± 0.04*
0.5 ± 0.04*
0.6 ± 0.05*
0.5 ± 0.02*
0.4 ± 0.03*
1.1 ± 0.09*
>50
11.2 ± 0.96
>50
-
20
-
0.9 ± 0.08
0.5 ± 0.04
0.7 ± 0.07
1.4 ± 0.1
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
5a
5b
5c
5d
5e
5f
19.5 ± 1.12
>50
11
-
4-OCH3C6H5-
-CH(C6H5)2
-CH2-C6H5
-C6H5
0.5 ± 0.04
0.4 ± 0.03
0.3 ± 0.03
0.5 ± 0.05
0.4 ± 0.03
0.3 ± 0.02
0.9 ± 0.09
>50
-
>50
-
5g
6a
6b
6c
6d
>50
-
4- NO2C6H5-
4-FC6H5-
-C5H4N
>50
-
5.2 ± 0.19
>50
15
-
3
4-OCH3C6H5-
0.3 ± 0.03*
>50
-
0.3 ± 0.02
6e
3
3
4
4
4
4
4
4
4
5
5
5
5
5
-CH(C6H5)2
-CH2-C6H5
-C6H5
>5*
0.2 ± 0.01ns
1.9 ± 0.12*
0.6 ± 0.04*
1.4 ± 0.13*
>5*
0.5 ± 0.03*
>5*
0.6 ± 0.05*
2.2 ± 0.19*
1.9 ± 0.15*
>5*
-
4.6 ± 0.19
>50
-
23
-
nt
-
6f
6g
7a
7b
7c
7d
7e
7f
0.2 ± 0.01
1.5 ± 0.9
0.5 ± 0.04
1.1 ± 0.99
nt
nc
nc
nc
nc
-
4-NO2C6H5-
4-FC6H5-
>50
-
23.3 ± 2.19
>50
17
-
-C5H4N
4-OCH3C6H5-
-CH(C6H5)2
-CH2-C6H5
-C6H5
>50
-
0.4 ± 0.03
nt
nc
-
-
-
8.6 ± 0.54
>50
13
-
0.5 ± 0.05
1.8 ± 0.14
1.6 ± 0.13
nt
nc
nc
nc
-
7g
8a
8b
8c
8d
8e
4-NO2C6H5-
4-FC6H5-
9.3 ± 0.84
>50
5
-
-C5H4N
>5*
>5*
>50
-
nt
-
4-OCH3C6H5-
>50
-
nt
-
8

5
5
-
-CH(C6H5)2
-CH2-C6H5
-
>5*
1.7 ± 0.16*
>50
>50
-
-
nt
1.4 ± 0.97
nt
-
nc
-
8f
8g
0.1 ± 0.01
4.1 ± 0.28
48
donepezil
Values are presented as mean ± S.D (n = 3) and analyzed by one-way ANOVA followed by Dunnett’s test,*p <
a
0.05 compared to standard donepezil, ns = Non-significant; Selective index ratio = (BChE IC50) / (AChE IC50).
nc = non-competitive; nt = not tested.
The enzyme inhibition activity of all the synthesized compounds was analyzed by concerning
the variations of alkyl linkers (n = 2, 3, 4, 5) and substituents attached to the terminal end of
piperazine moiety.
Variation of carbon chain linkers
The methylene linkers (n = 2-5) formed a bridge between 5,6-biphenyl-3-oxo-1,2,4-triazine
core group and various substituents of the piperazine nucleus and resulted compounds were
evaluated for ChE inhibition. The results indicated that lengthening of carbon chain increased
the lipophilic characteristics and hindered the freedom of C-C bond rotation [41, 42].
Overall, three carbon chain (n = 3) was found to be optimal linker for AChE inhibition.
The compounds (5f, 6f, 7f, and 8f) containing benzhydryl piperazine group at the terminal
end not showed any significant inhibitory activity with various linkers. The literature also
revealed that very high hydrophobicity hindered the binding of compounds at the active site
of AChE [43].
Variation of terminal piperazine functionalities
The compounds (6a-g) bearing propyl (n =3) linker showed a promising AChE inhibitory
activity. Thereby, our discussion is more focused on compounds (6a-g). The compound 6a
consisting of phenylpiperazine moiety displayed an acceptable AChE inhibitory activity (IC50
= 0.6 ± 0.05 µM). The electron-withdrawing (nitro, 6b; and fluoro, 6c) and electron donating
(methoxy, 6e) substituents at para position of phenylpiperazine ring increased the AChE
inhibition (6b, IC50; 0.5 ± 0.02 µM; 6c, IC50; 0.4 ± 0.03 µM; 6e, IC50; 0.3 ± 0.03 µM).
Interestingly, compound 6e having 4-methoxyphenyl group showed better AChE inhibition
9

compared to compound 6b and 6c. The improvement in AChE inhibitory potential might be
because of non-polar characteristics of OCH3 group which interacted with the hydrophobic
pocket of AChE [44]. In the comparison amongst compounds bearing different electron
withdrawing substituents, compound 6c having a fluoro substituent showed slightly better
inhibitory potential against the AChE compared to compound 6b with a nitro substituent. The
reason for better inhibitory activity could be the high electronegativity of fluorine atom that
modulates the lipophilicity of the molecule [45]. The compound 6d with 2-pyridyl piperazine
displayed a lower AChE inhibition (IC50; 1.1 ± 0.09 µM) compared to compounds 6a, 6b, 6c,
6e, and 6g.
Amongst all the tested derivatives, compound 6g bearing three carbon atoms linked with
benzylpiperazine terminal group showed potent inhibition against the AChE (IC50 = 0.2 ±
0.01µM). The AChE inhibitory activity of compound 6g also comparable with standard
donepezil (IC50 = 0.01 ± 0.002 µM) because of the benzyl group engaged at the bottom of the
enzyme gorge and better interacted with CAS residues of AChE [46].
All the synthesized compounds were further tested for their BChE inhibitory effect but failed
to exhibit any significant activity. The probable reason for this might be the wider BChE
gorge than AChE owing to the replacement of two aromatic amino acids by the smaller
aliphatic amino acids [47].
2.2.2 Enzyme kinetics study
The synthesized compounds (5a-g, 6a-e, 6g, 7a-c, 7e, 7g and 8a-b, 8g) were subjected to
enzyme kinetics studies, and the results are reported in Table 1. The substrate-dependent
enzyme kinetics elucidated the mode of enzyme inhibition for the most active compound 6g
using the Lineweaver-Burk reciprocal 1/Vmax Vs. 1/S plot, which suggested the non-
competitive type of enzyme inhibition of AChE (Ki = 0.2 ± 0.01 µM) (Fig. 2).
10

Fig. 2. Lineweaver-Burk plot showing non-competitive inhibition of compound 6g
2.2.3. Propidium iodide displacement assay
Propidium iodide assay was used to determine the displacement of propidium iodide (a
selective PAS-AChE inhibitor) from the propidium iodide-AChE enzyme complex [48]. The
binding of compounds at PAS of AChE leads to diminished fluorescence intensity with the
displacement of propidium iodide. All the tested derivatives showed substantial displacement
of propidium iodide (17.45-22.22%) except compounds 6b and 6d. The results also indicated
that compound 6g could efficiently bound to the PAS site of AChE (Table 2).
2.2.4 Parallel artificial membrane permeation assay (PAMPA-BBB)
The selected compounds (6a-e, and 6g) were screened through PAMPA-BBB assay [49].
Among of all the derivatives, standard donepezil and compound 6g showed Pe more than
(4.3926 x 10-6 cm/s) indicated their high BBB permeation (Table 2).
11

Table 2. Propidium iodide displacement and Permeability analysis using PAMPA-BBB
assay.
PAMPA-BBB
PAMPA-BBB
Prediction
Propidium Iodide
Displacement (%)a
permeability
Comp.
(CNS+b, CNS-c,
CNS±d)
Pe(exp) (10-6 cm s-1)
21.45 ± 1.72*
12.54 ± 0.87*
17.84 ± 1.38 ns
13.15 ± 0.79*
17.45 ± 0.80 ns
22.22 ± 1.11*
17.33 ± 1.38
3.29 ± 0.27*
4.63 ± 0.32*
4.21 ± 0.21*
4.48 ± 0.32*
4.85 ± 0.29*
6.93 ± 0.46ns
7.28 ± 0.43
CNS±
CNS+
CNS±
CNS+
CNS+
CNS+
CNS+
6a
6b
6c
6d
6e
6g
Donepezil
Values are presented as the mean ± S.D (n = 3) and analyzed by one-way ANOVA followed by Dunnett’s test,
* p < 0.05 compared to standard donepezil, ns = No significant;
aPropidium iodide displacement assay was performed on eeAChE.
b‘CNS+’ (prediction of high BBB permeation); Pe (10-6 cm s-1) > 4.3926.
c‘CNS-’ (prediction of low BBB permeation); Pe (10-6 cm s-1) < 1.7766.
d‘CNS±’ (prediction of uncertain BBB permeation); Pe (10-6 cm s-1) 4.3926 to 1.7766.
2.2.5 Neurotoxicity assay
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric
assay was used to determine the neurotoxicity against SH-SY5Y neuroblastoma cell lines
[50]. The compound 6g and donepezil showed the IC50; 83.78 ± 6.87 µM and IC50; 86.9 ±
7.42 µM, respectively (Table 3).
Table 3. In vitro neurotoxicity on SH-SY5Y cell lines
Mean ± SD
Comp.
(IC50)
6g
83.8 ± 6.87
86.9 ± 7.42
Donepezil
Values are presented as the mean ± S.D (n = 3).
12

2.2.6 In vivo behavioral studies
Owing to considerable in vitro anti-ChE activity, propidium iodide displacement activity, and
predicted high CNS permeability, the potential compound 6g was further evaluated for in
vivo learning and memory through scopolamine-induced mouse models [18].
2.2.6.1 Acute oral toxicity study
The acute toxicity of compound 6g was determined as per the OECD 423 guidelines on
healthy Swiss albino mice (25-30 gm). All the mice were observed for mortality up to 14
days post-treatment period. The dose of 500 mg/kg, p.o. of compound 6g was well tolerated
and suggested a significant margin of safety [51].
2.2.6.2 Y-maze test in mice
Y-maze test is a standard neurobehavioral experiment to evaluate the hippocampal-dependent
short-term memory in the rodents [52]. The compound 6g treated groups showed a dose-
dependent increase in spontaneous alteration rate. Interestingly, compound 6g (10 mg/kg)
attenuated the dysfunctions of alteration rate with a non-significant difference to the
donepezil-treated group (5 mg/kg). Further, spontaneous alteration rate significantly declined
in the scopolamine-treated group suggested the induction of memory impairment (Fig. 3A).
The total numbers of arm entries showed a non-significant difference amongst all groups,
indicated that scopolamine-treated group did not affect the locomotive behavior in mice (Fig.
3B). The results suggested that compound 6g reversed the scopolamine-induced cognitive
dysfunctions in mice.
13

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B
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Fig. 3. Effect of compound 6g (2.5, 5 and 10 mg/kg, p.o.) in the y-maze test. (A)
Spontaneous alterations rate; (B) Number of arm entries; Bars display values as mean ± SD
(n = 6) and analyzed by one-way ANOVA followed by Tukey’s test; ap<0.05 as compared to
b
c
d
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f
compared to donepezil; ep<0.05 as compared to compound 6g at dose of 2.5 mg/kg; p<0.05
as compared to compound 6g at dose of 5 mg/kg.
2.2.6.3 Passive avoidance test
The transfer latency time (TLT) was calculated to evaluate the memory impairment in mice.
There was no significant alteration observed in TLT during acquisition test compared to
retention test of the scopolamine-treated group. Whereas, the compound 6g-treated group
significantly increased the TLT in a dose-dependent manner during the retention test. The
effect of compound 6g at the dose of 10 mg/kg was comparable with the donepezil-treated
group (5 mg/kg).
14

250
200
150
100
50
Retention Test
*
*
*
*
*
Acquisition test
0
l
l
)
)
S
S
S
S
S
S
S
S
S
S
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S
ro
(
ro
t
(
+
+
+
+
+
+
+
+
+
+
)
t
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e
)
)
/kg /kg /kg /k
)
)
)
)
/kg /kg /kg /k
)
n
n
Co
g
g
Co
min
min
g
g
la
la
mg
m
mg mg
mg
m
mg mg
Vehicle
Vehicle
0
.
5
5
0
1
0
.
5
.
5
(
0
1
po
po
.
(
5
(
2
5
(
2
(
(
(
(
g
g
Sco
Sco
g
g
g
g
zil
zil
d 6
d 6
e
e
d 6
d 6
d 6
d 6
un
p
p
un
e
e
un
po
un
po
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un
un
n
n
po
po
po
m
m
Do
Do
m
m
m
m
Co
Co
Co
Co
Co
Co
Fig. 4. Effect of compound 6g in the passive avoidance test. Bars display values as mean ±
SD (n = 6) and analyzed by one-way ANOVA followed by Tukey’s Test *p< 0.05 compared
to the respective groups of acquisition test.
2.2.7 Neurochemical estimation of AChE
The ex vivo study was conducted to estimate the AChE in mice brain homogenates using
Ellman colorimetric assay. Compound 6g (10 mg/kg)-treated group showed a low rate of
hydrolysis compared to scopolamine-treated group and also reflected that compound 6g
inhibited the AChE and penetrated the BBB.
2.2.8 Biochemical estimation of the oxidative stress factors
Oxidative stress is an important damaging factor for neurodegenerative disorders [53]. The
compound 6g was investigated for the neuroprotective activity to address the oxidative stress
in animals with cognitive dysfunctions. The malondialdehyde (MDA), glutathione (GSH),
–
superoxide dismutase (SOD), catalase enzyme, and nitrite (NO2 ) levels were quantitatively
analyzed using mice brain homogenates.
15

B
40
30
20
10
0
C
A
20
15
10
5
20
15
10
5
a
a
a, b
a,b
a,b
a,b
a
0
l
)
0
o
g
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)
g
r
k
/
t
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)
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mine
)
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a
mine
a
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mg
i
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k
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0
n
o
m
g
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1
(
1
(
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a
l
o
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S
C
o
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5
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g
6
p
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6
d
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6
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n
n
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mp
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D
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p
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C
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E
6
4
2
0
a
100
80
60
40
20
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D
15
10
5
a,b
a
a,b
a,b
a
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)
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r
o
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mine
a
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mine
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mg
0
mine
l
C
mg
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a
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mg
0
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p
1
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o
1
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o
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p
o
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1
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o
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g
6
g
6
c
S
g
S
6
d
n
d
d
n
n
u
o
u
u
o
o
mp
o
mp
o
mp
o
C
C
C
Fig. 5. The results of ex vivo and biochemical analysis (A) AChE activity; (B)
Malonaldehyde (MDA); (C) Reduced glutathione (GSH); (D) Superoxide dismutase (SOD);
(E) Nitrite concentration and; (F) Catalase levels on scopolamine-induced of the cholinergic
deficit and oxidative stress. The values presented as mean ± SD (n = 3) and analyzed by one-
a
b
way ANOVA, followed by Tukey's test. p<0.05 as compared to control; p<0.05 as
compared to scopolamine.
2.2.8.1 2-Thiobarbituric acid reactive substances (TBRAS)
The TBARS assay was used to analyze the malondialdehyde (MDA) levels in the brain
homogenates. The compound 6g (10 mg/kg) treated group exhibited a significant reduction in
malondialdehyde (MDA) concentration compared to scopolamine-treated group (Fig. 5B).
2.2.8.2 Reduced glutathione estimation
Ellman’s method was used for the estimation of reduced glutathione (GSH). Glutathione is a
natural antioxidant tri-peptide thiol agent (L-glutamyl-L-cysteinylglycine) neutralizes the
generation of ROS during the cellular oxidative stress. The results revealed that compound 6g
(10 mg/kg) treated group showed a significantly higher GSH level compared to scopolamine-
treated group (Fig. 5C).
16

2.2.8.3 Superoxide dismutase (SOD) assay
-
The superoxide dismutase estimation was based on the production of superoxide (O2 )
-
radical. SOD generally consumes the O2 radicals of the biological system. The SOD level
significantly raised by the compound 6g (10 mg/kg) compared to scopolamine-treated groups.
(Fig. 5D).
–
2.2.8.4 Nitrite (NO2 ) assay
The Griess reagent was used to analyze the nitrite ion. The results showed that compound 6g
(10 mg/kg) treated group significantly reduced the nitrite concentration in the brain compared
to scopolamine-treated groups (Fig. 5E).
2.2.8.5 Catalase assay
Catalase assay signifies the decomposition of hydrogen peroxide (H2O2) into water (H2O) by
the catalase. Interestingly, Compound 6g (10 mg/kg) significantly averted the loss of catalase
activity compared to scopolamine-treated groups (Fig. 5F).
2.3. Computational Studies
2.3.1 In silico molecular docking studies
Molecular docking studies were performed to explore the binding affinity and binding pattern
of compound 6g in the active sites of AChE (PDB Code: 1EVE) using Glide module of
Schrödinger Maestro 2018-1. Initially, the prepared enzyme grid and docking protocols were
validated by extracting and re-docking of co-crystallized ligand (donepezil). Furthermore, the
poses of re-docked and co-crystallized ligands were compared using superposition tool, and
the RMSD value was observed within the range of 3Å (Fig. 6).
17

Fig. 6. Superposition of re-docked (red) and co-crystallized (blue) poses of donepezil on
AChE (PDB Code: 1EVE)
The docked scores of the compound 6g and donepezil were found to be -14.1 and -13.6
kcal/mole, respectively. The ligand interaction diagram of donepezil and AChE showed their
strong binding affinity with the active site residues of AChE. The benzylpiperidine nucleus of
donepezil interacted via π–cation interactions at the anionic subsite (Trp84, Phe330) and PAS
(Tyr334), and observed to fit completely into the CAS of AChE. Some polar interactions of
compound 6g were also observed with His440 and Ser200, whereas the indanone nucleus of
donepezil interacted effectively to the Trp279 of PAS via π–π stacking (Suppl. Fig. S1). The
active compound 6g and donepezil were superimposed and showed a similar type of
interaction at the active pocket of AChE (Suppl. Fig. S2). Docking analysis of compound 6g
revealed that benzylpiperazine moiety interacted with His440 and Ser200 through polar
interactions in the CAS. Interestingly, the most significant interaction found with the Trp84
of anionic subsite of AChE where the compound 6g exhibited the π-π stacking and cation-π
interaction with the Trp84. Also, the quaternary nitrogen showed cation-π interaction with the
18

Phe330, Phe331, and Phe334 (Fig. 7A and 7B). The biaryl nucleus of compound 6g further
exhibited π-π stacking interactions with amino acids Trp279 at the PAS of AChE (Table 4).
Fig. 7. The docking of compound 6g on AChE enzyme. (A) 3D interaction image showing
compound 6g in the ligand binding surface (yellow color) interacting with active site residues
of AChE; (B) 2D image showing active site interactions.
Table 4. In silico docking interaction analysis of compound 6g and donepezil on AChE.
Interacting residues#
Acyl
binding
pocket
Other
interacting
residues
Comp.
6g
Anionic
subsite
Oxyanion
hole
CAS
PAS
Ser200a,
His440a
Trp279b,
Tyr334f,
Tyr70c,
Tyr121c,
Asp72d
Glu199d, Phe288c, Gly117g,
Ser81a, Tyr130c,
Trp84b,f, Phe290c, Gly118g Leu282c, Ser286a,
Phe330f Phe331f
Ile287c, Arg289d,
Gly441g, Ile444c
Ser200a,
His440a
Trp279b,
Tyr334f,
Tyr70c,
Tyr121c,
Asp72d
Glu199d, Phe288c, Gly117g,
Tyr130c,
Donepezil
Trp84b,f, Phe290c, Gly118g Leu282c, Ser286a,
Phe330f Phe331c
Ile287c, Arg289d,
Gly441g, Ile444c
#All the interacting residues are within the 4Å distance with the ligand; polar interaction; π-π stacking
interaction; chydrophobic interaction; dcharged interaction; eH-bonding interaction; fπ-cation interaction;
gglycine interaction.
a
b
19

2.3.2. Molecular dynamics (MD) simulations
The stability of docked binding pose of the compound 6g-AChE complex was analyzed by
molecular dynamics simulation analysis using Desmond (DE Shaw Research). The molecular
dynamics simulations analyzed the stability of the docked complex in a flexible protein
environment in the presence of virtual water molecules. The root means square deviations
(RMSD) values were calculated for the docked complexes and compared with reference
protein backbone structures which were within the range (Fig. 8). Root mean square
fluctuations (RMSF) values also indicated stable trajectories of the ligand and protein
residues throughout the simulation analysis (Suppl. Fig. S3).
Fig. 8 RMSD graph of the compound 6g-AChE complex for the period of 20 ns dynamics
simulations.
The detailed interaction analysis was carried out using the protein-ligand histogram (Suppl.
Fig. S4), graphical (Fig. 9), and time-line representation (Fig. 10). The percentage of
interactions of the protein residues with compound 6g were identified using protein-ligand
contacts. The PAS region of Tyr334 formed π-cation interaction with compound 6g for 52%
time-scale. The nitrogen atom (N-4) of the piperazine ring of compound 6g displayed a
hydrogen bonding with the Asp72 residue. The phenyl ring of benzylpiperazine nucleus
20

formed π-π stacking interaction with the Trp84 at anionic subsite on 85% time-scale. The
piperazine -NH also showed π-cation interactions with the Trp84 and Phe330 at 87% and
97% time-scales, respectively.
Fig. 9. Graphical representation of compound 6g showing interacting residues for the
simulations of 20 nsec
In a nutshell, the biaryl structure of compound 6g showed the hydrophobic interaction with
Trp279, the phenyl group of benzylpiperazine moiety and the nitrogen atom of piperazine
exhibited π-π stacking interaction (85% time-scale) and cation-π interaction (87% time-scale)
with Trp84 of anionic subsite of the AChE, respectively. The quaternary nitrogen of
piperazine also depicted π-cation interaction with Tyr334 (52% time-scale) and Phe330
(97%).
21

Fig. 10. A timeline representation of the compound 6g-AChE
2.3.3. Drug-likeliness by QikProp module
Compound 6g was evaluated for drug-likeliness characteristics using the QikProp module of
Schrödinger, and the result was found comparable with standard donepezil Table 5. The
predicted QPlogKhsa values affirm their strong binding with plasma protein. The outcome of
Lipinski’s rule of five (mol_MW < 500, QPlog Po/w < 5, donorHB 0-6.0, accptHB 2.0-20),
along with the other predicted parameters (SASA 300-1000, QPlogBB -3.0 to 1.2,
QPlogPo/w -2.0 to 6.5) reflected that the compound 6g elicited ‘‘drug-like” characteristics.
Table 5. QikProp analysis of compound 6g
Donor Acceptor
HBb HBc
Qplog
Qpp
Qplog
Compound Mol_MWa
SASAd QplogBBe
Po/wf MDCKg Khsah
Donepezil
393.61
465.59
1
0
7.1
759.67 0.14
827.23 0.27
4.25
4.99
595.28
113.28
0.69
0.76
7
6g
aMol_MW- Molecular weight of the molecule (130 to 725).
bDonor HB- number of hydrogen bonds (0.0 to 6.0).
cAcceptor HB- number of hydrogen bonds (2.0 to 20.0).
dSASA- total solvent accessible surface area (SASA) in square angstroms using a probe with a 1.4 Å radius
(300 to1000).
eQPlogBB- predicted brain/blood partition coefficient (-3.0 to 1.2).
fQPlogPo/w- this gives the predicted octanol/water partition coefficient (-2.0 to 6.5).
gQPPMDCK- predicted MDCK cell permeability in nm/s using the Affix scale (< 25 is considered poor and
>500 is considered excellent).
hQPLog Khsa- prediction of binding to human serum albumin (-1.5 to 1.5).
22

3. CONCLUSION
A series of novel hybrids were designed and synthesized by combining the piperazine
nucleus with vicinal biphenyl-3-oxo-1,2,4-triazine through varied alkyl linkers. Amongst all
the synthesized compounds, 6a-g with propyl (n=3) linker displayed the better in vitro AChE
inhibitory potential. The benzylpiperazine tail containing compound 6g revealed the most
significant inhibitory potency (AChE, IC50 = 0.2 ± 0.01 μM) as standard donepezil (AChE,
IC50= 0.1 ± 0.002 μM) with non-competitive type of inhibition. Therefore, the AChE
inhibitory activity was more accountable with alkyl linkers and piperazine substituents.
Further, PI displacement assay showed significant displacement of propidium iodide with
compound 6g (22.22 ± 1.11 %) from PAS of AChE along with high BBB permeability.
Additionally, compound 6g exhibited the IC50; 83.8 ± 6.87 µM against the neuroblastoma cell
line as determined by MTT assay. In vivo behavioral studies in mice models revealed that
compound 6g (10 mg/kg) significantly reversed the scopolamine-induced cognitive
impairment comparable to that of standard donepezil (5 mg/kg). The ex vivo and biochemical
analysis of oxidative stress biomarkers indicated significant AChE inhibition and reversed the
scopolamine-induced oxidative stress by compound 6g. Furthermore, the molecular docking
and dynamics simulations studies substantiated our findings that compound 6g consensually
interacted with PAS and CAS residues of AChE. Overall, the novel derivatives of biphenyl-
3-oxo-1,2,4-triazine linked piperazine analogs as a propitious moiety to have their valuable
effect in the treatment of dementia.
4. EXPERIMENTAL
4.1. Chemistry
4.1.1 Instrumentation and chemicals
1
The H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker
Avance FT-NMR spectrophotometer in deuterated solvents (DMSO-d6 and CDCl3) with
23

tetramethylsilane (TMS) as an internal reference. The spectra were measured in chemical
shift (δ, ppm) and coupling constant (J, Hz). Elemental analyses (C, H, N) were performed
using EXETER CE-440 elemental analyzer. All the chemicals and solvents were bought from
Sigma-Aldrich, TCI chemicals, Avra synthesis, and were used without further purification.
Progress of chemical reactions was monitored on 60F254 sheets, precoated with silica gel,
thickness- 0.25mm, Merck, Germany. Product quantities were unoptimized which was
presented in the experimental section.
4.1.2 Synthesis of intermediate (4)
Compound 4 was synthesized according to previously reported procedures [30].
4.1.3. General procedure of synthesis of intermediates (5, 6, 7, and 8)
The intermediate 4 (10 mmol) was dissolved in 20 ml of dry N,N-Dimethylformamide
(DMF) in round bottom flask under the inert gas condition and stirred at 10-15 ºC on ice-cold
bath for 5 min. Accurate quantity (14.10 mmol) of potassium hydroxide (KOH) was added,
and precipitation of compound 4 in the form of potassium salt was observed after the 30 min.
The reaction mixture was kept at room temperature and bromochloro alkane (13.08 mmol)
was added very slowly. After completion of reaction, DMF was evaporated under vacuo to
afford the crude product, which was dissolved in ethyl acetate, washed with water (2×100ml)
and passed through the anhydrous sodium sulfate (Na2SO4) to remove the residual water. The
solvent was evaporated to obtain a crude mixture, which was purified using the silica gel
(100-200 mesh size) as the stationary phase and dichloromethane: methanol (4:1) as mobile
phase through glass column chromatography to afford intermediates (5, 6, 7, and 8).
5,6-diphenyl-3-oxo-2-(2-chloroethyl)-(2H)-1,2,4-triazine (5)
Yield: 82%.1H NMR (500 MHz, δH, CDCl3): 7.48-7.32 (m, 10H), 4.53 (t, 2H, J = 7.0 Hz),
3.74 (t, 2H, J = 6.3 Hz). 13C NMR (125 MHz, δC, CDCl3): 166.4, 153.4, 142.2, 134.1, 133.1,
24

131.2, 129.8, 129.4, 128.7, 128.5, 128.3, 49.8, 41.0. Anal. C17H14ClN3O: C, 65.49; H, 4.53;
N, 13.48; Found: C, 65.39; H, 4.51; N, 13.50.
5,6-diphenyl-3-oxo-2-(3-chloropropyl)-(2H)-1,2,4-triazine (6)
Yield: 81%.1H NMR (500 MHz, δH, CDCl3): 7.49-7.30 (m, 10H), 4.44 (t, 2H, J = 7.0 Hz),
13
3.69 (t, 2H, J = 6.3 Hz), 2.46 (p, 2H, J = 6.5 Hz). C NMR (125 MHz, δC, CDCl3): 166.5,
153.2, 142.6, 134.9, 133.5, 131.5, 129.8, 129.3, 128.8, 128.5, 128.2, 50.9, 41.8, 30.8. Anal.
C18H16ClN3O: C, 66.36; H, 4.95; N, 12.90; Found: C, 66.39; H, 4.93; N, 12.92.
5,6-diphenyl-3-oxo-2-(4-chlorobutyl)-(2H)-1,2,4-triazine (7)
Yield: 86%. 1H NMR (500 MHz, δH, CDCl3): 7.51- 7.28 (m, 10H), 4.32 (t, 2H, J = 7.0 Hz),
13
3.64 (t, 2H, J = 6.5 Hz), 3.50 (t, 1H, J = 6.8 Hz), 2.17-2.11 (m, 2H), 2.05-1.99 (m, 1H). C
NMR (125 MHz, δC, CDCl3): 166.3, 153.3, 142.5, 134.9, 133.0, 131.4, 129.9, 129.3, 128.8,
128.5, 128.2, 52.3, 44.2, 29.5, 25.6. Anal. C19H18ClN3O: C, 67.16; H, 5.34; N, 12.37; Found:
C, 67.20; H, 5.31; N, 12.39.
5,6-diphenyl-3-oxo-2-(5-chloropentyl)-(2H)-1,2,4-triazine (8)
Yield: 84%. 1H NMR (500 MHz, δH, CDCl3): 7.51- 7.29 (m, 10H), 4.29 (t, 2H, J = 7.0 Hz),
3.58 (t, 1H, J = 6.0, 6.5 Hz), 3.45 (t, 1H, J = 6.3 Hz), 1.99 (q, 2H, J = 7.5 Hz), 1.88 (t, 1H, J =
13
7.0), 1.71-1.61 (m, 3H). C NMR (125 MHz, δC, CDCl3): 166.2, 153.3, 142.4, 135, 134.1,
131.4, 129.9, 129.3, 128.8, 128.5, 128.2, 53.0, 44.7, 32.0, 27.4, 23.9. Anal. C20H20ClN3O: C,
67.89; H, 5.70; N, 11.89; Found: C, 67.91; H, 5.67; N, 11.92.
4.1.4 General procedure of synthesis of compounds (5a-g, 6a-g, 7a-g, and 8a-g)
5.13 mmol of intermediates (5, 6, 7, and 8) and various piperazine substituents (6.64 mmol),
potassium iodide (7.51 mmol) and anhydrous potassium carbonate (7.51 mmol) were
suspended into the dry acetonitrile (20 ml) and refluxed for 13 h. The solvent was removed
under reduced pressure to get the crude product. The crude product was dissolved in ethyl
25

acetate (2×100ml) and passed through anhydrous sodium sulfate (Na2SO4). The organic
phase was removed from the crude mixture and further purified by column chromatography
on 100-200 mesh size silica gel using dichloromethane: methanol (4:1) as the mobile phase to
afford pure compounds (5a-g, 6a-g 7a-g, and 8a-g).
5,6-diphenyl-3-oxo-2-(2-(4-phenylpiperazin-1-yl)ethyl)-1,2,4-triazine (5a)
Yield: 71%, 1H NMR (500 MHz, δH, CDCl3): 7.43-7.29 (m, 12H), 6.96-6.84 (m, 3H), 4.31 (t,
13
2H, J = 7.0 Hz), 3.35 (t, 2H, J = 5.0 Hz), 2.89-2.78 (m, 4H), 2.68-2.61 (m, 4H); C NMR
(125 MHz, δC, CDCl3): 166.1, 153.1, 143.0, 133.9, 133.1, 131.3, 129.1, 129.2, 129.1, 128.5,
128.1, 120.1, 116.2, 54.2, 52.3, 51.5, 48.3. Anal. C27H27N5O: C, 74.12; H, 6.22; N, 16.01;
Found: C, 74.15; H, 6.20; N, 16.04.
5,6-diphenyl-3-oxo-2-(2-(4-(p-nitrophenyl)piperazin-1-yl)ethyl)-1,2,4-triazine (5b)
Yield: 77%. 1H NMR (500 MHz, δH, DMSO-d6): 8.05-8.01 (m, 4H), 7.49-7.24 (m, 6H), 7.11-
6.92 (m, 4H), 4.39-4.21 (m, 2H), 3.43-3.36 (m, 2H), 2.87-2.77 (m, 4H), 2.67-2.58 (m, 4H);
13C NMR (125 MHz, δC, DMSO-d6): 166.3, 155.2, 149.9, 142.4, 139.7, 137.6, 137.1, 135.3,
129.2, 128.4, 128.1, 127.9, 127.8, 126.1, 113.5, 112.5, 112.7, 57.1, 52.4, 48.4, 46.1, 45.3
Anal. C27H26N6O3: C, 67.21; H, 5.43; N, 17.42; Found: C, 67.19; H, 5.45; N, 17.40.
5,6-diphenyl-3-oxo-2-(2-(4-(p-fluorophenyl)piperazin-1-yl)ethyl)-1,2,4-triazine (5c)
1
Yield: 69%. H NMR (500 MHz, δH, CDCl3): 7.49-7.29 (m, 10H), 6.95-6.91 (m, 2H), 6.88-
6.81 (m, 2H), 4.35 (t, 2H, J = 7.0 Hz), 3.24-3.11 (m, 4H), 2.81-2.54 (m, 6H). 13C NMR (125
MHz, δC, CDCl3): 166.3, 157.9 (JCF = 241 Hz), 153.2, 147.2, 142.1, 135.1, 134.3, 131.2,
129.8, 129.1, 128.7, 128.4, 128.1, 117.6 (JCF = 23 Hz), 115.3 (JCF = 8.8 Hz), 55.3, 53.2, 52.1,
49.1. Anal. C27H26FN5O: C, 71.19; H, 5.75; N, 15.37; Found: C, 71.21; H, 5.77; N, 15.35.
5,6-diphenyl-3-oxo-2-(2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)-1,2,4-triazine (5d)
26

1
Yield: 74%. H NMR (500 MHz, δH, CDCl3): 8.31 (m, 1H), 7.50- 7.26 (m, 11H), 6.72-6.62
(m, 2H), 4.33 (t, 2H, J = 7.3 Hz), 3.41-3.31 (m, 4H), 2.86-2.41 (m, 6H). 13C NMR (125 MHz,
δC, CDCl3): 166.1, 159.2, 153.2, 147.9, 142.2, 137.4, 135.2, 134.3, 131.4, 129.7, 129.1,
128.8, 128.4, 128.1, 113.1, 107.1, 58.3, 53.0, 45.1, 28.2. Anal. C26H26N6O: C, 71.21; H, 5.98;
N, 19.16; Found: 71.24; H, 5.96; N, 19.18.
5,6-diphenyl-3-oxo-2-(2-(4-(p-methoxyphenyl)piperazin-1-yl)ethyl)-1,2,4-triazine (5e)
1
Yield: 67%. H NMR (500 MHz, δH, DMSO-d6): 7.51-7.28 (m, 10H), 6.85-6.72 (m, 4H),
4.32 (t, 2H, J = 7.3 Hz), 3.71 (s, 3H, -OCH3), 3.56-3.40 (m, 2H), 2.87-2.71 (m, 4H), 2.65-
13
2.51 (m, 4H). C NMR (125 MHz, δC, DMSO-d6): 166.4, 153.3, 153.1, 144.6, 142.2, 135.8,
134.1, 130.3, 129.7, 129.3, 129.3, 128.5, 128.4, 117.3, 114.7, 57.2, 55.4, 53.1, 52.2, 49.6.
Anal. C28H29N5O2: C, 71.93; H, 6.25; N, 14.98; Found: C, 71.95; H, 6.21a; N, 15.00.
5,6-diphenyl-3-oxo-2-(2-(4-benzhydrylpiperazin-1-yl)ethyl)-1,2,4-triazine (5f)
Yield: 73%. 1H NMR (500 MHz, δH, CDCl3): 7.56- 7.15 (m, 20H), 4.29 (t, 2H, J = 6.8 Hz),
4.11 (s, 1H), 3.52-3.41 (m, 4H), 2.84- 2.73 (m, 4H), 2.61-2.54 (m, 2H). 13C NMR (125 MHz,
δC, CDCl3): 166.1, 153.1, 143.0, 142.3, 134.9, 134.1, 131.2, 130.0, 130.2, 128.7, 128.3,
128.0, 126.5, 76.4, 55.3, 53.2, 52.1. Anal. C34H33N5O: C, 77.39; H, 6.30; N, 13.27; Found: C,
77.45; H, 6.27; N, 13.31.
5,6-diphenyl-3-oxo-2-(2-(4-benzylpiperazin-1-yl)ethyl)-1,2,4-triazine (5g)
Yield: 73%. 1H NMR (500 MHz, δH, CDCl3): 7.50- 7.12 (m, 15H), 4.33 (t, 2H, J = 7.0 Hz),
13
3.45-3.38 (m, 6H), 2.87- 2.72 (m, 4H), 2.67-2.56 (m, 2H). C NMR (125 MHz, δC, CDCl3):
166.2, 153.4, 143.1, 142.31 134.9, 134.3, 131.2, 130.5, 130.1, 128.3, 128.1, 128.0, 126.4,
62.9, 55.5, 53.4, 52.5, 48.9. Anal. C28H29N5O: C, 74.47; H, 6.47; N, 15.51; Found: C, 74.53;
H, 6.45; N, 15.48.
5,6-diphenyl-3-oxo-2-(3-(4-phenylpiperazin-1-yl)propyl)-1,2,4-triazine (6a)
27

Yield: 70%, 1H NMR (500 MHz, δH, CDCl3): 7.43-7.24 (m, 12H), 6.91-6.86 (m, 3H), 4.33 (t,
2H, J = 7.0 Hz), 3.25 (t, 4H, J = 5.0 Hz), 2.88 (d, 4H, J = 5.0 Hz), 2.79 (t, 2H, J = 7.3 Hz),
2.27 (t, 2H, J = 7.3 Hz). 13C NMR (125 MHz, δC, CDCl3): 166.5, 153.2, 142.7, 134.9, 134.0,
131.4, 129.8, 129.3, 129.1, 128.5, 128.2, 120.3, 116.4, 54.8, 52.2, 51.4, 48.2, 29.3. Anal.
C18H16ClN3O: C, 74.47; H, 6.47; N, 15.51; Found: C, 74.51; H, 6.43; N, 15.50.
5,6-diphenyl-3-oxo-2-(3-(4-(p-nitrophenyl)piperazin-1-yl)propyl)-1,2,4-triazine (6b)
1
Yield: 74%. H NMR (500 MHz, δH, CDCl3): 8.11-8.09 (m, 1H), 7.47-7.15 (m, 12H), 6.79-
6.78 (m, 1H), 4.49-4.37 (m, 3H), 3.38 (s, 2H), 2.72-2.60 (m, 5H), 2.32-2.02 (m, 3H), 1.63-
1.61 (m, 1H); 13C NMR (125 MHz, δC, CDCl3): 166.6, 154.8, 149.2, 142.8, 139.2, 137.6,
137.1, 135.3, 129.8, 128.8, 128.5, 127.9, 127.8, 125.9, 114.0, 112.6, 55.4, 52.4, 51.0, 46.1,
29.6; Anal. C28H28N6O3: C, 67.73; H, 5.68; N, 16.92; Found: C, 67.76; H, 5.65; N, 16.96.
5,6-diphenyl-3-oxo-2-(3-(4-(p-fluorophenyl)piperazin-1-yl)propyl)-1,2,4-triazine (6c)
Yield: 71%. 1H NMR (500 MHz, δH, CDCl3): 7.41- 7.26 (m, 10H), 6.95-6.92 (m, 2H), 6.84-
6.82 (m, 2H), 4.37 (t, 2H, J = 7.0 Hz), 3.06 (t, 4H, J = 4.5 Hz), 2.61-2.57 (m, 6H), 2.30-2.17
(m, 6H, J = 6.8 Hz). 13C NMR (125 MHz, δC, CDCl3): 166.2, 156.1, 153.4, 147.9, 142.2,
135.0, 134.1, 131.3, 129.8, 129.2, 128.8, 128.5, 128.1, 117.8 (JCF-O = 30.0Hz), 115.5 (JCF-m
=
88.5 Hz), 55.6, 53.1, 52.0, 50.2, 25.2; Anal. C28H28FN5O: C, 71.62; H, 6.01; N, 14.91; Found:
C, 71.65; H, 6.03; N, 14.94.
5,6-diphenyl-3-oxo-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-1,2,4-triazine (6d)
Yield: 75%. 1H NMR (500 MHz, δH, CDCl3): 8.17 (s, 1H), 7.47- 7.25 (m, 11H), 6.62-6.60 (d,
2H, J = 7.0 Hz), 4.36 (t, 2H, J = 6.5 Hz), 3.50 (s, 4H), 2.57 (s, 6H), 2.18 (t, 2H, J = 6.8 Hz).
13C NMR (125 MHz, δC, CDCl3): 166.2, 159.4, 153.4, 147.8, 142.3, 137.4, 135.0, 134.1,
131.3, 129.8, 129.2, 128.8, 128.5, 128.1, 113.3, 107.1, 55.6, 52.9, 51.9, 45.2, 29.6; Anal.
C27H28N6O: C, 71.66; H, 6.24; N, 18.57; Found: C, 71.70; H, 6.25; N, 18.59.
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5,6-diphenyl-3-oxo-2-(3-(4-(p-methoxyphenyl)piperazin-1-yl)propyl)-1,2,4-triazine (6e)
Yield: 72%. 1H NMR (500 MHz, δH, CDCl3): 7.47-7.24 (m, 10H), 6.85-6.79 (m, 4H), 4.34 (t,
2H, J = 6.5 Hz), 3.73 (s, 3H, -OCH3), 3.03 (brs, 4H), 2.60-2.56 (m, 6H), 2.16 (t, 2H, J = 6.8
Hz). 13C NMR (125 MHz, δC, CDCl3): 166.2, 153.7, 153.4, 145.6, 142.3, 135.0, 134.1, 131.3,
129.8, 129.2, 128.9, 128.8, 128.5, 128.1, 118.2, 114.3, 55.6, 55.5, 53.2, 52.0, 50.6, 25.2;
Anal. C29H31N5O2: C, 72.33; H, 6.49; N, 14.54; Found: C, 72.35; H, 6.46; N, 14.53.
5,6-diphenyl-3-oxo-2-(3-(4-benzhydrylpiperazin-1-yl)propyl)-1,2,4-triazine (6f)
Yield: 72%. 1H NMR (500 MHz, δH, CDCl3): 7.53- 7.16 (m, 20H), 4.33 (t, 2H, J = 7.0 Hz),
4.13 (s, 1H), 2.55-2.38 (m, 9H), 2.13 (q, 2H, J = 6.9 Hz), 1.68 (s, 1H). 13C NMR (125 MHz,
δC, CDCl3): 166.0, 153.4, 142.8, 142.1, 135.1, 134.2, 131.3, 129.9, 129.1, 128.8, 128.4,
128.1, 127.9, 126.8, 76.3, 55.6, 53.3, 52.0, 25.3. Anal. C35H35N5O: C, 77.60; H, 6.51; N,
12.93; Found: C, 77.63; H, 6.53; N, 12.90.
5,6-diphenyl-3-oxo-2-(3-(4-benzylpiperazin-1-yl)propyl)-1,2,4-triazine (6g)
Yield: 72%. 1H NMR (500 MHz, δH, CDCl3): 7.50- 7.22 (m, 15H), 4.31 (brs, 2H), 3.44 (brs,
2H), 2.51-2.45 (m, 8H), 2.12-2.02 (m, 2H), 1.42-1.37 (m, 2H). 13C NMR (125 MHz, δC,
CDCl3): 166.1, 153.4, 142.2, 139.2, 137.8, 135.0, 134.1, 131.3, 129.9, 129.2, 128.8, 128.5,
127.0, 114.1, 62.9, 55.6, 52.9, 52.0, 29.7. Anal. C35H35N5O: C, 77.60; H, 6.51; N, 12.93;
Found: C, 77.63; H, 6.53; N, 12.90.
5,6-diphenyl-3-oxo-2-(4-(4-phenylpiperazin-1-yl)butyl)-1,2,4-triazine (7a)
Yield: 60%. 1H NMR (500 MHz, δH, CDCl3): 7.51- 7.26 (m, 12H), 6.94 (d, 2H, J = 7.5 Hz),
6.94 (t, 1H, J = 7.0 Hz), 4.32 (t, 2H, J = 6.8 Hz), 3.21 (m, 4H), 2.62 (m, 4H), 2.49 (t, 2H, J =
7.5 Hz), 2.02 (t, 2H, J = 6.8 Hz), 1.28 (m, 2H). 13C NMR (100 MHz, δC, CDCl3): 166.2,
153.3, 151.3, 142.4, 135.0, 134.1, 131.4, 129.9, 129.2, 129.1, 128.9, 128.5, 128.2, 58.0, 53.4,
29