Development of cyanopyridine-triazine hybrids as lead multitarget anti-Alzheimer agents

Article abstract of DOI:10.1016/j.bmc.2016.04.041

A series of new cyanopyridine-triazine hybrids were designed, synthesized and screened as multitargeted anti-Alzheimer's agents. These molecules were designed while using computational techniques and were synthesized via a feasible concurrent synthetic ro

Full text of DOI:10.1016/j.bmc.2016.04.041

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of cyanopyridine–triazine hybrids as lead multitarget
anti-Alzheimer agents
Mudasir Maqbool a, Apra Manral b, Ehtesham Jameel c, Jitendra Kumar a, Vikas Saini b,
Ashutosh Shandilya d, Manisha Tiwari b, , Nasimul Hoda a, , B. Jayaram d,e,f
⇑
⇑
a Department of Chemistry, Jamia Millia Islamia (Central University), Jamia Nagar, New Delhi 110025, India
b Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi 110007, India
c Department of Chemistry, B. R. Ambedkar Bihar University, Muzaffarpur 842001, Bihar, India
d Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
e Kusuma School of Biological Sciences, IIT Delhi, New Delhi 110016, India
f Supercomputing Facility for Bioinformatics & Computational Biology, IIT Delhi, New Delhi 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new cyanopyridine–triazine hybrids were designed, synthesized and screened as multitar-
geted anti-Alzheimer’s agents. These molecules were designed while using computational techniques
and were synthesized via a feasible concurrent synthetic route. Inhibition potencies of synthetic com-
pounds 4a–4h against cholinesterases, Ab1–42 disaggregation, oxidative stress, cytotoxicity, and neuro-
protection against Ab1–42-induced toxicity of the synthesized compounds were evaluated. Compounds
4d and 4h showed promising inhibitory activity on acetylcholinesterase (AChE) with IC50 values 0.059
Received 6 March 2016
Revised 20 April 2016
Accepted 21 April 2016
Available online xxxx
Keywords:
and 0.080 lM, respectively, along with good inhibition selectivity against AChE over butyryl-
Alzheimer’s disease
Acetylcholinesterase
Triazine
Molecular docking
Butyrylcholinesterase
Ab1–42 disaggregation
cholinesterase (BuChE). Molecular modelling studies revealed that these compounds interacted simulta-
neously with the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The mixed type
inhibition of compound 4d further confirmed their dual binding nature in kinetic studies. Furthermore,
the results from neuroprotection studies of most potent compounds 4d and 4h indicate that these deriva-
tives can reduce neuronal death induced by H2O2-mediated oxidative stress and Ab1–42 induced cytotox-
icity. In addition, in silico analysis of absorption, distribution, metabolism and excretion (ADME) profile of
best compounds 4d and 4h revealed that they have drug like properties. Overall, these cyanopyridine–tri-
azine hybrids can be considered as a candidate with potential impact for further pharmacological devel-
opment in Alzheimer’s therapy.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
neuronal degeneration, and ultimately death of the patient. It is a
chronic disorder that cannot be prevented, cured or slowed hence
Alzheimer’s disease (AD) is an age-related neurodegenerative
disorder, characterized by progressive cognitive impairment,
dementia and neuropsychiatric symptoms.1,2 It starts with minus-
cule changes of hippocampal synaptic adequacy, simultaneous
is the third leading cause of death after cardiovascular diseases and
cancer worldwide.
The complete etiology of the disease is still not clear. However,
the synaptic dysfunction is believed to be caused due to extracel-
lular amyloid-b (Ab) aggregation,3 produced by the proteolytic
cleavage of a transmembrane protein amyloid precursor protein
Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; eelAChE, electric
eel acetylcholinesterase; BuCh, butyrylcholine; BuChE, butyrylcholinesterase;
eqBuChE, equine serum butyrylcholinesterase; ChE, cholinesterase; AD, Alzheimer’s
disease; Ab, amyloid beta; AChEI, acetylcholinesterase inhibitor; BChEI, butyryl-
cholinesterase inhibitor; PAS, peripheral anionic site; CAS, catalytic active site;
TcAChE, Torpedo Californica acetylcholinesterase; ROS, reactive oxygen species;
SAR, structure activity relationship; SEM, scanning electron microscope; TEM,
transmission electron microscopy; ThT assay, thioflavin T assay; equiv, equivalent.
(APP) by the b- and
c-secretases. Intracellular tangle formation
due to hyperphosphorylation of microtubule associated protein
tau4 which in turn is governed by the overexpression of glycogen
synthase kinase beta (GSK-3b),5 also accounts for the synaptic dys-
function. These two protein misfoldings are considered to be the
main hallmarks of this ailment. Another popular physiological tar-
get in AD is cholinergic pathway that tides symptoms of dementia
and learning difficulties to the significant decrease of acetylcholine
⇑
Corresponding authors.
E-mail addresses: mtiwari07@gmail.com (M. Tiwari), nhoda@jmi.ac.in (N. Hoda).
http://dx.doi.org/10.1016/j.bmc.2016.04.041
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
level. AChE, a key member of serine hydrolase family, catalyses the
breakdown of acetylcholine (ACh) an important neurotransmitter.
Its catalytic triad Ser-His-Glu is located at the bottom of a deep and
narrow gorge mainly covered with the aromatic residues which are
The monosubstituted triazines were synthesized at below 0 °C, dis-
ubstituted at room temperature and the targeted compounds were
obtained at higher temperature, i.e., 110 °C. All the newly synthe-
sized compounds were purified by column chromatography and
were characterized by different spectroscopic techniques—1H
NMR, 13C NMR, ESI MS and elemental analysis.
considered to be responsible for cation–p interaction with the sub-
strate Ach.6,7 Peripheral anionic site (PAS) of AChE constitutes Tyr
121, Tyr70 and Trp279 residues, present at the opening of the
gorge and guides the substrate approaching the active site. In addi-
tion, Ab binds to the peripheral anionic site (PAS) of the AChE
which enhances the rate of Ab fibril formation.8,9 Therefore, due
to their non-classical roles, cholinesterase inhibitors could also
be useful as potential disease-modifying drugs.10,11
Compound
4a
R1
R2
NH2
H2N
CF3
CF3
On the other hand, evasion of Ab stockpile is considered to be
the potentially influential step approaching the treatment of
AD.12 In addition to this, oxidative stress occurs in the early break-
through of AD.13 Brain aging is directly correlated to the AD devel-
opment which in turn is believed to be due to a gradient between
production of reactive oxygen species (ROS) and antioxidant
defences.14 Several evidences suggest that oxidative stress has a
prominent role in the AD buildup. Therefore, therapeutics targeting
clearance or prevention of the free radicals in the brain would be
beneficial against AD.
Till date drugs like tacrine, donepezil, rivastigmine, galan-
thamine, caproctamine and memantine targeting cholinesterases,
cholesterol esterase, and lipase are used to treat AD but these drugs
only curtail the symptoms and do not produce a complete cure.
These drugs are also recognized to have many side effects includ-
ing hepatotoxicity, gastrointestinal disturbance, dizziness, diar-
rhoea, vomiting and nausea.15,16
1,3,5-Triazine scaffold has been serving medicinal chemists for
a long time for the development of antifungal,17 anticancer,18 anti-
malarial,19 antiviral,20 antibacterial agents.21 The construction of
triazine and its derivatives could be easily achieved by routine
chemical reactions.
Keeping the above aspects into consideration, here we have
designed and synthesized a series of eight new cyanopyridine–tri-
azine hybrids as persuasive multifunctional agents for the treat-
ment of AD. All the synthesized compounds were assessed for
their inhibitory activity towards cholinesterases and their Ab
anti-aggregating activity. Further, the mechanism of AChE inhibi-
tion was investigated by kinetic studies. Selected cholinesterase
inhibitors were subsequently examined for neuroprotective prop-
erties in SH-SY5Y neuronal cells. In order to obtain a better under-
standing of possible interactions with biological targets, molecular
docking studies were also performed.
NH2
NH2
NH2
NH2
NH2
4b
4c
4d
4e
4f
CH3
NH2
F
CF3
CF3
F
NH2
Cl
F
NH2
CF3
F
F
NH2
NH2
O
NH2
NH2
4g
4h
Cl
CH3
NH2
F
NH2
CF3
O
2. Results and discussion
2.1. Chemistry
2.2. Design of multifunctional ligands
The target molecules were synthesized via multiple steps as
depicted in Schemes 1 and 2. The mono and di-substituted triazine
compounds (2a–2h and 3a–3h) were synthesized according to the
reported literature procedure.22 2,4,6-Trichloro-1,3,5-triazine was
first treated with 3-(trifluoromethyl)aniline (2a–2e, 2h), 2-fluo-
roaniline (2f), 3-chloro-4-fluoroaniline (2g) at ꢀ10 °C in THF and
K2CO3. The mono-substituted triazine compounds were further
treated with different amines to obtain disubstituted compounds
(3a–3h). On the other hand, 2-(piperazin-1-yl)nicotinonitrile (6)
was synthesized as shown in Scheme 2.23 Finally, the di-substi-
tuted triazine compounds (3a–3h) were treated with compound
(6) in presence of K2CO3 and 1,4-dioxane at 110 °C to obtain the
targeted compounds as tri-substituted triazines (4a–4h). The reac-
tions involved in the synthesis of the target compounds were tem-
perature dependent nucleophilic aromatic substitution reactions.
In order to develop a persuasive drug candidate for multi-
faceted AD, we undertook structure based drug design approach.
Triazine was selected as preferred scaffold, because its roughly pla-
nar structure was expected to intercalate between beta-amyloid
sheets and was expected to enhance the beta-amyloid disaggrega-
tion. It was also expected to engage efficiently with the active site
residues of AChE via weak non-covalent interactions like H-bond-
ing, p–p stacking interaction, and alkyl–p-interaction. In addition,
the molecules substituted with p-anisidine are known to have neu-
roprotective and radical scavenging capability.24 Inclusion of
piperazine was a natural choice to connect two electrophilic cen-
tres. Nitrogen of piperazine was expected to be in protonated state
and hence may engage in
p–cation interactions with the residues
of aromatic gorge. Pardock module of Sanjeevini (SCFBIO, IITD)25,26
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
R2
N
R1
R2
N
R1
Cl
N
Cl
Cl
N
R1
(i)
(ii)
(iii)
6
+
N
N
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
3
1
2
N
4(a-h)
Scheme 1. Reagents and conditions: (i) aromatic amines, tetrahydrofuran (THF), ꢀ110 °C, 1–4 h, K2CO3, N2 (ii) aromatic amines, tetrahydrofuran (THF), rt, 6–12 h, K2CO3, N2
(iii) 1,4-dioxane, reflux, 12–16 h, K2CO3, N2.
A docking study was performed on reported crystal structure of
AChE which was retrieved from RCSB (PDB ID 1EVE). Compounds
4d and 4h were predicted to show better interaction with AChE.
In Figure 1a aromatic ring of Trp279 and Tyr334 was expected to
H
N
Cl
N
(a)
interact with 3-cholo-4-fluoroaniline and triazine moiety via
p–p
N
N
N
N
stacking, likewise Phe330 and Phe331 simultaneously with 3-(tri-
fluoromethyl)aniline of compound 4d. In Figure 1b Phe330 and
Phe331 was expected to engage with p-anisidine ring of compound
4h through aryl–aryl interaction. Similarly Trp279 and Tyr334 was
supposed to interact with 3-(trifluoromethyl)aniline and triazine
rings, respectively. In addition to this, a strong hydrogen bond
was assumed between the oxygen of p-anisidine and hydrogen of
His440 with donor–acceptor distance of 2.42 Å.
5
6
Scheme 2. Reagents and conditions: (a) Piperazine, K2CO3, 1,4-dioxane, reflux, 3–
4 h, 5 = 2-chloronicotinonitrile.
a comprehensive drug design suite was selected for docking and
scoring. The computational binding free energy of synthesized
compounds 4a–4h with AChE and BuChE is mentioned in Table 1.
The results of docking study revealed that the dual binding
The Binding free energy (DG in kcal/mol) of compounds 4d and 4h
activity for selected compound may be due to
p–p (aromatic)
with AChE was found to be ꢀ14.41 and ꢀ14.13, respectively, sug-
gesting a very high binding-affinity between the ligands and the
enzyme. All the compounds 4a–4h showed more inhibition selec-
tivity against AChE over BuChE.
and hydrogen bonding interactions with CAS and PAS of AChE.
Docking studies were further extended to the reported crystal
structure of Human BuChE (PDB ID 4TPK) with the synthesized
compounds. The docked structures of most two potent compounds
(4d and 4h) are given in Figure 2a and b, respectively. However, the
calculated free binding energies of the synthesized compounds 4a–
4h suggested their selectivity towards AChE over BuChE as shown
in Table 1.
Table 1
The calculated free energies of selected derivatives in molecular docking using Par
DOCK27
Compound Calculated free energy
(Kcal/mol) AChE
Calculated free energy
(Kcal/mol) BuChE
2.3. Biology
4a
4b
4c
4d
4e
4f
ꢀ12.76
ꢀ12.35
ꢀ13.20
ꢀ14.41
ꢀ12.59
ꢀ12.12
ꢀ13.01
ꢀ14.13
ꢀ12.14
ꢀ11.61
ꢀ11.85
ꢀ13.40
ꢀ10.61
ꢀ10.34
ꢀ11.48
ꢀ10.94
2.3.1. Inhibitory activity against cholinesterases
The inhibitory activity of synthetic compounds (4a–4h) against
AChE (from electric eel) and BuChE (from equine serum) was eval-
uated using the method of Ellman et al.,28 with tacrine and done-
pezil as a positive control. Their IC50 values and selectivity index
for AChE over BuChE were summarized as shown in Table 2. Most
of the target compounds achieved IC50 values in sub-micromolar to
nanomolar range with better inhibitory potency towards AChE
4g
4h
Bolded values represent the most active compounds 4d and 4h.
Figure 1. (a) and (b) are the docked structures of compounds 4d and 4h, respectively, with AChE. Tyr residues is shown in blue, Phe in yellow, His in purple, Ser in green, and
Trp in magentas colours. The stacking between two aryl centres and H-bonding interactions are shown by red coloured dashed lines.
p–p
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 2. (a) and (b) are the docked structures of compounds 4d and 4h, respectively, with BuChE. Tyr residues is shown in blue, His in purple, Ser in green, Leu in dim grey,
Ala in orange, Asn in cyan, Asp in forest green, Thr in red, Glu in dark green, and Trp in magentas colours. The
red coloured dashed lines.
p–p stacking interaction between two aryl centres is shown by
Table 2
In vitro inhibition of AChE and BuChE, selectivity index and oxygen radical
absorbance capacity (ORAC, Trolox equivalents) of 4a–4h, donepezil, and tacrine
Compounds AChEa IC50
M)
BuChEb IC50
M)
Selectivityc BuChE/ ORACd
AChE
(l
(l
4a
0.528 0.04 5.245 0.13
9.93
1.03 0.02
4b
4c
4d
4e
4f
4g
4h
Donepezil
Tacrine
0.153 0.01 2.214 0.005 14.47
0.072 0.02 2.849 0.09 39.5
0.059 0.003 3.603 0.07 61.06
1.72 0.05
2.30 0.14
2.39 0.08
2.45 0.04
2.92 0.23
1.89 0.07
3.46 0.15
n.t
0.084 0.004 4.894 0.07 58.26
1.412 0.02 4.132 0.007
1.701 0.08 5.365 0.16
2.9
3.1
0.080 0.005 2.015 0.08 25.2
0.038 0.001 3.14 0.2 82.6
0.13 0.21 0.054 0.85 0.40
n.t
Bolded values represent the most active compounds 4d and 4h.
n.t—Not tested.
a
50% inhibitory concentration (means SD of three experiments) of AChE from
electric eel.
Figure 3. Lineweaver–Burk plots resulting from subvelocity curve of AChE activity
with different substrate concentrations (0.05–0.50 mM) in the absence and
presence of 20 nM, 40 nM and 80 nM of compound 4d.
b
50% inhibitory concentration (means SD of three experiments) of BuChE from
equine serum.
c
SI selectivity index for AChE; IC50 eq BuChE/eeIC50 AChE.
Data are expressed as lmol of trolox equivalent/lmol of tested compound.
d
(IC50 = 0.528 lM) which was nearly 9 folds lower than most potent
compound 4d. It was also worth noting that all the target com-
pounds exhibited greater selectivity towards AChE over BuChE
with best derivative 4d attained selectivity ratio of 61-fold. More-
over, inhibitory potency of 4d was found to be comparable to stan-
over BuChE. With the aim of optimizing the inhibitory activity of
the synthesized compounds on ChEs, substituents with different
electronic properties were varied on core triazine scaffold. Within
the series, it was observed that compounds featuring 3-(trifluo-
romethyl)aniline attached to triazine moiety (compounds 4a–4e
and 4h), showed stronger inhibition of AChE than 2-fluoro and
3-chloro-4-fluoro containing aniline substituents, i.e., 4f
dard donepezil (IC50 = 0.038
lM) and better than tacrine
(IC50 = 0.038 M). Further, docking studies were carried out with
l
cholinesterases to bring more clarity in understanding their mode
of action.
(IC50 = 1.412 lM) and 4g (IC50 = 1.701 lM), respectively.
Within the sub-group of triazines containing 3-(trifluo-
romethyl)aniline, it was observed that inhibitory activity against
AChE was improved as electron withdrawing properties of 5-(sub-
stituted)aniline moiety increased. Thus, the trend observed was as
follows: 4c [5-(2,4-difluoro)aniline] > 4e [5-(4-fluoro)aniline] > 4h
[5-(4-methoxy)aniline] > 4b [5-(4-methyl)aniline], respectively.
Interestingly, compound 4d bearing 3-chloro-4-fluoroanaline pos-
2.3.2. Kinetic study of AChE
To gain further insight into the mechanism of action on AChE of
the potent inhibitor 4d, Lineweaver–Burk double reciprocal plots
were generated. The interception of the lines in the Lineweaver–
Burk plot above the x-axis with both increased slope (decreased
Vmax) and intercepts (higher Km) at increasing concentration of
the inhibitor, which indicated a mixed-type inhibition. Based on
their kinetic studies, we concluded that compound 4d might be
able to interact with both the catalytic active site (CAS) and periph-
eral anionic site (PAS) of AChE (Fig. 3).
sessed highest inhibitory potency (IC50 = 0.059 lM) despite being
lesser electronegative than compound 4c. One likely explanation
could be the presence of –Cl group might have contributed in sta-
bilizing the 3-D structure of the pharmacophore and facilitating its
interaction with the active site of the enzyme. Furthermore, the
replacement of substituted aniline group with cyclopropylamine
in compound 4a lead to a significant loss of inhibitory potency
2.3.3. Inhibition of self-mediated Ab1–42 aggregation
Targeting the self-induced Ab1–42 aggregation represents an
emerging approach for discovering drug candidates for AD.
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
of which could interfere with Ab aggregation process.31 Therefore,
the three most potent compounds of the series against Ab1–42
Table 3
Inhibition of self-induced Ab1–42 aggregation by target compounds 4a–4h and
reference compound curcumin
aggregation 4d (83.7%, IC50 = 10.1
lM), 4h (82.4%, IC50 = 10.9 lM)
Compound Ab1–42 aggregation inhibition (%)a Ab1–42 aggregation IC50 M)b
(l
and 4c (81.2%, IC50 = 11.3 M) have corresponding higher cholines-
l
terase inhibitory activity as well. Hence, on the basis of in vitro,
kinetic and the molecular docking results, we can conclude that
the target compounds act as dual binding site AChE inhibitors,
endowed with Ab anti-aggregating properties.
4a
4b
4c
4d
4e
4f
60.7 0.44
70.5 0.61
81.2 0.82
83.7 1.13
80.4 0.66
68.5 0.33
63.5 0.54
82.4 1.08
22.4 0.18
16.7 0.11
11.3 0.12
10.1 0.09
12.8 0.15
17.4 0.12
18.9 0.13
10.9 0.15
22.84 0.16
2.3.4. Antioxidant activity
4g
4h
The antioxidant activities of compounds 4a–4h were evaluated
by the well-established ORAC-FL method.32,33 Peroxyl radicals
were thermally generated from 2,2-azobis (amidinopropane) dihy-
drochloride and reacted with fluorescein to form nonfluorescent
products at 535 nm. The antioxidant capacity of compounds 4a–
4h was determined by their competition with fluorescein in the
radical capture, using a fluorescence microplate reader (Table 1).
The ability of compounds 4a–4h to scavenge radicals at concentra-
Curcumin 55.3 0.87
Bolded values represent the most active compounds 4d and 4h.
a
Inhibition of self-induced Ab1–42 aggregation (25
by thioflavin-T based fluorescence method (means SD of three
experiments).
lM) by tested inhibitors at
25 lM
b
The concentration (lM) required for 50% inhibition was determined from dose–
response curves. Data are expressed as means SD of three independent
experiments.
tions between 1 and 10 lM was compared with that of the highly
potent compound Trolox and is expressed as its equivalent. All the
target compounds exhibited significant radical-capture capacities
ranging from 1.03 to 3.46 times the Trolox value. Compounds 4h
however, exhibited the most potent antioxidant activity within
the series with ORAC-FL values of 3.46 trolox equivalents followed
by compound 4f with 2.92 trolox equivalents, respectively. The
presence of common structural feature, i.e., methoxy substituted
moieties, in compounds 4h and 4f appears to be favourable to
the compound’s overall radical scavenging ability.
Ab1–42 has a high tendency to form fibrils and aggregates. Also, its
oligomers are neurotoxic and cause membrane disruption in neu-
ronal cells.29 To investigate the activity of our compounds to inhi-
bit the self-mediated Ab1–42 aggregation, the thioflavin-T (ThT)
fluorescence assay was performed,30 As shown in Table 2, most
of our compounds showed better inhibitory activity of Ab1–42
aggregation (1.09–1.5 folds) at 25 lM compared to the reference
compound curcumin. The plausible reason for their remarkable
anti-aggregation activity was the presence of core triazine moiety,
through which the pharmacophore acquire a nearly planer struc-
ture which in turn enables it to fit between the beta-amyloid
sheets and thus produced a beta-amyloid disaggregating effect.
Within the group, a similar trend could be seen in the activities
of compounds 4a–4h shown in Table 3, as was observed in choli-
nesterase inhibition assay. This might be due to the involvement
of peripheral anionic site (PAS) site of AChE enzyme, the blockade
2.3.5. Inhibition of Ab1–42 aggregation formation monitored by
transmission electron microscopy (TEM)
Finally, transmission electron microscopy (TEM) was
employed34 to monitor and clarify the effect of two most potent
compounds 4d and 4h on Ab1–42 aggregation along with curcumin
as control.35 As shown in Figure 4b, after incubation at 37 °C for
Figure 4. TEM images for Ab-induced aggregation and test compound induced Ab disaggregation. The incubations were carried out with following reagents:
(a) 50 M Ab1–42 alone at 0 h (b) 50 M Ab1–42 alone at 48 h, 37 °C (c) 50 M Ab1–42 and 25 M curcumin at 48 h, 37 °C (d) 50 M Ab1–42 and 25 M compound 4d at
48 h, 37 °C (e) 50 M Ab1–42 and 25 M compound 4h at 48 h, 37 °C.
l
l
l
l
l
l
l
l
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
110
100
90
200
4d
4h
###
*
*
*
**
150
**
100
50
80
70
0
25
50
75
100
0
concentration (μM)
H2O2(200 μm)
est compound (μm)
-
-
+
-
+
5
+
10
+
20
+
5
+
+
Figure 5. Effect of compounds 4d and 4h on the viability of SH-SY5Y cells. The cells
were incubated with the indicated concentrations of the tested compounds for 48 h.
The cell viability was assessed by MTT assay. Percentages of the cell viability are
presented as mean SEM from 3 independent experiments.
T
10 20
4d
4h
Figure 6. Neuroprotection against H2O2 toxicity in SH-SY5Y cells. Compounds 4d
and 4h were tested for neuroprotective activity against H2O2 toxicity in neurob-
lastoma cell cultures. Results are expressed as percent viability compared to
untreated control cells. All data were expressed as mean SD of three experiments
and each included triplicate sets. #p <0.05, ##p <0.01, ###p <0.001 versus control;
*p <0.05, **p <0.01, ***p <0.001 versus H2O2 alone. Statistical analysis was performed
using one way ANOVA followed by Bonferroni test.
48 h, Ab1–42 alone aggregated into mature, denser and bulky aggre-
gates as compared to Ab1–42 alone (Fig. 4a) kept at 0 °C. However,
disaggregation effects were clearly visible upon addition of com-
pounds 4d and 4h (25 lM each), as fewer and slender Ab fibrils
were observed (Fig. 4d and e), respectively, as compared to Ab1–42
alone (Fig. 4b) and standard curcumin (Fig. 4c). The results of TEM
are consistent with ThT binding assay results which suggested that
compounds 4d and 4h could inhibit Ab aggregation noticeably
through direct interaction with Ab or by blocking PAS site of AChE.
neurotoxicity in SH-SY5Y cells. However, compounds treatment
(20 lM) blocked these morphological changes and significantly
prevented the cell death induced by H2O2 toxicity in these cells
(Fig. 7c and d). These results indicate that the neuroprotective
action of our synthesized compounds might come from radical
scavenging action.
2.3.6. Cytotoxicity of synthetic compounds in SH-SY5Y cells and
neuroprotective effect on H2O2-induced oxidative cell damage
in SH-SY5Y cells
To determine the potential cytotoxic effects of triazine deriva-
tives on neuronal cell line SH-SY5Y, two most potent compounds
4d and 4h were selected for treatment, with different concentra-
2.3.7. Neuroprotection against Ab1–42-induced toxicity
Ab-induced apoptotic neuronal cell death is a critical event in
the pathology of AD. The toxicity caused by Ab42 is complex, which
includes the generation of abnormally high concentration of reac-
tive oxygen species, activating the release of damaging cytokines
tions ranging from 1 to 100 lM. After exposing the cells to these
compounds for 48 h, the cell viability was evaluated by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.36,37
The Figure 5 shows a dose-dependent effect of tested compounds
(e.g., interleukin-1, interleukin-6, TNF-a), and causing mitochon-
drial dysfunctions.38 A separate set of experiments were conducted
to assess the neurotoxic concentration of Ab1–42 in SH-SY5Y neu-
roblastoma cells, using cell viability assay (Fig. 8). The neuropro-
tective potential of compounds of the representative compounds
4d and 4h on Ab1–42 induced neurotoxicity were evaluated at three
on the viability of SH-SY5Y cells, for instance, 4d (1
l
l
l
M:
M:
M:
97.1 3.5%;
5
l
M: 95.4 3.6%; 10
M: 88.6 3.1%; 75 M: 84.3 2.6% and 100
M: 98.1 2.5%;
M: 93.8 2.5%; 50
M: 86.3 3.6% and 100 M: 83.3 2.5%), respectively. The
lM: 95.7 5.4%; 25
92.8 2.5%; 50
l
l
80.3 2.5%) and 4h (1
l
5
lM: 96.4 3.6%;
10
75
l
l
M: 94.7 3.4%; 25
l
l
M: 89.6 2.1%;
different concentrations, i.e., 5, 10 and 20
lM. Treatment of cells
l
with Ab1–42 (25 M) for 48 h, resulted in marked decrease in cell
l
results demonstrated that new compounds did not exhibit cyto-
toxic effect on the cells in the range of concentrations, which also
covers the highest concentration (25 lM) used in the experiment.
Oxidative stress plays an important role in neuronal degenera-
tion. Oxidative damage marked by lipid peroxidation, nitration,
reactive carbonyls, and nucleic acid oxidation is increased in vul-
nerable neurons in AD.15 Hence, the intracellular antioxidant activ-
viability (47.2%, p <0.001) as compared to untreated control. The
co-treatment of cells with compounds ameliorated Ab1–42 induced
neurotoxicity. As can be seen in Figure 8, the selected compounds
exhibited neuroprotective effects at concentrations ranging from 5
to 20
capability.
Compound 4h exhibited a significant effect in a dose dependant
manner, i.e., 5 M, p <0.05; 10 M p <0.01 and 20 M, p <0.001.
lM, with both compounds showing the highest protective
ity of selected compounds 4d and 4h (5, 10 and 20
lM) against
l
l
l
H2O2-induced ROS in SH-SY5Y cells were assessed by DCFH-DA flu-
While 4d showed weaker effect at lower concentration however,
orescent assay. As illustrated in Figure 6, exposure of SH-SY5Y cells
displayed similar neuroprotective potential at the higher dose con-
to H2O2 (200
lation by about 1.58 folds, p <0.001. Treatment with compounds 4d
and 4h, produced a moderate effect at lower doses 10 M, p <0.05
while their protective ability against H2O2-induced ROS formation
l
M) for 24 h, increased the intracellular ROS accumu-
centration of 10
compound 4h.
l
M (p <0.01) and 20
l
M (p <0.001) as observed in
l
Furthermore, microscopic observation revealed a marked
change in the morphology of Ab1–42 treated cells as compared to
control cells (Fig. 9b). After Ab1–42 treatment, cell loss with visibly
shrink cell body, fading synapses, loss of adherence and
fragmented or rounded floating cells which signifies onset of
apoptosis or necrosis. However, compounds treatment signifi-
cantly restored these alterations in SH-SY5Y cells (Fig. 9c and d).
These results suggested that these novel compounds exerted
significantly improved at 20
levels of ROS.
lM (p <0.01) without affecting basal
Furthermore, microscopic observations showed that a large
number of H2O2-treated cells became rounded, vacuolated and
floated which clearly indicated apoptosis or necrosis in these cells.
All these morphological alterations clearly indicated H2O2 induced
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Figure 7. Phase-contrast micrographs showing H2O2-induced neurotoxicity and neuroprotection of 4d and 4h in SH-SY5Y cells. (a) Cells without treatment showed healthy
shapes. (b) H2O2 alone (200 M) induced neurotoxicity. (c) Compound 4d (20 M) was given for 24 h with H2O2 (200 M) at 37 °C. (d) Compound 4h (20 M) was given for
M) at 37 °C and co-treatment showed neuroprotection.
l
l
l
l
24 h with H2O2 (200
l
affect biological functions (ADME). In the present study, to predict
the drug-likeness of the targeted compounds, about 45 physically
significant descriptors and pharmacologically relevant properties
of these compounds were calculated using QikProp module of
the Schrödinger program.39 A few significant indicators of their
pharmacokinetic profiles were considered and are reported in
Table 4. Fundamental physiochemical features of CNS active drugs
are mainly related to their ability to penetrate the blood–brain bar-
rier (BBB) and exhibit CNS activity. Our results indicated that com-
pounds showed drug like characteristics based on Lipinski’s rule of
five (mol_MW < 500, QPlogPo/w < 5, donorHB 6 5, accptHB 6 10).
Based on the predicted values for QPlogBB, QPlog Po/W and CNS
activity, all compounds might be able to penetrate into the CNS.
PSA is another important predictor for BBB. The calculated theoret-
ical PSA values for all compounds may support their ability to pen-
etrate the blood–brain barrier. Among calculated parameters,
QPPCaco, QPlogBB, QPLogKhsa, and QPPMDCK mainly account for
the ability of the compound’s distribution across BBB (QPPMDCK)
and gut-blood barrier (QPPCaco). The amount of protein binding
of CNS drugs is an important consideration which tends to be
rather high. The predicted values of QPLogKhsa for all compounds
indicate their strong binding with plasma protein. In addition, the
results showed that all compounds displayed excellent oral
absorption. The predicted ADME properties revealed that all com-
pounds possess appropriate pharmacokinetic profiles required to
penetrate BBB and therefore, could be considered as good candi-
date for drug development.
120
***
***
**
80
40
**
*
###
0
Aβ1-42 (25μm)
est compound (μm)
-
-
+
-
+
5
+
10
+
20
+
5
+
+
T
10 20
4d
4h
Figure 8. Neuroprotection against Ab-induced toxicity. Compounds 4d and 4h
were tested for neuroprotective activity against Ab toxicity in SH-SY5Y neuroblas-
toma cell cultures. Results are expressed as percent viability compared to untreated
control. All data were expressed as mean SD of three experiments and each
included triplicate sets. #p <0.05, ##p <0.01, ###p <0.001 versus control; *p <0.05,
**p <0.01, ***p <0.001 versus Ab1–42 alone. Statistical analysis was performed using
one way ANOVA followed by Bonferroni test.
marked neuroprotection against Ab-induced neurotoxicity. Thus,
the protective action against the Ab1–42 peptide of our synthetic
compounds might be enhanced by the anti-Ab aggregation action.
3. Conclusion
2.4. Pharmacokinetic properties and drug likeness of
compounds 4a–4h
In summary, eight new triazine derivatives were synthesized
and characterized as multifunctional anti-Alzheimer agents. All of
these synthetic compounds showed potent AChE inhibitory activity
and good selectivity for AChE over BuChE. Our synthetic derivatives
also exhibited potent radical scavenging activity. Compounds 4d
and 4h exhibited highest anti-Ab1–42 aggregation potency with
A drug candidate should have desirable biopharmaceutical
properties such as good solubility and good permeability, as well
as optimal ADME properties. Hence, physicochemical indicators
based on in silico models derived from in vitro and in vivo data,
are increasingly used during the early stages of drug discovery to
provide a comprehensive understanding of the key properties that
IC50 values of 10.1 and 10.9
lM, respectively. In addition, TEM
studies have also confirmed the anti-Ab1–42 aggregation property
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8
M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 9. Phase-contrast micrographs showing Ab-induced neurotoxicity and neuroprotection of 4d and 4h in SH-SY5Y cells. (a) Cells without treatment showed healthy
shapes. (b) Ab alone (25 M) induced neurotoxicity. (c) Compound 4d (20 M) was given for 24 h with Ab (25 M) at 37 °C. (d) Compound 4h (20 M) was given for 24 h with
Ab (25 M) at 37 °C.
l
l
l
l
l
Table 4
Predicted ADMET properties of the target compounds 4a–4h
Compound Mol_MW
HB
HB
QPlog
Rotatable PSA
bonds (7–200)
(ꢀ2 to 6.5) (0–15)
SASA
CNS
QPPcaco
QPlogBB
QPPMDCK QPlog % human
(130–725) donors acceptors Po/W
(300–1000) (ꢀ2 to 2) (<25 poor,
(ꢀ3 to 1.2) (<25 poor, KhSa oral
(0–6)
(2–20)
>500 great)
>500
(ꢀ1.5 absorption
great)
to 1.5) (>80% high,
<25% poor)
4a
4b
4c
4d
4e
4f
481.482
531.542
553.496
569.951
535.506
497.534
515.979
547.541
2
2
2
2
2
2
2
2
7.5
7.5
7.5
7.5
7.5
8.25
7.5
5.076
6.155
6.259
6.539
6.085
5.189
5.861
5.937
5
5
5
5
5
6
5
6
91.668
89.742
89.65
89.734
89.745
97.542
89.742
98.033
822.889
894.235
877.029
893.439
871.157
853.357
872.936
898.928
ꢀ1
ꢀ1
0
0
0
ꢀ1
ꢀ1
ꢀ1
820.768
889.014
890.058
888.832
889.652
935.115
888.284
888.48
ꢀ0.794
ꢀ0.806
ꢀ0.594
ꢀ0.535
ꢀ0.669
ꢀ0.979
ꢀ0.806
ꢀ0.863
1759.931 0.792 95.867
1918.451 1.16 89.849
5182.969 1.082 90.468
7667.097 1.156 92.094
3475.774 1.047 89.441
775.624 0.797 100
4g
4h
1739.827 1.056 88.121
1917.601 1.009 88.565
8.25
MW: molecular weight, HBA: hydrogen-bond acceptor atoms, HBD: hydrogen-bond donor atoms, PSA: polar surface area, SASA: total solvent accessible surface area,
QPlogPo/w: Predicted octanol/water partition coefficient, CNS: Predicted central nervous system activity, QPPCaco: Caco-2 cell permeability in nm/sec, QPPMDCK: Predicted
apparent MDCK cell permeability in nm/sec, QPlogBB: brain/blood partition coefficient, QPlogKhsa: binding to human serum albumin, Percent Human-oral Absorption:
human oral absorption on 0–100% scale.
of compounds 4d and 4h. The molecular modelling studies indi-
cated that our synthetic derivatives have significant binding affinity
with both CAS and PAS of the AChE. In addition, our synthetic com-
pounds showed a neuroprotective effect against both oxidative
stress induced by H2O2 and Ab1–42 toxicity. Taken together, the data
show that triazine derivatives can be considered very promising
lead compounds for treatment of AD.
nated as s (singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), br (broad), m (multiplet, for unresolved lines). LCMS of
the compounds were carried out on applied biosystem (absciex
2000 triple quad). Column chromatography was performed on col-
umns packed with alumina from Merck (70–230 mesh). Aluminum
oxide thin-layer chromatography (TLC) cards from Merck (alu-
minum oxide-precoated aluminum cards with fluorescent indicator
detectable at 254 nm) were used for TLC. Developed plates were
visualized by a Spectroline ENF 260C/FE UV apparatus. Organic
solutions were dried over anhydrous sodium sulfate. Evaporation
of the solvents was carried out on a Büchi rotavapor R-100
equipped with a Büchi V-100 vacuum controller. Elemental analy-
ses were obtained in an Elementar Vario analyser. Elemental anal-
yses of the compounds were found to be within 0.4% of the
theoretical values. The purity of tested compounds was >95%.
4. Experimental
4.1. Chemistry
All reagents and solvents were commercially available and used
as purchased without further purification. Melting points (mp)
were determined on a Stuart Scientific SMP1 apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker 400 MHz FT spectrometer. Proton chem-
ical shifts are reported in ppm (TMS, d 0.00) or with the solvent ref-
erence relative to TMS employed as the internal standard (CDCl3, d
7.26; DMSO-d6 d 2.54) and multiplicities of NMR signals are desig-
4.1.1. General procedure for the synthesis of mono-substituted
triazine (2a–2h)22
The synthesis of mono-substituted triazine is depicted in
Scheme 1. 2,4,6-trichloro-1,3,5-triazine (5.03 g; 1.1 equiv) was
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M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
dissolved in anhydrous THF at ꢀ10 °C and K2CO3 (3.43 g; 1 equiv)
was added. Appropriate aromatic amines (4 g; 1 equiv) were dis-
solved in THF and were added drop wise to the reaction mixture
for half an hour with constant stirring. The progress of the reaction
was monitored by thin layer chromatography. After the completion
of the reaction, the reaction mixture was dried with a rotatory
evaporator. The reaction was quenched with water and extracted
with ethylacetate (2 ꢁ 50 ml). The organic phase was washed with
brine solution, dried over Na2SO4 and evaporated at reduced pres-
sure to get the crude products. The crude products were purified by
silica gel column chromatography eluted with petroleum ether and
EtOAc (2–3%) to give the pure compounds 2a–2h, which were used
for the next step reaction.
114.39, 95.11, 72.74, 60.41, 47.86, 43.03, 23.42, 21.02, 14.17,
7.12. LCMS: (ESI, m/z): [M+H]+ Calcd for C23H22F3N9 481.9, found:
482.1. Elemental Anal.: Calcd C, 57.37; H, 4.61; N, 26.18, found C,
57.49; H, 4.80; N, 26.07.
4.1.5. Synthesis of 2-(4-(4-(p-toluidino)-6-(3-(trifluoromethyl)
phenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)nicotinonitrile
(4b)
Solid, off white, (Rf = 0.2 in ethylacetate), yield = 76%, mp 178–
180 °C, 1H NMR (300 MHz, CDCl3) d 8.37 (dd, J = 4.8, 1.8 Hz, 1H),
8.06 (s, 1H), 7.80 (dd, J = 7.8, 2.1 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H),
7.41 (d, J = 8.4 Hz, 3H), 7.33 (s, 1H), 7.27 (s, 1H), 7.13 (d,
J = 8.1 Hz, 3H), 6.80 (1H), 3.99 (4H), 3.79 (4H), 2.32 (s, 3H). 13C
NMR (75 MHz, CDCl3) d 165.12, 164.38, 164.21, 160.65, 151.90,
143.87, 139.66, 135.98, 132.96, 131.32, 129.40, 125.91, 122.69,
120.76, 119.09, 117.97, 116.81, 114.46, 95.21, 47.87, 43.20, 29.69,
20.80. LCMS: (ESI, m/z): [M+H]+ Calcd for C27H24F3N9 531.2, found:
532.2. Elemental Anal.: Calcd C, 61.01; H, 4.55; N, 23.72, found C,
61.23; H, 4.69; N, 23.62.
4.1.2. General procedure for the synthesis of di-substituted
triazine (3a–3h)22
A solution of appropriate aromatic amine (0.87 g; 1 equiv) in
THF (5 ml) was added drop wise to the stirred solution of mono-
substituted triazine (2.5 g; 1 equiv) and K2CO3 (1.1 g; 1 equiv) in
anhydrous DMF (5 ml) at 0 °C. Then the reaction mixture was con-
tinued to stir at room temperature for 6–12 h. The progress of the
reaction was monitored by thin layer chromatography. After the
completion of the reaction, the reaction mixture was quenched
with water and extracted with ethylacetate (3 ꢁ 50 ml). The
organic phase was washed with brine solution and then dried over
Na2SO4 and evaporated to dryness. The compounds 3a–3h were
also purified by column chromatography eluted with petroleum
ether and EtOAc (6–8%) and were used for the final step.
4.1.6. Synthesis of 2-(4-(4-(2,4-difluorophenylamino)-6-(3-
(trifluoromethyl)phenylamino)-1,3,5-triazin-2-yl)piperazin-1-
yl)nicotinonitrile (4c)
Solid, white, (Rf = 0.2 in ethylacetate), yield = 69%, mp 170–
172 °C, 1H NMR (300 MHz, CDCl3) d 8.38 (d, J = 4.8 Hz, 1H), 8.17
(d, J = 6.3 Hz, 1H), 8.07 (s, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.56 (d,
J = 7.2 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 12.6 Hz, 2H),
7.19 (s, 1H), 6.90 (dd, J = 2.1, 10.8 Hz, 2H), 6.82 (dd, J = 7.8,
5.1 Hz, 1H), 4.00 (s, 4H), 3.79 (s, 4H). 13C NMR (75 MHz, CDCl3) d
165.05, 164.42, 163.58, 160.68, 151.91, 143.87, 139.43, 136.83,
131.39, 129.27, 127.45, 123.46, 122.79, 119.39, 117.93, 116.86,
114.58, 111.03, 110.72, 108.46, 103.63, 101.74, 95.32, 47.87,
43.19. LCMS: (ESI, m/z): [M+H]+ Calcd for C26H20F5N9 553.1, found:
554.2. Elemental Anal.: Calcd C, 56.42; H, 3.64; N, 22.78, found C,
56.54; H, 3.73; N, 22.56.
4.1.3. General procedure for the synthesis of tri-substituted
triazine (4a–4h)
2-(Piperazin-1-yl)nicotinonitrile (6) was synthesized while
reacting 2-chloronicotinonitrile with piperazine in presence of
base as K2CO3.23 0.56 g (1.2 equiv) of (6) was dissolved in 1,4-diox-
ane and K2CO3 (0.38 g; 1.1 equiv) was added at room temperature.
Di-substituted triazine compounds (3a–3h) (1 g; 1 equiv) dis-
solved in 1,4-dioxane were added drop wise for 30 minutes at
room temperature. After the addition, the reaction mixtures were
heated under reflux for 12–16 h. The progress of the reactions were
monitored by thin layer chromatography. After the completion of
the reaction, the reaction mixtures were filtered and the residues
were washed with 1,4-dioxane. The filtrates obtained were vac-
uum dried. The crude tri-substituted compounds obtained were
purified using column chromatography eluted with petroleum
ether and EtOAc (60–70%) to give the pure compounds 4a–4h.
2,4,6-Trichloro-1,3,5-triazine undergoes temperature depen-
dent nucleophilic aromatic substitution reaction in which all the
three chlorine atoms were substituted step by step with different
nucleophiles. The first two steps, occurred below ꢀ10 °C with
1 equiv of base (K2CO3) and at room temperature with 1–1.2 equiv
K2CO3, respectively. Third substitution occurred at 110 °C in pres-
ence of base K2CO3 and 1,4-dioxane. All the synthesized com-
pounds were purified by column chromatography using EtOAc/
Pet. ether as eluent and characterized by various spectroscopic
techniques.
4.1.7. Synthesis of 2-(4-(4-(3-chloro-4-fluorophenylamino)-6-
(3-(trifluoromethyl)phenylamino)-1,3,5-triazin-2-yl)piperazin-
1-yl)nicotinonitrile (4d)
Solid, white, (Rf = 0.3 in ethylacetate), yield = 75%, mp 175–
177 °C, 1H NMR (300 MHz, CDCl3) d 8.39 (d, J = 4.8 Hz, 1H), 8.03
(s, 1H), 7.82 (dd, J = 7.8, 2.1 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.60
(d, J = 7.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 6.9 Hz, 3H),
7.09 (d, J = 8.7 Hz, 1H), 6.82 (dd, J = 7.5, 4.8 Hz, 1H), 4.00 (t,
J = 4.5, 4H), 3.79 (t, J = 5.7, 4H). 13C NMR (75 MHz, CDCl3) d
164.84, 163.73, 160.67, 151.90, 143.85, 139.25, 135.26, 130.95,
129.33, 123.00, 122.64, 120.68, 120.06, 119.97, 119.58, 119.53,
117.88, 117.01, 116.59, 116.29, 114.64, 95.39, 47.83, 43.28. LCMS:
(ESI, m/z): [M+H]+ Calcd for C26H20ClF4N9 569.1, found: 570.3. Ele-
mental Anal.: Calcd C, 54.79; H, 3.54; N, 22.12, found C, 56.94; H,
3.70; N, 22.03.
4.1.8. Synthesis of 2-(4-(4-(4-fluorophenylamino)-6-(3-(trifluo-
romethyl)phenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)nico-
tinonitrile (4e)
4.1.4. Synthesis of 2-(4-(4-(cyclopropylamino)-6-(3-(trifluoro-
methyl)phenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)nicotino-
nitrile (4a)
Sticky semisolid which later on solidified, light brownish,
(Rf = 0.2 in ethylacetate), yield = 72%, mp 165–167 °C, 1H NMR
(300 MHz, CDCl3) d 8.37 (s, 2H), 8.07 (s, 1H), 7.79 (d, J = 7.2 Hz,
1H), 7.50 (d, J = 6.9 Hz, 1H), 7.33 (1H), 6.79 (d, J = 4.8 Hz, 1H),
5.69 (s, 1H), 3.97 (8H), 2.80 (s, 1H), 2.05 (s, 1H), 1.26 (s, 1H), 0.80
(s, 2H), 0.54 (s, 1H). 13C NMR (75 MHz, CDCl3) d 171.19, 160.63,
151.87, 143.85, 140.24, 129.04, 122.18, 118.58, 117.98, 116.54,
Solid, snow white, (Rf = 0.3 in ethylacetate), yield = 77%, mp
139–142 °C, 1H NMR (300 MHz, DMSO) d 9.60 (s, 1H), 9.35 (s,
1H), 8.44 (dd, J = 4.8, 1.8 Hz, 1H), 8.24 (s, 1H), 8.12 (d, J = 7.5 Hz,
1H), 7.98 (s, 1H), 7.74 (s, 2H), 7.52 (s, 1H), 7.29 (d, J = 7.8 Hz, 1H),
7.13 (s, 2H), 6.96 (dd, J = 7.5, 4.8 Hz, 1H), 3.94 (s, 4H), 3.74 (s,
4H). 13C NMR (75 MHz, DMSO) d 164.53, 164.03, 160.14, 159.09,
155.93, 152.05, 144.36, 140.94, 136.18, 129.53, 129.00, 126.10,
123.20, 121.94, 117.85, 115.89, 115.05, 114.86, 114.76, 94.43,
47.43, 42.59. LCMS: (ESI, m/z): [M+H]+ Calcd for C26H21F4N9
Please cite this article in press as: Maqbool, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.041

10
M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
535.1, found: 536.2. Elemental Anal.: Calcd C, 58.32; H, 3.95; N,
23.54, found C, 58.56; H, 3.99; N, 23.33.
4.3. Biological assays
4.3.1. Inhibition of AChE and BuChE
4.1.9. Synthesis of 2-(4-(4-(2-fluorophenylamino)-6-(4-methox-
yphenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)nicotinonitrile
(4f)
AChE and BuChE inhibitory activities of the test compounds
were determined by the modified Ellman’s method. Briefly, Stock
solutions of tested compounds (10 mM) were prepared in ethanol
and diluted using 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to afford a
Solid, off white, (Rf = 0.2 in ethylacetate), yield = 70%, mp 185–
187 °C, 1H NMR (300 MHz, CDCl3) d 8.37 (dd, J = 4.5, 1.8 Hz, 1H),
8.31 (d, J = 8.1 Hz, 1H), 7.81 (dd, J = 7.5, 1.8 Hz, 1H), 7.46 (d,
J = 8.7 Hz, 2H), 7.26 (s, 2H), 7.11 (d, J = 8.1 Hz, 3H), 6.90 (d,
J = 9.0 Hz, 2H), 6.80 (dd, J = 7.8, 4.8 Hz, 1H), 3.99 (s, 3H), 3.82
(8H). 13C NMR (75 MHz, CDCl3) d 165.05, 160.65, 155.94, 151.91,
143.89, 131.64, 124.13, 124.08, 122.96, 122.85, 122.61, 122.08,
118.00, 115.00, 114.74, 114.45, 114.06, 110.14, 95.18, 55.54,
47.90, 43.20. LCMS: (ESI, m/z): [M+H]+ Calcd for C26H24FN9O
497.2, found: 498.1. Elemental Anal.: Calcd C, 62.77; H, 4.86; N,
25.34, found C, 62.90; H, 4.97; N, 25.19.
final concentration range between (0.01–100)
lM. Enzyme solu-
tions were prepared by dissolving lyophilized powder in double-
distilled water. The assay solution consisted of 1 mL of 0.1 M phos-
phate buffer KH2PO4/K2HPO4, 25
l
L of AChE (0.22 U/mL, E.C.
L of BuChE (0.06 U/mL, E.C.
L of various concentrations
3.1.1.7, from electric eel) or 25
l
3.1.1.8, from equine serum) and 100
l
of test compounds which was allowed to stand for 5 min before
100 L of 0.01 M DTNB were added. A positive control of donepezil
was used in the same range of concentrations. The reaction was
started by addition of 20 L of the 0.075 M substrate solution
l
l
(acetylthiocholine/butyrylthiocholine) and exactly 2 min after sub-
strate addition the absorption was measured at 25 °C at 412 nm.
The non-enzymatic hydrolysis of acetylthiocholine/butyrylthio-
choline iodide was also measured in enzyme-free assay systems,
and the results were employed as blank. In control experiments,
inhibitor-free assay systems were utilized to measure the full
activity. The percent inhibition was calculated by the following
expression: (1 – Ai/Ac) ꢁ 100, where Ai and Ac are the absorbances
obtained for AChE in the presence and absence of the inhibitors,
respectively, after subtracting the respective background. Each
experiment was performed in triplicate, and the mean standard
deviation was calculated. Data from concentration-inhibition
experiments of the inhibitors were calculated by nonlinear regres-
sion analysis, using the Graph Pad Prism 5 program.
4.1.10. Synthesis of 2-(4-(4-(p-toluidino)-6-(3-chloro-4-fluoro-
phenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)nicotinonitrile
(4g)
Solid, off white, (Rf = 0.2 in ethylacetate), yield = 77%, mp 195–
197 °C 1H NMR (300 MHz, CDCl3) d 8.37 (d, J = 4.8 Hz, 1H), 7.82
(d, J = 7.8 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.26 (s, 2H), 7.15 (d,
J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 1H), 7.03 (s, 1H), 6.80 (dd,
J = 7.8, 4.8 Hz, 1H), 3.98 (t, J = 4.2 Hz,4H), 3.79 (t, J = 3 Hz, 4H),
2.33 (s, 3H). 13C NMR (75 MHz, CDCl3) d 165.07, 164.10, 160.65,
155.54, 151.91, 143.89, 135.96, 135.70, 132.96, 129.43, 122.25,
120.74, 119.48, 117.98, 116.46, 116.17, 114.48, 95.22, 47.89,
43.15, 29.70, 20.84.LCMS: (ESI, m/z): [M+H]+ Calcd for C26H23ClFN9
515.1, found: 516.0. Elemental Anal.: Calcd C, 60.52; H, 4.49; N,
24.43, found C, 60.69; H, 4.58; N, 24.35.
4.3.2. Kinetic study of AChE
4.1.11. Synthesis of 2-(4-(4-(4-methoxyphenylamino)-6-(3-(tri-
fluoromethyl)phenylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)
nicotinonitrile (4h)
Kinetic study of AChE was performed using a reported
method.43 Test compound was added into the assay solution and
pre-incubated with the enzyme at 37 °C for 15 min, followed by
the addition of substrate. Kinetic characterization of the hydrolysis
of ATC catalysed by AChE was carried out spectrometrically at
412 nm. A parallel control was made with the assay solution of
no inhibitor for each times. The plots were assessed by a weighted
least square analysis that assumed the variance of V to be a con-
stant percentage of V for the entire data set. Slopes of these recip-
rocal plots were then plotted against the concentration of the
inhibitors in a weighted analysis and Ki was determined as the
ratio of the replot intercept to the replot slope.
Solid, snow white, (Rf = 0.3 in ethylacetate), yield = 68%, mp
179–180 °C 1H NMR (300 MHz, CDCl3) d 8.37 (d, J = 2.4 Hz, 1H),
8.08 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.42
(d, J = 8.7 Hz, 3H), 7.26 (s, 1H), 7.15 (s, 1H), 6.89 (d, J = 8.7 Hz,
3H), 6.80 (s, 1H), 3.99 (s, 3H), 3.81 (8H). 13C NMR (75 MHz, CDCl3)
d 165.13, 164.53, 164.24, 160.67, 156.00, 151.90, 143.88, 139.68,
131.56, 131.32, 130.90, 122.61, 119.10, 117.98, 116.69, 114.46,
114.12, 95.21, 95.04, 55.51, 47.89, 47.70, 43.16. LCMS: (ESI, m/z):
[M+H]+ Calcd for C27H24F3N9O 547.2, found: 548.2. Elemental
Anal.: Calcd C, 59.23; H, 4.42; N, 23.02, found C, 59.46; H, 4.60;
N, 22.89.
4.3.3. Thioflavin T (ThT) assay
Commercially available peptides were first treated with hex-
afluoroisopropanol (HFIP) at 5 mg/ml to avoid self-aggregation.
The clear solution containing the dissolved peptide was then ali-
quoted in micro centrifuge tube. The HFIP was allowed to evapo-
rate under a stream of nitrogen until a clear film remained in the
test tube. The pre-treated Ab1–42 samples were then dissolved in
DMSO in order to have a stable stock solution (Ab = 1 mM). For
the inhibition of self-mediated Ab1–42 aggregation experiment,
the Ab stock solution was diluted with 50 mM phosphate buffer
4.2. Docking protocol
Molecular docking and Scoring of ligands with AChE and BuChE
were carried out using Pardock module of Sanjeevini,25,26 a compre-
hensive drug design suite, based on physicochemical descriptors. It
integrates several protocols and handles protein and ligands at
atomic level where the hydrogen added protein is prepared in a
force field compatible manner. Ligand was considered in its input
pose for the calculation. Addition of hydrogen to the ligand was
achieved utilizing xleap of AMBER40 and geometry optimization
and partial charge calculations were performed using AM1-BCC
procedure.
The docked structures are energy minimized using the sander
module of AMBER and scored using scoring function Bapp41 which
is an all atom based scoring function, constituting electrostatics,
van der Waals, hydrophobicity, loss of conformational entropy of
protein side chains after complexation. Docked structures were
visualized using Chimera.42
(pH 7.4) to 25
25 M, final concentration) with or without the tested compound
(25 M) was incubated at 37 °C for 48 h. To quantify amyloid fibril
lM before use. A mixture of the peptide (10 lL,
l
l
formation, the thioflavin-T fluorescence method was used. Blanks
containing 50 mM phosphate buffer (pH 7.4) instead of Ab with
or without inhibitors were also carried out. After incubation, sam-
ples were diluted to a final volume of 200
NaOH buffer (pH 8.0) containing thioflavin-T (5
l
L with 50 mM glycine–
M). Then the flu-
l
orescence intensities were recorded five minutes later (excitation,
450 nm; emission, 485 nm). The percent inhibition of aggregation
Please cite this article in press as: Maqbool, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.041

M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
was calculated by the expression (1 ꢀ IFi/IFc) ꢁ 100% in which IFi
and IFc are the fluorescence intensities obtained for Ab in the pres-
ence and absence of inhibitors after subtracting the background,
respectively. Each measurement was run in triplicate.
measured using a microculture plate reader with a test wavelength
of 570 nm and a reference wavelength of 630 nm. Results are
expressed as the mean SD of three independent experiments.
4.3.7. Ab1–42-induced cytotoxicity
4.3.4. Oxygen radical absorbance capacity (ORAC-FL) assay
Ab1–42 peptides were first dissolved in hexafluoroisopropanol to
1 mg/ml, sonicated, incubated at room temperature for 24 h and
lyophilized. The resulting Ab1–42 peptide film was dissolved with
dimethylsulfoxide and stored at ꢀ20 °C until use. SH-SY5Y cells
were harvested from flasks and plated in 96-well polystyrene
plates with approximately 2 ꢁ 104 cells per well. Plates were incu-
bated at 37 °C for 24 h to allow cells to attach. Ab1–42 with or with-
out compounds 4d and 4h were diluted with fresh medium and
added to individual wells. The plates were then incubated for an
additional 48 h at 37 °C. Cell viability was determined using MTT
assay and expressed as a percentage of control cells. Image micro-
graphs were also taken with the help camera attached to the
microscope (Olympus, Japan) to assess morphological alterations
in SH-SY5Y cells after 48 h of treatment.
The reaction was carried out in 75 mM phosphate buffer (pH
7.4), and the final reaction mixture was 200
lL. Antioxidant
(20 L) and FL (120 L; 70 mM, final concentration) solutions were
l
l
placed in a black 96-well microplate (96F untreat, Nunc). The mix-
ture was preincubated for 15 min at 37 °C, and then AAPH solution
(60 lL, 12 mM, final concentration) was added rapidly using a mul-
tichannel pipette. The microplate was immediately placed in the
reader and the fluorescence recorded every minute for 80 min
(excitation, 485 nm; emission, 520 nm). Samples were measured
at different concentrations (1–25
phosphate buffer instead of the tested compound and eight cali-
bration solutions using Trolox (1–25 M), final concentration) as
lM). A blank (FL+AAPH) using
l
antioxidant were also carried out in each assay. A blank using
phosphate buffer instead of the tested compound was also carried
out. All of the reaction mixtures were prepared in triplicate, and at
least three independent runs were performed for each sample. The
antioxidant curves (fluorescence vs time) were normalized to the
curve of the blank. The area under the fluorescence decay curve
(AUC) was calculated using the following equation:
4.3.8. ROS measurements under H2O2-induced cellular stress
ROS assay was performed in living cells as previously described
by Wang and Zhu.44 SH-SY5Y cells were seeded at 2 ꢁ 104 cells per
well in 96-well plates for neuroprotection activity assay. Briefly,
intracellular ROS production was measured from SH-SY5Y cells
by treating them with fluorescent probe 20,70-dichlorofluorescein
AUC ¼ 1 þ i¼80ðfi=f0Þ
X
diacetate (DCFH-DA) for 20 min after 24 h of H2O2 (200
ment in the presence or absence of compounds 4d and 4h (5, 10
and 20 M). As a nonpolar compound, DCFH-DA crosses cell mem-
lM) treat-
i¼1
where f0 is the initial fluorescence reading at 0 min and fi is the flu-
orescence reading at time i. The net AUC was calculated by the fol-
lowing equation: AUCsample ꢀ AUCblank. The regression equations
between the net AUC and the Trolox concentrations were calcu-
lated. The ORAC-FL value for each sample was calculated using
the standard curve, and the ORAC-FL value of each tested compound
is thus expressed as Trolox equivalents.
l
brane and cellular esterases cleaved diacetate groups of DCFH-DA
to form non-fluorescent 20,70-dichlorofluorescein (DCFH). In the
presence of intracellular ROS, DCFH is oxidized very quickly to
highly fluorescent DCFH. The total fluorescence was measured
using ELISA plate reader (Tecan infinity 20) at an emission wave-
length of 488 nm and an excitation wavelength of 524 nm. Image
micrographs were also taken with the help camera attached to
the microscope (Olympus, Japan) to assess morphological alter-
ations in SH-SY5Y cells after 24 h of treatment.
4.3.5. TEM assay
Ab1–42 peptide (Sigma) stock was diluted with 20 mM phos-
phate buffer (pH 7.4) at 4 °C to 40 mM before use. For the inhibi-
tion of Ab1–42 aggregation experiment, Ab1–42 was incubated with
in the presence and absence of compounds 4d, 4h and Curcumin
at 37 °C for 48 h. The final concentration of Ab1–42 and compounds
4.4. In silico ADME property prediction
ADME (absorption, distribution, metabolism and excretion)
properties of compounds were predicted using QikProp program,
v. 3.5 (Schrödinger, LLC, New York, USA).39 This gave an estimate
of the physicochemical properties and the bioavailability of the
compounds. Parameters such as polar surface area (PSA), solvent
accessible surface area (SASA), CNS activity (Predicted central ner-
vous system activity, QPPCaco (Predicted apparent Caco-2 cell per-
meability in nm/s. Caco-2 cells is a model for the gut blood barrier),
QPlogBB (Predicted brain/blood partition coefficient), QPPMDCK
(Predicted apparent MDCK cell permeability in nm/s. MDCK cells
are considered to be a good mimic for the blood–brain barrier),
QPLogKhsa (Prediction of binding to human serum albumin) and
percent human oral absorption (Predicted human oral absorption
on 0–100% scale) were calculated. The acceptability of the com-
pounds based on the Lipinski’s rule of five were also estimated
from the results.
were 50 lM and 25 lM, respectively. Aliquots (10 lL) of the sam-
ples were placed on a carbon coated copper/rhodium grid for 2 min
at room temperature. Each grid was stained with uranyl acetate
(1%) for 2 min. Excess staining solution was removed and the spec-
imen was transferred for imaging with transmission electron
microscopy (JEOL JEM-1400).
4.3.6. Cell toxicity assay
The toxicity effect of 4d and 4h on human neuroblastoma cells
(SH-SY5Y) cells was examined according to the previous method.37
The SH-SY5Y cells were cultured in Dulbecco’s minimum essential
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1% penicillin–streptomycin solution (10,000 units of peni-
cillin and 10 mg of streptomycin in 0.9% NaCl) at 37 °C in a humid-
ified atmosphere containing 5% CO2. The cells were plated in 96-
well plate at a density 2000 cells/well and cultured for 24 h. Next,
cells were exposed to the tested compounds at different concentra-
Acknowledgements
tions (0.1–100 lM) for 48 h. Stock solution of tested compound
was prepared in DMSO and diluted in complete medium to give
final concentrations. Cytotoxicity assay was performed after 48 h
incubation with compounds in triplicates. The MTT reagent was
added to each well for additional 4 h followed by solubilisation
of formazan crystals in DMSO. The absorbance of each well was
M.M. is thankful to University Grants Commission (UGC),
Government of India for financial assistance through Central
University Ph.D. Students Fellowship. J.K. thanks to Council of Sci-
entific and Industrial Research (CSIR) for financial assistance
through SRF.
Please cite this article in press as: Maqbool, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.041

12
M. Maqbool et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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tra of new compounds) associated with this article can be found, in
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These data include MOL files and InChiKeys of the most important
compounds described in this article.
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Please cite this article in press as: Maqbool, M.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.04.041