Design and development of 1,3,5-triazine-thiadiazole hybrids as potent adenosine A2A receptor (A2AR) antagonist for benefit in Parkinson's disease

Article abstract of DOI:10.1016/j.neulet.2020.135222

Various studies showed adenosine A₂A receptors (A₂ARs) antagonists have profound therapeutic efficacy in Parkinsons Disease (PD) by improving dopamine transmission, thus being active in reversing motor deficits and extrapyramidal symptoms related to the disease. Therefore, in the presents study, we have showed the development of novel 1,3,5-triazine-thiadiazole derivative as potent A₂ARs antagonist. In the radioligand binding assay, these molecules showed excellent binding affinity with A₂AR compared to A₁R, with significant selectivity. Results suggest, compound 7e as most potent antagonist of A₂AR among the tested series. In docking analysis with A₂AR protein model, compound 7e found to be deeply buried into the cavity of receptor lined via making numerous interatomic contacts with His264, Tyr271, His278, Glu169, Ala63, Val84, Ile274, Met270, Phe169. Collectively, our study demonstrated 1,3,5-triazine-thiadiazole hybrid as a highly effective scaffold for the design of new A₂A antagonists.

Full text of DOI:10.1016/j.neulet.2020.135222

Journal Pre-proof
Design and development of 1,3,5-triazine-thiadiazole hybrids as potent
adenosine A2A receptor (A2AR) antagonist for benefit in Parkinson’s
Disease
Anoop Masih, Saumya Singh, Amol Kumar Agnihotri, Sabeena Giri,
Jitendra Kumar Shrivastava, Nidhi Pandey, Hans Raj Bhat, Udaya
Pratap Singh
PII:
S0304-3940(20)30492-4
DOI:
https://doi.org/10.1016/j.neulet.2020.135222
NSL 135222
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Neuroscience Letters
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Revised Date:
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22 May 2020
25 June 2020
27 June 2020
Please cite this article as: Masih A, Singh S, Agnihotri AK, Giri S, Shrivastava JK, Pandey N,
Bhat HR, Singh UP, Design and development of 1,3,5-triazine-thiadiazole hybrids as potent
adenosine A2A receptor (A2AR) antagonist for benefit in Parkinson’s Disease, Neuroscience
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Design and development of 1,3,5-triazine-thiadiazole hybrids as potent
adenosine A2A receptor (A2AR) antagonist for benefit in Parkinson's Disease
Short Title: 1,3,5-triazine as A2A receptor antagonist.
Anoop Masih,1 Saumya Singh,1 Amol Kumar Agnihotri,1 Sabeena Giri,1 Jitendra Kumar
Shrivastava,1 Nidhi Pandey,2 Hans Raj Bhat,3 Udaya Pratap Singh1*
1 Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences, Sam
Higginbottom University of Agriculture, Technology & Sciences, Allahabad, Uttar Pradesh,
India
2 Department of Medicine and Health Sciences, University Rovira i Virgili, Tarragona 43007,
Spain
3 Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam 786004
*Address for correspondence
Udaya Pratap Singh
Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences,
Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, Uttar
Pradesh, India.
Email: udaysingh98@gmail.com; udaya.singh@shiats.edu.in
Graphical abstract
Highlights
1

 Discovery of 1,3,5-triazine-thiadiazole as novel A2A receptor antagonist.
 Compound 7e as most potent A2A receptor antagonist.
 Compound 7e found deeply buried into the active site of A2A receptor.
Abstract
Various studies showed adenosine A2A receptors (A2ARs) antagonists have profound
therapeutic efficacy in Parkinsons Disease (PD) by improving dopamine transmission, thus
being active in reversing motor deficits and extrapyramidal symptoms related to the disease.
Therefore, in the presents study, we have showed the development of novel 1,3,5-triazine-
thiadiazole derivative as potent A2AARs antagonist. In the radioligand binding assay, these
molecules showed excellent binding affinity with A2AR compared to A1R, with significant
selectivity. Results suggest, compound 7e as most potent antagonist of A2AR among the tested
series. In docking analysis with A2AR protein model, compound 7e found to be deeply buried
into the cavity of receptor lined via making numerous interatomic contacts with His264,
Tyr271, His278, Glu169, Ala63, Val84, Ile274, Met270, Phe169. Collectively, our study
demonstrated 1,3,5-triazine-thiadiazole hybrid as a highly effective scaffold for the design of
new A2A antagonists.
Keywords: Parkinson’s disease, adenosine A2A receptor, 1,3,5-triazine, antagonist, docking.
1. Introduction
Parkinson's disease (PD) is a long term, degenerative neurological disease mainly
affecting motor function of the body due to loss of nerve cells producing dopamine [1]. Thus,
depletion of dopamine results in tremor, rigidity, bradykinesia and postural instability. The
current therapeutic approach to treat PD are mainly based on the dopaminergic drugs designed
to replace the action of dopamine in the deplete striatum [2–4]. Despite their clinical benefit
against PD, these drugs are associated with problematic adverse effects, such as, dyskinesia,
sedation and compulsive behaviour and increases as disease progresses [5].
In recent year targeting the adenosine A2A receptor (A2AR) has emerged as a promising
strategy for the treatment of Parkinson's diseases (PD) [6]. It belongs to the four subtypes of
A-family of G-protein-coupled receptors (GPCRs), for instance, A1, A2A, A2B and A3, which
elicit their specific function after interaction with neurotransmitter adenosine. The striatum of
brain is the location of A2AR, where it regulates the motor activity by modulating the dopamine
2

D2 receptor activation [7,8]. Consequently, inhibition of the interaction of adenosine with the
A2AR by antagonists may provide a potential treatment for Parkinson’s disease by improving
dopamine transmission, thus being active in reversing motor deficits and extrapyramidal
symptoms related to the disease [9]. Various A2AR antagonists have made their way to the
clinical trials and advanced up to Phase III (Fig. 1) [10]. Despite their high potency, these
inhibitors are associated with various drawbacks such as poor solubility and high toxicity.
Among the heterocyclic molecules, 1,3,5-triazine is well known for diverse array of
biological activities [11]. Since last decade, our group has been working on the development
of novel derivatives of 1,3,5-triazine against cancer [12], diabetes, bacteria [13–15], fungus
[16], malaria [17–19] and against cystic fibrosis [20]. Particularly against PD, ZM241385, a
non-xanthine, 1,3,5-triazine based competitive A2AR antagonist showed high affinity in the
nano-molar range while it has low affinity at the other adenosine receptor subtypes (A1, A2B,
and A3) [21–23]. Tozadenant, an another A2A receptor antagonist shown promising result in
PD and advanced to Phase III trials [24–26]. However, the trial has been stopped due to death
of five enrolled patients. Thus, discovery of novel A2A receptor antagonist is worth to be
investigated. The present study was undertaken to develop potent and effective 1,3,5-triazine-
thiadiazole based A2AR antagonist inspired by ZM241385 and tozadenant for possible benefit
in PD (Fig. 1).
2. Experimental
The chemical used in present study were obtained Sigma Aldrich, USA, unless otherwise
stated. The chemical used in the present study was procured from Sigma Aldrich (USA). The
13
spectra of 1H NMR and C NMR were recorded on Bruker Avance 400 and Bruker Avance
100 Spectrophotometer, respectively. The chemical shifts are expressed in parts per million
(ppm), and coupling constants are expressed in Hertz (Hz). The Low-resolution mass spectrum
(MS) was recorded on a Waters ZQ LC/MS single quadrupole system equipped with an
electrospray ionization (ESI) source. The elemental analysis of the final derivatives was
performed on Vario Elemental analyser. The Thin-layer chromatography was performed on
0.25 mm Merck silica gel plates (60F-254) and visualized under UV light.
2.1. The synthesis of compound 2, 3, 5 (a-f) and 6 (a-f) was performed as per the earlier
reported procedures.[14, 27]
2.2. General procedure for the synthesis of title 1,3,5-triazine-thiadiazole derivatives 7 (a-f)
3

Compound 3 (0.1 mol) was added into 50 mL of 1,4-dioxane at temperature 120–145 ̊C. A
solution of substituted thiadiazole 6 (a–f) (0.1 mol) in 35 mL of 1,4-dioxane was added slowly
to above solution and stirred for 90 min followed by drop-wise addition of K2CO3 (0.1 mol).
The reaction mixture was refluxed for 8-9 h at 135–145 ̊C. The product was filtered and washed
with cold water and re-crystallized with ethanol to afford the corresponding pure products 7
(a-f).
2.2.1.
4-(2-((4-amino-6-((5-phenyl-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7a)
o
Yield: 86%; MP: 219-220 C; MW: 406.47; Rf: 0.79; FT-IR (νmax; cm-1 KBr): 3429 (OH
stretching), 3328 (NH2 stretching), 3279 (N–H stretching), 3083 (C-H stretching, Aromatic),
1668 (C=N Aromatic), 1618 (C=C stretching), 1543 (N–H bending, NH), 1464 (CH2 bending,
Aliphatic), 1151 (C-C stretching, Aromatic), 1038 (C–N stretching), 642 (C–S stretching), 605;
1H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.06 (s, 1H, Ar-OH), 8.04 (d, 2H, J = 1.47 Hz,
Ar-H), 7.56 (d, 2H, J = 1.11 Hz, Ar-H), 7.43 (t, 1H, J = 1.4 Hz, Ar-H), 7.05 (d, 2H, J = 1.08
Hz, Ar-H), 6.97 (s, 2H, NH2), 6.71 (d, 2H, J = 2.53 Hz, Ar-H), 3.95 (s, 2H, 2 × NH)), 3.43 (t,
13
2H, J = 6.69 Hz, CH2), 2.91 (t, 2H, J = 6.17 Hz, CH2); C-NMR (100MHz, DMSO-d6) δ
ppm:182.7, 174.4, 165.9, 159.4, 155.9, 152.8, 133.7, 132.1, 130.9, 130.2, 129.3, 128.8, 115.9,
44.5, 35.2; Mass: 407.48 (M+1); Elemental analysis for C19H18N8OS: Calculated: C, 56.14; H,
4.46; N, 27.57; Found: C, 56.18; H, 4.42; N, 27.58.
2.2.2.
4-(2-((4-amino-6-((5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7b)
o
Yield: 79%; MP: 245-246 C; MW: 440.91; Rf: 0.69; FT-IR (νmax; cm-1 KBr): 3424 (OH
stretching), 3317 (NH2 stretching), 3281 (N–H stretching), 3074 (C-H stretching, Aromatic),
1689 (C=N Aromatic), 1631 (C=C stretching), 1139 (C-C stretching, Aromatic), 1516 (N–H
bending, NH), 1459 (CH2 bending, Aliphatic), 1048 (C–N stretching), 793 (C-Cl stretching),
645 (C–S stretching); 1H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.10 (s, 1H, Ar-OH), 7.74
(d, 2H, J = 1.35 Hz, Ar-H), 7.68 (d, 2H, J = 1.49 Hz, Ar-H), 7.05 (d, 2H, J = 1.05 Hz, Ar-H),
6.96 (s, 2H, NH2), 6.71 (d, 2H, J = 2.58 Hz, Ar-H), 3.99 (s, 2H, 2 × NH)), 3.36 (t, 2H, J = 6.67
Hz, CH2), 2.74 (t, 2H, J = 6.12 Hz, CH2);13C-NMR (100MHz, DMSO-d6) δ ppm:182.7, 174.4,
165.8, 159.5, 155.9, 152.6, 134.4, 132.2, 131.7, 130.5, 129.4, 128.8, 115.3, 44.8, 35.6 ; Mass:
441.94 (M+1); Elemental analysis for C19H17ClN8OS: Calculated: C, 51.76; H, 3.89; N, 25.41;
Found: C, 51.75; H, 3.92; N, 25.40.
4

2.2.3.
4-(2-((4-amino-6-((5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7c)
o
Yield: 84%; MP: 224-225 C; MW: 424.46; Rf: 0.62; FT-IR (νmax; cm-1 KBr): 3431 (OH
stretching), 3323 (NH2 stretching), 3288 (N–H stretching), 3079 (C-H stretching, Aromatic),
1694 (C=N Aromatic), 1635 (C=C stretching), 1139 (C-C stretching, Aromatic), 1512 (N–H
bending, NH), 1462 (CH2 bending, Aliphatic), 1162 (C-F stretching), 1042 (C–N stretching),
643 (C–S stretching); 1H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.08 (s, 1H, Ar-OH), 7.72
(d, 2H, J = 1.49 Hz, Ar-H), 7.34 (d, 2H, J = 1.28 Hz, Ar-H), 7.03 (d, 2H, J = 1.05 Hz, Ar-H),
6.93 (s, 2H, NH2), 6.71 (d, 2H, J = 2.52 Hz, Ar-H), 3.97 (s, 2H, 2 × NH)), 3.39 (t, 2H, J = 6.62
Hz, CH2), 2.75 (t, 2H, J = 6.08 Hz, CH2);13C-NMR (100MHz,DMSO) δ,ppm:182.7, 174.5,
165.9, 162.9, 159.2, 155.8, 152.9, 132.4, 130.2, 129.5, 129.1, 116.2, 115.8, 44.5, 35.3, ; Mass:
425.49 (M+1); Elemental analysis for C19H17FN8OS: Calculated: C, 53.76; H, 4.04; N, 26.40;
Found: C, 53.79; H, 4.02; N, 26.38.
2.2.4.
4-(2-((4-amino-6-((5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7d)
o
Yield: 68%; MP: 256-257 C; MW: 485.36; Rf: 0.75; FT-IR (νmax; cm-1 KBr): 3435 (OH
stretching), 3329 (NH2 stretching), 3284 (N–H stretching), 3075 (C-H stretching, Aromatic),
1687 (C=N Aromatic), 1628 (C=C stretching), 1139 (C-C stretching, Aromatic), 1512 (N–H
bending, NH), 1461 (CH2 bending, Aliphatic), 1053 (C–N stretching), 764 (C-Br stretching),
632 (C–S stretching); 1H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.04 (s, 1H, Ar-OH), 7.82
(d, 2H, J = 1.51 Hz, Ar-H), 7.72 (d, 2H, J = 1.48 Hz, Ar-H), 7.05 (d, 2H, J = 1.02 Hz, Ar-H),
6.95 (s, 2H, NH2), 6.69 (d, 2H, J = 2.51 Hz, Ar-H), 3.98 (s, 2H, 2 × NH), 3.41 (t, 2H, J = 6.58
Hz, CH2), 2.72 (t, 2H, J = 6.03 Hz, CH2);13C-NMR (100MHz, DMSO-d6) δ ppm:182.7, 174.4,
165.8, 159.5, 155.9, 152.8, 132.6, 132.3, 132.1, 130.2, 129.7, 123.2, 115.7, 44.6, 35.3 ; Mass:
486.39 (M+1); Elemental analysis for C19H17BrN8OS: Calculated: C, 47.02; H, 3.53; N, 23.09;
Found: C, 47.01; H, 3.54; N, 23.07.
2.2.5.
4-(2-((4-amino-6-((5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7e)
o
Yield: 86%; MP: 261-263 C; MW: 451.47; Rf: 0.81; FT-IR (νmax; cm-1 KBr): 3436 (OH
stretching), 3325 (NH2 stretching), 3289 (N–H stretching), 3078 (C-H stretching, Aromatic),
1692 (C=N Aromatic), 1634 (C=C stretching), 1548 (NO2 stretching), 1141 (C-C stretching,
Aromatic), 1512 (N–H bending, NH), 1457 (CH2 bending, Aliphatic), 1056 (C–N stretching),
644 (C–S stretching); 1H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.07 (s, 1H, Ar-OH), 8.26
(d, 2H, J = 1.94 Hz, Ar-H), 7.78 (d, 2H, J = 1.56 Hz, Ar-H), 7.01 (d, 2H, J = 1.03 Hz, Ar-H),
5

6.94 (s, 2H, NH2), 6.72 (d, 2H, J = 2.54 Hz, Ar-H), 3.96 (s, 2H, 2 × NH)), 3.44 (t, 2H, J = 6.52
Hz, CH2), 2.78 (t, 2H, J = 6.05 Hz, CH2);13C-NMR (100MHz, DMSO-d6) δ ppm:182.3, 174.8,
165.8, 159.5, 155.4, 152.9, 147.9, 139.5, 132.1, 130.4, 128.5, 124.4, 115.9, 44.4, 35.5, ; Mass:
452.47 (M+1); Elemental analysis for C19H17N9O3S: Calculated: C, 50.55; H, 3.80; N, 27.92;
Found: C, 50.58; H, 3.75; N, 27.95.
2.2.6.
4-(2-((4-amino-6-((5-(p-tolyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,5-triazin-2-
yl)amino)ethyl)phenol. (7f)
o
Yield: 72%; MP: 237-238 C; MW: 420.50; Rf: 0.84; FT-IR (νmax; cm-1 KBr): 3438 (OH
stretching), 3327 (NH2 stretching), 3295 (N–H stretching), 3081 (C-H stretching, Aromatic),
2958 (alkyl C-H stretching), 1693 (C=N Aromatic), 1621 (C=C stretching), 1143 (C-C
stretching, Aromatic), 1512 (N–H bending, NH), 1458 (CH2 bending, Aliphatic), 1052 (C–N
1
stretching), 643 (C–S stretching); H-NMR (400MHz, DMSO-d6, TMS) δ ppm: 9.04 (s, 1H,
Ar-OH), 7.74 (d, 2H, J = 1.57 Hz, Ar-H), 7.24 (d, 2H, J = 1.16 Hz, Ar-H), 7.03 (d, 2H, J =
1.02 Hz, Ar-H), 6.97 (s, 2H, NH2), 6.72 (d, 2H, J = 2.51 Hz, Ar-H), 3.98 (s, 2H, 2xNH)), 3.42
(t, 2H, J = 6.51 Hz, CH2), 2.72 (t, 2H, J = 6.07 Hz, CH2);2.23 (s, 3H, CH3); 13C-NMR (100MHz,
DMSO-d6) δ ppm:182.7, 174.3, 165.9, 159.5, 155.8, 152.9, 132.2, 131.8, 130.6, 130.4, 129.5,
127.4, 115.9, 44.5, 35.3, 21.3 ; Mass: 421.51 (M+1); Elemental analysis for C20H20N8OS:
Calculated: C, 57.13; H, 4.79; N, 26.65; Found: C, 57.11; H, 4.80; N, 26.64.
2.3. Physicochemical Property
All compounds were analyzed for physiochemical properties, such as logP value, solubility in
water, hydrogen bonder donar/acceptor, molecular weight and total polar surface area (TPSA)
via molispiration (http://www.molispiration.com) software.
2.5. Radioligand binding assays
[3H]ZM241385 and [3H]DPCPX binding assays for adenosine A2AR and A1Rs, respectively,
were performed in HEK293 cells. Briefly, 10 µg HEK293 cell membranes isolated from stably
transfected HEK293 cells with human A2AR and A1R cDNA were incubated with different
concentrations (1 pM to 1 lM) of compound 7e and 1 nM [3 H]ZM241385 in 200 µl incubation
buffer containing 50 mM Tris–Cl, 1 mM EDTA, pH 7.4 and 2.5 U/ml adenosine deaminase.
Adenosine A1R assays were performed on 10 µg of HEK293 cell membranes expressing
human adenosine A1Rs and 1 nM [3H]DPCPX in 200 µl incubation buffer. Reactions were
carried out for 60 min at 26 ̊C and were terminated by rapid filtration over 96-well plates
6

equipped with GF/B filters (Millipore, USA). Filters were washed three times with 300 µl of
cold washing buffer containing 50 mM Tris–Cl, 10 mM MgCl2, pH 7.4, air dried, and
radioactivity retained on filters were counted in 1450 LSC & Luminescence counter (Wallac
Microbeta Trilux, Perkin–Elmer, USA). Nonspecific binding for adenosine A2AR and A1R
were determined in the presence of 50 µM NECA and 50 µM CPA, respectively. Assays were
performed in duplicates and compounds were tested twice. Data were fitted in one site
competition-binding model for IC50 determination using the program GraphPad Prism 4.0
(GraphPad Software, Inc., San Diego, CA) and Ki values were calculated using Cheng and
Prusoff formula.
2.6. Molecular docking analysis
The X-ray crystal structure of human A2A adenosine receptor co-crystallized with ligand
ZM241385 at 2.6 Å resolution (PDB: 3EML) was downloaded from protein data bank. All
molecular modelling calculations and docking studies were carried out using BIOVIA
Discovery Studio software (Dassault Systèmes BIOVIA, Discovery Studio Modeling
Environment, Release 2018, San Diego, USA: Dassault Systèmes, 2016) running on a
Windows 7 PC.
Binding site sphere determination
The protein–ligand complex obtained from the protein data bank was prepared for docking as
follows. Initially, co-crystallized ligand and water molecules were deleted from the protein file.
Automatic protein preparation module was used for preparation of protein for docking using
CHARMm forcefield. The binding site sphere has been defined automatically by the software
over the co-crystallized ligand ZM241385.
Preparation of target compounds for docking
The compound to be dock was exported to Discovery Studio 3.0 for docking after sketching
on Chem Draw 10.0. The hydrogen atoms were added to the exported compound and their
standard geometry, optical isomers and 3D conformations were automatically generated.
Docking analysis
Docking was performed using CDOCKER protocol in BIOVIA Discovery Studio software
keeping the parameters at default. The best scoring pose of the docked compounds was
recognized. Receptor–ligand interactions of the complexes were examined in 2D and 3D styles.
7

Results and discussion
Chemistry
The synthesis of title molecules was achieved via three synthetic pathways as shown in scheme
1. Initially, in step 1, mono-substituted 4,6-dichloro-1,3,5-triazin-2-amine (2) was achieved by
reaction of 2,4,6-trichloro-1,3,5-triazine (1) with ammonia in presence of activating base. The
compound 2 was further reacted with 4-(2-aminoethyl) phenol in presence of triethylamine and
DMF to afford compound 3. In the step 2, different substituted methyl benzoate 4 (a-f) was
reacted with thiosemicarbazide to furnish corresponding thiosemicarbazide derivatives 5 (a-f),
which on subsequent reaction with sulphuric acid to form different substituted thiadiazoles 6
(a-f). In the final step, compound 3 was allowed to react with substituted thiadiazoles 6 (a-f)
to afford title compounds 7(a-f) in good yield.
All the synthesized compounds were characterized by different spectroscopic methods, such as
FT-IR, 1H NMR, 13C NMR, Mass and elemental analysis. The FT-IR peaks observed at 3424-
3438 cm-1 is due to the presence of OH group linked to phenyl ring. Absorption bands at 3323-
3328 cm-1 suggested as stretching vibration of NH2 group at 1,3,5-triazine. Another peak at
3279-3295 cm-1 attributed to NH group at 1,3,5-triazine ring. Furthermore, absorption bands
appeared at 3074-3083 cm-1 due to stretching vibration of C-H of aromatic ring. Another peak
at 1668-1693 cm-1 corresponds to stretching vibrations of aromatic C=N group. Triazine C-N
group appeared at 1038-1056 cm-1. The aromatic NO2 group appeared at 1548 cm-1. Another
peak at 1162 cm-1 corresponds to the presence of fluoro linked to phenyl ring. The phenyl
bromine group appears at 764 cm-1. The thiadiazole C-S group appears at 632-645 cm-1. The
1H-NMR spectrums of final synthesized compounds 7(a-f) shown singlet peak at 9.04-9.10 due
to the presence of aromatic hydrogen. The proton in the phenyl ring appeared as doublet as
8.26-7.82 ppm. The 1,3,5-triazine NH2 proton appears at 6.93-6.97 ppm as singlet. The proton
in the triazine ring linked with NH group appeared at 3.95-3.99 as singlet peak. Furthermore,
the resonance at 3.44-3.36 ppm suggested the presence of C-H proton of aliphatic chain with
triplet peak. Another, aliphatic CH2 proton appears at 2.72-2.91 ppm as triplet peak. The 13C-
NMR resonance peaks appear at 182.9- 159.1 ppm due the 1,3,5-triazine ring carbon atom. The
thiadiazole carbon appeared at 152.4-152.7 ppm. Furthermore, the resonance at 44.1-35.2 ppm
due to the presence of carbon of aliphatic CH2 side chain. Another resonance peak appears at
155.7- 115.8 ppm due phenyl ring carbon atoms. The mass spectroscopy and elemental analysis
data were found in aggrement with the proposed structure of compounds 7 (a-f).
8

Physiochemical properties
The physiochemical and molecular properties such as, partition coefficient (LogP), total polar
surface area (TPSA), and molecular weight (m.w.) has a significant impact on both
pharmacokinetic and pharmacodynamic process, as well as drug safety [28]. LogP value
signifies unique hydrophobic/lipophilic balance of the molecule and numerous studies
suggested a strong correlation exist between lipophilicity and activity, where lipophilic nature
of the compound has an easy access to CNS via increased permeability to BBB (blood brain
barrier) [29]. On the other hand, TPSA is defined as the surface sum over all polar atoms or
molecules, primarily oxygen and nitrogen, which also counting their attached hydrogen atoms.
The molecules with a polar surface area of greater than 140 angstroms squared tend to be poor
at permeating cell membranes. The molecular weight is another parameter which significantly
affect the drug action, and higher molecular weight have less chances to reach CNS [30].
Therefore, in the present study the designed molecules were assessed for determination of their
molecular and physicochemical properties, and it has been found that none of the synthesized
molecules disobeyed the Lipinski’s rule of 5 (Ro5). These results suggest that designed
molecules have easy access to CNS.
Radioligand binding assay
The results of the radioligand binding assay of the synthesized molecules are tabulated in table
1. It has been found that designed compounds showed considerable binding affinities against
both A2AR and A1R receptor subtypes. Among the tested derivative, compound 7a showed least
binding affinity against both the tested receptors. However, upon insertion of para-chloro (7b),
the binding affinity was found to be boosted significantly against A2AR, with mild improvement
against A1R receptor. The selectivity index was found reduced in the case of compound 7c
having para-fluoro with mild reduction in binding affinity against A2AR. Moreover, compound
7c showed mild improvement in affinity against A1R. In the case of compound 7d (para-
bromo), the binding affinity was found reduced significantly against both the tested receptors
with a mild drop in SI also. Among the tested analogues, compound 7e (para-nitro) was found
most potent for both receptor sub-types with more selectivity towards A2AR as confirmed by
SI. However, the binding affinity was found reduced significantly with drop in SI in the case
of compound 7f having para-methyl group. The structure-activity relationship of the designed
analogues sugest that, electron withdrawing substituent found more potent than their electron
donating counterparts. Among electron withdrawing groups, non-halogen substituent (NO2)
9

found more active than halogen counterparts for both iso-forms of receptor. Moreover, the
activity was found significantly reduced in the case of non-substituted derrivative, suggesting
substitution favours bioactivity..
Table 1: Radioligand binding and selectivity study of target compounds.
Entry
7a
Substituent
H
Ki (A2AR) nM Ki (A1R) nM
A1R / A2AR
5.6
127.2
44.6
59.3
71.2
32.1
89.2
712.7
520.4
456.5
505.2
322.3
569.1
4-Cl
11.66
6.69
7b
7c
4-F
4-Br
7.09
7d
7e
4-NO2
4-CH3
10.04
6.38
7f
Docking analysis
In current scenario of drug discovery, molecular docking is one of the most widely accepted
tool and it have irreplaceable role in structure-based drug design. It allows predicting the
binding-conformation of small molecule ligands to the appropriate target binding site [31]. In
view of excellent binding affinity of compound 7e against A2A receptor, it is necessary to
perform docking analysis with A2A protein model to predict its orientation in receptor cavity.
The X-ray crystal structure of A2AR (pdb id: 3EML) co-crystallized to ZM241385 at 3.5 Å was
selected to perform docking with compound 7e. To perform docking analysis, the ligand
ZM241385 was removed and protein was prepared according to the default protocol of
Discovery Studio 3.0. The ligand 7e was minimized and allowed to be docked into the active
site of A2A receptor via CDOCKER protocol. Analysis of docking results as shown in Fig. 2
and 3, compound 7e found to be efficiently dock into the active site of A2A receptor having
perpendicular binding pocket lined by His264, Glu169, Asn253 and Val84. As shown in Fig.
4, the phenyl-thiadiazole core of the molecule 7e was found to be deeply buried into the cavity
of receptor lined with Cys262, Thr256 and Pro260, via formation of H-bond with His264 and
pi-anion with Glu169. The 1,3,5-triazine central core forms two H-bond with Tyr271 via
engaging amine and its neighbouring nitrogen. It also bind with Met270 via pi-alkyl interaction,
which further found similar in the case of thiadiazole nucleus. The phenyl containing hydroxy
group form numerous pi-bonds with neighbouring residues, such as, pi-pi stacking with Phe168
10

and Pi-alkyl with Ile274 and Val84. Moreover, the terminal hydroxy group of compound 7e
form one H-bond with His278. Compound 7e also showed scoring function with A2A receptor,
Table 2. Collectively, our docking results confirmed that compound 7e bind efficiently with
A2A receptor probably due to excellent docking interaction and scoring functions (Table 2).
Table 2: Interacting residues and scoring of compound 7e in A2A receptor.
Compound
Interacting residues with type of interaction
Scoring function
CDOCKER
Pi-
Pi-Pi
CDOCKER
H-bond
Pi-Anion
Glu169
Interaction
energy
Alkyl
stacking energy:
7e
Ala63,
Val84,
Ile274,
Met270
His264,
Tyr271,
His278
Phe169
45.66
46.21
Figure 2
Figure 3
Figure 4
Conclusion
This paper report results for our research program aiming to identify potent A2A receptor
antagonists. The present study demonstrated the development of novel 1,3,5-triazine-
thiadiazole hybrids as potent and selective A2A receptor antagonists via integration of
computational tools, synthetic chemistry and pharmacological assays. Currently, we are
working on the structural optimization of this novel scaffold to provide insights into the
structural requirements for future development of new anti-parkinsonian agents.
Credit author statment
Anoop Masih, Saumya Singh, Amol Kumar Agnihotri, Sabeena Giri, Jitendra Kumar
Shrivastava, Nidhi Pandey: Performed experiments, data analysis and interpretation,
statistical analysis and docking analysis.
Hans Raj Bhat: Data analysis and interpretation; Writing - review & editing.
Udaya Pratap Singh: Conceptualization Funding acquisition; Investigation; Writing - review
& editing.
11

All authors approved the final version of the manuscript.
Conflict of Interest
Authors declare no conflict of interest.
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Figure captions
Scheme 1: Synthesis of 1,3,5-triazine-thiadiazole hybrid derivatives. Reagents and condition:
a) liq. NH3, 0
̊
̊
c) reflux; d) H2SO4, heating, 50-70
̊
̊
15

Figure 1: Various A2A receptor antagonists.
Figure 2: Orientation of compound 7e after docking into the active site of A2A receptor (Ligand
7e shown as ball and stick, protein shown as solid ribbon; pdb: 3EML).
16

Figure 3: Docked pose of compound 7e along with neighbouring and interacting amino acid
residues of A2A receptor.
17

Figure 4: 2-D interaction diagram of compound 7e in the active site of A2A receptor.
18