Synthesis, adenosine receptor binding and molecular modelling studies of novel thieno[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1111/cbdd.13155

A series of new molecules containing a thieno[2,3-d]pyrimidine scaffold was synthesized and characterized by adopting an efficient synthetic scheme. The effect of a free or substituted amino group at 2-position as well as an oxo-group, imidazole or 1,2,4-triazole ring at 4-position of the scaffold on the affinity and selectivity towards adenosine receptors (ARs) was evaluated. Compounds 17–19 with a free amino group at 2-position along with the presence of an imidazole/1,2,4-triazole ring at 4-position of the scaffold showed selective binding affinities for hA_2A AR, whereas carbamoylation of the amino group at 2-position (in the presence of an oxo-group at 4-position of the scaffold) increased the affinity and selectivity of certain compounds (7–10) for hA₃ AR. Molecular dynamic simulation study of one of the most active compound 8 (K_i hA₁?>?30?μm, hA_2A?=?0.65?μm, and hA₃?=?0.124?μm) revealed the role of important amino acid residues for imparting good affinity towards hA₃ and hA_2A ARs. Molecular docking studies were carried out for other compounds using the crystal structure of hA_2A AR and a homology model of hA₃ AR to rationalize their structure–activity relationships. The molecular docking results were in agreement with the experimental binding affinity data of ARs.

Full text of DOI:10.1111/cbdd.13155

DR PRAN KISHORE DEB (Orcid ID : 0000-0002-8650-2874)
Article type : Research Letter
Synthesis, Adenosine Receptor Binding and Molecular Modeling
Studies of Novel Thieno[2,3-d]pyrimidine Derivatives
1
*
2
1
Pran Kishore Deb, Raghuprasad Mailavaram, Balakumar Chandrasekaran, Venkata Rao
1
1
3
3
4
Kaki, Rajwinder Kaur, Sonja Kachler, Karl-Norbert Klotz, Raghuram Rao Akkinepally **
1
Pharmaceutical Chemistry Division, University Institute of Pharmaceutical Sciences and
UGC Centre of Advanced Study in Pharmaceutical Sciences (UGC-CAS), Panjab University,
*
Chandigarh-14, India; Faculty of Pharmacy, Philadelphia University-Jordan, P.O BOX (1),
Philadelphia
University
(19392)
-
Jordan;
pdeb@philadelphia.edu.jo;
prankishore1@gmail.com
2
Pharmaceutical Chemistry Division, Sri Vishnu College of Pharmacy, Vishnupur,
Bhimavaram-02, Andhra Pradesh, India;
3
Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9,
9
*
7078 Würzburg, Germany;
4
* Medicinal Chemistry Division, University College of Pharmaceutical Sciences, Kakatiya
University, Warangal-09, India; raghumed@gmail.com
Running Title: Thieno[2,3-d]pyrimidines as ARs antagonists
ABSTRACT
A series of new molecules containing a thieno[2,3-d]pyrimidine scaffold was synthesized and
characterized by adopting an efficient synthetic scheme. The effect of a free or substituted
amino group at 2-position as well as an oxo-group, imidazole or 1,2,4-triazole ring at 4-
position of the scaffold on the affinity and selectivity towards adenosine receptors (ARs)
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13155
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were evaluated. Compounds 17-19 with a free amino group at 2-position along with the
presence of an imidazole/1,2,4-triazole ring at 4-position of the scaffold showed selective
binding affinities for hA2A AR whereas carbamoylation of the amino group at 2-position (in
presence of an oxo-group at 4-position of the scaffold) increased the affinity and selectivity
of certain compounds (7-10) for hA
3
AR. Molecular dynamics simulation study of one of the
most active compound 8 (K hA > 30 µM, hA = 0.65 µM, and hA = 0.124 µM) revealed
i
1
2A
3
3
the role of important amino acid residues for imparting good affinity towards hA and hA2A
ARs. Molecular docking studies were carried out for other compounds by using the crystal
structure of hA2A AR and a homology model of hA AR to rationalize their structure activity
3
relationships. The molecular docking results were in agreement with the experimental
binding affinity data of ARs.
Key Words: Thieno[2,3-d]pyrimidine, Adenosine receptors binding, Homology modeling,
Molecular docking, Molecular dynamics.
INTRODUCTION
Adenosine receptors (ARs), classified as A , A , A and A subtypes, belong to the super
1
2A
2B
3
[1]
family of G-protein coupled receptors (GPCRs). These receptors have received great
interest as targets for therapeutic intervention in many pathological conditions by virtue of
their multifarious involvement in a variety of cellular processes. The A and A ARs are
1
3
coupled to the G
and A2B ARs stimulate adenylate cyclase via a G
different second messenger systems have been described, including the stimulation of
phospholipase C (A , A2B, and A ) as well as the activation and inhibition of potassium and
calcium channels (A ), respectively.
i
and G
o
proteins, respectively, which inhibit adenylate cyclase, while A2A
s
protein. Additional interactions with
1
3
[2]
1
In particular, A
peripheral tissues. The antagonists of A
stimulatory drugs and for the treatment of cognitive disorders. They are also currently
1
ARs are expressed in the central nervous system (CNS) as well as in
1
ARs have been found to be useful as CNS-
[
3]
4]
[
utilized in development of drugs for the treatment of asthma, cardiac arrhythmia and
[
5,6]
congestive heart failure, renal failure, renal dysfunction, nephritis, hypertension etc.
The
A2A receptor antagonists may be useful for the treatment of neurodegenerative movement
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disorders such as Parkinson’s and Huntington’s disease, and dystonia’s such as restless leg
syndrome, dyskinesias such as those caused by prolonged use of neuroleptic and
[
7]
dopaminergic drugs. A2B receptor antagonists are found to be useful for the treatment of
[
4,5,8,9]
type 2 diabetes and asthma.
CVT-6883, a potent and selective A2B receptor antagonist
was found to be efficacious in various animal models of asthma, chronic obstructive
pulmonary disease (COPD) and pulmonary fibrosis and reported as the first A2B specific
[
8,9]
antagonists entered in clinical trials.
Highly selective A AR antagonists are being investigated as potential antiasthmatic, anti-
3
[10]
inflammatory, antiglaucoma, and cerebroprotective agents. They have also been described
as potential therapeutics in the treatment of glioblastoma multiforme, colon cancer, and renal
[4,5,10,11]
injury.
specifically in the treatment of inflammation and asthma, is yet to be established. In recent
years, much effort have been directed toward searching for potent and selective human A
However, the potential role of these receptor modulators concerning
[4]
3
adenosine receptor antagonists but only a few such compounds are currently undergoing
clinical trials for the treatment of several diseases like inflammation, rheumatoid arthritis,
[10,11]
cancer and glaucoma respectively.
This is because of their undesirable side effects due to
the wide distribution of ARs, low water solubility of the compounds, or lack of effects in
[
10,11]
some cases perhaps due to low receptor density in the targeted tissue.
Hence, the design and development of potent and selective AR antagonists has been the
subject of intense medicinal chemistry research. Furthermore, the pyrimidine core, being part
of the heterocyclic moiety of the endogenous ligand of these receptors (adenosine), is a
[
4,5,10,11]
recurrent substructural motif within bi- and tricyclic ARs antagonists.
Based on these
series of
and pyrido[2,3-
[
12]
observations, we earlier designed,
synthesized and reported
a
[13]
[14]
triazolothienopyrimidines,
2-amino[1,2,4]triazolo[1,5-c]quinazolines
[15]
d]pyrimidines as possible ARs antagonists. In continuation to our ongoing efforts, herewith
we are reporting the synthesis and evaluation of a series of new thieno[2,3-d]pyrimidine
derivatives as potent ARs antagonists.
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RESULTS AND DISCUSSION
Chemistry
As illustrated in Scheme 1, all the compounds 2-(substituted)aminothieno[2,3-d]pyrimidin-
4(3H)-ones (4-14) and 2,4-(disubstituted)aminothieno[2,3-d]pyrimidines (17-21) were
[
16]
synthesized from 2-amino-3-carbethoxythiophenes 1-3 which on a reaction with excess of
cyanamide in the presence of concentrated hydrochloric acid underwent cyclization to form
2-aminothieno[2,3-d]pyrimidin-4(3H)-one (4-6) quantitatively. When we attempted to
[
17]
synthesize these compounds (4-6) by using known procedure,
the product yield was found
°
to be very low in our hand (15%). Our method of carrying this reaction between 60-70 C was
found to be facile, economic, less time consuming and superior as compared to earlier
reported
procedures.
Compounds
4-6
on
a
further
reaction
with
acetyl/benzoyl/trifluoroacetyl/sulfonyl chlorides in basic medium (pyridine) resulted in the
formation of target compounds 7-14 in quantitative yields. The chlorination of compounds 4
and 6 with excess of phosphorus oxychloride (POCl ) resulted in the formation of respective
3
chloro-derivatives (15 and 16) in 20-25% yield. The intermediate compounds 5, 6 and 16 are
reported and their physical as well as spectral data corroborated well with literature
[17-19]
reports.
The compounds 15 and 16 on further reaction with 1H-imidazole/1,2,4-triazole
in basic medium (pyridine) underwent nucleophilic substitution reaction to form the target
compounds 17-19 in quantitative yields. Furthermore, the compound 19 on reaction with
benzoyl chloride / 4-methylphenylsulfonyl chloride in pyridine resulted in the formation of
the compounds 20 and 21 in quantitative yield. All the synthesized compounds were
1
13
characterized by IR, H NMR, C NMR, MS and elemental analysis. Figures S1-S13
1
13
represents the H-NMR, C-NMR, MS-ESI, and LCMS spectra of some of the representative
synthesized compounds 4-6, 8, 13, 15, 17, and 18, respectively.
Biological Activity
The newly synthesized compounds (4-14 and 17-21) were screened to evaluate their binding
1
affinity as well as selectivity towards human A , A2A, and A
3
ARs (expressed in CHO cells)
3
3
3
by using three different radioligands [ H]CCPA (A
1
), [ H]NECA (A2A) and [ H]HEMADO
3
(A ) respectively. The AR binding affinity data of all the compounds 4-14 and 17-21 are
presented in Table 1. A possible agonistic activity of these compounds towards hA2B AR was
determined by stimulation of adenylyl cyclase activity, through the measurement of the
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inhibition of NECA-stimulated adenylyl cyclase activity an antagonistic appearance should
be discovered. Data are not shown in Table 1 as none of the compounds showed measurable
interaction with the hA AR in an agonistic or antagonistic fashion (EC or IC values > 60
2
B
50
50
µM).
All the derivatives exhibited poor affinity as well as selectivity towards hA AR. The most
1
active compounds toward hA
1
AR (4, 6, 14 and 18) shown potency (K
ARs.
i
) at the range of 4.4-
1
0.4 µM without exhibiting any selectivity against hA2A, and hA
3
Concerning hA2A AR, compound 8 was found to be the most active and selective against hA
AR, but having less affinity as well as selectivity as compared to hA AR (K hA > 30 µM,
hA2A = 0.65 µM and hA = 0.124 µM). Similarly, compounds 12-14 with benzamido,
1
3
i
1
3
trifluoroacetamide and sulfonamide linkage at 2-position (C
affinity towards hA2A AR with less affinity as well as selectivity against hA
whereas compound 11 with acetamide moiety (K hA > 100 µM, hA2A = 4.58 µM, and hA
.93 µM) was found to have moderate affinity towards hA2A AR with better selectivity
against hA AR and less selectivity against hA AR. Compounds 4 and 6 with a free amino
group at 2-position (C ) of the thieno[2,3-d]pyrimidine-4(3H)-one scaffold were found to
have poor affinity as well as selectivity towards hA2A AR as compared to hA and hA ARs.
Interestingly, introduction of an imidazole/1,2,4-triazole moiety at 4-position (C ) while
retaining a free amino group at 2-position (C ) of the thieno[2,3-d]pyrimidine scaffold
nullified the affinity of compounds 17-19 towards hA AR and hence exhibited enhanced
hA2A AR selectivity versus hA and hA ARs with moderate selectivity against hA AR.
2
) of the scaffold showed poor
1
and hA ARs,
3
i
1
3
=
1
1
3
2
1
3
4
2
3
2B
3
1
At the hA
3
AR subtype, the compounds 8, 10 and 12 with benzamide moiety at 2-position of
the scaffold showed better affinity for hA AR with good selectivity against hA , hA and
3
1
2A
hA2B ARs as compared to acetamide and sulfonamide analogues 7, 9, 11, 13, 14, 20 & 21,
respectively. It is interesting to observe that the replacement of methyl groups at 5- and 6-
positions with a cyclopentyl and cyclohexyl ring while retaining the benzamido group at 2-
position of the thienopyrimidinone scaffold resulted in enhanced selectivity (with little
decrease in affinity) towards hA
3
AR against hA1, and hA2A ARs for compound 10 (K
i 1
hA >
3
0 µM, hA2A > 30 µM, and hA = 0.59 µM) and 12 (K hA = 15.5 µM, hA = 18.1 µM, and
3
i
1
2A
hA = 0.32 µM) as compared to the compound 8. Similarly, introduction of a triazole moiety
3
at 4-position and the replacement of cyclopentyl ring with a cyclohexyl ring while retaining
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the benzamido group at 2-position for compound 20 caused a further decrease in affinity
towards hA AR without losing selectivity against hA1, and hA2A ARs (K hA > 30 µM, hA2A
30 µM, and hA3 = 4.8 µM). The introduction of trifluoroacetamide and p-
3
i
1
>
methylbenzenesulfonamido groups at the 2-position of the thienopyrimidinone scaffold while
retaining the cyclohexyl ring in compounds number 13 and 14 also resulted in decreased
affinity as well as hA AR selectivity against hA and hA ARs. In case of sulfonamide
3
1,
2A
derivatives, the presence of 1,2,4-triazolo moiety at 4-position of the thienopyrimidine
scaffold further decreased the affinity towards hA AR in compound 21 (K hA > 30 µM,
hA2A > 30 µM, and hA = 9.63 µM) without compromising the selectivity as compared to
3
i
1
3
i 1 3
compound 14 (K hA = 8.01 µM, hA2A = 8.20 µM, and hA = 1.67 µM).
In case of acetamide derivatives (7, 9 and 11), the replacement of methyl groups from 5- and
-positions of the compound 7 (K hA > 30 µM, hA2A > 30 µM, and hA = 0.524 µM) with
alicyclic cyclopentyl ring reduced the affinity towards hA AR for compound 9 (K hA > 100
µM, hA2A > 30 µM and hA = 1.348 µM) while retaining the selectivity whereas the fusion of
cyclohexyl ring with thienopyrimidine moiety induced moderate affinity towards hA2A AR
with compromised selectivity for compound 11 (K hA > 100 µM, hA2A = 4.58 µM and hA
1.93 µM).
6
i
1
3
3
i
1
3
i
1
3
=
Molecular Modeling
Molecular docking studies were carried out for compounds 4-14 and 17-21 to evaluate their
hypothetical binding mode as well as to rationalize their structure-activity relationships by
using the X-ray crystal structure of human A2A AR (PDB ID: 4EIY) and homology model of
hA
Schrodinger. Docking studies using the homology models of A
performed as these compounds (4-14 and 17-21) did not show any noteworthy experimental
binding interactions with these receptors. Homology model of A AR was constructed by
3
AR respectively by employing the GLIDE XP and Induced Fit Docking (IFD) module of
1
and A2B ARs were not
3
using the X-ray crystal structure of human A2A AR (PDB ID: 4EIY) as an appropriate
template employing the Prime module of Schrodinger.
3
Docking simulation at the hA2A and hA ARs revealed that the binding mode of all the
compounds (4-14 and 17-21) varied depending on the substituents at 2 and 4-position of the
thieno[2,3-d]pyrimidine scaffold. Mainly hydrogen bonding (H-bonding) and hydrophobic
interactions were found to play an important role for the binding affinity of ligands. The best
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pose of ligand-receptor complex of the most potent compound 8 obtained after IFD
simulation against hA2A and hA ARs were further subjected to molecular dynamics
simulation study for 50 ns to explore the stability and binding interaction of compound 8 with
hA2A and hA ARs respectively by using Desmond software. In case of molecular dynamic
simulation of A AR, the major stability contribution was observed from Asn236 which
3
3
3
formed stable hydrogen bonding interaction with the cyclic NH at 3-position and the carbonyl
group (C=O) of the amide substituent at 2-position of the thieno[2,3-d]pyrimidine scaffold
for 83% and 61% of the simulation time as shown in Figure 1B and S14E. Similarly, Leu232
was also found to form a stable hydrogen bonding interaction with the NH group of the amide
substituent for 74% of the simulation time. The amino acid residue Phe168 formed a stable π-
π stacking interaction with the thienopyrimidine moiety for 59% of the simulation time. Two
of the amino acid residues Val169 and Ser181 participated in the formation of bridge H-
bonding through water molecules with the carbonyl group (C=O) at 4-position of the scaffold
as well as with the carbonyl group (C=O) of the amide substituent respectively. The Root
Mean Square Fluctuation (RMSF) characterized the local changes along the protein chain
where it has been observed that the secondary structure elements like alpha helices and beta
strands were more rigid than the unstructured part of the protein and thus showed less
fluctuation as compared to loop region (Figure S14D). The protein-ligand Root Mean Square
3
Deviation (RMSD) between compound 8 and hA AR was found to be of 1-3 Å during the
entire simulation run demonstrating that the protein had undergone minor conformational
change during the simulation as well as compound 8 and the receptor were very compatible
with each other (Figure S14F). Molecular dynamic simulation study of compound 8 inside
the hA2A AR (Figure 1A, S14A-C) revealed that the compound occupied a similar position as
3
in the hA AR and formed stable hydrogen bonding interaction between the cyclic NH as
well as amide NH with Glu169 (95% of the simulation time). The amino acid residues
Phe168 and His264 also formed vital π-π stacking interaction with the thienopyrimidine
moiety and the phenyl ring respectively for 39% of the simulation time. Further, the RMSD
between compound 8 and the A2A AR was also found to be stable (1-3 Å) during the entire
simulation run. This has led to the poor selectivity of compound 8 over A2A AR.
3
The binding mode of the other benzamide analogues 10 and 12 to hA AR showed that the
increase in bulkiness by fusing cyclopentyl and cyclohexyl moiety with the thiophene ring of
the scaffold caused little change in their binding orientation which resulted in the formation
of additional π-π stacking interaction with His95. The compounds 10 and 12 could not form
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H-bonding interactions with Val169 and Leu232, respectively, which could be the reason for
its decrease in affinity for hA AR as compared to compound 8. Interestingly, only compound
2 was found to form an additional H-bonding interaction between the carbonyl group (C=O)
3
1
of the benzamide moiety with the OH group of Ser233 which could be the reason for its
better affinity as compared to the compound 10 (Figure 1D) Both the cyclic NH at 3-position
and amide NH at 2-position of the scaffold were also found to form H-bonding interaction
with the carbonyl group (C=O) of backbone amino acid residue Leu90. In case of compound
20, introduction of a triazole ring at 4-position while retaining the benzamide substituent at 2-
position of the scaffold caused change in binding orientation and rotation of the scaffold
towards counter-clockwise direction where it could form only two π-π stacking interactions
between the thienopyrimidine scaffold and the Phe168 as well as between the phenyl ring and
His95, respectively and a moderate H-bonding interaction with Asn236 which resulted in the
further loss in affinity towards hA
3
AR. Binding interactions of these compounds 10, 12 and
20 inside the binding pocket of hA2A AR revealed that they anchored inside the binding
pocket by forming a π-π stacking interactions between phenyl ring of the benzamide moiety
with the phenyl ring of Tyr271 except compound 12 which also formed an additional π-π
stacking interaction between thienopyrimidine moiety and the phenyl ring of Phe168 (Figure
1C). Further, these compounds (10, 12 and 20) could not form any distinguished interaction
with the crucial amino acid residues Glu169 and Asn253, respectively, which probably
resulted in the lack of their notable affinity towards hA2A AR.
The 4-methylbenzenesulfonamide analogues (14 and 21), were found to be embed inside the
hydrophobic pocket of A AR where the compound 14 could form only one H-bonding
3
interaction between the cyclic amide group and Asn236 but failed to form π-π stacking
interactions with Phe168 and His95 which could be the reason for its lower affinity for hA
3
AR. In case of compound 21, a π-π stacking interaction was observed between the 1,2,4-
triazole ring and Try251. Binding interaction of both these compounds inside the binding
pocket of hA2A AR showed that the compound 14 embedded inside the hydrophobic pocket,
forming H-bonding interaction with Asn236 whereas the compound 21 anchored away from
the main binding pocket and failed to interact with any crucial amino acid residues required
for binding affinity.
3
The binding interaction of compound 7 with hA AR (Figure S15B) showed that the
heterofused thiophene ring formed a π-π stacking interaction with the phenyl ring of Phe168
This article is protected by copyright. All rights reserved.

1
and the cyclic N -atom of the thienopyrimidine scaffold formed a moderate H-bond
interaction with Asn236. Both the cyclic NH at 3-position and amide NH at 2-position of the
scaffold were found to form H-bonding interaction with Leu90 instead of Asn236 which
could be the reason for its lower affinity as compared to compound 8. In case of hA2A AR,
the compound 7 (Figure S15A) formed H-bonding interaction between the cyclic NH at 3-
position and amide NH at 2-position with Asn236 but it could not form vital H-bonding and
π-π stacking interactions with the crucial amino acid residues Glu169 and Phe168,
respectively, which could be the reason for its lack of notable affinity for hA2A AR. Similar
binding interactions were also observed for other acetamide derivatives (compounds 9 and
3
11) and trifluoroacetamide derivative (compound 13) with hA2A and hA ARs, respectively.
The compound 17 (with unsubstituted amino group) anchored well inside the binding pocket
of A2A AR by forming significant H-bonding interaction between its NH group and the
2
crucial amino acid residues Glu169 and Asn253 respectively. The heterofused thiophene
moiety formed a vital π-π stacking interaction with Phe168 whereas the 1,2,4-triazole ring
3
formed a moderate π-π stacking interaction with Tyr271 while (Figure 1E). In case of A AR
°
(
Figure 1F) the free NH
2
group allowed the scaffold to rotate by 90 in clockwise direction
within the binding pocket as compared to other amide derivatives such as compounds 7 and
2 (as shown in Figure 1D and 1F). As a result it could only form H-bonding interactions
between the NH group and cyclic N-atom (N ) of the thienopyrimidine moiety with Asn236
while losing essential binding interactions with other crucial amino acid residues such as
Leu90, and Phe168, respectively, which could explain its loss of affinity towards hA AR.
1
2
3
3
Similar observations were also evident for compounds 18 and 19 with a free NH group at 2-
2
position of the scaffold which made them more selective towards A2A AR as compared to
other receptors. The compounds 4-6 with a free amino group at 2-position and an oxo-group
at 4-position were found to be highly non-selective for all the receptors and could not form
essential binding interactions with the crucial amino acid residues required for higher
activity. The above observations revealed that the compounds with carbamoyl substitution at
NH group at C position of the pyrimidine ring (compounds 7-12) exhibited better affinity
2
2
3
with selectivity towards hA AR against other ARs as compared to compounds with an
unsubstituted NH group. Further, the presence of imidazole/1,2,4-triazole ring at 4-position
2
along with a free amino group at 2-position of the thieno[2,3-d]pyrimidine scaffold made the
compounds 17-19 more selective towards hA2A AR as compared to other adenosine receptors.
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CONCLUSIONS
In conclusion, we have synthesized and characterized a series of thieno[2,3-d]pyrimidine
derivatives by using an appropriate synthetic scheme. The AR binding affinity data of all the
compounds showed that the thieno[2,3-d]pyrimidine scaffold with amide linkage (7-14, 20
2 3
and 21) at 2-position (C ) exhibited good affinity as well as selectivity towards hA AR as
compared to compounds with un-substituted amino group (4-6 and 17-19). Further,
introduction of imidazole or triazolo moieties at 4-position with free amino group at 2-
position of the scaffold drastically narrowed down or nullified the affinity of compounds 17-
1
9 towards hA
against hA AR. Interestingly, none of the compounds showed any noteworthy affinity
towards hA AR (except compounds 4 and 6 with moderate affinity, K 4.4 µM and 6.8 µM,
3
AR and enhanced the affinity towards hA2A AR with moderate selectivity
1
1
i
respectively). Similarly, none of the compounds showed detectable interaction with hA2B AR.
Molecular dynamics simulation study of the most active compound 8 revealed that the amino
acid residues Phe168, Val 169, Asn236 and Leu232 played an important role by forming π-π
stacking interaction and H-bonding interactions for imparting good affinity inside the binding
3
pocket of hA AR; whereas hydrophobic and H-bonding interactions with the amino acid
residues Phe168, Glu169 and His264, respectively, played a crucial role for binding to the
hA2A AR. Further, molecular docking studies for other compounds rationalized their structure
activity relationships. The preliminary results of these novel series of compounds
demonstrate that the thieno[2,3-d]pyrimidine can be considered as a promising scaffold for
further optimization in search of potential antagonists with better affinity and selectivity
3
towards hA2A and hA ARs.
ACKNOWLEDGEMENTS
One of the authors (PKD) thanks Council of Scientific and Industrial Research (CSIR), New
Delhi for awarding the Senior Research Fellowship (SRF) [F. No. 09/135/(0534)/2008/EMR-
I]. We would also like to thank Mr. Vinod Devaraji, and Dr. Ravikumar, application
scientists, Schrodinger, India for supporting us with the evaluation license, and sincere
guidance to carry out the homology modeling, docking and molecular dynamics simulation
studies.
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
FIGURE LEGEND
Figure 1: Explicit molecular dynamics study of compound 8 in 50 ns: (A) Ligand interaction
pattern showing percentage of contacts with amino acid residues of A2A AR. (B) Ligand
interaction pattern showing percentage of contacts with amino acid residues of A AR.;
3
Ligand-receptors binding interactions generated after Glide docking studies (IFD): (C)
Hypothetical binding mode of compound 12 in inside the binding site of hA2A AR. (D)
Hypothetical binding mode of compound 12 in inside the binding site of hA
Hypothetical binding mode of compound 17 in inside the binding site of hA2A AR. (F)
Hypothetical binding mode of compound 17 in inside the binding site of hA AR. Ligands are
3
AR. (E)
3
represented in tube format and amino acid residues are represented in thin tube format.
Nonpolar H-atoms are omitted to provide clear visualization of the binding interactions
(poses).
Scheme
Scheme 1. (a) NH CN, conc. HCl, 60-70 °C, 6h; (b) R COCl / R SO Cl anhydrous pyridine,
2
3
3
2
3
0-5 °C, 1-5h; (c) POCl , 110 °C, 2-8h; (d) 1,2,4-triazole/imidazole, anhydrous pyridine, 90
°
C; (e) R COCl / R SO Cl, anhydrous pyridine, 0-5 °C, 2-5h.
3 3 2
Table
Table 1: Binding affinity (K
i 1
) of synthesized compounds (4-14 and 17-21) at hA , hA2A and
hA ARs and selectivity against hA and hA ARs.
3
1
2A
SUPPORTING INFORMATION
Appendix S1. Methods and materials (details) for synthesis and characterization data of
compounds, biological activity and molecular modeling studies.
1
13
Figures: S1-S13 Representative H-NMR, C-NMR, MS-ESI, and LCMS spectra of
synthesized compounds 4-6, 8, 13, 15, 17, and 18, respectively.
Figure S14: Explicit molecular dynamics study of compound 8 in 50 ns: Root Mean Square
Fluctuation (RMSF), bar-diagram for interactions, Root Mean Square Deviation (RMSD) of
3
ligand with A2A and A ARs.
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Figure S15: (A) Hypothetical binding mode of compound 7 in inside the binding site of hA2A
AR. (B) Hypothetical binding mode of compound 7 in inside the binding site of hA AR.
3
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1
1
8. K.M. Abu-Zied, A.E.M. Gaafar, A.S. Aly, Phosphorus Sulfur and Silicon. 2007, 182,
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4
O
O
O
2
OC H5
NH
N
(b)
NH
N
(a)
R1
R1
R1
R2
NH2
NHR
3
NH2
S
R2
S
R2
S
(
1-3)
(4-6)
(7-14)
(
c)
N
X
Cl
S
N
N
N
(d)
N
N
R1
R2
R1
R2
NH2
NH2
S
(17-19)
(15-16)
(
e)
Comp. No.
R1
R2
R3
X
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
-CH3
-CH3
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
N
C
N
N
N
N
-(CH ) -
-(CH ) -
-CH3
-(CH ) -
-(CH ) -
-CH3
-CH3
-(CH ) -
-(CH ) -
-(CH ) -
-(CH ) -
-(CH ) -
-(CH ) -
-CH3
2
3
X
N
2
4
N
R1
S
-CH3
2
3
R2
2
4
N
NHR3
-CH3
-CH3
-COCH3
-COC H5
-COCH3
-COC H5
-COCH3
-COC H5
-COCF3
-SO -p-CH -C H
--
--
--
--
--
6
(
20-21)
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
6
2
4
2
4
6
2
4
2
4
2 3 6 4
-CH3
-(CH ) -
-CH3
2
4
-CH3
-(CH ) -
-(CH ) -
-(CH ) -
-(CH ) -
2
4
2
4
-COC H5
-SO -p-CH -C H
2 3 6 4
2
4
6
2
4
Scheme 1. (a) NH CN, conc. HCl, 60-70 °C, 6h; (b) R COCl/R SO Cl anhydrous pyridine, 0-5 °C, 1-5h;
2
3
3
2
(
c) POCl , 110 °C, 2-8h; (d) 1,2,4-triazole/imidazole, anhydrous pyridine, 90 °C;
3
(
e) R COCl/R SO Cl, anhydrous pyridine, 0-5 °C, 2-5h.
3
3
2
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Table 1: Binding affinity (K
i
) of synthesized compounds (4-14 and 17-21) at hA
1
, hA2A and
hA ARs and selectivity against hA
3
1
and hA2A ARs.
N
X
O
N
R1
S
R
1
NH
NHR3
NH
R2
R
2
S
N
N
NHR
3
(4-14)
(17-21)
Selectivity
i
K , µM (95 % Cl)
CN
R
1
R
2
R
3
X
hA
hA2A
1
/
hA
hA
1
/
hA2A
/
hA3
a
b
c
hA
1
hA2A
hA
3
3
4
5
6
7
8
9
-CH3
-(CH
-(CH
-CH3
-CH
-(CH
-CH3
H
H
H
-
4.40
3.80-5.09)
22.6
(17.5-29.0)
6.56
(5.45-7.76)
3.55
0
.19
> 1.0
.72
0.68
> 8.44
6.5
3.5
(
(
2
)
3
4
-
-
-
>
30
> 30
> 8.44
9.1
(2.74-4.61)
2
)
-
6.90
6.09-7.81)
9.59
(8.15-11.3)
1.06
(0.984-1.14)
0.524
0
-CH3
-CH
-COCH3
-COC
-COCH
-COC H
-
>
>
30
30
> 30
> 1.0
> 46
> 3.3
> 1.0
> 22
> 57
> 240
> 74
> 57
> 52
48
> 57
5.24
> 22
> 57
2.4
(
0.444-0.619)
3
3
6
H
5
5
5
-
0.650
0.585-0.722)
0.124
(0.100-0.154)
1.35
(
2
)
3
-
3
-
>
100
30
> 30
(1.09-1.66)
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
7
8
9
0
1
-(CH ) -
-
0.529
0.348-0.805)
1.93
(1.43-2.61)
0.320
(0.241-0.425)
1.71
(1.17-2.49)
1.67
(1.51-1.85)
> 30
2
3
4
4
6
>
> 30
4.58
4.02-5.21)
18.1
(
-(CH
2
)
)
-
-
-COCH
-COC
-COCF3
3
-
>
100
(
-(CH
2
6
H
-
15.5
13.0-18.6)
10.2
6.14-17.0)
8.01
4.42-14.5)
40.8
24.9-67.0)
10.4
6.42-16.7)
29.7
0
.86
> 0.34
.98
57
(
(
(
(
(
(
(9.09-35.9)
30
8.21
-(CH ) -
-
2
4
>
6.0
> 18
4.9
-(CH
2
)
4
-
-SO
2
-p-CH
3
-C
6
H
5
-
0
4.8
(6.64-10.10)
-CH
3
-CH
3
H
N
C
N
N
N
5.69
(5.02-6.44)
4.06
(3.24-5.09)
4.55
(4.19-4.94)
> 30
7
.2
.6
.5
> 1.4
> 0.35
> 0.99
> 6.3
> 3.1
> 0.19
> 0.14
> 0.15
> 6.3
> 3.1
-(CH ) -
H
> 30
> 30
4.80
2
4
4
4
4
2
-(CH
-(CH
-(CH
2
)
)
)
-
-
-
H
6
27.5-32.0)
> 30
2
6 5
-COC H
>
>
1.0
1.0
(3.34-6.90)
2
-SO
2
-p-CH
3
-C
6
H
5
> 30
> 30
9.63
(7.63-12.2)
a
b
c
3
Displacement of specific [ H]-CCPA binding at human A AR expressed in CHO cells (n = 3-6).
Displacement of specific [ H]-NECA binding at human A2A AR expressed in CHO cells (n = 3-6).
Displacement of specific [ H]-HEMADO binding at human A
1
3
3
3
AR expressed in CHO cells (n = 3-6).
Data are expressed as geometric means, with 95% confidence limits in parentheses.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.