Design and synthesis of novel xanthine derivatives as potent and selective A2B adenosine receptor antagonists for the treatment of chronic inflammatory airway diseases

Article abstract of DOI:10.1016/j.ejmech.2017.04.014

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A_2B adenosine receptor (A_2BAdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory cond

Full text of DOI:10.1016/j.ejmech.2017.04.014

Accepted Manuscript
Design and synthesis of novel xanthine derivatives as potent and selective A
adenosine receptor antagonists for the treatment of chronic inflammatory airway
diseases
2B
Sujay Basu, Dinesh A. Barawkar, Vidya Ramdas, Meena Patel, Yogesh Waman, Anil
Panmand, Santosh Kumar, Sachin Thorat, Minakshi Naykodi, Arnab Goswami, B.
Srinivasa Reddy, Vandna Prasad, Sandhya Chaturvedi, Azfar Quraishi, Suraj Menon,
Shalini Paliwal, Abhay Kulkarni, Vikas Karande, Indraneel Ghosh, Syed Mustafa,
Siddhartha De, Vaibhav Jain, Ena Ray Banerjee, Sreekanth R. Rouduri, Venkata P.
Palle, Anita Chugh, Kasim A. Mookhtiar
PII:
S0223-5234(17)30268-4
10.1016/j.ejmech.2017.04.014
EJMECH 9361
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 November 2016
Revised Date: 1 April 2017
Accepted Date: 7 April 2017
Please cite this article as: S. Basu, D.A. Barawkar, V. Ramdas, M. Patel, Y. Waman, A. Panmand,
S. Kumar, S. Thorat, M. Naykodi, A. Goswami, B.S. Reddy, V. Prasad, S. Chaturvedi, A. Quraishi, S.
Menon, S. Paliwal, A. Kulkarni, V. Karande, I. Ghosh, S. Mustafa, S. De, V. Jain, E.R. Banerjee, S.R.
Rouduri, V.P. Palle, A. Chugh, K.A. Mookhtiar, Design and synthesis of novel xanthine derivatives as
potent and selective A adenosine receptor antagonists for the treatment of chronic inflammatory
2B
airway diseases, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.014.
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ACCEPTED MANUSCRIPT
Graphical abstract
Optimization
29
46
hA2B binding Ki = 13.0 nM
hA2B functional Ki = 8.0 nM
No CYP and hERG liability, not cytotoxic,
%F = 76
hA2B binding Ki = 3.6 nM
hA2B functional Ki = 8.8 nM
%F = 42
Showed efficacy in Ovalbumin treated mice
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Design and Synthesis of Novel Xanthine Derivatives as Potent and Selective A2B Adenosine
Receptor Antagonists for the treatment of Chronic Inflammatory Airway Diseases
Sujay Basu , Dinesh A. Barawkar, Vidya Ramdas, Meena Patel, Yogesh Waman, Anil Panmand,
Santosh Kumar, Sachin Thorat, Minakshi Naykodi, Arnab Goswami, B. Srinivasa Reddy,
Vandna Prasad, Sandhya Chaturvedi, Azfar Quraishi, Suraj Menon, Shalini Paliwal, Abhay
Kulkarni, Vikas Karande, Indraneel Ghosh, Syed Mustafa, Siddhartha De, Vaibhav Jain, Ena
Ray Banerjee, Sreekanth R. Rouduri, Venkata P. Palle, Anita Chugh, Kasim A. Mookhtiar
Advinus Therapeutics Ltd., Drug Discovery Facility, Quantum Towers, Plot-9, Phase-I, Rajiv
Gandhi Infotech Park, Hinjawadi, Pune 411 057, India
Abstract. Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through
activation of A2B adenosine receptor (A2BAdoR). Selective antagonists have been shown to attenuate
airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification
of novel, potent and selective A2BAdoR antagonist may be beneficial for the potential treatment of
asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several
prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-
ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding
affinity (Ki) of 62 nM but was non-selective for A2BAdoR over other AdoRs. Incorporation of
Corresponding author. Tel.: +91 20 66539600; fax: +91 20 66539620.
E-mail address: sujay.basu@advinus.com
Corresponding author. Tel.: +91 20 66539600; fax: +91 20 66539620.
E-mail address: kasim.mookhtiar@advinus.com
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substituted phenyl on the terminal acetylene increased the binding affinity (Ki) significantly to <10 nM.
Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were
explored to improve the potency, selectivity for A2BAdoR and the solubility. In general, compounds
with meta-substituted phenyl provided better selectivity for A2BAdoR compared to that of para-
substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or
cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of
xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to
compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high
human A2BAdoR affinity (Ki = 13 nM), selectivity against other AdoR subtypes and with good
pharmacokinetic properties. It was found to be a potent functional A2BAdoR antagonist with a Ki of 8
nM in cAMP assay in hA2B-HEK293 cells and an IC50 of 107 nM in IL6 assay in NIH-3T3 cells.
Docking study was performed to rationalize the observed affinity data. Structure-activity relationship
(SAR) studies also led to identification of compound 36 as a potent A2BAdoR antagonist with Ki of 1.8
nM in cAMP assay and good aqueous solubility of 529 µM at neutral pH. Compound 46 was further
tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in
mice.
Keywords: Adenosine; cAMP; Bioavailability; Propargyl; Heteroaryl; Human liver microsomes
1. Introduction
Adenosine plays numerous important physiological roles via a family of four G-protein coupled
receptors (GPCR), namely A1, A2A, A2B and A3 adenosine receptors (AdoRs) [1]. A1 and A2AAdoRs are
stimulated by low nanomolar adenosine concentrations, while micromolar concentrations of adenosine
are required for the activation of A2B and A3AdoRs [2]. The low affinity AdoRs (A2B and A3) are
activated only under stressful conditions wherein adenosine levels are elevated to micromolar
concentrations. Increased levels of adenosine have been reported under pathophysiological conditions,
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such as hypoxic or ischemic conditions, after massive cell death, or as a consequence of inflammatory
processes [3,4]. Rodent and human A2BAdoRs share 86-87% amino acid sequence homology [5]. The
A2BAdoRs show a ubiquitous distribution, with highest levels observed in the cecum, colon, bladder,
mast cells, hematopoietic cells, and lung, while lower levels are detected in other organs, such as brain
and liver [6].
Apart from the differences in affinity to adenosine, the receptors also differ in their signaling
mechanism. A1 and A3AdoRs mediate inhibition of adenylate cyclase through Gi coupled pathway,
whereas A2B and A2A stimulate adenylate cyclase activity via Gs coupling and result in increased
intracellular cAMP levels [5]. In addition, coupling of A2A and A2BAdoRs to phospholipase C via Gq,
resulting in mobilization of intracellular calcium and direct coupling to calcium channels (stimulation of
calcium influx), have also been reported [7-9].
Adenosine has been shown to cause mast cell degranulation [10], vasodilation [11], chloride secretion
in epithelial cells [12, 13], growth inhibition of smooth muscle cells [14], and stimulation of glucose
production in rat hepatocytes [15] and stimulate VEGF secretion [16] via activation of A2BAdoR.
A2BAdoR antagonists, therefore, can potentially be used as therapeutic agents for the treatment of
asthma and chronic obstructive pulmonary disease [4, 17-19] type II diabetes [15], cystic fibrosis [12],
inflammatory bowel disease [13] and cancer [20].
Adenosine induces bronchoconstriction in asthmatic patients but not in normal people [21, 22]. In
addition, adenosine levels are elevated in the bronchoalveolar lavage fluid and exhaled breath
condensates of asthmatic patients [23, 24] and adenosine deaminase deficient mice show evidence of
bronchioli remodelling and emphysema-like lesions [25,26]. The mechanism of this adenosine-mediated
damage lies with the activation of A2BAdoR on human mast cells, which in turn leads to mast cell
degranulation and release of inflammatory cytokines IL-4, IL-8 and IL-13 [27]. A2BAdoR activation also
increases the expression and release of IL-6 and TNF-α in bronchial epithelial cells [28] and this
indicates that A2BAdoR play a key role in the pathogenesis of inflammatory lung disease. There is also
evidence that A2BAdoR is the predominant AdoR expressed in bronchial smooth muscle cells, and its
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activation increases the expression and release of IL-6 and monocytic chemotactic peptide-1(MCP-1)
[29] and is implicated in airway remodeling. In human lung fibroblasts, activation of A2BAdoR induces
the release of IL-6 and differentiation of fibroblasts to myofibroblasts [30].
On account of all the evidence that implicates A2BAdoR activation in inflammatory lung diseases such
as asthma, COPD and fibrosis, a lot of research has been carried out with the goal of identifying potent
and selective A2BAdoR antagonists to provide a novel approach to the management and treatment of
asthma and COPD.
The alkylxanthines theophylline 1 and enprofylline 2 (Figure 1), are weak, non-selective AdoR
antagonists used therapeutically for the treatment of asthma [31, 32]. The bronchodilating effect of 1 and
2 has been attributed to a selective antagonism of A2BAdoR, which prompted several groups to design
and test a large number of xanthine derivatives in the search for new, more potent and selective A2B
AdoR antagonists. During the past decade, several structure-activity relationship studies of A2BAdoR
antagonists have been published [33-37]. Though several potent and selective A2BAdoR antagonists like
MRS-1754 (3), MRE2029-F20 (4) have been reported, these compounds were highly lipophilic,
possessing very low solubility and poor oral bioavailability [38, 39].
A potent and selective A2BAdoR antagonist CVT-6883 (5) [36, 40] entered into clinics and later
discontinued from Phase I clinical trials. It has shown inhibition of airway inflammation and airway
reactivity induced by allergen or AMP in mouse model of allergic asthma [41]. In addition, compound 5
attenuated pulmonary inflammation, fibrosis, and alveolar airspace enlargement and reduced elevations
of pro-inflammatory cytokines and chemokines, as well as mediators of fibrosis [42].
In our earlier communication we had reported a new series of xanthine derivatives [43]. The objective
of present study was to identify and develop novel, potent and selective A2BAdoR antagonists combined
with good aqueous solubility and improving in vivo half life. As we were interested in synthesizing and
testing novel xanthine derivatives, first we explored different propargylated aryl and heteroaryl groups at
C8 position of xanthine and identified 8-(1-prop-2-ynyl-1H-pyrazol-4-yl)-xanthine as a potential core
for SAR studies. Herein, we discuss the structure-activity relationship (SAR) studies carried out on 8-(1-
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prop-2-ynyl-1H-pyrazol-4-yl)-xanthine towards identification of novel, potent and selective A2B AdoR
antagonists with good oral bioavailability.
O
O
H
N
O
N
H
N
H
N
HN
N
O
O
N
N
N
O
N
N
O
N
CN
O
N
H
1 Theophylline
2 Enprofylline
3 MRS-1754
O
O
H
H
N
N
N
N
N
N
N
H
N
N
N
O
O
N
O
O
N
N
O
O
CF3
4 MRE2029F20
5 CVT-6883
Figure 1. Representative Structures of Xanthine Based A2BAdoR Antagonists
2. Results and discussion
2.1. Chemistry
The key intermediates 8-phenyl-, 8-isoxazolyl-, 8-(1H-pyrazol-3-yl)- and 8-(1H-pyrazol-4-yl)-1,3-
dipropyl xanthines were prepared by classical method starting from 1,3-dipropyl-5,6-diaminouracil 6
(Scheme 1) [36]. The diamino uracil 6 was coupled with phenyl carboxylic acid 7a, isoxazole carboxylic
acid 7b, pyrazole-3- or pyrazole-4-carboxylic acids 7c or 7d, using N-(3-dimethylaminopropyl)-N-
ethylcarbodiimide hydrochloride (EDCI) in methanol at room temperature to obtain corresponding
carboxamide derivatives 8a-d. Subsequent ring closure of these derivatives was performed by refluxing
8a-d with 10% sodium hydroxide in methanol to afford 8-substituted xanthine derivatives 9a-d. The N-
7 position of 9a-d was protected with 2-(trimethylsilyl)ethoxymethylchloride (SEM-Cl) using potassium
5

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carbonate in dimethylformamide to furnish 10a-d which on debenzylation using hydrogen and 10%
Pd/C provided the key intermediates 11a-c with “O” and 11d with “N” as a handle for further
derivatization [36]. These compounds were alkylated with various substituted (3-bromo-prop-1-ynyl)-
benzene derivatives using potassium carbonate in acetone at reflux temperature to provide the silyl
protected xanthine derivatives which on silyl deprotection with 2N HCl furnished the final compounds
12- 40 in good yields (28-99%) Scheme 1.
In a similar fashion, various N-1, N-3 differentially substituted xanthine derivatives were prepared by
starting from 1-ethyl-3-propyl-5,6-diaminouracil 41a or 1H-3-propyl-5,6-diaminouracil 41b [44]. The
key intermediates, 3-ethyl-1-propyl-8-(1H-pyrazol-4-yl)-xanthine 42a or 1-propyl-8-(1H-pyrazol-4-yl)-
xanthine 42b were prepared (Scheme 2) by following the synthetic sequence as described in Scheme 1.
The alkylation of 42a or b with various phenyl substituted propargyl bromides using standard alkylation
conditions followed by N-7 silyl deprotection with 2N HCl furnished 43-56 in good yields (11-85%).
Propargyl and 2-butyne substituted 8-(1H-pyrazol-4-yl)-xanthine derivatives 59 and 60 were prepared
(Scheme 3) by alkylation of intermediate 11d with corresponding bromo compounds using potassium
carbonate in acetone at reflux condition, followed by N-7 deprotection of the resulting silyl protected
compounds 57 and 58 with 2N HCl in ethanol at reflux condition. Compounds substituted with basic
amino moiety on terminal acetylene 61-66 were prepared (Scheme 4) by treating the 1,3-dipropyl-8-(1-
prop-2-ynyl-1H-pyrazol-4-yl)-xanthine 57 with formaldehyde solution and acyclic or heterocyclic
amines in presence of CuI in dimethylsulfoxide at room temperature followed by N-7 silyl deprotection.
The compounds, 8-{1-[3-(1-Hydroxy-cycloalkyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-xanthine derivatives,
67 and 68 were also obtained in a similar fashion. The reaction of intermediate 57 with cyclopentanone
or cyclohexanone in the presence of lithiumhexamethyl disilazide in tetrahydrofuran at low temperatures
followed by N-7 silyl deprotection furnished the compounds 67 and 68, Scheme 4.
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Scheme 1. Synthesis of compounds 12-40 a
O
O
N
O
N
O
N
H
N
O H
H
N
Ar/Het
NH2
NH2
N
N
N
a
b
Ar/Het
Ar/Het
+
O
N
O
O
NH2
O
6
7a-d
8a-d
9a-d
Ar/Het = -Phenyl, Isoxazole, Pyrazole
O
O
Si
O
Si
O
N
O
N
H
N
N
c
d
e, f
N
N
N
N
N
N
A
Ar/Het
Ar/Het
N
O
N
R
O
O
Br
10a-d
11a-d
12-40
O
N
R
O
O
R
12-16
17-21
N
N
A
N
N
R
O
R
22-23
24-40
aReagents and conditions: (a) EDCI, MeOH, r.t.; (b) 10% NaOH, MeOH, reflux; (c) SEM-Cl, K2CO3,
DMF, r.t.; (d) H2, 10% Pd/C, MeOH:DCM (2:1), r.t.; (e) K2CO3, acetone, 50 °C; (f) 2N HCl, ethanol, 80
°C
Scheme 2. Synthesis of compounds 43-49 and 50-56 a
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Si
O
O
O
NH2
NH2
N
N
N
N
Scheme 1
NH
N
O
N
R1
O
N
R1
41a, R1=Et
41b, R1=H
42a, R1=Et
42b, R1=SEM
Br
R
O
H
N
R
N
N
N
a, b
N
O
N
R1
43-49, R1=Et
50-56, R1=H
aReagents and conditions: (a) K2CO3, acetone, 50 °C; (b) 2N HCl, ethanol, 80 °C
Scheme 3. Synthesis of compounds 59-60 a
a
b
11d
R2=H; Me
59, R2=H
57, R2=H
60, R2=Me
58, R2=Me
8

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aReagents and conditions: (a) K2CO3, acetone, 50 °C; (b) 2N HCl, ethanol, 80 °C
Scheme 4. Synthesis of compounds 61-68 a
Si
O
O
N
O
N
H
N
i) a or b
ii) c
N
N
N
N
N
N
N
N
N
O
O
R2
61-68
57
aReagents and conditions: (a) 37% HCHO solution, amine, CuI, DMSO, r.t.; (b) LHMDS,
cyclopentanone or cyclohexanone, THF, -78 to 20 °C; (c) 2N HCl, ethanol, 80 °C
2.2. Biological activity
As we were interested in synthesizing and testing novel derivatives of xanthine with propargylated aryl
and heteroaryl groups at C8 position, we first explored different aryl and heteroaryl groups at the C8
position of xanthine. Herein, we discuss the structure-activity relationship (SAR) of compounds 12-25
with phenyl, isoxazolyl, 3-pyrazolyl and 1H-4-pyrazolyl substitution at the C8 position (Table 1).
Compounds 12-16 having phenyl, pyridyl (structure not shown), and compounds 22, 23 having 3-
pyrazolyl substitutions at C8 position were found to be inactive or showed low binding affinity for
A2BAdoR, indicating that phenyl, pyridyl and 3-pyzolyl are not suitable at C8 of xanthine. These
compounds also showed poor solubility. When isoxazolyl was placed at C8 of xanthine, compounds 17
and 18 with small substitution like m-F and p-F on terminal phenyl ring showed moderate binding
affinity of 55 and 15 nM, respectively, for A2BAdoR. Compounds 19-21 with larger substituents such as
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trifluoroalkyl or trifluoroalkoxy substitution on terminal phenyl ring were inactive or showed low
affinity for A2BAdoR. 4-Pyrazolyl, between C8 of xanthine and m-F and p-F (24 and 25) substituted
terminal phenyl ring had high binding affinity of 6.7 and 5.9 nM, respectively, for A2BAdoR (Table 1).
Based on the binding affinity of compounds 12-25 for A2BAdoR, it was concluded that 4-pyrazolyl ring
is the best structural requirement group at the C8 position. Hence, 8-(1-propargyl-4-pyrazolyl)-1,3-
disubstituted xanthine derivative was selected for further SAR studies to identify a potent, selective and
soluble A2BAdoR antagonist.
The binding selectivity of 8-(1-propargyl-4-pyrazolyl)-xanthine analogs 24 and 25 over other AdoR
subtypes A1AdoR and A2AAdoR is shown in Table 2. These compounds were not very selective for
A2BAdoR. Therefore, to increase the selectivity, we explored various substitutions on the terminal
phenyl ring, including electron donating groups such as methyl and methoxy, electron withdrawing
groups such as trifluoromethyl, trifluoromethyloxy, and cyano and polar carboxylic acids and hydroxyl
groups with the intention to improve solubility. The data for these compounds is tabulated in Table 2.
The binding affinity of compounds 24-40 clearly indicates that substituted phenyl groups are well
tolerated on the acetylene moiety, majority of compounds showing high binding affinity Ki of <10 nM
for A2BAdoR, irrespective of the nature of substitution (e.g. electron donating or withdrawing group),
and irrespective of the position of substitution (ortho, meta or para on phenyl ring), as outlined in Table
2. Compound 26, with ortho substitution of trifluoromethyl, was found to be relatively less potent (Ki =
29 nM), and in general, not much difference was observed between meta and para substitutions in terms
of binding affinity for A2BAdoR. However, majority of compounds with para substitution showed
modest selectivity over A2AAdoR with low or no selectivity over A1AdoR (except compounds 32 and
37). Compounds 25, 28, 30, and 40 exhibited similar binding affinity for A2B and A1AdoR. Majority of
compounds with meta substitutions were selective for A2BAdoR over both A1 and A2AAdoRs with
Majority of the compounds exhibited highl selective over A3AdoR (≤30% inhibition at 1 µM) except for
compound 38. Among these compounds, 29, 36, and 39 were identified as the best compounds with
high affinity and selectivity for A2BAdoR. It is interesting to note that polar susbstitution like carboxylic
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acid and ester groups 36-40 were well tolerated in terms of potency for A2BAdoR and selectivity over
other AdoRs. Also, compound 36 with m-carboxylic acid substitution endowed with better aqueous
solubility of 529 µM at pH 7.4.
To further improve the selectivity over other AdoR subtypes, we have explored different substitutions
at N-3 position [44]. Table 3 shows follow-up SAR where the 4-pyrazolyl at C8 and propyl at N-1 side
chain was fixed and the N-3 side chain was varied with ethyl (43-49) or H (50-56). Unexpectedly,
compounds 48, 49 and 56 having carboxylic acid substitution showed lower binding affinity for
A2BAdoR compared to the corresponding 1,3-dipropyl derivative 36 and 40. Consistent with dipropyl
derivatives, even in the case of 1-propyl-3-ethyl-xanthine derivatives, all para substituted compounds
44, 45, 47, 51, 53 and 55 exhibited modest selectivity over A2AAdoR and low selectivity over A1AdoR,
whereas all the meta substituted compounds 46, 52, and 54 had better selectivity over both the receptors.
An exception was observed for m-fluoro substituted compounds 43 and 50. Poor selectivity observed in
the case of compounds 43 and 50 could be due to the presence of a smaller group like fluoro. All these
compounds (43-56) retained high selectivity over A3AdoR (≤30% inhibition at 1 µM).
Compared to meta trifluoromethoxy phenyl substituted dipropyl xanthine derivative 29, corresponding
1-propyl-3-ethyl-xanthine derivative 46 had improved selectivity as shown in Table 3. N1-
monopropylated xanthine derivatives 52 and 54 had better binding affinity as well as selectivity than
corresponding 1,3-dipropyl or 1-propyl-3-ethyl xanthine derivatives. However these compounds
suffered from poor solubility issues.
In parallel, we pay attention towards replacement of highly lipophilic terminal aryl ring with the
intension to achieve better solubility for the insoluble xanthine derivatives in combination with potency
and selectivity. We replaced terminal aryl ring with small lipophilic group like H or methyl and
explored polar substitutions, e.g., alcohol or basic functions on the terminal acetylene moiety as in
compounds 61-68. First, we evaluated 8-(1-prop-2-ynyl-1H-pyrazol-4-yl)-1,3-dipropyl xanthine 59 and
8-(1-But-2-ynyl-1H-pyrazol-4-yl)-1,3-dipropyl xanthine 60, where terminal phenyl was replaced by “H”
and methyl respectively for binding affinity and selectivity for A2BAdoR. These compounds had modest
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affinity of Ki = 62 and 23 nM, respectively for A2BAdoR but were poorly selective (Table 4). This
indicated clear need of optimization of terminal substituent for potency as well as selectivity.
Compounds 61-65 with substitutions like diethylamine, pyrrolidine, piperidine, morpholine and N-
methyl piperazine showed low affinity for A2BAdoR and were non-selective over other AdoR subtypes
(Table 4). However, compound 66 substituted with 4-(3-trifluoromethyl-phenyl)-piperazine showed
high affinity (Ki = 12 nM) for A2BAdoR, although it had only modest selectivity over A1AdoR.
Compounds 67 and 68 with tertiary alcohol function also were of low affinity towards A2BAdoR.
In terms of affinity and selectivity, clear SAR was demonstrated with focused optimization of 8-(1-
prop-2-ynyl-1H-pyrazol-4-yl)-1,3-dipropyl xanthine analogs. Compounds with substituted phenyl on
terminal propyne group showed high affinity compared to other non-aryl substitutions. Low or no
selectivity over A1 and A2A AdoRs, as observed with most of the para-substituted compounds and
compounds with smaller groups like fluoro (irrespective of substitution at N-3 position), could be
attributed to the presence of rigid propargyl group between C8-pyrazolyl ring and terminal phenyl ring,
which makes the right hand side of these compounds close to linear structures in these cases. Bringing a
bulkier substitution at the meta position on the phenyl ring improved the selectivity for A2BAdoR over
other AdoR subtypes.
Among all compounds, compounds 29, 36 and 46 were identified as the best in terms of binding affinity,
selectivity and hence were taken forward for detailed profiling. Functional potency of these compounds
was determined in hA2BHEK-293 cells using cAMP assay and in NIH-3T3 cells using IL-6 assay.
Compounds 29, 36 and 46 were found to be potent A2BAdoR antagonists and inhibited 5′-N-
ethylcarboxamidoadenosine (NECA)-induced increase in cAMP with Ki of 8.8, 1.8 and 8 nM,
respectively, in hA2BHEK-293 cells. The compounds 29, 36 and 46 also inhibited NECA induced IL-6
release in NIH-3T3 cells with IC50 of 90, 2000 and 107 nM, respectively.
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Table 1. Binding Affinity of Xanthine Derivatives with C8 variation for A2BAdoR
O
O
H
H
N
N
O
N
N
N
O
R
N
O
N
O
N
O
N
R
17-21
12-16
N
O
N
O
R
H
N
H
N
N
N
N
N
N
N
O
O
N
O
N
22-23
R
24-25
Compd
R
hA2B Ki (nM)a
m-F
p-F
> 1 µM
NRb
NRb
NRb
>600
55
12
13
14
15
16
17
18
19
20
21
22
23
24
p-CF3
m-OCF3
p-OCF3
m-F
p-F
15
m-CF3
p-CF3
m-OCF3
m-CF3
p-OCF3
m-F
>600
NR
>600
NR
NR
6.7
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p-F
5.9
25
a
NR- no response. Binding affinity for hA2BAdoR was determined by using HEK-A2B cells with [3H]-
MRS-1754 as the radioligand, All data points were evaluated in triplicates. Each compound was
evaluated at 8 concentrations with each data point in triplicates for Ki determination.
Table 2. Binding Affinity of Xanthine Derivatives with Different Substitution on Terminal Phenyl for
A2B, A1, A2A and A3AdoRs
O
R
H
N
N
N
N
N
O
N
24-40
Ki (nM) % Inhibition at
% Inhibition at
1 µM
Compd
R
a
hA2B
0.1 µM
a
a
a
hA1
hA2A
hA3
m-F
6.7
5.9
29
87
105
52
43
84
39
92
52
14
57
70
64
66
29
64
30
27
51
84
0
19
25
32
12
6
24
25
26
27
28
29
30
31
32
33
34
p-F
o-CF3
m-CF3
p-CF3
4.9
1.5
3.6
5.5
3.7
14
m-OCF3
p-OCF3
m-CN
37
0
p-CN
29
55
33
14
9
m-CH3
p-CH3
5
6.5
0
14

ACCEPTED MANUSCRIPT
m-OCH3
m-CO2H
p-CO2H
o-CO2Me
m-CO2Et
p-CO2Et
6.3
5.3
17.6
12
57
20
19
37
29
80
74
32
44
41
29
19
21
0
35
36
37
38
39
40
25
43
0
7.8
3.5
27
aBinding affinities were determined by using membrane preparations from HEK-293 cells
overexpressing the relevant human AdoR isoform. All data points were evaluated in triplicates. Each
compound was evaluated at 8 concentrations with each data point in triplicates for Ki determination
[45].
Table 3. Binding Affinity of N-1, N-3 Differentially Substituted Xanthine Derivatives for A2B, A1,
A2A and A3AdoRs
43-49, R1=Et
50-56, R1=H
% Inhibition at
0.1 µM
%
at 1 µM
Inhibition
Compd
Ki (nM)
hA2B
R
a
a
a
a
hA1
hA2A
hA3
m-F
p-F
7.4
7.3
12
73
92
66
17
58
23
43
44
45
46
56
21
37
33
14
0
p-CF3
m-OCF3
13
15

ACCEPTED MANUSCRIPT
p-OCF3
m-CO2H
p-CO2H
m-F
9.9
190
130
3
84
NT
NT
53
49
NT
NT
60
32
NT
NT
0
47
48
49
50
51
52
53
54
55
56
p-F
1.6
2.2
1.3
2.8
1.6
48
77
55
0
m-CF3
p-CF3
26
39
0
89
39
15
0
m-OCF3
p-OCF3
m-CO2H
14
33
88
46
17
NT
NT
NT
NT- not tested. aBinding affinities were determined by using membrane preparations from HEK-293
cells overexpressing the relevant human AdoR isoform. All data points were evaluated in triplicates.
Each compound was evaluated at 8 concentrations with each data point in triplicates for Ki
determination[45].
Table 4. Binding Affinity of Xanthine Derivatives with Different Substitution on Acetylene for A2B,
A1, A2A and A3AdoRs
O
H
N
N
N
R2
N
N
O
N
59-68
% Inhibition
% Inhibition
at 1 µM
Compd
Ki (nM)
hA2B
at 0.1 µM
R
a
a
a
a
hA1
hA2A
hA3
16

ACCEPTED MANUSCRIPT
H
62
23
56
42
69
60
32
59
60
-CH3
NT
N
200
120
71
66
59
67
69
59
61
62
N
N
N
140
330
83
64
28
55
74
39
73
73
51
79
63
64
65
O
N
N
N
N
12
69
10
31
66
CF3
HO
HO
79
NT
NT
NT
NT
NT
NT
67
68
100
a
NT- not tested. Binding affinities were determined by using membrane preparations from HEK-293
cells overexpressing the relevant human AdoR isoform. All data points were evaluated in triplicates.
Each compound was evaluated at 8 concentrations with each data point in triplicates for Ki
determination [45].
2.3. Drug Metabolism and Pharmacokinetic Studies
17

ACCEPTED MANUSCRIPT
Based on affinity, potency and selectivity compound 29, 36 and 46 were progressed for drug metabolism
and pharmacokinetics (DMPK) evaluation and summarized in Table 5 [43]. All the three compounds
were metabolically stable in mouse, rat and human liver microsomes and improved solubility. When
dosed orally to mice at 10 mg/Kg, compound 46 showed better pharmacokinetics compared to
compound 29, with Cmax of 13 µM, systemic exposure AUC(0-t) of 60 µM.h, t1/2 of 6 h and oral
bioavailability of 76%, Compound 36 displayed poor systemic exposure with high clearance in mice
when dosed orally at 10 mg/Kg dose, and was found to be a substrate for Pgp (B-A/A-B = 12).
Furthermore, compound 46 did not show any CYP liability (IC50 >10 µM), cytotoxicity in MDCK and
HepG2 cells (IC50 >100 µM), or any significant hERG inhibition (IC50 > 25 µM).
Table 5. Oral Pharmacokinetic Profile of Compounds 29, 36 and 46 in C57BL/6J Mice at 10mg/Kga
AUC(0-t)
(µM.h)
Solubility
Compd
t1/2 (h)
%F
Cmax (µM)
(µM)b
6.6
1.9
13
67
2.2
60
5
42
10
76
<6
529
21
29
36
46
1.8
6.1
aPO formulation for 29 and 46: DMAC(10%), CrEL(10%), PEG300(10%), MQ water (qs), pH ~9; for
36: DMSO(10%), CrEL(10%), PEG300(10%), MQ water (qs), pH ~7; bAqueous solubility at pH 7.4
2.4. Molecular Docking analysis
As compound 46 showed good binding affinity, selectivity and acceptable pharmacokinetic properties,
we were interested to understand the binding mode of this compound (46) in conjunction with reference
compound 5. Molecular docking analysis revealed that both the compounds had similar binding mode
with good overlay and forming same intermolecular interactions with the A2BAdoR binding pocket
residues formed by trasnsmembrane region TM3, TM5, TM6 and TM7 and extracellular loop ECL2
(Figure 2a). Moreover, both the compounds showed comparable docking score (Gold Chemscore DG
was -26.03 and -26.88 for compounds 5 and 46, respectively). It was identified that compound 46 is an
18

ACCEPTED MANUSCRIPT
extended analog of 5, where acetylene linker of the former is mimicking the northern phenyl ring of the
later (Figure 2a). Both the compounds xanthine ring showed hydrogen bonding with a key residue
Asn254 (TM6) and <pi>-<pi> interaction with Phe173 (ECL2). N-1 ethyl and N-3 propyl of the
compounds accommodate well in the hydrophobic pocket. Northern phenyl ring of the compounds
formed hydrophobic interaction with the ECL2 residue Leu172, whereas CF3 group exposed in the bulk
solvent. 2D interaction diagram of compound 46 is illustrated in Figure 2b. This analysis demonstrates
the
important
molecular
recognition
interactions
required
for
the
A2BAdoR
activity.
Figure 2. (a) Overlay of docking poses of the reference compound 5 (magenta) and the
synthesized novel active compound 46 (yellow) in the homology modeled A2BAdoR binding site
(cyan cartoon representation). (b) 2D interaction diagram of compound 46 (hydrophobic residues
are shown in green color and polar residues in pink).
2.5. In vivo efficacy study
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Compound 46, when evaluated for in vivo efficacy (Figure 3), showed reduction of increased
inflammatory cells in ovalbumin-induced allergy model in mice, and significantly inhibited NECA-
induced airway hyper-responsiveness [43].
A) Effect on NECA Induced Airway Hyper-
B) Effect of Total Cell Number in BALf
responsiveness
300
150
250
125
200
*
100
150
*
75
*
100
50
*
*
*
50
0
25
0
*
*
100
*
*
*
200
300
400
NECA (µg/ml)
-50
Sentinel
Alum
Vehicle
OVA + CVT-6883; 3mg/kg, IP,BID
OVA + Compound-46; 3 mg/kg, IP,BID
OVA + Vehicle
OVA + CVT 6883; 3mg/kg, IP, BID
OVA+ Compound 46; 3mg/kg, IP, BID
OVA + Dexa; 3mg/kg, PO, BID
Figure 3: Effect of Compound 46 on A) NECA-induced airway hyper-responsiveness and B) total cell count
in BALf (bronchoalveolar lavage fluid) in Ovalbumin treated mice. Data is presented as mean ± SEM.
*significantly different compared to Vehicle treated group.
3. Conclusion
A focused SAR study on 8-(1-prop-2-ynyl-1H-pyrazol-4-yl)-xanthine derivatives led to the
identification of novel, potent and selective A2BAdoR antagonists. Compound 46, 3-ethyl-1-propyl-8-
{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-xanthine emerged as the best
compound in terms of affinity and selectivity over other subtypes for A2BAdoR, solubility and PK. SAR
study also led to identification of potent A2BAdoR antagonist 36 with good aqueous solubility.
20

ACCEPTED MANUSCRIPT
Compound 46 showed high binding affinity (Ki = 13 nM) and great selectivity over other AdoR
subtypes. It was also potent with Ki of 8 nM in cAMP assay and IC50 107 nM in the IL-6 assay, and
showed good PK profile. Furthermore, compound 46 showed significant in vivo efficacy in the
ovalbumin-induced allergic asthma model in mice. Further development around this series is in progress
and will be reported in a due course of time.
4. Experimental section
4.1. General
Commercial chemicals and solvents were of reagent grade and were used without further purification.
Anhydrous solvents were used without further drying. The following abbreviations are used for reagent
and solvents: DCM-dichloromethane; DMF-dimethyl formamide; DMSO-dimethyl sulfoxide; EtOAc-
ethyl acetate; EtOH-Ethanol; and MeOH-methanol. Globe chemie silica gel (100-200 or 230-400 mesh)
was used for column chromatography. Analtech thin layer chromatography plates (20 x 20 cm, 2000
microns) were used for preparative thin layer chromatography. Proton NMR (1H NMR) spectra were
recorded on a Varian 400 spectrometer (400 MHz). Solutions were typically prepared in either
deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD) or deuterated chloroform
(CDCl3). Chemical shifts are reported in δ units (parts per million) downfield from tetramethylsilane and
are assigned as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q) and multiplets (m),
and broad (br). Coupling constants (J) are reported in Hertz (Hz). Mass spectra (MS) were recorded on
Agilent 6110. HPLC were recorded on Agilent RRLC using Eclipse XOB-C18 (250x4.6) mm Su
column and 0.05 % fumaric acid (aq.) and acetonitrile as mobile phase with flow rate of 1ml/min at 30
°C for run time of 17 minutes and the HPLC purity is ≥95% unless otherwise stated.
4.2. Synthesis
21

ACCEPTED MANUSCRIPT
Preparation of 1,3-Dipropyl-8-{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-3,7-
dihydro-purine-2,6-dione (29).
Preparation of 3-(3-Trifluoromethoxy-phenyl)-prop-2-yn-1-ol: A mixture of propargyl alcohol (1.0g,
18mmol), 1-Iodo-3-trifluoromethoxy-benzene (5.14g, 18mmol), copper iodide (0.342g, 1.8mmol),
dichlorobis (triphenylphosphine) palladium (II) (0.632g, 0.9 mmol), diethylamine (30ml) was degassed
for 10min. and stirred for 20h at 25-25 °C. Excess of diethyl amine was distilled off under vacuum. The
residue was diluted with water (50ml) and extracted with ethyl acetate (3 X 50ml). The organic layer
was washed with brine solution and dried over Na2SO4. The solvent was evaporated and the crude
product was purified by column chromatography (10% Ethyl acetate in hexane) to obtain pure 3-(3-
Trifluoromethoxy-phenyl)-prop-2-yn-1-ol (3.8g, 100 %). 1HNMR (CDCl3): δ 1.71 (t, J= 6Hz, 1H); 4.52
(d, J= 6Hz, 2H); 7.20 (d, J=7.2Hz, 1H); 7.28-7.39 (m, 3H).
Preparation of 1-(3-Bromo-prop-1-ynyl)-3-trifluoromethoxy-benzene: 3-(3-Trifluoromethoxy-phenyl)-
prop-2-yn-1-ol (3.9g, 18mmol) was taken in DCM (50ml). The reaction mixture was then cooled to -5-0
°C. Tribromo phosphine (1ml, 11mmol) was added slowly at -5-0 °C and stirred at the same temperature
for 3h. The reaction mixture was quenched with saturated NaHCO3 solution and the organic layer was
separated. The aqueous layer was extracted with DCM (2 X 40ml). The combined organic layer was
washed with saturated brine solution, dried over Na2SO4, and evaporated under vacuum to get 1-(3-
1
Bromo-prop-1-ynyl)-3-trifluoromethoxy-benzene (2.97g, 60%). HNMR (CDCl3): δ 4.12 (s, 2H); 7.20
(d, J= 6.8Hz, 1H); 7.30 (s, 1H); 7.34-7.39 (m, 2H); MS (m/z ) (M++1): 279.3.
Preparation of 1, 3-Dipropyl-8-{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-7-
(2-trimethylsilanyl-ethoxymethyl)-3, 7-dihydro-purine-2, 6-dione: A mixture of 1, 3-Dipropyl-8-(1H-
pyrazol-4-yl)-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-purine-2,6-dione 11d36 (1.0g, 2.3mmol),
1-(3-Bromo-prop-1-ynyl)-3-trifluoromethoxy-benzene (0.829g, 3.0mmol), K2CO3 (0.731mg, 5.3mmol)
and acetone (50ml) were refluxed at 80 oC for 5h. The reaction mixture was cooled to room temperature
and the solid K2CO3 was filtered off and evaporated the organic solvent under vacuum. The residue
obtained was purified by column chromatography (2% methanol in dichloromethane) to get pure 1,3-
22

ACCEPTED MANUSCRIPT
Dipropyl-8-{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-7-(2-trimethylsilanyl-
1
ethoxymethyl)-3,7-dihydro-purine-2,6-dione (1.5g, 100%). HNMR (DMSO d6): δ -0.01 (s, 9H); 0.94-
1.03 (m, 8H); 1.68-1.73 (m, 2H); 1.80-1.87 (m, 2H); 3.83 (t, J= 8Hz, 2H); 4.01 (t, J= 7.6Hz, 2H); 4.13
(t, J= 7.2Hz, 2H); 5.26 (s, 2H); 5.81 (s, 2H); 7.35-7.42 (m, 4H); 8.19 (s, 1H); 8.40 (s, 1H); MS (m/z )
(M++1): 631.4
Preparation of 1,3-Dipropyl-8-{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-3,7-
dihydro-purine-2,6-dione: 1,3-Dipropyl-8-{1-[3-(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-
4-yl}-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-purine-2,6-dione (1.4g, 2.2mmol) in EtOH
(40mL) was treated with 2N HCl (20mL) and heated at 80 °C for 2h. The reaction mixture was
concentrated in vacuo, and the residue was triturated with diethyl ether to afford 1,3-Dipropyl-8-{1-[3-
(3-trifluoromethoxy-phenyl)-prop-2-ynyl]-1H-pyrazol-4-yl}-3,7-dihydro-purine-2,6-dione
(0.915g,
80%) as a white solid. M.P. 199-200 oC; 1HNMR (DMSO d6): δ 0.85-0.91 (m, 6H); 1.54-1.60 (m, 2H);
1.69-1.75 (m, 2H); 3.85 (t, J= 7.2Hz, 2H); 3.98 (t, J= 7.2Hz, 2H); 5.42 (s, 2H); 7.45-7.50 (m, 2H); 7.54-
7.56 (m, 2H); 8.12 (s, 1H); 8.53 (s, 1H); 13.59 (s, 1H); 13C NMR (DMSO-d6, 100MHZ): δ 153.8, 150.6,
148.4, 148.2, 145.0, 138.3, 131.0, 130.9, 129.7, 124.0, 123.4, 122.1, 118.7, 112.7, 106.3, 85.3, 83.2,
44.3, 42.1, 41.7, 20.8, 11.2, 11.0, MS (m/z ) (M++1): 501.2; HPLC purity 100%.
4.2.1.
Radioligand Binding for Adenosine Receptors A1, A2A, A2B and A3.
Human adenosine receptor (A1, A2A, A2B and A3) cDNA was stably transfected into HEK-293 cells
(referred to as HEK-A1, HEK-A2A, HEK-A2B, HEK-A3 cells). The HEK-293 cell was obtained from
ATCC. The cells monolayer was washed with PBS once and harvested in a buffer containing 150 mM
NaCl, 1 mM EDTA, 50 mM Tris, pH 7.4 (10 mM EDTA, 10 mM HEPES, pH 7.4 for HEK-A3) at
1500 rpm for 5 min at room temperature. The cell pellet was incubated in sonication buffer containing
1 mM EDTA, 5 mM Tris, pH 7.4 (1 mM EDTA, 10 mM HEPES, pH-7.4 for HEK-A3) for 10 mins at
4 oC followed by sonication on ice for 6 min. The lysate was centrifuged at 1000 x g for 10 min at 4
oC and the pellet was discarded. The supernatant was centrifuged at 49,000 x g for 45 min at 4 oC.
23

ACCEPTED MANUSCRIPT
The resultant protein pellet was resuspended in sonication buffer supplemented with 1 U/ml adenosine
deaminase (ADA, Roche) and incubated for 30 min at room temperature with constant mixing. The
protein was washed twice with same buffer at 49,000 x g for 45 min at 4 oC and the final protein was
stored in 50 mM Tris, pH 7.4 supplemented with 1 U/mL ADA and 10 % sucrose (1 mM EDTA, 5
mM Tris, pH 7.4, 1 U/ml ADA and 10% sucrose for HEK-A3). The protein concentration was
estimated by Bradford assay and aliquots were stored at -80 oC.
The binding affinity and selectivity of test compounds was determined using radioligand binding
assays. DPCPX (8-Cyclopentyl-1,3-dipropylxanthine, for A1AdoR), Preladenant [2-(2-furanyl)-7-(2-
(4-(4-(2-methoxyethoxy)phenyl)-1-piperazinyl)ethyl)-7H-pyrazolo(4,3-e)(1,2,4)triazolo(1,5-
c)pyrimidine-5-amine, for A2AAdoR], 3-Ethyl-3,9-dihydro-1-propyl-8-[1-[[3-
(trifluoromethyl)phenyl]methyl]-1H-pyrazol-4-yl]-1H-purine-2,6-dione (CVT-6883, A2BAdoR) and 3-
Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-
119, A3AdoR) were used as internal standards. Competition radioligand binding assays were started by
mixing 1 nM [3H]-DPCPX (A1), 1 nM [2-3H]-4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-
a}{1,3,5,}triazin-5-yl amino]ethyl)phenol ([3H]ZM241385) (A2A),35 1.6 nM radiolabelled N-(4-
Cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide
([3H]-MRS-1754) (A2B)36 or 2 nM [3H]-HEMADO (A3) with various concentrations of test
compounds (and the respective membranes in assay buffer containing 50 mM Tris pH 7.4, 1 mM
EDTA (A1), 50 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 U/ml ADA (A2A, A3) or 50 mM
Tris pH 6.5, 5 mM MgCl2, 1 mM EDTA (A2B) supplemented with 1 U/ml ADA. The assays were
incubated at room temperature for 90 min with gentle agitation, stopped by filtration using a Harvester
(Molecular Devices), and washed four times with ice-cold 50 mM Tris (pH 7.4). Nonspecific binding
was determined in the presence of 100 µM NECA. The affinities of compounds (i.e., Ki values) were
calculated using GraphPad software.
24

ACCEPTED MANUSCRIPT
cAMP Assays: The functional activity of compounds was determined by evaluating the changes in
cAMP levels post agonist treatment in HEK-293 cells over-expressing human A2BAdoR. A 24 hour
culture of cells was treated with 1 U/mL of ADA in DMEM supplemented with 10% FBS for 90
minutes at 37oC and 5% CO2. Cells were harvested using cell dissociation buffer (Sigma), washed with
incomplete DMEM and incubated with increasing concentrations of test compound for 15 minutes in
DMEM supplemented with 1U/ml ADA at room temperature. The cAMP levels were induced using
A2BAdoR agonist NECA for 15 minutes. The levels of cAMP were detected and estimated as per
manufacturer’s instruction using the Cisbio HTRF cAMP kit. The FRET between d2-labeled cAMP and
cryptate labeled anti-cAMP antibody was read in HTRF mode in a Flex Station III (Molecular Devices)
using an excitation maximum of 313 nm and emission maxima of 620 nm and 665 nm. Data was
analyzed using GraphPad Prism software to determine IC50 and Ki values.
4.2.2. Molecular Modeling
Homology model development
In the absence of any experimentally (X-ray or NMR) determined three dimensional (3d) structure of
human A2BAdoR, homology model of this receptor was built using MOE program [46] by
implementing the standard protocol. On the basis of lowest E value, good Z score and highest
percentage identity (61%) with the query protein, PDB ID: 4EIY (X-ray crystal structure of A2AAdoR
with small molecule antagonist ZM241385) [47] was found to be the most suitable template for
building the homology model of human A2BAdoR. Among the several other crystal structures of
A2AAdoR in the Protein Data Bank (PDB), this structure was particularly selected due to its high
resolution and better overall B-factor. Initially, 100 models were generated and the best model was
selected on the basis of Generalized Born/Volume Integral (GB/VI) score, Cα RMSD with the
template protein, contact energy and packing score. The best model was checked for its
stereochemistry using Ramachandran plot. Model refinement and minimization was done using
Amber99 (R–Field) force field. The template ligand ZM241385 coordinates were transferred in the
homology modeled structure of A2BAdoR in order to define the binding site.
25

ACCEPTED MANUSCRIPT
Molecular Docking
Ligands were built in MOE, hydrogens were added, partial charges were assigned and minimized
using MMF94X force field. Molecular docking was performed by GOLD 5.2.2. (Genetic Optimization
for Ligand Docking) program from Cambridge Crystallographic Data Centre (CCDC), UK [47] using
standard protocol as mentioned elsewhere. The binding site of modeled A2BAdoR for the purpose of
docking was defined by selecting the bound ligand (ZM241385). All the atoms within 6 Å range from
the centre of bound ligand were selected in order to cover all the residues of active site. Subsequently,
all the H-bond donors/acceptors were forced to be treated as solvent accessible. A maximum of 10
docking GA runs per molecule were performed, allowing all the poses to generate and sorted by using
ChemScore fitness scoring function. All other parameters were kept at their default values for the
docking. Most plausible docking poses for the compounds were selected by visual inspection on the
basis of binding orientation, key interactions and Gold Chemscore DG.
Appendix A. Supplementary data
The contents of supporting information include the following: (1) experimental procedures and
characterization (3) 1H NMR/13C NMR spectra of selected compounds
Acknowledgements
Authors are grateful to the senior management of the Advinus Group for its support and encouragement.
Analytical departments are acknowledged for their help during this work. We thank Dr. Anup Ranade
for managing intellectual property - Advinus publication no. ADV-A-040
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