A2Badenosine receptor antagonists: Design, synthesis and biological evaluation of novel xanthine derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.11.007

A_2BAdoR is a low affinity adenosine receptor that functions by Gs mediated elevation of cAMP and subsequent downstream signaling. The receptor has been implicated in lung inflammatory disorders like?COPD and asthma. Several potent and selective

Full text of DOI:10.1016/j.ejmech.2016.11.007

Accepted Manuscript
A
adenosine receptor antagonists: Design, synthesis and biological evaluation of
2B
novel xanthine derivatives
Sujay Basu, Dinesh A. Barawkar, Vidya Ramdas, Yogesh Waman, Meena Patel, Anil
Panmand, Santosh Kumar, Sachin Thorat, Rajesh Bonagiri, Dilip Jadhav, Partha
Mukhopadhyay, Vandna Prasad, B. Srinivasa Reddy, Arnab Goswami, Sandhya
Chaturvedi, Suraj Menon, Azfar Quraishi, Indraneel Ghosh, Sushant Dusange,
Shalini Paliwal, Abhay Kulkarni, Vikas Karande, Rhishikesh Thakre, Gaurav Bedse,
Sreekanth Rouduri, Jayasagar Gundu, Venkata P. Palle, Anita Chugh, Kasim A.
Mookhtiar
PII:
S0223-5234(16)30945-X
10.1016/j.ejmech.2016.11.007
EJMECH 9040
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2016
Revised Date: 3 November 2016
Accepted Date: 4 November 2016
Please cite this article as: S. Basu, D.A. Barawkar, V. Ramdas, Y. Waman, M. Patel, A. Panmand, S.
Kumar, S. Thorat, R. Bonagiri, D. Jadhav, P. Mukhopadhyay, V. Prasad, B.S. Reddy, A. Goswami,
S. Chaturvedi, S. Menon, A. Quraishi, I. Ghosh, S. Dusange, S. Paliwal, A. Kulkarni, V. Karande, R.
Thakre, G. Bedse, S. Rouduri, J. Gundu, V.P. Palle, A. Chugh, K.A. Mookhtiar, A adenosine receptor
2B
antagonists: Design, synthesis and biological evaluation of novel xanthine derivatives, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.007.
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Graphical abstract
31
65
hA2B binding Ki = 1.0 nM
hA2B functional Ki = 0.9 nM
A1/A2B = 108 fold
hA2B binding Ki = 1.5 nM
hA2B functional Ki = 4.0 nM
A1/A2B = 118 fold
%F = 27
%F = 65
Showed efficacy in Ovalbumin treated mice
1

ACCEPTED MANUSCRIPT
A2B Adenosine Receptor Antagonists: Design, Synthesis and Biological Evaluation of Novel
Xanthine Derivatives
Sujay Basu , Dinesh A. Barawkar, Vidya Ramdas, Yogesh Waman, Meena Patel, Anil Panmand,
Santosh Kumar, Sachin Thorat, Rajesh Bonagiri, Dilip Jadhav, Partha Mukhopadhyay, Vandna
Prasad, B Srinivasa Reddy, Arnab Goswami, Sandhya Chaturvedi, Suraj Menon, Azfar Quraishi,
Indraneel Ghosh, Sushant Dusange, Shalini Paliwal, Abhay Kulkarni, Vikas Karande,
Rhishikesh Thakre, Gaurav Bedse, Sreekanth Rouduri, Jayasagar Gundu, Venkata P. Palle,
Anita Chugh, Kasim A. Mookhtiar
Departments of Discovery Chemistry, Discovery Biology and DMPK, Advinus Therapeutics Ltd.,
Hinjawadi, Pune-411 057, India
ABSTRACT:
A2BAdoR is a low affinity adenosine receptor that functions by Gs mediated elevation of cAMP and
subsequent downstream signaling. The receptor has been implicated in lung inflammatory disorders like
COPD and asthma. Several potent and selective A2BAdoR antagonists have been reported in literature,
however most of the compounds suffer from poor pharmacokinetic profile. Therefore, with the aim to
identify novel, potent and selective A2BAdoR antagonists with improved pharmacokinetic properties, we
Corresponding author. Tel.: +91 20 66539600; fax: +91 20 66539620.
E-mail address: kasim.mookhtiar@advinus.com
Corresponding author. Tel.: +91 20 66539600; fax: +91 20 66539620.
E-mail address: sujay.basu@advinus.com
1

ACCEPTED MANUSCRIPT
first explored more constrained form of MRS-1754 (4). To improve the metabolic stability, several
linker modifications were attempted as replacement of amide linker along with different phenyl or other
heteroaryls between C8 position of xanthine head group and terminal phenyl ring. SAR optimization
resulted in identification of two novel A2BAdoR antagonists, 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-
pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-dipropyl-xanthine
(31)
and
8-(1-{2-Oxo-2-[4-(3-
trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-xanthine (65), with high
binding affinity (Ki = 1 and 1.5 nM, respectively) and selectivity for A2BAdoR with very good functional
potency of 0.9 nM and 4 nM, respectively. Compound 31 and 65 also displayed good pharmacokinetic
properties in mice with 27% and 65% oral bioavailability respectively. When evaluated in in vivo mice
model of asthma, compound 65 also inhibited airway inflammation and airway reactivity in ovalbumin
induced allergic asthma at 3 mpk dose.
Keywords: Adenosine receptors; Xanthine; 2-Oxopyrrolidine; Mouse liver microsomes;
Pharmacokinetics
1. Introduction
Adenosine is an endogenous purine nucleoside which also functions as a signaling molecule through
specific cell membrane receptors belonging to GPCR family. There are four known subtypes of
adenosine receptors (AdoRs) refered to as A1, A2A, A2B, and A3.1 AdoRs exhibit high sequence
homology but differ in affinity for adenosine, tissue distribution and effector coupling, resulting into
each receptor subtype having a unique pharmacological profile.2,3 Thus different AdoRs offers broad
therapeutic potentials including cell growth, asthma, immune system, inflammatory diseases and CNS
disorders etc.4-6
Many cell types that play important role in the pathogenesis of inflammatory lung disease are known to
express adenosine receptors. These cell types include various inflammatory cells, such as mast cells,
eosinophils, lymphocytes, neutrophils macrophages, and structural cells in the lung, such as bronchia
2

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epithelial cells, smooth muscle cells, lung fibroblasts and endothelial cells.7-9 A2BAdoR activation
mediates the release of IL-19 and TNF-α in bronchial epithelial cells10 and increases the expression and
release of IL-6 and monocytic chemotactic peptide-1 (MCP-1)11 in bronchial smooth muscle cells. In
human lung fibroblasts, activation of A2BAdoR induces the release of IL-6 and differentiation of
fibroblasts to myofibroblasts.12 This indicates that adenosine via its receptor A2BAdoR plays a key role
in the pathogenesis of inflammatory lung disease; therefore, antagonists of the A2BAdoR would provide
a novel approach towards the management and treatment of asthma and chronic obstructive pulmonary
disease (COPD). Literature also provides evidence for the immune independent role of A2BAdoR
wherein blockade of A2BAdoR resulted in significant reduction of metastasis.13 A2BAdoR was also
shown to control cellular proliferation via HIF-1α activation, indicating that A2BAdoR may be a key
regulator of tumoral progression in oral squamous cell carcinoma.14 A2BAdoR antagonist CVT-6883
(Figure 1) has been shown to significantly inhibit in vivo growth of tumor in melanoma, lung and colon
cancer models.15
The alkylxanthines, theophylline 1 and enprofylline 2 (Figure 1), are being used therapeutically for the
treatment of asthma for several decades.16, 17 Theophylline (1), 1,3-dimethyl xanthine, is a PDE IV
(phosphodiesterase IV) inhibitor and a nonselective AdoR antagonist that has a Ki of 9 µM for the
A2BAdoR and has a low therapeutic index due to both CNS and cardiac side effects. Enprofylline (2), a
3-propyl xanthine derivative, has moderate affinity for the A2BAdoR (Ki = 4.7 µM) and also has
moderate selectivity against the other AdoR subtypes. The bronchodilating effect of 1 and 2 has been
attributed to antagonism of A2BAdoR, which prompted several groups to design and test a large number
of xanthine derivatives in search for new, more potent and selective A2BAdoR antagonists which could
provide an enhanced therapeutic index. CV Therapeutics reported the discovery of potent and selective
A2BAdoR antagonist 3 (CVT-6883), 18, 19 which inhibited pulmonary inflammation and injury in the lung
of adenosine deaminase-deficient mice and in a bleomycin-induced fibrosis model.20 In addition,
compound 3 attenuated the airway reactivity induced by 5′-N-ethylcarboxamidoadenosine (NECA),
3

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AMP, or allergen in sensitized mice.21 The compound however was discontinued from Phase I clinical
trials.
Apart from CVT, while several other research groups have reported a number of high-affinity A2BAdoR
antagonists, only a few show high affinity and selectivity for the A2BAdoR over A1, A2A, and
A3AdoRs.22-24 Jacobson and co-workers reported a highly potent and selective A2B antagonist MRS-
1754 (4. Figure-1).26, 27 Later, Baraldi and co-workers reported the 8-(5-pyrazolyl)-xanthines represented
by structure 528. Both the compounds, 3 and 4, contain the amide functionality in their linker part. Most
of these compounds show poor pharmacokinetic (PK) properties.26, 27 Earlier attempts, by other research
groups, to improve metabolic stability and oral bioavailability of tool compounds such as 4 and 5
resulted only in compounds with enhanced metabolic stability but did not yield compounds with good
pharmacokinetic profile.29, 30
To achieve our goal and identify potent and selective A2BAdoR antagonists with improved oral
bioavailability, we decided to constrain the longer and flexible amide linker on right hand side of the
molecules 4 or 5. Herein, we describe design, synthesis and focused SAR efforts. Systematic
optimization enabled us to identify a suitable linker modification which improved the metabolic stability
while retaining the binding affinity and selectivity of compounds.
O
O
N
H
N
O
N
H
N
H
N
HN
N
N
N
N
N
N
O
N
N
O
O
CF3
3 CVT-6883
1 Theophylline
2 Enprofylline
O
O
H
H
N
N
N
N
N
N
O
O
H
N
N
N
N
O
O
N
O
O
O
CN
N
H
O
4 MRS-1754
5 MRE2029F20
4

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Figure 1. Representative Structures of Xanthine Based A2BAdoR Antagonists
2. Results and discussion
2.1. Chemistry
The key intermediates 8-pyrazoloxanthines 10a,b were prepared by the classical method starting from
1,3-disubstituted-5,6-diaminouracil 6a,b (Scheme 1).18 The diamino uracil 6 was coupled with pyrazole
carboxylic acid 7 using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDCI) in
methanol at room temperature to obtain carboxamide derivatives 8a,b. Subsequent ring closure of these
derivatives was performed by refluxing with 10% sodium hydroxide in methanol to afford 8-substituted
xanthine derivatives 9a,b. Silyl protection of N-7 position of 9a,b with 2-
(trimethylsilyl)ethoxymethylchloride (SEM-Cl) using potassium carbonate followed by debenzylation
using hydrogen and 10% Pd/C afforded the key intermediates 10a,b.
5-Oxopyrrolidine xanthine derivatives 24-36 (Scheme-2) were prepared by alkylation of 1,3-dipropyl-
8-pyrazolyl xanthine derivative 10a with different mesylates derived from N-phenyl substituted 5-
oxopyrrolidines 11-23, which were synthesized by literature procedure,31 using potassium carbonate in
acetone at reflux temperature followed by de-protection of silyl group with 2N HCl in ethanol at reflux
temperature. In a similar fashion, compounds 37-42 were prepared by alkylation of 1-propyl-3-ethyl-8-
pyrazolo xanthine derivative 10b with appropriate mesylates.
2-Oxopyrrolidine xanthine derivatives 46-48 (Scheme-2) were prepared by alkylation of 1,3-
dipropyl-8-pyrazolyl xanthine derivative 10a with mesylates of 2-oxopyrrolidine 43-45, which were
synthesized by literature procedure,32 using potassium carbonate in acetone at reflux temperature
followed by N-7 deprotection with 2N HCl in ethanol at reflux temperature.
Pyrolidinyl xanthine derivatives 54-58 (Scheme-2) were prepared by alkylation of 10a with
pyrrolidine derivatives 49-53,32 as described above. Piperazinyl xanthine derivatives 64-68 were
prepared by alkylation of 1,3-dipropyl-8-pyrazolo xanthine derivative 10a with piperazinyl chlorides 59-
5

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63, which were synthesized by classical methods, using potassium carbonate in acetone at reflux
temperature, followed by N-7 de-protection.
5-oxopyrrolidone xanthine derivatives 70, 72 and 74 (Scheme-3) with different linker groups were
prepared by alkylation of 1,3-dipropyl-8-pyrazolo xanthine derivative 10a with 69, 71, or 73, using
potassium carbonate in acetone at reflux temperature or by using cesium carbonate in acetonitrile at
room temperature, followed by N-7 deprotection.
5-oxopyrrolidone xanthine derivatives 94-105 with a combination of methylene oxy linker and
different rings at C8 of xanthine head group were prepared in a linear method as described in (Scheme
4). Alkylation of hydroxy esters 79-81 with different hydroxymethyl pyrrolidinones 75-78 under
Mitsunobu conditions followed by conventional ester hydrolysis provided the carboxylic acids 82-93.
Amide coupling of 82-93 with 6a using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
(EDCI) in methanol at room temperature and subsequent ring closure of the carboxamide derivatives by
refluxing with 10% sodium hydroxide in methanol afforded xanthine derivatives 94-105 with methylene
oxy linker.
Scheme 1a
O
N
O
O
OH
H
N
NH2
NH2
N
N
N
a
N
O
+
N
O
N
O
N
NH2
R1
R1
8a,b
7
6a, R1=Pr
6b, R1=Et
O
O
Si
H
N
O
N
N
b
c, d
N
N
N
N
N
O
N
NH
N
O
N
R1
R1
9a,b
10a,b
6

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aReagents and conditions: (a) EDCI, MeOH, r.t.; (b) 10% NaOH, MeOH, reflux; (c) SEM-Cl, K2CO3,
DMF, r.t.; (d) 10% Pd/C, MeOH:DCM (2:1), r.t.
Scheme 2a
O
OMs
O
N
H
N
O
N
N
N
R
11-23
a, b
N
R
N
O
N
R1
24-36, R1=Pr
37-42, R1=Et
Si
N
O
OMs
N
H
N
O
O
N
N
N
O
R
R
N
N
N
N
43-45
N
O
N
NH
R1
O
a, b
O
N
R1
46-48, R1=Pr
10a, R1=Pr
10b, R1=Et
O
OMs
N
H
N
N
N
N
R
49-53
a, b
N
R
N
O
N
R1
54-58, R1=Pr
O
N
Cl
O
N
N
N
N
N
O
R
59-63
a, b
N
O
N
N
R1
N
R
64-68, R1=Pr
aReagents and conditions: (a) K2CO3, DMF, 80 °C or acetone, 50 °C; (b) 2N HCl, Ethanol, 80 °C
7

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Scheme 3a
O
O
H
N
OMs
CF
3
N
N
N
N
N
O
N
69
O
N
a, b
70
CF
3
Si
N
O
OMs
O
H
N
O
CF
3
N
O
N
O
N
N
CF
3
N
N
N
N
71
N
O
N
N H
O
N
a, b
72
10a
O
N
O
N
H
N
CF
3
OH
N
N
O
N
N
O
Br
73
O
N
c, b, d
74
CF
3
aReagents and conditions: (a) K2CO3, DMF, 80 °C; (b) 2N HCl, Ethanol, 80 °C; (c) Cs2CO3,
acetonitrile, r.t.; (d) NaBH4, THF, r.t.
8

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Scheme 4a
O
OH
A
HO
a, b
O
O
O
O
OH
A
+
N
N
R
O
R
82-93
79-81
75-78
O
NH2
N
O
N
H
N
O
N
NH2
O
N
N
A
O
R
6a
N
O
c, d
94-105
aReagents and conditions: (a) DIAD, PPh3, Et3N, Toluene, 80 °C; (b) LiOH, THF:MeOH:H2O, r.t. (c)
EDCI, MeOH, r.t.; (d) 10% NaOH, MeOH, 80 °C.
2.2. Biological activity
A series of xanthine derivatives with constrained form of amides in the linker part were prepared with
the goal of identifying new, potent, and selective A2BAdoR antagonists with improved pharmacokinetic
properties compared to earlier reported xanthine derivatives with more flexible amide linker like MRS-
1754 (4, hA2B Ki: 2 nM) or MRE-2029F20 (5, hA2B Ki: 5.5 nM). Both the possible options of cyclization
of amide bond to result into pyrrolidone’s and pyrrolidinyl (without oxo group) offering different degree
of constrain were explored. This effort provided 5-Oxopyrrolidine (24-42), 2-Oxopyrrolidine (46-48)
and pyrrolidinyl (54-58) xanthine derivatives with various substitutions on terminal phenyl ring. The
binding affinities of these compounds for all four adenosine receptors are listed in Tables 1-5. Most of
the pyrrolidone xanthine derivatives 24-42 and 46-48 showed good affinity with Ki of <30 nM for
A2BAdoR regardless of the nature of substitutions like electron withdrawing groups such as –F, -CF3, -
OCF3 and –CN, or electron donating groups such as -Me, and -OMe on phenyl ring attached to
9

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pyrrolidone moiety. However, compounds 28 and 46 with m-fluoro substitution showed modest binding
affinity of 41 and 43 nM, respectively, for A2BAdoR. Compound 35 with charged carboxylic acid
substitution at para position showed low binding affinity of 77 nM for A2BAdoR (Table 1). In general,
para substituted compounds displayed relatively higher binding affinity than the corresponding meta
substituted compounds. Compound 31 showed highest affinity of 1 nM for A2BAdoR. Most of the 5-
oxopyrrolidine derivatives (24-42) showed high selectivity over A1AdoR and A3AdoR, and modest
selectivity over A2AAdoR. Compound 40 showed better selectivity for A1, A2A and A3AdoRs than
compound 31. Both compounds 40 and 31 have 5-oxopyrrolidine and terminal para trifluoromethyl
phenyl substituent in common, with only noticeable difference of 1-propyl-3-ethyl xanthine and 1,3-
dipropyl xanthine moieties resepectively, indicating probable role of N1 and N3 xanthine substitution in
selectivity. 2-Oxopyrrolidine derivatives 46-48 showed high selectivity over A2AAdoR and A3AdoR and
modest selectivity over A1AdoR. This suggests that 5-oxo and 2-oxo pyrrolidine xanthine derivatives
bind differently with A1AdoR and A2AAdoR. However, there was no significant difference observed
between 5-oxopyrrolidinyl and pyrrolidinyl xanthine derivatives 54-58 in terms of binding affinity and
selectivity for A2BAdoR over other AdoR subtypes (Table 1).
Most of these compounds were metabolically unstable in mouse liver microsomes (MLM) and human
liver microsomes (HLM) (Table-1). Structure-activity relationship (SAR) in terms of metabolic stability
demonstrated that meta-substituted compounds were relatively more unstable than the corresponding
para-substituted compounds.
To improve metabolic instability of these compounds, analogs of metabolically unstable compound 30
(Table-1) with various linker modifications were explored. Compounds 70, 72 and 74 were synthesized
with extended linker or substituted linker and tested for binding affinity and metabolic stability (Table-
2). These compounds retained the binding affinity for A2BAdoR with little compromise on selectivity
over A2AAdoR (70 and 74) but without any improvement on metabolic stability. More drastic
modifications of linker were also explored to find out its role on metabolic stability. Piperazinyl
xanthine derivatives 64-68 showed high affinity for A2BAdoR (Table-3) and modest selectivity over
10

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A1AdoR and A2AAdoR. Like other analogs, these compounds also showed high selectivity over
A3AdoR. Most of the piperazinyl analogs were metabolically unstable in mouse and human liver
microsomes. Considering its high affinity and selectivity for A2BAdoR over other AdoR subtypes,
compound 65 was further tested for its functional potency in cAMP assay using hA2B-HEK293 cells.
Compound 65 was found to be a potent A2BAdoR antagonist with Ki of 4 nM (Table-5).
To render metabolic stability to these compounds, it was decided to further try ether linker or
methylene oxy linker between xanthine and 5-oxopyrrolidine moiety where C-8 pyrazole is replaced by
phenyl, pyridyl and isoxazolyl group (Table-4). For this purpose, compounds with phenyl (94-97),
pyridyl (98-101) and isoxazolyl (102-105) derivatives with ethyl ether linker were prepared. These
compounds showed reasonable binding affinity for A2BAdoR. To our delight, compounds (96, 97, 100,
101 and 105) with oxy methylene linker were found to be metabolically stable. Compounds 94-101,
showed a high binding affinity (Ki <10 nM) for A2BAdoR, but lost selectivity for A2AAdoR.
Additionally, these compounds suffered from solubility issues. Isoxazole derivatives 102-104 have
shown low affinity for A2BAdoR. However, compound 105 had high binding affinity (12.8 nM) and high
selectivity for A2BAdoR over other AdoR subtypes as well as good metabolic stability.
Based on the overall profile in terms of high binding affinity, selectivity over other AdoR’s and
metabolic stability, best compounds 31, 38, 40, 42, 48, 101 and 105 (as racemic mixture) and 65 were
chosen for further profiling. The binding affinity Ki of these compounds for A1AdoR and A2AAdoRs
were determined in addition to their functional potency in cAMP assay using hA2B-HEK293 cells.
Compounds 31, 40, and 65 had good functional potency of 0.9, 8 and 4 nM respectively (Table-5) and
>100 fold binding selectivity over other AdoRs (except for compound 65’s selectivity over A2AAdoR).
Compounds 48, 101 and 105 were functionally less potent with Ki of 191, 71 and 540 nM, respectively,
while 38, 42 and 48 were only modestly selective for A1AdoR and A2AAdoRs.
11

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Table 1. Binding Affinity of Pyrrolidinyl xanthine Derivatives with Different Substitution on Terminal
Phenyl for A2B, A1, A2A and A3AdoRs.
O
O
N
O
H
N
H
N
H
N
N
N
N
N
N
N
N
N
N
N
O
N
N
N
O
N
O
O
R1
N
N
N
O
24-36, R1=Pr
37-42, R1=Et
54-58
46-48
R
R
R
MRb
(nmol/min/mg)
%Inhibition
0.1 µM
at
%Inhibition
Ki (nM)
hA2B
Compd R
at
1 µM
a
a
hA3
a
a
hA1
hA2A
MLM HLM
m-CH3
10.7
8
29
33
22
38
32
31
25
49
20
23
18
nt
0
0
0.46
0.25
0.41
0.22
0.35
0.19
0.61
0.16
0.57
0.24
0
0.18
nt
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
p-CH3
m-OCH3
p-OCH3
m-F
37
46
51
68
42
48
52
49
24
20
nt
0
21.6
9
11
27
17
14
31
0
0.18
0.19
0.02
0.06
0.18
0.02
0.23
0.13
0.15
nt
41
9.7
6.5
1
p-F
m-CF3
p-CF3
m-OCF3
p-OCF3
p-CN
9
0
4
0
23
77
19
24
12
4.5
2
p-CO2H
2,4-diF
m-F
nt
0
nt
13
30
22
18
45
37
33
23
0.48
0.29
0.15
0.49
0.03
0.06
0
0
p-F
0
m-CF3
0
0.12
12

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p-CF3
4
6
4
4
0.16
0.25
0.07
nt
0.08
0.4
40
41
42
46
47
48
54
55
56
57
58
m-OCF3
p-OCH3
m-F
17.5
16
43
6
15
15
nt
16
21
nt
3
14
nt
17
32
2
0.11
nt
m-CF3
p-OCF3
p-CH3
58
51
26
43
21
38
23
30
7
0.34
0.03
0.38
0.67
0.43
0
0.2
8
0.02
0.66
0.78
0.37
0.01
0.16
12
13.7
13
3.6
14
51
69
53
57
26
m-OCH3
p-OCH3
m-CF3
p-OCF3
12
3
2
2
0.15
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.30 bMetabolic Rate (MR) <0.1 nmol/min/mg is considered metabolically stable.
Table 2. Binding Affinity of Pyrrolidone xanthine Derivatives with Linker Modification for A2B, A1,
A2A and A3AdoRs.
MRb
(nmol/min/mg)
%Inhibition
0.1 µM
at
%Inhibition
Ki (nM)
Compd
at
1
µM
a
a
hA2B
A3
a
a
hA1
hA2A
MLM HLM
2.6
4
40
17
12
65
15
60
29
18
27
0.3
0.14
0.2
70
72
74
0.59
1.2
0.22
0.19
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.33
bMetabolic Rate (MR) <0.1 nmol/min/mg is considered metabolically stable.
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Table 3. Binding Affinity of Piperazinyl xanthine Derivatives with Different Substitution on Terminal
Phenyl for A2B, A1, A2A and A3AdoRs.
O
N
N
N
N
O
N
O
N
N
N
R
64-68
MRb
(nmol/min/mg)
%Inhibition at
0.1 µM
%Inhibition
Ki (nM)
hA2B
Compd R
at
1
µM
a
a
hA3
a
a
hA1
hA2A
MLM
HLM
p-CH3
5
58
32
53
45
56
58
37
71
56
72
11
0
nt
nt
64
65
66
67
68
m-CF3
p-CF3
1.5
0.9
10.7
1.8
0.04
nt
0.16
nt
11
0
2,4-diF
0.23
nt
0.11
nt
3-Cl, 4-F
17
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.33 bMetabolic Rate (MR) <0.1 nmol/min/mg is considered metabolically stable.
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Table 4. Binding Affinity of 5-oxopyrrolidinyl xanthine derivatives with methylene oxy linker for A2B,
A1, A2A and A3 AdoRs.
94-105
=
=
=
94-97
98-101
102-105
MRb
(nmol/min/mg)
%Inhibition at
0.1 µM
Ki
(nM)
%Inhibiti
on
at
Compd A
R
a
a
1 µM hA3
a
a
hA2B
hA1
hA2A
MLM HLM
Phenyl
m-F
p-F
9
33
29
1
89
89
80
60
85
80
71
46
nt
19
44
13
23
53
53
26
0
nt
nt
94
Phenyl
Phenyl
Phenyl
Pyridyl
Pyridyl
Pyridyl
Pyridyl
5.4
nt
nt
95
m-CF3 7.5
0.06
0.01
nt
0.09
0.05
nt
96
p-CF3
m-F
p-F
1
29
17
30
4
97
4.6
4
98
nt
nt
99
m-CF3 8.7
0
0.05
0.02
nt
100
101
102
103
104
105
p-CF3
3
25
nt
0
0
Isoxazolyl m-F
Isoxazolyl p-F
90
210
nt
7
nt
0
nt
nt
Isoxazolyl m-CF3 69
0
0
5
nt
nt
Isoxazolyl p-CF3 12.8
0
18
0
0.04
0.07
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.33 bMetabolic Rate <0.1 nmol/min/mg is considered metabolically stable.
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Table 5. Functional Potency, A2B, A1, A2AAdoR Affinity, and A2BAdoR Selectivity Over Other AdoRs
of Selective Compounds.
Binding Affinity Ki (nM)
A2B Selectivity
cAMP Ki (nM)
Compd
a
a
a
hA2B*
hA2B
hA1
hA2A
A1/A2B A2A/A2B
0.9
4
1
108
346
670
711
340
177
124
192
460
486
36
108
28
124
16
31
12
4
38
8
167
44
115
30
40
14
191
4
16
6
42
56
6
48
1.5
3
57
118
>266
328
38
65
71
540
>800 53
17
101
105
12.8
4200 2900
226
* Functional activity for hA2BAR was determined by inhibiting NECA induced cAMP levels in
HEK-A2B cells. Each compound was evaluated in triplicates at 12 concentrations. The mean Ki
from two independent experiments has been reported.a Binding affinities were determined by
using membrane preparations from HEK-293 cells overexpressing the relevant human AdoR
isoform. Each compound was evaluated at 8 concentrations with each data point in triplicates.33
2.3. DMPK
To identify a lead, compound 31, 40 and 65 were shortlisted for further profiling as they achieved our
predetermined targets for affinity, potency, selectivity and metabolic stability. The compounds 31, 40
and 65 were evaluated for drug metabolism and pharmacokinetics (DMPK) properties (Table 6) before
studies in in vivo models for efficacy. Of the three compounds, 65 showed best pharmacokinetic profile
(Table-6) with Cmax of 44 µM, oral exposure AUC(0-t) of 51 µM.h, t1/2 of 1.9 h and oral bioavailability
of 65%. In addition, compound 65 did not posssess any CYP liability (IC50 >25 µM) or cytotoxicity
(IC50 >100 µM) in MDCK and HepG2 cells.
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Table 6. Oral Pharmacokinetic Profile of Compounds 31, 40 and 65 in C57BL/6J Mice at 10 mg/Kga
Compd
t1/2(h)
%F
Cmax(µM) AUC(0-t)(µM.h)
16.7
3.8
44
21
2.9
51
2.4
0.9
1.9
27
11
65
31
40
65
aPO formulation for 31: DMAC(20%), PG(10%), PEG400(10%), MQ water(qs), pH ~9; for 40:
NMP(10%), DMAC(10%), PG(10%), PEG400(10%), Normal saline(qs), pH ~10; 65: DMSO(20%),
CrEL(10%), PEG300(10%), MQ water(qs)
2.4. In vivo efficacy study
Compound 65 was tested for efficacy in mice model of ovalbumin induced allergic asthma. Compound
65, at 3 mg/kg dose, significantly inhibited airway hyper-responsiveness induced by NECA (400 µg/ml)
(Figure 6A) and showed significant inhibition of total cell count (Figure 6B) in BALf (bronchoalveolar
lavage fluid).
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A) Effect of NECA Induced Airway Hyper responsiveness
B) Effect of Total Cell Number in BALf
Figure 1: Effect of Compound 65 on A) NECA-induced airway hyper-responsiveness and B) total cell count in
BAL fluid in Ovalbumin treated mice. Data is presented as mean ± SEM. *significantly different compared to
Vehicle treated group.
3. Conclusion
SAR study to improve oral bioavailability of tool compounds such as MRS-1754 (4) and MRE-2029-
F20 (5) led to identification of xanthine derivatives with constrained amide group as novel, potent and
selective A2BAdoR antagonists. Systematic optimization helped in arriving at a suitable linker
modification which improved the metabolic stability while retaining the binding affinity and selectivity
of compounds (31, 38, 40, 42, 48, 65, 101 and 105), for A2BAdoR, though these compounds had low
functional potency. Compounds 31 and 65 were identified with high binding affinity (Ki of 1 and 1.5
nM) and selectivity for A2BAdoR with very good functional potency of 0.9 and 4 nM, respectively.
Compound 31 and 65, on further evaluation, displayed good pharmacokinetic properties in mice with
27% and 65% oral bioavailability respectively. Compound 65 showed significant inhibition of NECA
induced airway hyper-responsiveness as well as total cell count in BALf, in ovalbumin induced allergic
asthma in mice thereby improving airway reactivity. Considering the overall profile, compounds 31 and
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65 may serve as leads for further optimization to identify a therapeutically useful A2BAdoR antagonist
for airway inflammation.
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 have been 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.
4.2. Synthesis
4.2.1.
Preparation of 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-
pyrazol-4-yl}-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (31): A mixture of 1, 3-dipropyl-8-(1H-
pyrazol-4-yl)-7-(2-trimethylsilanyl-ethoxymethyl)-3, 7-dihydro-purine-2, 6-dione 10a18 (0.20g,
0.46mmol), methanesulfonic acid 5-oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl ester
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1831,32 (0.202g, 0.60mmol), K2CO3 (0.145g, 0.69mmol) and DMF (2ml) were heated at 75-80 °C
for 16h. The reaction mixture was cooled to 20-25 °C and quenched with water (10ml). The aqueous
layer was extracted with ethyl acetate, the organic layer was washed with saturated brine solution,
dried over Na2SO4 and evaporated to obtain crude product (0.3g). The crude product was purified by
preparative TLC to obtain 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-
pyrazol-4-yl}-1,3-dipropyl-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-purine-2,6-dione (0.255g,
1
82%) as a light yellow oil. HNMR(DMSO d6): δ (-0.12) (s, 9H); 0.87-0.92 (m, 8H); 1.57-1.62 (m,
2H); 1.70-1.76 (m, 2H); 2.74-2.80 (m, 1H); 3.01-3.05 (m, 1H); 3.65-3.69 (m, 2H); 3.74-3.78 (m, 2H);
3.87 (t, J= 7.2Hz, 2H); 3.97-4.02 (m, 3H); 4.41 (d, J= 7.2Hz, 2H); 5.73-5.87 (m, 2H); 7.73 (d, J=
8.4Hz, 2H); 7.85 (d, J= 8.8Hz, 2H); 8.03 (s, 1H); 8.46 (s, 1H); (M++1): 674.
A mixture of 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-
dipropyl-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-purine-2,6-dione (0.255 g, 0.3mol), 2N HCl
(3ml), ethanol (2ml) were heated at 85 °C for 3h. The mixture was cooled to 10-15 °C. The solid
obtained was filtered and washed with water (1ml), diethyl ether (1ml) to obtain 8-{1-[5-Oxo-1-(4-
trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-dipropyl-3,7-dihydro-purine-2,6-
dione (0.158 g, 93%) as a white solid. M.P.: 234-235 °C; 1H NMR (DMSO d6): δ 0.87-0.92 (m, 6H);
1.57-1.62 (m, 2H); 1.70-1.76 (m, 2H); 2.46-2.49 (m, 1H); 2.71-2.78 (m, 1H); 3.1-3.19 (m, 1H); 3.74-
3.78 (m, 1H); 3.87 (t, J= 7.2Hz, 2H); 3.97-4.02 (m, 3H); 4.37 (d, J= 7.2Hz, 2H); 7.73 (d, J= 8.4Hz,
2H); 7.85 (d, J= 8.8Hz, 2H); 8.12 (s, 1H); 8.47 (s, 1H); 13.55 (s, 1H); (M++1): 544; HPLC: 99 %.
4.2.2.
Preparation of 8-(1-{2-Oxo-2-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-
pyrazol-4-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (65)
Preparation of 2-Chloro-1-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl] ethanone: To a mixture of 1-
(3-Trifluoromethyl-phenyl)-piperazine (10g, 43.47mmol), triethyl amine (13.29g, 130.41mmol) in
o
dichloromethane (100ml), chloroacetyl chloride (4.9g, 43.47mmol) was added at 0 C. The mixture
was stirred for 3h at room temperature. The reaction was quenched with water and the compound was
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extracted with dichlorormethane. The organic layer was washed with water and dried over sodium
sulphate, filtered and evaporated to give crude oil. The crude compound was purified by column
chromatography using silica gel (230-400mesh) and 0.5% methanol in dichloromethane to get pure 2-
1
Chloro-1-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl] ethanone (6g, 45%) as brown oil. HNMR
(DMSO d6): δ 3.18-3.32 (m, 4H); 3.55-3.67 (m, 4H); 4.47 (s, 2H); 7.08-7.13 (m, 1H); 7.12-7.28 (m,
2H); 7.41-7.49 (m, 1H); (M++1): 307.
To a mixture of 1,3-Dipropyl-8-(1H-pyrazol-4-yl)-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-
purine-2,6-dione 10a (7.44g, 17.22mmol), dry K2CO3 (7.14g, 51.66mmol) in acetone (50ml), 2-
Chloro-1-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl] ethanone (5.27g, 17.22mmol) was added and
the mixture was stirred for 4h at reflux temperature. The reaction mixture was filtered off and the
filtrate was evaporated to obtain the crude product which was purified by column chromatography
using silica gel (230-400mesh) and 1% methanol in DCM to get pure 8-(1-{2-Oxo-2-[4-(3-
trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-7-(2-trimethylsilanyl-
1
ethoxymethyl)-3,7-dihydro-purine-2,6-dione (10.2g, 82%) as an off white sticky solid. HNMR
(DMSO d6): δ (−0.12)(s, 9H); 0.80-0.93 (m, 8H); 1.52-1.64 (m, 2H); 1.67-1.79 (m, 2H); 3.19-3.39
(m, 4H); 3.60-3.73 (m, 6H); 3.80-3.92 (t, J=7.4Hz, 2H); 3.95-4.05 (t, J =7.4Hz, 2H); 5.35 (s, 2H);
5.76 (s, 2H); 7.08-7.16 (m, 1H); 7.19-7.32 (m, 2H); 7.40-7.50 (m, 1H); 7.99 (s, 1H); 8.3 (s, 1H);
(M++1): 703.3.
A mixture of 8-(1-{2-Oxo-2-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-
1,3-dipropyl-7-(2-trimethylsilanyl-ethoxymethyl)-3,7-dihydro-purine-2,6-dione (10.2g, 14.24mol),
MeOH (500ml) and 2N HCl (250ml) were taken and heated at 75 oC for 3h. The reaction mixture was
cooled to RT. The organic volatiles were evaporated to half of its volume and the solid product was
filtered off, washed with water to get crude solid. The crude compound was purified by column
chromatography using silica gel (230-400mesh) and 2% methanol in DCM to get pure 8-(1-{2-Oxo-2-
[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-3,7-dihydro-
o
1
purine-2,6-dione, 65 (4.2g, 50%) as an off white solid. M.P. 228-230 C; H NMR (DMSO
21

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d6): δ 0.83-0.95 (m, 6H); 1.50-1.64 (m, 2H); 1.66-1.81 (m, 2H); 3.21-3.39 (m, 4H); 3.58-3.72 (m, 4H);
3.85 (t, J=7.4Hz, 2H); 3.98 (t, J=7.4Hz, 2H); 5.31 (s, 2H); 7.08-7.15 (m, 1H); 7.19-7.32 (m, 2H);
7.42-7.70 (m, 1H); 8.01(s, 1H); 8.31 (s, 1H); 13.54 (s, 1H); (M++1): 573.3; HPLC Purity 100%.
4.3. Biological studies
4.3.1.
Radioligand Binding for A2B Adenosine Receptor: Human A2B adenosine receptor cDNA
was stably transfected into HEK-293 cells and the cells were maintained in DMEM supplemented with
10% FBS and antibiotics. For membrane preparation, cells were harvested by trypsinization with
0.25% Trypsin-EDTA (Sigma), and washed in 1X PBS at 1500 rpm for 5 minutes at room
temperature. The cells were washed in wash buffer containing 150 mM NaCl, 1 mM EDTA, 50 mM
Tris (pH-7.4) at 1500 rpm for 10 minutes at room temperature and incubated in sonication buffer
containing 1 mM EDTA, 5 mM Tris (pH 7.4) for 10 min at 4oC. The cells were sonicated on ice for 6
min with six intermittent pulses of 9 seconds each and centrifuged at 1000 x g for 10 minutes at 4oC.
The pellet was discarded and the supernatant was centrifuged at 49,000 x g for 45 minutes at 4oC. The
protein pellet was resuspended in buffer containing 1 mM EDTA, 5 mM Tris (pH-7.4), and 1 U/ml
adenosine deaminase (ADA) and incubated for 30 minutes at room temperature. The lysate was
washed twice with buffer containing 1 mM EDTA, 5 mM Tris (pH-7.4) at 49,000 x g for 45 minutes
at 4 oC and the protein pellet was resuspended in 50 mM Tris, pH-7.4 supplemented with 1 U/ml ADA
and 10% sucrose. The protein content was estimated using BCA method (Pierce).
Competition assays were initiated by mixing 1.6 nM [3H]-MRS-1754 with various concentrations of
test compounds and 10 µg membrane protein in reaction buffer (50 mM Tris pH 6.5, 5 mM MgCl2, 1
mM EDTA) supplemented with 1 U/ml ADA. The assay reactions were incubated for 90 minutes at
room temperature and stopped by filtration using 96 well-plate harvester (Molecular Devices) and
washed four times with ice cold 50 mM Tris (pH 7.4). Non specific binding was determined in
presence of 200 µM NECA. Radioligand binding was determined using Liquid Scintillation Counter
(Perkin Elmer) and the IC50 and affinities of compounds (i.e. Ki values) were calculated using
GraphPad prism software.
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4.3.2.
Radioligand Binding for A1, A2A and A3 Adenosine Receptors: Human A1, A2A and A3
adenosine receptor cDNAs were stably transfected into HEK-293 cells. Membranes for A1 and A2A
AdoR were prepared as described for A2BAdoR. For preparation of membranes from A3AdoR cells,
the wash buffer was modified to 10 mM HEPES, pH 7.4, 10 mM EDTA while the sonication buffer
was modified to 10 mM HEPES, pH 7.4, 1 mM EDTA. For competition radioligand binding assays,
1 nM [3H]-DPCPX, 2 nM [3H]-ZM-241385 and 2 nM [3H]-HEM-ADO were used for A1AdoR,
A2AAdoR and A3AdoR respectively. The reactions were carried out in presence of 1 U/ml ADA in 50
mM Tris pH 7.4, 1 mM EDTA (for A1 and A2A AdoR) or 50 mM Tris pH 7.4, 1 mM EDTA, 10 mM
MgCl2 (for A3 AdoR). The membrane preparations were incubated with varying concentrations of test
compounds and radioligand for 90 min at room temperature with constant shaking and the unbound
ligand were separated using filtration method as described above. Non specific binding was
determined in presence of 100 µM NECA. The bound radioligand were determined using liquid
scintillation counting and the IC50 and binding affinities were determined using GraphPad prism
software.
4.3.3.
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 A2B AdoR.
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.
23

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4.3.4.
Metabolic Stability assay protocol: The assay was carried out by incubating test
compound with species-specific liver microsomes (0.125 mg liver microsomal protein/mL), in 50 mM
phosphate buffer (pH 7.4), and NADPH (1 mM) for up to 30 minutes at 37 oC (final volume 100 µL).
o
The mixtures containing all components except NADPH were pre-incubated at 37 C for 5 min, and
the reactions were started by the addition of NADPH. The final concentration of the organic solvent in
the incubations was 0.1%. All incubations were performed in duplicate. The reactions were terminated
at 0, 10, 20 and 30 min time-points with the addition of 100 µL of acetonitrile containing internal
standard. Following protein precipitation and centrifugation, supernatant (100 µL) was transferred to a
separate 96-well plate and analyzed by LC/MS/MS. Metabolic stability of a test compound, measured
as the rate of disappearance of the parent (termed metabolic rate, MR), was calculated from the peak
area ratios of the test compound at each time point as nmol/min/mg microsomal protein.
4.3.5.
Pharmacokinetic Experiments: Pharmacokinetic experiments were carried-out in male
C57BL/6J mice (8–12 weeks age, weighing 25 ± 5 g). Plasma exposures determined after intravenous
(IV) and oral (PO) administration. Animals were housed in cages under the supply of filtered
pathogen-free air and ad libitum water. Studies were conducted after overnight fasting (~ 6 h pre dose)
and food was returned 2 h post-dose for oral group whereas IV group animals had free access to food
and water. For PO dosing, drug in suspension was administered via oral gavage at doses 10 mg/kg (n =
4) using oral feeding tube (Vehicle: 31: DMAC(20%), PG(10%), PEG400(10%), MQ water(qs), pH
~9; for 40: NMP(10%), DMAC(10%), PG(10%), PEG400(10%), Normal saline(qs), pH ~10; 65:
DMSO(20%), CrEL(10%), PEG300(10%), MQ water(qs). Following dose administration, blood
samples were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 h for PO group. Blood samples were collected
into micro-centrifuge tubes (sparse sampling design, approximately 0.100 µl) containing 10 µL
disodium EDTA (20 mg/mL) as anticoagulant. The blood samples collected were thoroughly mixed
and immediately centrifuged at 6000 rpm for 5 minutes and the supernatant plasma was separated into
fresh micro-centrifuge tubes. The plasma and formulation aliquote were stored below -70 0C until bio-
analysis. An aliquot of the plasma samples was extracted by protein precipitation and following
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centrifugation, an aliquot of the supernatant was injected on to the LC/MS/MS to determine the
plasma concentration.A 50 ꢀL aliquot of sample was deprotinized with a 150 ꢀL aliquot of acetonitrile
containing internal standard. After centrifugation at 10,000×g for 10 min, the supernatant was
transferred to a separate tube and a 50 ꢀL aliquot was injected directly onto the reversed-phase LC
column. Data analysis: Plasma pharmacokinetic parameters were calculated using the non-
compartmental analysis tool of WinNonlin Professional software (Version 5.2.1). Pharmacokinetic
parameters were determined from individual animals in each group.
4.3.6.
In vivo efficacy study: Animals: Female BALB/c mice, 6 to 8 weeks of age were obtained
from Advinus Banglore. Mice were fed a standard mice chow and housed under controlled
environmental conditions. Animals were given water and food ad libitum. All experimental protocols
were pre-approved by the Institutional Animal Ethics Committee (IAEC). Study Design: Mice were
intra-peritoneally sensitized on days 1 and 5 with 10 µg of ovalbumin (Sigma 9006-59-01) in 4 mg of
Imject alum (Pierce Laboratories, Rockford, IL). Non-sensitized control animals only received 4 mg
of Imject Alum.
Treatment Groups: On day 12, both non-sensitized and sensitized mice were divided into following
groups:
•
•
•
•
•
Sentinel
Alum
Ovalbumin + Vehicle
Ovalbumin + Compound 65
Ovalbumin + CVT-6883
Compound 65 and CVT-6883 were administered at 3 mg/kg dose BID. All the treatments were
administered intra-peritoneally (IP).
Challenge with Ovalbumin aerosol: Required amount of Ovalbumin was weighed and dissolved in
required volume of saline. On day 12, mice were dosed with their respective treatments. One hour after
the treatment, mice were placed in a Plexiglas chamber (Buxco mass dosing system) and challenged
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with 0.5% aerosolized ovalbumin using an ultrasonic nebulizer (Aerogen) for 60 min. Five hours after
the ovalbumin challenge, mice were again treated with their respective treatments. On day 13 and 14,
mice were challenged and treated in the same way as on day 12.
Determination of airway responsiveness: On day 15, mice were dosed with respective treatments and
one hour later ovalbumin-induced airway hyper-responsiveness to adenosine analog NECA i.e. 5′-(N-
Ethylcarboxamido) adenosine (12.5 ug/ml-400 ug/ml) was assessed with whole body plethysmograph
(Buxco Max II, Troy, NY, USA). The response time for Penh measurement was 2 min followed by
baseline of 1 min. Airway responsiveness was expressed as percentage increase in Penh compared to
0.9% saline challenge (as baseline) for each dose.
Assessment of airway inflammation: After measurement of Penh, bronchoalveolar lavage fluid (BALf)
was collected. The total cell count was measured using Guava cytometer.
Statistical Analyses: All values are expressed as mean ± SEM. Analysis of variance was used to
determine the levels of significance between groups. Single pairs of groups were compared by one-way
ANOVA followed by Dunnett’s multiple comparison tests while multiple comparisons were done using
Bonferroni’s test with Prism GraphPad 4.0. P value of < 0.05 was considered statistically significant.
Appendix A. Supplementary data
The contents of supporting information include the following: (1) extensive schemes, (2) experimental
procedures for intermediates and final compounds, (3) analytical and spectral characterization data.
Acknowledgements
Authors are grateful to the senior management of the Advinus Group for its support and encouragement.
Analytical departments are being acknowledged for their help during this work. We thank Dr. Anup
Ranade for managing intellectual property - Advinus publication no. ADV-A-037
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