Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1receptor antagonists and their biological evaluation

Article abstract of DOI:10.1016/j.bmc.2017.02.029

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.

Full text of DOI:10.1016/j.bmc.2017.02.029

Accepted Manuscript
Design and synthesis of novel, potent and selective hypoxanthine analogs as
adenosine A1 receptor antagonists and their biological evaluation
Summon Koul, Vidya Ramdas, Dinesh A. Barawkar, Yogesh B. Waman, Neela
Prasad, Santosh Kumar Madadi, Yogesh D. Shejul, Rajesh Bonagiri, Sujay
Basu, Suraj Menon, Srinivasa B. Reddy, Sandhya Chaturvedi, Srinivas Rao
Chennamaneni, Gaurav Bedse, Rhishikesh Thakare, Jayasagar Gundu, Sumit
Chaudhary, Siddhartha De, Ashwinkumar V. Meru, Venkata Palle, Anita
Chugh, Kasim A. Mookhtiar
PII:
S0968-0896(16)31209-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.029
BMC 13558
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 November 2016
8 February 2017
10 February 2017
Please cite this article as: Koul, S., Ramdas, V., Barawkar, D.A., Waman, Y.B., Prasad, N., Kumar Madadi, S.,
Shejul, Y.D., Bonagiri, R., Basu, S., Menon, S., Reddy, S.B., Chaturvedi, S., Rao Chennamaneni, S., Bedse, G.,
Thakare, R., Gundu, J., Chaudhary, S., De, S., Meru, A.V., Palle, V., Chugh, A., Mookhtiar, K.A., Design and
synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their
biological evaluation, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.029
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Design and synthesis of novel, potent and selective
hypoxanthine analogs as adenosine A1 receptor
antagonists and their biological evaluation
Leave this area blank for abstract info.
Summon Koul*, Vidya Ramdas, Dinesh A. Barawkar, Yogesh B. Waman, Neela Prasad, Santosh
Kumar Madadi, Yogesh D. Shejul, Rajesh Bonagiri, Sujay Basu, Suraj Menon, Srinivasa B. Reddy,
Sandhya Chaturvedi, Srinivas Rao Chennamaneni, Gaurav Bedse, Rhishikesh Thakare, Jayasagar
Gundu, Sumit Chaudhary, Siddhartha De, Ashwinkumar V. Meru, Venkata Palle, Anita Chugh, Kasim
A. Mookhtiar
Cy
III
Compound
14
23
33
38
Cy
X
O
O
O
Absent
16
A1 Binding Ki (nM)
23
6.6
57

Design and synthesis of novel, potent and selective hypoxanthine analogs as
adenosine A1 receptor antagonists and their biological evaluation
Summon Koul*, Vidya Ramdas, Dinesh A. Barawkar, Yogesh B. Waman, Neela Prasad,
Santosh Kumar Madadi, Yogesh D. Shejul, Rajesh Bonagiri, Sujay Basu, Suraj Menon,
Srinivasa B. Reddy, Sandhya Chaturvedi, Srinivas Rao Chennamaneni, Gaurav Bedse,
Rhishikesh Thakare, Jayasagar Gundu, Sumit Chaudhary, Siddhartha De, Ashwinkumar
V. Meru, Venkata Palle, Anita Chugh, Kasim A. Mookhtiar
Drug Discovery Unit, Advinus Therapeutics Ltd., Hinjewadi, Pune-411 057, India
*Corresponding author:
Summon Koul
Advinus Therapeutics Ltd, Plot No. 9, Rajiv Gandhi Park, Phase I,
Hinjewadi, Pune, Maharashtra – 411 058
summon.koul@advinus.com
Abstract: Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl
hypoxanthine analogs from a common intermediate III. Several potent and selective compounds
were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the
compounds 14 with acceptable PK parameters was selected for testing in in-vivo primary acute
diuresis model. The compound demonstrated significant diuretic activity in this model.
Keywords: Adenosine, Rolofylline, BG-9928, diuresis, hypoxanthine, glomerular
1. Introduction
Adenosine is an endogenous signaling nucleoside distributed in a wide variety of tissues and
plays a key role in a variety of physiological processes.1-4 The effects elicited by adenosine are
mediated by its interactions with four receptor subtypes named A1, A2A, A2B and A3. These
receptors belong to the superfamily of G protein coupled receptors and modulate a number of
effector systems, including adenylate cyclase, potassium and calcium channels, phospholipase C
or D, and guanylate cyclase.5 Activation of A1 and A3AR leads to an inhibition of adenylyl
cyclase activity, while activation of A2A andA2BAR causes a stimulation of adenylyl cyclase.
Different therapeutic applications have been identified in preclinical and clinical studies for
A1AR antagonists, which are effective as potassium-sparing diuretic agents with kidney-
protecting properties.6 The ability of A1R antagonists to induce diuresis and natriuresis while not
compromising glomerular filtration rate is an attractive therapeutic option for the treatment of
fluid retention disorders, e.g. in kidney disease and heart failure, especially in conditions

associated with diuretic resistance. Adenosine, via A1 receptors (A1R), also serves as a mediator
for triggering the hepatorenal reflex leading to renal water and sodium retention.7, 8 . Blockade of
hepatic and renal A1R could therefore provide a new therapeutic option for sodium and water
9
retention in conditions such as liver cirrhosis as well. Berthold Hocher and his coworkers have
demonstrated long term safety and efficacy of A1R-specific antagonist SLV329 in an animal
model of liver cirrhosis.10 In clinics, SLV329 is known to increase renal sodium and water
11, 12
excretion in patients with liver cirrhosis without affecting the glomerular filtration rate.
13
Despite progression of A1 receptor antagonists to clinics (including a large Phase 3 trial),
development of clinically successful A1 receptor antagonists has been hampered by safety
concerns. Seizures have been reported in patients treated with Rolofylline [14]. Adenosine via A1
receptors located in central nervous system modulates seizure threshold [15]. Selective renal A1
receptor antagonism with compounds that do not cross blood brain barrier may offer an
opportunity to develop safe A1 receptor antagonists. This provides impetus to discover and
develop novel selective A1 receptor antagonists for therapeutic intervention.
A1R antagonists can be mainly divided into two structural subgroups, xanthines and non-
16
17
xanthines. The compounds, Rolofylline (1) and BG-9928 (2) that entered Phase III clinical
trials (Fig. 1) are xanthine based A1R antagonists. SLV320 (3), another A1R antagonist in clinical
trials is an adenine analog.18 Other non xanthine based antagonists are pyrazolo pyridine
derivatives 19, 20 and quinoxaline analogs.21
1
2
3
Figure 1. A1R antagonists that entered clinical trials.
As described above, xanthine and adenine analogs are well known in literature as potent A1R
antagonists that follow a particular metabolic pathway as shown in Fig. 2. However,
hypoxanthine analogs that fall between adenine and xanthine on the pathway have not been much
explored in general as adenosine receptor antagonists. Hence, we rationalized that hypoxanthines
can provide us with a novel chemotype forA1R antagonism. Herein we report discovery of novel
N-1 propyl-C-8 cyclopentyl hypoxanthine analogs that are potent and selective A1R antagonists.

O2, H2O
xanthine
oxidase
H2O
NH3
Xanthine
Hypoxanthine
Adenine
H2O2
Figure 2. Metabolic Pathway of Adenine to Xanthine.
The structure activity relationship studies, pharmacokinetic properties of selected compounds and
their in-vivo efficacy results are also described
2. Results and Discussion
2.1. Chemistry
Multipronged approach was used to synthesize a library of about 40 compounds from
Intermediate III (Scheme 2). Intermediate III was prepared from 5, 6-diamino-3-propyl-1H-
pyrimidine-2, 4-Dione 22, 23 (I) in two steps by EDCI coupling of I with cyclopentane carboxylic
acid followed by concerted cyclization and chlorination with POCl3 (Scheme 1)
a
b
III
I
II
Scheme 1. General synthesis of common intermediate, III. Reagents and conditions: (a) EDCI.HCl,
MeOH, 0 0C → rt; (b) POCl3, PCl5, reflux overnight, 19% over 2 steps.
Compounds 4-7, 8a and 9a were synthesized in moderate yields in one step by heating
Intermediate III with the corresponding aliphatic or aromatic amine in N-methyl pyrrolidine in
presence of diisopropylethylamine. 8a and 9a on base mediated hydrolysis afforded acids 8 and 9
in good yields. Intermediate III on treatment with the appropriately substituted anilines and
phenols in N-methyl pyrrolidine at elevated temperatures afforded compounds 10-13 and 14-28
respectively in overall good yields. Compounds 29-35 were prepared in moderate yields from III
by heating it with corresponding alcohol in presence of sodium hydride. Finally compounds 36-
41 were synthesized by palladium catalyzed Suzuki coupling of III with corresponding boronic
acids using sodium bicarbonate as a base.

4; R1 = p-CF3C6H4, 5; R1 = p-CH3C6H4
6; R1 = -CH2OH, 7; R1 = m-ClC6H4
10; R2 = Ph, 11; R2 = p-FC6H4
12; R2 = m-CF3C6H4, 13; R2 = m-FC6H4
a
c
8a; R1 = p-CO2MeC6H4, 9a; R1 = -CO2Et
b
b
9; R1 = -COOH
8; R1 = p-CO2HC6H4
d
f
14; R3 =3,4-difluoro C6H3, 15; R3 = m-OMeC6H4
16; R3 = m-CF3C6H4, 17; R3 = p-CONH2C6H4
18; R3 =4-Me-3-pyridine, 19; R3 =5-chloro-3-pyridine
20; R3 = 5-indole, 21; R3 = p-SO2NH2C6H4
22; R3 = m-CH2COOHC6H4' 23; R3 = 2-Me-3-pyridine;
24; R3 = 7-indole, 25; R3 = 4-indole;
III
36; Ar = m-CF3C6H4, 37; Ar = p-CF3C6H4
38; Ar = m-OMeC6H4, 39; Ar = m-COOHC6H4,
40; Ar = m-OHC6H4, 41; Ar = m-CONH2C6H4
e
26; R3 = 5-Me-2-pyridine,27; R3 = 2-chloro-3-pyridine
28; R3 = m-OHC6H4
29 R4 = Cyclopentyl, 30; R4 = N-methyl-3-pyrrolidine
31; R4 = 4-pyran, 32; R4 = 3-Tetrahydrofuran,
33; R4 = 3-hydroxy-cyclopentane; 34; R4 = (3S)-Tetrahydrofuran
35; R4 = (3R)-Tetrahydrofuran
Scheme 2. General synthesis of A1R antagonists 4-41. Conditions: (a) Corresponding amines, N-methyl
o
o
pyrrolidine, DIPEA, 130 C, 2 h, 15-30%; (b) NaOH, EtOH/THF/H2O (1:1:1), 50-60 C, 12 h; (c)
o
Corresponding anilines, N-methyl-pyrrolidine, 150 C, 12 h, 47%; (d) Corresponding phenols, N-methyl-
pyrrolidine, 130 oC, 12 h, 52%; (e) Corresponding alcohols, NaH (60% dispersion in mineral oil), 120 oC –
130 oC, 3 h, 22-34%; (f) Corresponding boronic acids, Pd (PPh3)4, NaHCO3, THF, 100 oC, 12 h, 20-40%.
2.2. Structure Activity Relationship
Compounds 4 and 5 with hydrophobic substituents at para positions of N-benzyl group, were
identified as the most potent (Ki = 23 and 27 nM) and selective A1R antagonists (Table 1). Meta
substitution on the aryl ring (compound 7) retained potency against A1R (Ki = 120 nM) though
found to be less potent as compared to compounds 4 and 5. Substituting the para position with
hydrophilic carboxylic group as in compound 8 resulted in drastic loss in potency. Compounds 6
and 9 with aliphatic ethanol amine and N-acetic acid moieties lost potency against A1R though 9
was found to be slightly more potent in comparison to its aromatic counterpart 8.
Table 1
In-vitro data of C-2 substituted N-benzyl and N-alkyl analogs
Compound
R1
hA1 Ki (nM) / %
% Inhibition at 1 µM conca

inhibition at 1µM conca
23
hA2B
0%
hA2A
0%
hA3
23%
4
5
6
27
6%
0%
5%
24%
0%
-CH2OH
36%
120
31%
nt
nt
nt
nt
7
8
16%
31%
54%
72%
9
-COOH
9%
0%
0%
All data points were evaluated in triplicates. Each compound was evaluated at 8 concentrations with each
data point in triplicates for Ki determination
aBinding affinities were determined by using membrane preparations from HEK-293 cells overexpressing
the relevant human AdoR subtype.
nt - not tested.
Compounds 10-13 with substituted anilines at C-2 position were found to be less potent as
compared to N-benzyl analogs (Table 2). In this series, compound 12 with meta-CF3 substitution
was identified as the most potent compound (Ki = 370 nM) against A1R.
Table 2
In- vitro data of C-2 substituted N-aryl analogs
Compound
R2
hA1 Ki (nM)
> 1000
% Inhibition at 1 µM conc.a
hA2B
hA2A
hA3
nt
nt
nt
10
> 1000
370
6%
5%
0%
24%
nt
11
12
27%

4500
16%
31%
nt
13
All data points were evaluated in triplicates. Each compound was evaluated at 8 concentrations with each
data point in triplicates for Ki determination
aBinding affinities were determined by using membrane preparations from HEK-293 cells overexpressing
the relevant human AdoR subtype.
nt - not tested.
A set of about 15 O-aryl and heteroaryl analogs 14-28 were prepared with different hydrophobic
and hydrophilic substitutions at meta and para positions to understand detailed SAR for potency
and selectivity against A1R (Table 3). Compounds 14-16 with aryl rings substituted at meta
positions with hydrophobic groups were found to be the most potent compounds in both A1R
binding (Ki = 12-23 nM) and functional assays (Ki = 2-13 nM). However, even though
compounds 14 and 16 with 3,4-difluoro and 3-trifluoromethyl groups respectively were selective
against other adenosine receptor subtypes, compound 15 with 3-methoxy group showed less
selectivity against A2B (70% inhibition at 1 µM) and A3 (Ki = 430 nM) subtypes. Compounds 17,
21 and 22 with meta or para substituted hydrophilic groups on aryl rings lost potency against A1R
(Ki > 1000 nM) though 3-hydroxyl substituted analog 28 was an exception. 28 was found to be
potent (Ki = 10.5 nM) in A1 binding assay but less selective against other adenosine receptor
subtypes. Since this compound was expected to show high clearance in vivo due to Phase 2
metabolism, we shifted our focus to the indole bioisosteric replacements of phenol. Thus,
compounds 20, 24 and 25 with 5-, 7- and 4-indolyl moieties respectively were synthesized to
understand the impact of different orientations of indole nitrogen on A1 potency. All three
compounds retained potency against A1R and compound 24 with 7-indolyl substitution was
identified as the most potent compound in A1 binding and functional assays. Then our focus
shifted to understand the effect of C-2 substituted pyridinoloxy groups on A1R potency.
Compounds with para substitutions with respect to either pyridine nitrogen (compound 26, Ki >
1000 nM)) or point of attachment at C-2 (compound 18, Ki = 630 nM) seemed to lose potency
against A1R. All other pyridine derivatives having meta or ortho substitutions with respect to
pyridine nitrogen or point of attachment at C-2 were found to be potent against A1R. The most
potent compounds identified in this series were 23 [Ki (binding) = 6.6 nM, Ki (functional) = 0.28
nM)] and 27 (Ki = 3.6 nM) both of which had substitutions ortho to pyridine nitrogen and point of
attachment at C-2. In binding assay, compound 23 was found to have less selectivity against A2B
(91% inhibition at 1 µM) though in functional assay, the compound showed about 2000 fold
selectivity for A1 over A2B.

Table 3
In-vitro data of C-2 substituted O-aryl analogs
% Inhibition at 1 µM conca
hA1 Ki (nM)a
23
Compound
R3
A1 cAMP Ki (nM)
13
hA2B
hA2A
hA3
IC50
>
nr
IC50 >
14
15
16
17
18
19
20
10,000 nM
10,000 nM
12.5
19
2.3
5.9
nt
70%
0%
nt
36%
34%
nt
IC50: 430
nM
35%
nt
> 1,000
630
nt
nt
nt
nt
12
2.2
nt
37%
0%
22%
0%
76%
34%
36
> 1,000
> 1,000
6.6
nt
nt
nt
nt
nt
nt
21
22
23
24
nt
nt
91%b
42%
0.28
5.6
33%
25%
35%
40%
5.5

74
> 1,000
3.6
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
25
26
27
28
nt
nt
nt
10.5
53%
81%
69%
All data points were evaluated in triplicates. Each compound was evaluated at 8 concentrations with each
data point in triplicates for Ki determination
aBinding affinities were determined by using membrane preparations from HEK-293 cells overexpressing
the relevant human AdoR subtype.
b% inhibition at 30 µM concentration.
nt - not tested.
nr – no response at highest tested concentration.
In case of C-2 substituted O–alkyl analogs, similar trend in SAR was observed as seen in
previous series (Table 4). All compounds with carbocyclic or heterocyclic substitutions at C-2
were found to be potent against A1R though compound 30 with N-methyl pyrrolidine substitution
was found to be less potent. In this series, compounds 29 [Ki (binding) = 14 nM; Ki (functional) =
13 nM] and 31 [Ki (binding) = 20 nM; Ki (functional) = 12 nM] were identified as the most
potent compounds. However, compound 29 was found to be less selective against other three
adenosine receptor subtypes while compound 31 was selective against A3R. Racemic compound
32 with tetrahydrofuryl moiety at C-2 (Ki = 59 nM) and its two enantiomers, 34 (Ki = 85 nM) and
35 (Ki = 50 nM) retained similar potency against A1R
Table 4
In-vitro data of C-2 substituted O-alkyl analogs
hA1 Ki (nM)a
14
Compound
R4
A1 cAMP Ki (nM) % Inhibition at 1 µM conca
hA2B
hA2A
hA3
13
92%
85%
79%
29

> 1,000
20
nt
nt
nt
nt
30
31
12
56%
82%
0%
59
57
nt
nt
nt
nt
32
33
9.6
35%
0%
37%
85
50
nt
nt
nt
nt
nt
nt
nt
nt
34
35
All data points were evaluated in triplicates. Each compound was evaluated at 8 concentrations with each
data point in triplicates for Ki determination
aBinding affinities were determined by using membrane preparations from HEK-293 cells overexpressing
the relevant human AdoR subtype.
nt - not tested.
In case of C-2 substituted aryl analogs, compounds 36 and 38 with meta substituted aryl moieties
were found to be potent against A1R while compound 37 with para substitution lost A1 potency to
some extent. Compound 36 was also selective against other adenosine receptor subtypes.
Compounds 39 and 41 with hydrophilic substitutions drastically lost potency against A1.
Compound 40 with meta hydroxyl group, however, retained very good A1R potency both in
binding (Ki = 1 nM) and functional (Ki = 0.2 nM) assays though it was found to be less selective
against A2B and A2A receptor subtypes. This SAR confirms that hydrophilic carboxyl and amide
functionalities are not well tolerated for A1R activity across the series
Table 5
In-vitro data of C-2 aryl analogs

Compound
Ar
hA1 Ki (nM) /%
A1 cAMP Ki (nM)/% Inhibition at 1 µM conca
inhibition at 1µM conca
Ki (nM)
hA2B
hA2A
hA3
100%
12
14%
0%
0%
36
70%
16
nt
12%
1100
0%
5%
37
38
15
10,000
690
< 50%b
nr
nt
nt
nt
39
1
0.2
nt
100%
nt
74%
nt
nt
nt
40
41
> 1000
All data points were evaluated in triplicates. Each compound was evaluated at 8 concentrations with each
data point in triplicates for Ki determination
aBinding affinities were determined by using membrane preparations from HEK-293 cells overexpressing
the relevant human AdoR subtype.
b% inhibition at 30 µM concentration.
nt - not tested.
nr – no response at highest tested concentration.
Based on diverse SAR explored at C-2 position of hypoxanthine core (Tables 1-5),
potent and selective representative compounds, 14, 23, 33 and 38 were selected for
further profiling. The compounds were found to have good to moderate stability in liver
microsomes and were free from any CYP liability. Compounds 14 and 38 did not show
any hERG liability in binding assay (Table 6)
Table 6
In-vitro and ADME data of Compounds 14, 23, 33 and 38
Parameter
A1AdoR (binding) Ki
(nM)
14
23
33
38
23
6.6
57
16
A1AdoR
cAMP
13
0.28
9.6
15

(functional) Ki (nM)
Selectivity against other
A2A: 2000 nM
A2B: 49%
A2A :0%
A2B: 41%
A3 : 6%
A2A : >10 µM
A2B: 1.1 µM
A3 : 650 nM
adenosine
subtypes
receptor
(Ki/%
> 10 µM
A2B cAMP Ki =
560 nM
inhibition at 1 µM)
A3 : 16%
b
MRa,
nmol/min/mg 0.08/0.05; 75/82
0.07/0.06; 78/79
0.10/0.12; 68/60
0.05/0.03; 82/97
protein (RLM/HLM);
% remaining at 30 min
(+NADPH)
hERG (% inhibition) @
5, 25 uM concs
2, 4
7, nt, 0, 10, 34
99/99
nt
nt
9, 72
0, 7, nt, 1, 5
95/95
CYP 450 % Inhibition
at 5 uM; 1A2, 2C9,
2C19, 2D6, 3A4
0, 4, 10, 0, 0
5, 4, 3, 0, 0
nt / 80
Plasma Protein
binding at 5 uM
(Mouse/Rat)
%
nt / 77
Thermodynamic
77, 23, 17
2152, 43, 38
2589, 276, 182
614, 63, 54
Solubility (µM) @ pH
2, 4, 7.4
nt- not tested.
aMetabolic Rate <0.1 nmol/min/mg is considered metabolically stable.
bTestosterone [MR (RLM) > 1.52, 2% remaining at 30 min (+NADPH); MR (HLM): 0.05, 84% remaining
after 30 min (+NADPH)] and Verapamil [MR (RLM): 0.1, 68% remaining at 30 min (+NADPH); MR
(HLM): 0.06, 78% remaining after 30 min (+NADPH)] have been used as positive controls in microsomal
metabolic stability studies.
RLM: Rat liver microsomes.
HLM: Human liver microsomes.
2.3. In-vivo Pharmacokinetics in Male Wistar Rats
We next determined the pharmacokinetic (PK) profile of these four compounds, 14, 23,
33 and 38 in male Wistar rats. The results are summarized in Table 7. Compound 14

showed reasonable half life of 2 hours with low clearance. Compounds 23 and 33 were
found to have high clearance and very low half life. To address the high clearance of
these compounds, PK studies of compounds 23 and 33 were carried out in presence and
absence of 1-aminobenztriazole (ABT) which is a non-specific CYP inhibitor. In these
studies 23 in presence of ABT was found to show 6 fold increase in exposure and 3 fold
increase in half life with 6 fold decrease in clearance while in case of compound 33,
exposure and half life increased 3-4 fold and clearance was reduced by 4 fold suggesting
that these compounds are undergoing CYP and non-CYP based Phase 1/2 metabolism.
To find metabolic soft spots of these compounds, metabolite identification studies were
carried out. Compound 23 appears to undergo hydroxylation at N-1 propyl and C-8
cyclopentyl ring followed by dehydration. Similarly, it was proposed that compound 33
undergoes N- oxidation and C-8 cyclopentyl ring hydroxylation.
Table 7
Pharmacokinetic (PK) profilea of 14, 23, 33 and 38 in male Wistar rats
Compound
Route of
14
23
33
38
administration
Dose (mg/kg)
IV
3
PO
5
IV
3
IV
3
IV
3
PO
5
Cmax (µM)
Tmax (h)
NA
NA
9.71 ± 1.8
NA
NA
NA
NA
1.74 ± 0.8
0.3 ± 0.11
1.6 ± 0.83
NA
1.63 ± 0.75
NA
NA
AUC0-t (µM.h)
Vss (L/kg)
10.18 ± 3.53 3.19 ± 1.13
2.7 ± 0.9
2.0 ± 0.6
3.0 ± 0.3
0.8 ± 0.2
3.53 ± 0.33
1.2 ± 0.66
1.73 ± 0.62
NA
NA
NA
27
CL (mL/min/kg) 13.61 ± 4.42
55 ± 16
0.5 ± 0.05
NA
48 ± 5
0.3 ± 0.04
NA
39.22 ± 3.59
0.49 ± 0.35
NA
NA
NA
27
T1/2 (h)
%F
2.06 ± 0.61
NA
aValues indicate mean for n = 4.
NA: Not applicable.
Compound 14: IV - NMP-10%,PEG-400 (20%), normal saline q.s pH adjusted to 12 with 1N NaOH; PO
– NMP (10%), Labrasol (30%), citrate buffer q.s (pH 7.0).
Compounds 23 and 33: NMP-5%, PEG-300 (10%), 0.1 M Citrate buffer q.s. (pH 5.5).
Compound 38: NMP-10%, PEG-400 (20%), Citrate buffer q.s, pH 8.0 (pH 4.2).
2.3. In-vivo Brain Disposition of Compounds 14 and 38 in Male Wistar Rats

Since our objective was to identify selective A1 receptor antagonists that do not cross
blood brain barrier, we carried out in-vivo brain disposition studies of representative
compounds 14 and 38 in male Wistar rats after oral administration of compounds at 5
mg/kg dose (Table 8). Compound 14 showed negligible (3-5% of plasma) concentrations
in brain while as compound 38 showed brain concentrations below quantification limit.
Table 8
In-vivo Brain and Plasma Concentrationsa of 14 and 38 in male Wistar Rats.
Compound
14
38
Dose
5 mg/kg, PO
Time (h)
1
2
4
1
2
4
Concentrations in Plasma (µM)
Concentrations in Brain (µM)
Brain/Plasma Ratio
1.64 0.94 1.81 0.67
0.09 0.05 0.08 BQL BQL BQL
0.05 0.05 0.05 NA NA NA
0.54 0.04
aValues indicate mean for n = 2 per time point
BQL: Below Quantification level (10 ng/mL)
NA: Not applicable
Compound 14: NMP (10%) v/v, Labrasol- 30% v/v, citrate buffer pH 8.5
Compound 38: NMP (10%) v/v, polyethylene glycol-400 (PEG-400) - 20% v/v, citrate buffer pH 8.5
2.4. In-vivo Pharmacodynamics of Compound 14 in Acute Diuretic Model
Since compound 14 showed negligible brain concentrations and longer half life as well as
better exposure in PK studies, this compound was selected for assessment of oral activity
in an acute rat diuresis model at 3, 10 and 30 mg/kg doses. Compound 14 showed
significant diuresis at 10 and 30 mg/kg doses and natriuresis at all the three doses as
compared to the vehicle treated group (Figure 3). These results were comparable with
those obtained with Rolofylline treated animals at 0.1 mg/kg dose. There was no
significant kaliuresis observed with compound 14 treated groups compared to vehicle.
Also there was no significant difference in serum Na+ and K+ levels as compared to
vehicle group.

Urine Na +
*
6
5
4
3
2
1
0
*
Urine volume
40
30
20
10
0
*
*
*
*
*
Vehicle-1
Rolofylline (0.1 mg/kg,PO)
Vehicle-2
Compound-14 (3 mg/kg,PO)
Compound-14 (10 mg/kg,PO)
Compound-14 (30 mg/kg,PO)
Urine K +
0.8
0.6
0.4
0.2
0.0
The data represent mean ± S.E.M., (n=6 per group).
One-way ANOVA followed by Tukey's Multiple
Comparison Test, *P <0.05 vs. vehicle.
Figure 3. Effect of compound 14 on acute diuretic activity in Wistar Rats.
3. Conclusions
In summary, we have reported herein the discovery of a series of novel hypoxanthine
based antagonists which demonstrated good in-vitro A1R antagonism and acceptable
selectivity against other adenosine receptor subtypes. Four compounds 14, 23, 33 and 38
were evaluated for their pharmacokinetic properties in Wistar rats. Compounds 14 and 38
were further profiled in brain disposition study. Compound 14 showed reasonable
exposure, half life and negligible brain exposure. This warranted the evaluation of this
compound in rat PD model in which 14 exhibited significant diuretic activity at 10 mg/kg
dose.
4. Materials and methods
The purity of compounds was determined by HPLC and all compounds had purities of
1
>95% unless otherwise stated. H nuclear magnetic resonance (NMR) spectra were
recorded in the deuterated solvents specified on a Varian 400 spectrometer operating at
400 MHz. The signal of the deuterated solvent was used as internal reference. Chemical

shifts (δ) are given in ppm and are referenced to residual not fully deuterated solvent
signal. Coupling constants (J) are given in Hz. Chemical shifts are reported in parts per
million (δ) from the tetramethylsilane resonance in the indicated solvent (TMS: 0.0 ppm).
Data are reported as follows: chemical shift, multiplicity (br=broad, s=singlet, d=doublet,
t=triplet, q=quartet, quint=quintet, m=multiplet) and integration. Mass spectra were
determined by using Agilent MSD/VL spectrometer or Agilent 1200SL-6110 LC/MS
(ESI) system using positive-negative switching.
4.1. Chemical synthesis
4.1.1. N-(6-amino-2, 4-dioxo-3-propyl-1H-pyrimidin-5-yl) cyclopentanecarboxamide
(II)
To a solution of 5, 6-diamino-3-propyI-lH-pyrimidine-2, 4-dione (0.6 g, 2.72 mmol) in
methanol (50 mL) was added cyclopentane carboxylic acid (0.31 g, 2.72 mmol). The
reaction mixture was cooled to 0°C and then l-ethyl-3(3'-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI.HC1) (0.78 g, 4.1 mmol) was added. The resulting
reaction mixture was stirred at room temperature overnight. The reaction mixture was
concentrated under reduced pressure. The residue was suspended in water. The solid was
filtered and washed thoroughly with water followed by diethyl ether. The product
obtained was dried under high vacuum. The crude product II (0.40 g) was used for the
next step without further purification.
4.1.2. 2-chloro-8-cyclopentyl-1-propyl-7H-purin-6-one (III)
To a suspension of cyclopentane carboxylic acid (6-amino-2,4-dioxo-3-propyl- 1,2,3,4-
tetrahydro-pyrimidin-5-yl)-amide (0.40 g, crude) obtained from step 1 in phosphorus
oxychloride (25 ml) was added phosphorus pentachloride (0.10 g) and the resulting
reaction mixture was refluxed overnight. Phosphorus oxychloride was evaporated under
reduced pressure. The residue was slowly quenched with water. Ethyl acetate was added
and the organic layer was separated and washed thoroughly with water followed by brine.
The ethyl acetate layer was dried over anhydrous sodium sulphate and concentrated under
vacuum. The crude product was purified by preparative TLC using dichloromethane/
methanol (9:1) as the solvent system to give 0.075 g (19% over two steps) of III as a
white solid. III: mp: 204-206 oC; LCMS: m/z 281.3 [M++1]; 1HNMR (400 MHz, DMSO-

d6) δ 0.9 (t, J = 8 Hz, 3H), 1.59-1.82 (m, 8H), 1.99 (m, 2H), 3.15 (t, J = 8 Hz, 1H), 4.12
(t, J = 8 Hz, 2H)
4.1.3. General Synthesis of Compounds 4-8a, 9a
To a solution of III (1 equiv., 0.10 g) in N-methyl-2-pyrrolidone (10 equiv., 0.3 mL) was
added corresponding amine (1.2 equiv.) followed by diisopropylethylamine (3 equiv., 0.2
mL) and the reaction mixture was heated at 130 °C for 2 hours. The reaction mixture was
then diluted with water and extracted with ethyl acetate. Ethyl acetate layer was dried
over anhydrous sodium sulphate and concentrated under vacuum. The crude products so
obtained were purified by preparative TLC using varying proportions of methanol in
dichloromethane as the solvent systems to give the desired products compounds 4-8a and
9a in moderate to good yields
4.1.3.1. 8-cyclopentyl-1-propyl-2-[[4-(trifluoromethyl) phenyl] methylamino]-7H-
purin-6-one (4)
o
Yield: 0.091 g, 61%; mp: 235-237 C; LC-MS: m/z 420.0 [M+ +1]; HPLC RT: 19.781
min; Purity: 94%; 1HNMR (400 MHz, DMSO-d6) δ 0.92 (t, J = 8 Hz, 3H), 1.58-1.74 (m,
8H), 1.89 (br. s, 2H), 2.96-3.04 (m, 1H), 3.98 (br. s, 2H), 4.64 (br. s, 2H), 7.52 (s, 2H),
7.64-7.69 (m, 2H), 12.1 (br. s, 1H)
4.1.3.2. 8-Cyclopentyl-2-(4-methyl-benzylamino)-1-propyl-1,7-dihydro-purin-6-one
(5)
o
Yield: 0.077 g, 59%; mp: 228-230 C; LC-MS: m/z 366.1 [M+ +1]; HPLC RT: 18.002
1
min; Purity: 98%; HNMR (400 MHz, CD3OD) δ 0.90 (t, J = 8 Hz, 3H), 1.56-1.58 (m,
4H), 1.69-1.75 (m, 4H), 1.91 (br.s, 2H), 2.26 (s, 3H), 2.99 (m, 1H), 3.97 (br.s, 2H), 4.51
(br.s, 2H), 7.11 (d, J = 8 Hz, 2H), 7.18 (d, J = 8Hz, 2H), 12.41 (br.s, 1H)
4.1.3.3. 8-Cyclopentyl-2-(2-hydroxy-ethylamino)-1-propyl-1,7-dihydro-purin-6-one
(6):
o
Yield: 0.051 g, 47%; mp: 257-259 C; LC-MS: m/z 306.3 [M+ +1]; HPLC RT: 10.740
min; Purity: 96%; 1HNMR (400 MHz, DMSO-d6) δ 0.88 (br.s, 3H), 1.54-1.77 (br.s, 8H),
1.93 (br.s, 2H), 3.14 (br.s, 1H), 3.55 (br.s, 3H), 3.90 (br.s, 2H), 4.72 (br.s, 1H), 6.86 (br.s,
1H), 12.15 (br.s, 1H)
4.1.3.4. 2-(3-Chloro-benzylamino)-8-cyclopentyl-1-propyl-1, 7-dihydro-purin-6-one
(7)

o
Yield: 0.072 g, 51%; mp: 163-165 C; LC-MS: m/z 387.9 [M+ +2]; HPLC RT: 18.540
min; Purity: 96.9%; 1HNMR (400 MHz, CD3OD) δ 1.00 (t, J = 8 Hz, 3H), 1.69-1.81 (m,
8H), 2.05 (br.s, 2H), 3.01 (m, 1H), 4.04 (t, J = 8 Hz, 2H), 4.55 (br.s, 2H), 7.21-7.36 (m,
4H)
4.1.3.5. 4-[(8-Cyclopentyl-6-oxo-1-propyl-6,7-dihydro-1H-purin-2-ylamino)-methyl]-
benzoic acid methyl ester (8a)
Yield: 0.087 g, 60%; mp: 161-163 oC; LC-MS: m/z 410.0 [M+ +1]; HPLC RT: 16.50 min;
1
Purity: 96%; HNMR (400 MHz, CD3OD) δ 1.01 (t, J = 8 Hz, 3H), 1.68-1.83 (m, 8H),
2.04-2.08 (m, 2H), 3.14 (t, J = 8 Hz, 1H), 3.89 (s, 3H), 4.07 (t, J = 8 Hz, 2H), 4.73 (s,
2H), 7.47 (d, J = 8 Hz, 2H), 7.83 (d, J = 8 Hz, 2H)
4.1.3.6. (8-Cyclopentyl-6-oxo-1-propyl-6,7-dihydro-1H-purin-2-ylamino)-acetic acid
ethyl ester (9a)
o
Yield: 0.068 g, 55%; mp: 238-240 C; LC-MS: m/z 348.1 [M+ +1]; HPLC RT: 13.783
min; Purity: 93%; 1HNMR (400 MHz, DMSO-d6) δ 0.87 (t, J = 8 Hz, 3H), 1.19 (t, J = 8
Hz, 3H), 1.59 (br. s, 4H), 1.70-1.79 (m, 4H), 1.93 (br. s, 2H), 3.33 (br. s, 1H), 3.93 (s,
2H), 4.03 (t, J = 8 Hz, 2H), 4.1 (q, J = 8 Hz, 2H), 7.4 (br. s, 1H), 12.2 (br. s, 1H)
4.1.4. General synthesis of Compounds 8 and 9
To a solution of ester 8a or 9a (1 equiv., 0.05 g) in a mixture of MeOH/H2O/THF (1:1:1)
was added NaOH (3 equiv.) and the resulting reaction mixture was refluxed overnight.
The reaction mixture was concentrated under reduced pressure. The residue was
dissolved in water and neutralized with 1 N HCl and then extracted with ethyl acetate.
The ethyl acetate layer was dried over anhydrous sodium sulphate and concentrated under
vacuum. The crude product was purified by washing repeatedly with diethyl ether to give
the products in moderate to good yield
4.1.4.1. 4-[(8-cyclopentyl-6-oxo-1-propyl-6,7-dihydro-1H-purin-2-ylamino)-methyl]-
benzoic acid (8)
Yield: 0.015 g, 32%; mp: 204-206 oC; LC-MS: m/z 396.0 [M+ +1]; HPLC RT: 13.60 min;
1
Purity: 98%; HNMR (400 MHz, CD3OD) δ 1.00 (t, J = 8 Hz, 3H), 1.68-1.81 (m, 7H),
1.99 (s, 1H), 2.06-2.10 (m, 2H), 3.16 (t, J = 8 Hz, 1H), 4.06 (t, J = 8 Hz, 2H), 4.73 (s,
13
2H), 7.44 (d, J = 8 Hz, 2H), 7.95 (d, J = 8 Hz, 2H); CNMR (100 MHz, DMSO-d6) δ

11.00, 20.64, 25.01, 31.52, 41.41, 44.48, 126.65, 129.09, 129.36, 145.22, 151.43, 156.32,
159.45, 167.23
4.1.4.2. (8-Cyclopentyl-6-oxo-1-propyl-6,7-dihydro-1H-purin-2-ylamino)-acetic acid
(9)
o
Yield: 0.022 g, 48%; mp: 243-245 C; LC-MS: m/z 320.1 [M+ +1]; HPLC RT: 11.046
min; Purity: 92%; 1HNMR (400 MHz, DMSO-d6) δ 0.90 (t, J = 8 Hz, 3H), 1.56-1.73 (m,
4H), 1.74-1.88 (m, 4H), 1.89-2.01 (br. s, 2H), 3.01-3.10 (m, 1H), 3.2 (s, 1H), 3.93 (br. s,
3H), 7.25 (br. s, 1H), 12.2 (s, 1H)
4.1.5. General Synthesis of Compounds 10-13
To a solution of III (1 equiv., 0.10 g) in N-methyl-pyrrolidone (10 equiv., 0.3 mL) was
added aniline (10 equiv., 0.3 mL) and the resulting reaction mixture was heated at 150 °C
for 12 hours. After completion of reaction, the reaction mixture was cooled down to room
temperature, diluted with water and the product was extracted with ethyl acetate. The
ethyl acetate layer was dried over anhydrous sodium sulphate and concentrated under
reduced pressure. The crude products so obtained were purified by preparative TLC using
methanol in dichloromethane as solvent system to give the target compounds 10-13 in
good yields.
4.1.5.1. 2-anilino-8-cyclopentyl-1-propyl-7H-purin-6-one, (10)
o
Yield: 0.076 g, 63%; mp: 313-315 C; LC-MS: m/z 338.0 [M+ +1]; HPLC RT: 16.478
1
min; Purity: 97.8%; HNMR (400 MHz, CD3OD) δ 1.01 (s, J = 8 Hz, 3H), 1.68 (br. s,
2H), 1.73-1.83 (m, 6H), 2.04-2.10 (m, 2H), 3.15 (t, J = 8 Hz, 1H), 4.22 (t, J = 8 Hz, 2H),
7.13 (t, J = 8 Hz, 1H), 7.32 (t, J = 8 Hz, 2H), 7.49 (d, J = 8 Hz, 2H); 13CNMR (100 MHz,
DMSO) δ 11.02, 20.93, 25.03, 31.54, 41.99, 116.38, 123.77, 128.40, 139.63, 149.47,
152.39, 156.25, 158.93
4.1.5.2. 8-cyclopentyl-2-(4-fluoroanilino)-1-propyl-7H-purin-6-one (11)
o
Yield: 0.066 g, 52%; mp: 327-329 C; LC-MS: m/z 356.0 [M+ +1]; HPLC RT: 17.143
min; Purity: 99.2%; 1HNMR (400 MHz, CD3OD) δ 1.01 (t, J = 8 Hz, 3H), 1.67-1.68 (m,
2H), 1.73-1.82 (m, 6H), 2.03-2.08 (m, 2H), 3.14 (t, J = 8 Hz, 2H), 4.19 (t, J = 8 Hz, 2H),
7.06 (t, J = 8 Hz, 2H), 7.45-7.49 (m, 2H)
4.1.5.3. 8-cyclopentyl-1-propyl-2-[3-(trifluoromethyl) anilino]-7H-purin-6-one (12)
o
Yield: 0.071 g, 49%; mp: 312-314 C; LC-MS: m/z 406.0 [M+ + 1]; HPLC RT: 11.776

1
min; Purity: 92%; HNMR (400 MHz, DMSO-d6) δ 0.89 (t, J = 7.2 Hz, 3H), 1.58-1.78
(m, 8H), 1.93 (br s, 1H), 2.98-3.05 (m, 1H), 4.09-4.16 (m, 2H), 7.97-7.44 (m, 1H), 7.55
13
(t, J = 8.0 Hz, 1H), 7.81-7.88 (m, 2H); CNMR (100 MHz, DMSO-d6) δ 11.00, 20.96,
25.05, 31.63, 41.81, 119.42, 122.91, 125.62, 129.35, 129.49, 140.70, 148.82, 152.77,
155.89, 158.72
4.1.5.4. 8-cyclopentyl-2-(3-fluoroanilino)-1-propyl-7H-purin-6-one (13)
o
Yield: 0.078 g, 62%; mp: 300-302 C; LCMS: m/z 356.0 [M+ +1]; HPLC RT: 10.265
1
min; Purity: 97%; HNMR (400 MHz, DMSO-d6) δ 0.87 (t, J = 7.6 Hz, 3H), 1.55-1.80
(m, 8H), 1.92-1.97 (m, 2H), 3.03-3.07 (m, 1H), 4.13-4.17 (m, 2H), 6.83-6.87 (m, 1H),
7.30-7.35 (m, 2H), 7.47-7.49 (m, 1H), 8.6 (br. s, 1H), 12.4 (br. s, 1H)
4.1.6. General Synthesis of Compounds 14-28
To a solution of III (1 equiv., 0.10 g) in N-methyl-2-pyrrolidone (10 equiv., 0.3 mL) was
added K2CO3 (1.5 equiv., 0.074 g) followed by the corresponding phenol (1.1 equiv.) and
0
the reaction mixture was heated at 130 C for 12 hours. The reaction mixture was diluted
with ethyl acetate and water. The layers were separated and ethyl acetate layer was
washed with water. The ethyl acetate layer was dried over anhydrous sodium sulphate
and concentrated under reduced pressure. The crude product was purified by preparative
TLC using methanol in dichloromethane as solvent system to give the desired compounds
14-28 in good to moderate yields.
4.1.6.1. 8-cyclopentyl-2-(3,4-difluorophenoxy)-1-propyl-7H-purin-6-one (14)
o
Yield: 0.095 g, 71%; mp: 237-239 C; LC-MS: m/z 375.3 [M+ +1]; HPLC RT: 15.511
1
min; Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.94 (t, J = 8 Hz, 3H), 1.59-1.74
(m, 6H), 1.94 (br.s, 2H), 3.12 (m, 2H), 4.09 (br. s, 2H), 7.21 (d, J = 8 Hz, 1H), 7.53-7.65
(m, 2H), 12.74 (br.s, 1H); 13CNMR (100 MHz, DMSO-d6) δ 11.22, 21.52, 25.03, 31.82,
42.94, 112.44, 112.64, 117.69, 117.86, 119.06, 147.83, 148.02, 148.70, 150.47, 152.87,
153.55
4.1.6.2. 8-cyclopentyl-2-(3-methoxyphenoxy)-1-propyl-7H-purin-6-one (15)
o
Yield: 0.091 g, 69%; mp: 197-199 C; LC-MS: m/z 369.0 [M+ +1]; HPLC RT: 22.274
1
min; Purity: 96%; HNMR (400 MHz, CD3OD) δ 1.03-1.07 (t, J = 8 Hz, 3H), 1.69-1.72
(m, 2H), 1.77-1.89 (m, 6H), 2.05-2.12 (m, 2H), 3.15-3.21 (m, 1H), 3.82 (s, 3H), 4.25 (t, J
13
= 8 Hz, 3H), 6.81-6.89 (m, 3H); CNMR (100 MHz, DMSO-d6) δ 11.15, 21.52, 25.02,

31.35, 31.82, 42.83, 55.46, 107.99, 110.73, 111.58, 113.95, 130.02, 152.98, 153.32,
154.02, 159.38, 160.34
4.1.6.3. 8-cyclopentyl-1-propyl-2-[3-(trifluoromethyl)phenoxy]-7H-purin-6-one (16)
o
Yield: 0.075 g, 55%; mp: 222-224 C; LC-MS: m/z 382.1 [M+ +1]; HPLC RT: 15.511
min; Purity: 100%; 1HNMR (400 MHz, CD3OD) δ 1.04 (t, J = 8 Hz, 3H), 1.66-1.71 (m,
2H), 1.79-1.87 (m, 6H), 2.03-2.08 (m, 2H), 3.16 (m, 1H), 4.23-4.27 (m, 2H), 7.55 (d, J =
8 Hz, 1H), 7.61-7.67 (m, 3H)
4.1.6.4. 4-[(8-cyclopentyl-6-oxo-1-propyl-7H-purin-2-yl)oxy]benzamide (17)
Yield: 0.041 g, 30%; mp: 302-304 oC; LC-MS: m/z 382.1 [M+ +1]; HPLC RT: 15.51 min;
1
Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.95 (t, J = 7.2 Hz, 3H), 1.58-1.76 (m,
9H), 1.94 (br.s, 3H), 4.12 (br.s, 2H), 7.35-7.44 (m, 3H), 7.96-8.04 (m, 3H)
4.1.6.5. 8-cyclopentyl-2-[(6-methyl-3-pyridyl)oxy]-1-propyl-7H-purin-6-one (18)
Yield: 0.053 g, 42%; mp: 193-195 oC; LC-MS: m/z 354.1 [M+ +1]; HPLC RT: 16.54 min;
1
Purity: 98.98%; HNMR (400 MHz, DMSO-d6) δ 0.93 (t, J = 8 Hz, 3H), 1.55-1.57 (m,
2H), 1.65-1.74 (m, 6H), 1.90-1.95 (m, 2H), 3.09-3.11 (m, 2H), 3.32 (s, 3H), 4.10 (br. s,
2H), 7.36 (d, J = 8 Hz, 1H), 7.63-7.69 (m, 1H), 8.38-8.43 (dd, J = 8 Hz, 1H)
4.1.6.6. 2-[(5-chloro-3-pyridyl)oxy]-8-cyclopentyl-1-propyl-7H-purin-6-one (19)
o
Yield: 0.077 g, 58%; mp: 202-204 C; LC-MS: m/z 374.0 [M+ +1]; HPLC RT: 21.179
1
min; Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.93 (t, J = 8 Hz, 3H), 1.55-1.61
(m, 6H), 1.66-1.73 (m, 2H), 1.90-1.95 (m, 2H), 3.09-3.11 (m, 2H), 4.10 (t, J = 5.6 Hz,
2H), 8.14-8.21 (m, 1H), 8.58-8.63 (m, 2H)
4.1.6.7. 8-cyclopentyl-2-(1H-indol-5-yloxy)-1-propyl-7H-purin-6-one (20)
Yield: 0.033 g, 25%; mp: 352-354 oC; LC-MS: m/z 378.1 [M+ +1]; HPLC RT: 20.37 min;
1
Purity: 99.3%; HNMR (400 MHz, DMSO-d6) δ 0.97 ( t, J = 8 Hz, 3H), 1.57-1.581 (m,
8H), 1.90-1.95 (m, 2H), 3.00-3.10 (m, 1H), 4.12-4.17 (m, 2H), 6.45 (br.s, 1H), (td, J = 8
Hz, 4 Hz, 1H), 7.38 (br.s, 1H), 7.41-7.44 (m, 2H)
4.1.6.8. 4-[(8-cyclopentyl-6-oxo-1-propyl-7H-purin-2-yl) oxy] benzenesulfonamide
(21)
Yield: 0.033 g, 22%; mp: 268-270 oC; LC-MS: m/z 418.0 [M+ +1]; HPLC RT: 9.665 min;
1
Purity: 99.2%; HNMR (400 MHz, DMSO-d6) δ 0.95 (t, J = 8 Hz, 3H), 1.58 (m, 2H),

1.75 (m, 6H), 1.94 (m, 2H), 3.02-3.14 (m, 1H), 4.12 (m, 2H), 7.42-7.54 (m, 4H), 7.91-
7.93 (m, 2H)
4.1.6.9. 2-[3-[(8-cyclopentyl-6-oxo-1-propyl-7H-purin-2-yl) oxy] phenyl] acetic acid
(22)
o
Yield: 0.049 g, 35%; mp: 217-219 C; LC-MS: m/z 397.0 [M+ +1]; HPLC RT: 11.052
min; Purity: 97%; 1HNMR (400 MHz, DMSO-d6) δ 0.95 (t, J = 8 Hz, 3H), 1.58-1.74 (m,
8H), 1.94 (d, J = 8 Hz, 2H), 3.01-3.11 (m, 1H), 3.63 (s, 2H), 4.11 (m, 2H), 7.15-7.21 (m,
3H), 7.39-7.43 (m, 1H), 12.96 (br.s, 1H)
4.1.7.0. 8-cyclopentyl-2-[(2-methyl-3-pyridyl) oxy]-1-propyl-7H-purin-6-one (23)
o
Yield: 0.067 g, 53%; mp: 230-232 C; LC-MS: m/z 354.2 [M+ +1]; HPLC RT: 10.093
min; Purity: 96.4%; 1HNMR (400 MHz, DMSO-d6) δ 0.68 (t, J = 8 Hz, 3H), 1.30 (br.s,
2H), 1.40-1.50 (m, 6H), 1.65 (br.s, 2H), 2.08 (s, 3H), 2.80 (br.s, 1H), 3.87 (t, J = 8 Hz,
3H), 7.09 (t, 1H), 7.42 (br.s, 1H), 8.13 (br.s, 1H); 13CNMR (100 MHz, DMSO-d6) δ
11.17, 19.06, 21.57, 25.01, 31.66, 43.02, 122.81, 130.45, 146.51, 146.85, 150.98, 152.74,
154.20, 157.03
4.1.7.1. 8-cyclopentyl-2-(1H-indol-7-yloxy)-1-propyl-7H-purin-6-one (24)
o
Yield: 0.039 g, 29%; mp: 285-287 C; LC-MS: m/z 378.2 [M+ +1]; HPLC RT: 12.441
min; Purity: 100%; 1HNMR (400 MHz, DMSO-d6) δ 0.99 (t, J = 7.2 Hz, 3H), 1.56-1.91
(m, 10H), 2.99-3.11 (m, 1H), 4.17-4.22 (m, 2H), 6.50-6.53 (m, 1H), 6.94-7.05 (m, 2H),
7.35-7.37 (m, 1H), 7.48-7.51 (m, 1H), 11.25 (br. s, 1H), 12.78 (br. s, 1H)
4.1.7.2. 8-cyclopentyl-2-(1H-indol-4-yloxy)-1-propyl-7H-purin-6-one (25)
o
Yield: 0.037 g, 28%; mp: > 300 C (d); LC-MS: m/z 378.2 [M+ +1]; HPLC RT: 11.224
1
min; Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.97 (t, J =7.2 Hz, 3H), 1.52-1.92
(m, 10H), 2.96-3.09 (m, 1H), 4.16-4.20 (m, 2H), 6.19-6.23 (m, 1H), 6.85-6.90 (m, 2H),
7.09-7.13 (m, 1H), 7.31-7.35 (m, 1H), 11.35 (br. s, 1H), 12.77 (br. s, 1H)
4.1.7.3. 8-cyclopentyl-2-[(4-methyl-2-pyridyl) oxy]-1-propyl-7H-purin-6-one (26)
o
Yield: 0.069 g, 55%; mp: 287-289 C; LC-MS: m/z 354.0 [M+ +1]; HPLC RT: 19.865
1
min; Purity: 95.3%; HNMR (400 MHz, DMSO-d6) δ 7.42 (t, J = 8 Hz, 3H), 1.45-1.52
(m, 2H), 1.63-1.67 (m, 2H), 1.74-1.1.85 (m, 4H), 2.01 (m, 2H), 2.23 (s, 3H), 3.22 (t, J = 8
Hz, 2H), 6.32 (d, J = 8 Hz, 1H), 6.37 (s, 1H), 7.68-7.78 (m, 1H)
4.1.7.4. 2-[(2-chloro-3-pyridyl) oxy]-8-cyclopentyl-1-propyl-7H-purin-6-one (27)

o
Yield: 0.064 g, 48%; mp: 259-261 C; LC-MS: m/z 376.0 [M+ +2]; HPLC RT: 13.538
1
min; Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.96 (t, J = 8 Hz, 3H), 1.57-1.63
(m, 2H), 1.67-1.84 (m, 6H), 1.92-1.99 (m, 2H), 3.02-3.14 (m, 1H), 4.15 (t, J = 8 Hz, 2H),
7.60-7.64 (m, 1H), 8.02-8.09 (m, 1H), 8.41-8.43 (t, J = 4 Hz, 1H)
4.1.7.5. 8-cyclopentyl-2-(3-hydroxyphenoxy)-1-propyl-7H-purin-6-one (28)
Yield: 0.042 g, 33%; mp: 264-266 oC; LC-MS: m/z 355.1 [M+ +1]; HPLC RT: 9.892 min;
1
Purity: 99.4%; HNMR (400 MHz, DMSO-d6) δ 0.93 (t, J = 8 Hz, 3H), 1.57-1.60 (m,
2H), 1.67- 1.78 (m, 6H), 1.94 (m, 2H), 3.09 (m, 1H), 4.09 (t, J = 8 Hz, 2H), 6.65-6.72 (m,
3H), 7.21-7.25 (m, 1H), 9.95 (br. s, 1H), 12.93 (br. s, 1H)
4.1.8. General synthesis of Compounds 29-35
To a solution of III (1 equiv., 0.100 g) in corresponding alcohol (10 equiv.) was added
NaH (60% dispersion in mineral oil, 10 equiv., 0.51 g) under nitrogen atmosphere and the
reaction mixture was refluxed for 3 hours. After cooling to room temperature, the
reaction mixture was concentrated under reduced pressure. Water was added to the
residue and the products were extracted with ethyl acetate. The organic layer was dried
over anhydrous sodium sulphate and concentrated under reduced pressure. The crude
products were purified by preparative TLC using methanol in dichloromethane as solvent
system to give the desired compounds 29-35 in good to moderate yields
4.1.8.1. 2-(cyclopentoxy)-8-cyclopentyl-1-propyl-7H-purin-6-one (29)
o
Yield: 0.073 g, 62%; mp: 245-247 C; LC-MS: m/z 331.0 [M+ +1]; HPLC RT: 21.933
min; Purity: 99.6%; 1HNMR (400 MHz. CD3OD) δ 0.94 (t, J = 8 Hz, 3H), 1.64-2.11 (m,
18H), 3.12-3.35 (m, 2H), 3.99-4.03 (m, 2H)
4.1.8.2. 8-cyclopentyl-2-(1-methylpyrrolidin-3-yl)oxy-1-propyl-7H-purin-6-one (30)
o
Yield: 0.050 g, 41%; mp: 232-234 C; LC-MS: m/z 346.0 [M+ +1]; HPLC RT: 8.28 min;
Purity: 97.7%; 1HNMR (400 MHz, DMSO-d6) δ 0.92 (t, J = 7.2 Hz, 3H), 1.58-1.85 (m,
9H), 1.94-2.02 (m, 3H), 2.39-2.42 (m, 1H), 2.77 (s, 3H), 3.12-3.18 (m, 1H), 3.34-3.53
(m, 3H), 4.15-4.23 (m, 3H), 12.9 (br. s, 1H)
4.1.8.3. 8-cyclopentyl-1-propyl-2-tetrahydropyran-4-yloxy-7H-purin-6-one (31)
o
Yield: 0.073 g, 59%; mp: 236-238 C; LC-MS: m/z 347.3 [M+ +1]; HPLC RT: 10.256
min; Purity: 100%; 1HNMR (400 MHz, DMSO-d6) δ 0.86 (t, J = 7.6 Hz, 3H), 1.21-1.81

(m, 10H), 1.93-2.03 (m, 4H), 3.05-3.11 (m, 1H), 3.51-3.56 (m, 2H), 3.77-3.82 (m, 2H),
3.93 (t, J = 6.8 Hz, 2H), 5.20-5.22 (m, 1H), 12.8 (br. s, 1H)
4.1.8.4. 8-cyclopentyl-1-propyl-2-tetrahydrofuran-3-yloxy-7H-purin-6-one (32)
Yield: 0.064 g, 54%; mp: 243-245 oC; LC-MS: m/z 333.0 [M+ +1]; HPLC RT: 9.99 min;
Purity: 99.8%; 1HNMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 7.2 Hz, 3H), 1.52-1.59 (m,
4H), 1.69-1.78 (m, 4H), 1.94-1.96 (m, 2H), 2.05-2.12 (m, 1H), 2.17-2.25 (m, 1H), 3.04-
13
3.15 (m, 1H), 3.78-3.92 (m, 6H), 5.51-5.52 (m, 1H); CNMR (100 MHz, DMSO-d6) δ
11.12, 21.37, 24.97, 25.12, 31.44, 31.91, 32.39, 42.17, 66.40, 72.40, 78.72, 147.7, 152.48,
153.21, 154.83, 159.05
4.1.8.5. 8-cyclopentyl-2-(3-hydroxycyclopentoxy)-1-propyl-7H-purin-6-one (33)
Yield: 0.043 g, 35%; mp: 238-240 oC; LC-MS: m/z 347.0 [M+ +1]; HPLC RT: 9.625 min;
1
Purity: 97.6%; HNMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 7.2 Hz, 3H), 1.21 (s, 2H),
1.51-1.61 (m, 4H), 1.70-1.81 (m, 4H), 1.87-1.96 (m, 4H), 2.12-2.20 (m, 1H), 3.02-3.12
(m, 1H), 3.84-3.93 (m, 2H), 4.26-4.28 (m, 1H), 4.65-4.69 (m, 1H), 5.40-5.44 (m, 1H),
12.70 (m, 1H)
4.1.8.6. 8-cyclopentyl-1-propyl-2-[(3S)-tetrahydrofuran-3-yl] oxy-7H-purin-6-one
(34)
o
Yield: 0.059 g, 50%; mp: 230-232 C; LC-MS: m/z 333.2 [M+ +1]; HPLC RT: 10.315
1
min; Purity: 100%; HNMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 8 Hz, 3H), 1.53-1.80
(m, 8H), 1.94-2.48 (m, 5H), 3.07-3.11 (m, 1H), 3.78-3.91 (m, 5H), 5.51 (br. s, 1H)
4.1.8.7. 8-cyclopentyl-1-propyl-2-[(3R)-tetrahydrofuran-3-yl] oxy-7H-purin-6-one
(35)
Yield: 0.061 g, 52%; mp: 228-230 oC; LC-MS: m/z 333.2 [M+ +1]; HPLC RT: 9.639 min;
Purity: 98.4%; 1HNMR (400 MHz, CD3OD) δ 0.85 (t, J = 8 Hz, 3H), 1.22-1.25 (m, 3H),
1.55-1.64 (m, 3H), 1.70-1.82 (m, 3H), 1.93-2.00 (m, 2H), 2.08-2.13 (m, 1H), 2.21-2.27
(m, 1H), 3.11 (m, 1H), 3.79-3.93 (m, 5H), 5.52-5.54 (m, 1H)
4.1.9. General synthesis of Compounds 36-41
To a solution of III (1 equiv., 0.100 g) in THF was added sodium bicarbonate (4 equiv.,
0.12 g) dissolved in minimum amount of water followed by the corresponding boronic
acid (1.5 equiv.). The resulting reaction mixture was degassed for 10 minutes by purging
argon and then Pd [PPh3]4 (0.1 equiv., 0.041 g) was added. The reaction mixture was

refluxed for 12 hours. After completion of reaction, the reaction mixture was
concentrated under reduced pressure. The product was extracted with ethyl acetate, the
organic layer was washed with water followed by brine and dried over anhydrous sodium
sulphate. The organic layer was concentrated under reduced pressure to give the crude
products that were purified by preparative TLC using methanol in dichloromethane as
solvent system to afford desired products 36-41 in moderate to good yields
4.1.9.1. 8-cyclopentyl-1-propyl-2-[3-(trifluoromethyl) phenyl]-7H-purin-6-one (36)
Yield: 0.074 g, 53%; mp: 220-222 oC; LCMS: m/z 391.0 [M+ +1]; HPLC RT: 23.020 min;
1
Purity: 99%; HNMR (400 MHz, DMSO-d6) δ 0.63 (t, J = 7.6 Hz, 3H), 1.45-1.49 (m,
2H), 1.63 (br. s, 2H), 1.74 (br. s, 2H), 1.84 (br. s, 2H), 2.00 (br. s, 2H), 3.16-3.33 (m, 2H),
3.79 (t, J = 7.2 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.89-7.93 (m, 2H), 7.98 (s, 1H);
13CNMR (100 MHz, DMSO-d6) δ 10.84, 21.68, 25.07, 31.80, 46.59, 122.55, 125.05,
125.25, 126.08, 129.01, 129.33, 129.64, 132.29, 136.95, 153.60
4.1.9.2. 8-cyclopentyl-1-propyl-2-[4-(trifluoromethyl) phenyl]-7H-purin-6-one (37)
Yield: 0.077 g, 55%; mp: 253-255 oC; LCMS: m/z 391.0 [M+ +1]; HPLC RT: 23.224 min;
1
Purity: 97%; HNMR (400 MHz, DMSO-d6) δ 0.63 (t, J = 7.2 Hz, 3H), 1.44-1.50 (m,
2H), 1.63 (br. s, 2H), 1.74 (br. s, 2H), 1.80-1.87 (m, 2H), 2.00 (br. s, 2H), 3.18-3.33 (m,
1H), 3.81 (t, J = 6.8 Hz, 2H), 7.86 (dd, J = 7.6 Hz, 8 Hz, 4H)
4.1.9.3. 8-cyclopentyl-2-(3-methoxyphenyl)-1-propyl-7H-purin-6-one (38)
o
Yield: 0.061 g, 49%; mp: 195-197 C; LC-MS: m/z 353.0 [M+ +1]; HPLC RT: 19.958
1
min; Purity: 97.8%; HNMR (400 MHz, CD3OD) δ 0.74 (t, J = 7.6 Hz, 3H), 1.28 (m,
1H), 1.59-1.65 (m, 2H), 1.72-1.74 (m, 2H), 1.87-1.93 (m, 4H), 2.14 (d, J = 8 Hz, 2H),
3.85 (s, 3H), 3.93-3.97 (m, 2H), 7.07-7.13 (m, 3H), 7.44 (t, J = 8 Hz, 1H); 13CNMR (100
MHz, DMSO-d6) δ 10.97, 21.79, 25.09, 31.94, 46.52, 55.32, 113.66, 114.98, 120.16,
129.59, 137.26, 153.62, 154.57, 155.19, 156.31, 158.94, 159.77
4.1.9.4. 3-(8-cyclopentyl-6-oxo-1-propyl-7H-purin-2-yl) benzoic acid (39)
o
Yield: 0.034 g, 26%; mp: 305-307 C; LC-MS: m/z 367.0 [M+ +1]; HPLC RT: 16.234
1
min; Purity: 99.2%; HNMR (400 MHz, CD3OD) δ 0.74 (t, J = 7.2 Hz, 3H), 1.57-1.66
(m, 3H), 1.72-1.79 (m, 2H), 1.87-1.92 (m, 4H), 1.94-2.20 (m, 2H), 3.95 (t, J = 7.6 Hz,
2H), 7.66 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 8.18-8.22 (m, 2H)
4.1.9.5. 8-cyclopentyl-2-(3-hydroxyphenyl)-1-propyl-7H-purin-6-one (40)

o
Yield: 0.045 g, 37%; mp: 326-328 C; LC-MS: m/z 339.0 [M+ +1]; HPLC RT: 16.728
1
min; Purity: 99.8%; HNMR (400 MHz, CD3OD) δ 0.75 (t, J = 7.6 Hz, 3H), 1.59-1.65
(m, 2H), 1.71-1.74 (m, 3H), 1.87 (br. s, 4H), 2.13-2.15 (m, 2H), 3.94-3.98 (m, 2H), 6.90-
13
6.97 (m, 3H), 7.34 (t, J = 7.6 Hz, 1H); CNMR (100 MHz, DMSO-d6) δ 10.99, 21.84,
25.14, 31.98, 46.51, 113.71, 114.98, 116.28, 118.59, 129.59, 136.99, 153.69, 154.87
155.23, 157.20, 159.77
4.1.9.6. 3-(8-cyclopentyl-6-oxo-1-propyl-7H-purin-2-yl)benzamide (41)
Yield: 0.043 g, 33%; mp: 332-334 oC; LC-MS: m/z 366.0 [M+ +1]; HPLC RT: 14.39 min;
Purity: 95%; 1HNMR (400 MHz, CD3OD) δ 0.73 (t, J = 7.2 Hz), 1.28-1.33 (m, 3H), 1.35-
1.39 (m, 1H), 1.57-1.63 (m, 2H), 1.93 (br. s, 3H), 2.14-2.18 (m, 1H), 3.96 (t, J = 8 Hz,
2H), 7.65 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 8.07 (d, J = 9.2 Hz, 2H)
4.2. Biology
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
(10mM EDTA, 10mM HEPES, pH-7.4 for HEK- A3) at 1500 rpm for 5 minutes at room
temperature. The cell pellet was incubated in sonication buffer containing 1 mM EDTA,
5 mM Tris, pH 7.4 (1mM EDTA, 10mM HEPES, pH-7.4 for HEK-A3) for 10 mins at 4oC
followed by sonication on ice for 6 minutes. The lysate was centrifuged at 1000 x g for
10 minutes at 4oC and the pellet was discarded. The supernatant was centrifuged at
49,000 x g for 45 minutes at 4oC. The resultant protein pellet was resuspended in the
sonication buffer supplemented with 1 U/ml adenosine deaminase (ADA, Roche) and
incubated for 30 minutes at room temperature with constant mixing. The protein was
washed twice with same buffer at 49,000 x g for 45 minutes at 4oC 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 -80oC.

The binding affinity and selectivity of test compounds was determined using radioligand
binding assays. DPCPX, Preladenant, CVT-6883 and MRS-1191 were used as internal
3
standards. Competition radioligand binding assays were started by mixing 1 nM H-
3
3
DPCPX (for HEK-A1), 1 nM H-ZM241385 (HEK-A2A), 1.6 nM H-MRS-1754 (HEK-
A2B) or 2 nM 3H-HEMADO (HEK-A3) with various concentrations of test compounds and
the respective membranes in assay buffer containing 50 mM Tris pH 7.4, 1 mM EDTA
(HEK-A1), 50 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 1 U/ml ADA (HEK-A2A,
HEK-A3) or 50 mM Tris pH 6.5, 5 mM MgCl2, 1 mM EDTA (HEK-A2B) supplemented
with 1 unit/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 50mM 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.
4.2.2. cAMP Assay for Adenosine Receptor
The functional activity of test compounds on A1 adenosine receptor and selectivity
against adenosine receptor subtype A2B was determined using HTRF based cAMP assay
(Cisbio). Briefly, overnight seeded cultures (HEK-A1, HEK-A2B) were treated with 1U/ml
ADA for 90 minutes at 37oC and 5% CO2. Cell suspensions were treated with increasing
concentrations of test compounds for 15 minutes followed by treatment with agonists for
15 minutes (1 nM CPA for HEK-A1 and 70 nM NECA for HEK-A2B) at room temperature
with continuous mixing in incomplete DMEM supplemented with 1U/ml ADA. Rolipram
(20 µM) was included in the assay for A1 adenosine receptor. For functional activity of
HEK-A1, cell suspensions were further treated with forskolin for 30 minutes at room
temperature with constant mixing. cAMP levels were quantified using a Flex Station III
(Molecular Devices) at an excitation maximum of 313 nm and emission maxima of 620
nm and 665 nm. Data was analyzed using GraphPad Prism to generate IC50 and Ki.
4.3. Pharmacokinetics
4.3.1. General
Male Wistar rats (weighing 230 ± 15g) were obtained from in-house breeding facility,
Advinus Therapeutics Ltd., Pune, INDIA. The rats were grouped and housed in
polycarbonate cages with not more than 3 rats per cage and maintained under standard

laboratory conditions (temperature 25 ± 2oC) with dark/ light cycle (12 h). Rats were
maintained on T.2014C Global 14% protein rodent maintenance diets (Harlan, Teklad
diet, USA) and water ad libitum. All procedures described were reviewed and approved
by the Institutional Animal Ethics committee (IAEC) constituted by Committee for the
Purpose of Control and Supervision on Experiments (CPCSEA) on Animals, Govt. of
India.
Pharmacokinetic experiments were carried-out in male Wistar rats following Intravenous
(IV) and Per oral (PO) administration. The animals were fasted overnight before the start
of experimentation but had free access to water. For IV dosing, tested compounds were
dissolved in different vehicles and solution formulation (dose volume: 5 mL/kg and
formulation strength: 0.6 mg/mL) was administered to each rat at a dose of 3 mg/kg via
tail vein. For PO suspension formulation was administered to each rat at a dose of 5
mg/kg via oral gavage using oral feeding tube.
Study used serial sampling design (n=3/time point) with blood samples collected at 0.008
(IV only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h post dose into labeled micro-centrifuge tubes
contained K2EDTA as an anticoagulant. Tissue samples were washed with phosphate
buffer saline (pH 7.4), blotted dry and weighed. All samples were stored below -70°C
until bioanalysis. Blood samples were immediately centrifuged at 6000 RPM for 5
minutes to separate the plasma.
Tissue samples were homogenized in phosphate buffer saline by homogenizer (Polytron
PT 3100, Kinematica). Aliquot of plasma (100 µL) or tissue sample (300 µL) was
precipitated with 300 µL of acetonitrile containing internal standard was added to all the
samples and vortex mixed for 1 min on multiple vortex mixer and centrifuged at 8000
RPM for 8 minutes. Finally, supernatant (100 µL) was collected from each test sample
microcentrifuge tube and transferred into HPLC vials for LC/MS/MS to determine the
concentrations in plasma. Calibration standards and quality control (separate weighing)
samples were prepared in blank rat plasma or brain homogenates.
4.3.2. 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. The area under the
plasma concentration-time curve (AUC0-t & AUC0-inf), elimination half-life (T1/2), clearance

(CL) and volume of distribution (Vss) were calculated from intravenous group. The peak
plasma concentration (Cmax), time to achieve Cmax (Tmax), area under the plasma
concentration-time curve (AUC0-t & AUC0-inf), and oral bioavailability were calculated
from the oral groups.
4.3.3. In-vivo disposition study in brain
Rats were divided into two groups each consisting of six rats. The rats were fasted
overnight prior to dose administration and for 2 hours post dose but had free access to
water. Group 1 rats received oral solution administration of Compound 14 and group 2
received oral solution administration of Compound 38 at dose of 5 mg/kg (formulation
strength: 0.5 mg/mL). Oral solution formulation of 14 comprised of N-methyl-
pyrrolidinone (NMP) -10% v/v, Labrasol- 30% v/v, citrate buffer pH 8.5 quantity
sufficient. Oral solution formulation of 38 comprised of NMP -10% v/v, polyethylene
glycol-400 (PEG-400) - 20% v/v, citrate buffer pH 8.5 quantity sufficient. Study used
discrete sample design. Blood and brain were collected from two rats at 1, 2 and 8 h post
dose administration. Brain samples were washed with phosphate buffer saline (pH 7.4),
blotted dry and weighed. All samples were stored below -70°C until bioanalysis. Blood
samples were centrifuged at 6000 rpm for 5 min to obtain the plasma. Plasma and brain
samples were stored below -70 °C until bioanalysis.
Brain samples were transferred to the pre-labeled tubes, phosphate buffer saline was then
added into tubes in a ratio of 1:2 (for each 1 g of brain, 2 mL of phosphate buffer saline
was added). Samples were then homogenized by using homogenizer (polytron PT 3100,
Kinematica). Plasma samples were thawed at room temperature. Plasma and brain
homogenate samples were analyzed following protein precipitation with acetonitrile
containing internal standard. Quantitative bioanalysis of 14 and 38 in the plasma and
brain samples was performed using a fit for purpose liquid chromatography tandem mass
spectrometry (LC-MS/MS). The lower limit of quantification of both the compounds was
10 ng/mL (0.02 µM) in both the matrices.
4.4. Rat Oral Efficacy Screen
Male Wistar rats (n = 6; dose volume: 5mL/kg) with 250-300 g body weight were treated
with vehicle for Rolofylline (1) [DMAC (5%) + 1% 1N NaOH + Normal saline (qs)],
Rolofylline (0.1 mg/kg, PO), vehicle for compound 14 [NMP (10%), PEG -400 (20%),