Novel 8-(p-substituted-phenyl/benzyl)xanthines with selectivity for the A2A adenosine receptor possess bronchospasmolytic activity

Article abstract of DOI:10.1016/j.ejmech.2014.01.045

A new series of 8-(p-substituted-phenyl/benzyl)xanthines has been synthesized and evaluated in vitro for adenosine receptor binding affinity and in vivo for bronchospasmolytic effects. It was observed that the nature of substituent at para-position of 8-phenyl/benzyl group on the xanthine scaffold remarkably affects the binding affinity and selectivity of xanthine derivatives for various adenosine receptor subtypes and also their bronchospasmolytic effects. Newly synthesized 8-phenylxanthines displayed potent binding affinity and significant selectivity for A_2A receptors and also produced potent bronchospasmolytic effects. Replacement of phenyl ring with benzyl moiety at C₈ of xanthine skeleton resulted in notable reduction in adenosine receptor affinity and broncholytic effects.

Full text of DOI:10.1016/j.ejmech.2014.01.045

European Journal of Medicinal Chemistry 75 (2014) 327e335
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 8-(p-substituted-phenyl/benzyl)xanthines with selectivity for
the A2A adenosine receptor possess bronchospasmolytic activity
,
b
b
Rakesh Yadava, Ranju Bansal a , Sonja Kachler , K.N. Klotz
*
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
b Institut für Pharmakologie und Toxikologie, Universität Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 8-(p-substituted-phenyl/benzyl)xanthines has been synthesized and evaluated in vitro for
adenosine receptor binding affinity and in vivo for bronchospasmolytic effects. It was observed that the
nature of substituent at para-position of 8-phenyl/benzyl group on the xanthine scaffold remarkably
affects the binding affinity and selectivity of xanthine derivatives for various adenosine receptor sub-
types and also their bronchospasmolytic effects. Newly synthesized 8-phenylxanthines displayed potent
binding affinity and significant selectivity for A2A receptors and also produced potent bronchospasmo-
lytic effects. Replacement of phenyl ring with benzyl moiety at C8 of xanthine skeleton resulted in
notable reduction in adenosine receptor affinity and broncholytic effects.
Received 15 October 2013
Received in revised form
13 January 2014
Accepted 19 January 2014
Available online 30 January 2014
Keywords:
8-Phenylxanthines
Bronchospasmolytic activity
Adenosine receptors
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
their usefulness as in vivo research tools and their possible use as
therapeutic agents [11]. The incorporation of polar substituents
Asthma remains one of the most common respiratory diseases
with unmet medical needs. Enough evidence has accumulated in
support of the role of adenosine in pathogenesis of asthma [1,2].
Selective agonists or antagonists of adenosine receptors are being
explored by various research groups and pharmaceutical industry
in an attempt to generate novel antiasthmatic agents. All four
adenosine receptor (AR) subtypes (A1, A2A, A2B and A3) are
expressed in lungs and in inflammatory cells involved in asthma.
This has led to investigations into all AR subtypes as potential
therapeutic targets for the treatment of asthma [3e5]. As binding
studies in human lungs have indicated higher abundance of A2 than
other subtypes, therefore these receptors seem to play a more
prominent role in mediating the bronchoconstriction and proin-
flammatory effects of adenosine in lungs [6].
Substituted xanthines constitute one of the most persuasive
categories of adenosine receptor antagonists reported to date and
are known for variable potency and selectivity for adenosine re-
ceptor subtypes [7e9]. 8-Phenyltheophylline is the parent member
of a variety of potent adenosine receptor antagonists, e.g., MRS-
1754 (1) (Fig. 1) [9,10]. Although 8-phenyl substitution exhibits
maximum adenosine receptor antagonistic activity, it confers
extremely limited water solubility to xanthines, which restricts
improves the otherwise extremely limited water solubility of 8-
phenylxanthines and consequently increases their effectiveness
as potential therapeutic agents. Furthermore, appropriate sub-
stitutions on the 8-phenyl ring greatly affects the potency and
selectivity of xanthines towards AR subtypes and thus their phar-
macological effects [10,12,13].
In the light of these observations, we decided to study the
impact of substituting polar dialkylaminoethoxy substituents at
para position of the 8-phenyl ring of xanthines on adenosine re-
ceptor binding affinity and selectivity. In order to examine specific
structural features, which may be important for adenosine receptor
binding, it was also appealing to introduce a methylene spacer
between the aromatic unit and the C8 of the xanthine nucleus and
study the resulting effects on biological activity. As a continuation
of our earlier xanthine based research [10,14,15], a new series of 8-
(substituted-phenyl/benzyl)xanthines has been synthesized and
evaluated for their binding affinity for various adenosine receptor
subtypes and also for bronchospasmolytic effects.
2. Chemistry
The synthesis of various 8-(substituted-phenyl)xanthines has
been depicted in Schemes 1 and 2.
Substituted aromatic aldehydes 3e9 [16] were prepared by
treating 4-hydroxybenzaldehyde with hydrochloride of desired
* Corresponding author. Tel.: þ91 172 2541142; fax: þ91 172 2543101.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.045

328
R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
derivative 22 was thermally fused separately with powdered
H
N
O
N
imidazole and 1-(2-chlorobenzyl)piperazine at 160 ꢀC for 2 h to
CN
H
N
afford the corresponding xanthine congeners 24 and 25 (Scheme-
N
2). Imidazolyl protons appeared at
d 6.97, 7.03 and 7.52 and N-
O
O
methylene protons of eCH2eN< resonated downfield as a triplet at
3.99 for compound 24, while an 8-proton multiplet of piperazinyl
ring was seen at 2.56e2.60 ppm in the NMR spectrum of 25.
For the preparation of target 8-[4-(aminopropoxy)benzyl]xan-
thines, attempts to synthesize the starting substituted acidic
compound 29 by direct alkylation of 4-hydroxyphenylacetic acid
with 1-bromo-3-chloropropane remained unsuccessful. Therefore
an alternative synthetic route was adopted as shown in Scheme 3.
4-Hydroxyphenylacetic acid was esterified by heating under reflux
in methanol in presence of a catalytic amount of sulfuric acid to
afford an oily residue of methyl-4-hydroxyphenyl acetate (27).
Alkylation of the compound 27 was carried out by treating with 1-
bromo-3-chloropropane in ethyl methyl ketone using anhydrous
potassium carbonate to give the oily residue methyl-[4-(3-
chloropropoxy)phenyl]acetate (28). Alkaline hydrolysis of ester 28
led to the formation of the desired starting compound 29 as shown
in Scheme 3.
N
O
(1)
Fig. 1. Structure of xanthine based adenosine receptor antagonist MRS-1754 (1).
dialkylamino ethyl chloride, 1-bromo-3-chloropropane and cyclo-
pentyl bromide, respectively, in refluxing ethyl methyl ketone in
the presence of anhydrous potassium carbonate (Scheme-1).
Treatment of obtained aldehydes 3e9 with 5,6-diamino-1,3-
dimethyluracil (2) [17,18] in MeOH-AcOH (4:1) at room tempera-
ture resulted in the formation of corresponding benzylidene ad-
ducts 10e16, subsequent ring closure of which in refluxing thionyl
chloride afforded the target compounds 17e23 as shown in
Scheme-1. Characteristic NMR signals appeared at
d 2.93 [s, 6H, e
N(CH3)2] for 17, 1.32 [s, 6H, eN(CH2CH3)2] for 18, 2.98e3.06 (m, 4H,
eN(CH2)2, piperidine) for 19, 3.70 [t, 4H, O-(CH2)2, morpholine] for
20, 2.88 [s, 4H, eN(CH2)2, pyrrolidine] for 21, 3.77 ppm (t, 2H, e
CH2Cl) for 22 and a multiplet at 4.80e4.91 ppm for eOCH of
cyclopentyloxy ring substituted xanthine 23. Further, two separate
singlets for both methyl groups present at 1- and 3-position of
4-Chloropropoxyphenylacetic acid (29) was condensed with
5,6-diamino-1,3-dimethyluracil (2) in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) in methanol at room
temperature to yield 6-amino-5-[{4-(3-chloropropoxy)phenyl}
carboxacetamido]-1,3-dimethyluracil (30) as shown in Scheme 3.
1H NMR spectrum of carboxamide derivative 30 displayed a char-
acteristically downfield singlet integrating for two protons of NHe
purine nucleus were found at d w 3.4 and 3.6, and eOCH2e protons
resonated as a triplet around 4.3 ppm for all the compounds.
Keeping in mind the reported adenosine binding affinity of
imidazole [15] derived xanthines and antihistaminic properties of
aryl piperazine substituted 8-phenylxanthines, chloroalkoxy
COeCH2eAr group at
d 3.67, while protons of the free eNH2 group
resonated as a singlet at 5.87 ppm. Symmetric and asymmetric
stretching bands of free eNH2 group appeared at 3321 and
3200 cmꢁ1 in the IR spectrum of the compound 30.
O
O
NH2
N
N
N
O
MeOH/AcOH
O
R
+
O
R
RT
NH2
(10-16)
O
O
N
N
NH2
(2)
(3-9)
SOCl2
Reflux
O
N
H
N
N
OR
N
O
(17-23)
R
R
(3, 10, 17)
(6, 13, 20)
N
N
N
N
O
(7, 14, 21)
(4, 11, 18)
(5, 12, 19)
N
Cl
(8, 15, 22)
(9, 16, 23)
O
Scheme 1. Synthetic route to 8-(p-substituted phenyl)xanthine derivatives 17e23.

R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
329
(22)
Cl
N
HN
N
HN
N
Cl
O
N
N
O
N
N
N
H
N
H
N
N
N
O
O
N
O
N
O
(24)
(25)
Scheme 2. Synthesis of xanthine derivatives 24 and 25.
Cyclization of 30 on refluxing with a mixture of sodium hy-
droxide and dioxane resulted in the formation of desired xanthine
derivative 31 with a methylene spacer between C8 of the xanthine
nucleus and phenyl ring. The disappearance of absorption band of
free eNH2 in the IR spectrum at 3321 cmꢁ1 supported the forma-
tion of 8-[4-(3-chloropropoxy)benzyl]-1,3-dimethylxanthine (31).
The protons of the methylene linker and of eOCH2e group reso-
general, 8-phenyl substituted xanthines 17e25 displayed high
binding affinity for all the adenosine receptors with maximum af-
finity, and thus resulting in selectivity for the A2A subtype. The
binding selectivity for A2A is somewhat more pronounced versus A3
receptors compared to the A1 and A2B subtypes in most of the cases.
No significant difference was observed in binding affinity of 8-
alkylaminoethoxyphenyl substituted xanthines possessing open
(17, 18) and cyclized ring systems (19e21) in side chain towards
individual adenosine receptors. The maximum binding affinity of
these compounds was observed for A2A receptors, which is w5, 10,
and 20 times higher as compared to A1, A2B, and A3 receptors,
respectively. In comparison to amino substituted xanthines 17e21,
chloropropoxy phenyl substituted 22 (Ki ¼ 45 nM) and cyclo-
pentyloxy derivative 23 (Ki ¼ 72 nM) exhibited remarkable affinity
and selectivity for A2A receptors. Both the compounds were found
to possess w200e600 times higher binding for A2A in comparison
to other AR subtypes, but xanthine 23 was only 14 times more
selective for the A2A versus A2B subtype (Table 1). Imidazole (24)
and chlorobenzyl piperazine (25) derived xanthines displayed
some selectivity for A2A against A1 and A2B, which is little more
pronounced against A3 receptor subtype. The imidazolyl
substituted xanthine 24 was the most potent compound of the
series with Ki ¼ 42 nM for A2A receptors.
nated together as a multiplet at
d 4.1 ppm. To introduce various
polar functionalities, chloropropoxy substituted 8-benzylxanthine
was further fused with various heterocyclic amines like morpho-
line, imidazole and 1,4-dioxa-8-azaspiro[4.5]decane at 80e100 ꢀC
to obtain target 8-aminoalkoxybenzylxanthines 32e34.
8-[4-(3-Morpholinopropoxy)benzyl]-1,3-dimethylxanthine
(32) exhibited a broad singlet integrating for four protons of the e
Ne(CH2)2 and a triplet of eO-(CH2)2 protons at
respectively. Singlets of imidazolyl protons were found at
d
2.46 and 3.71 ppm,
6.93,
d
7.04 and 7.45 for compound 33, four protons of N-methylene
groups of 1,4-dioxa-8-azaspiro[4.5]decane and two protons of e
OCH2CH2CH2eN< appeared together as a multiplet at 2.52e
2.55 ppm in the 1H NMR spectrum of compound 34. Protons of the
methylene linker resonated as a singlet at w d 4.1 ppm for 8-benzyl
substituted xanthines.
Introduction of a methylene spacer between side chain and
purine core structure resulted in loss of binding affinity of 8-
benzylxanthine analogs 31 and 32 for all adenosine receptor sub-
types. However, introduction of an imidazole or dioxaazaspir-
odecan group in the side chain (33, 34) revived binding to A2A with
no measurable effect on binding to other subtypes (Table 1).
All the newly synthesized 8-phenylxanthines 17e25 displayed
significant protection against histamine aerosol induced broncho-
spasm in guinea pigs; effects being more prominent in comparison
to 8-benzyl congeners 31e34. The compounds showed more po-
tency in comparison to the standard drug theophylline as apparent
from enhanced time of onset of bronchospasm in case of test
compounds compared to theophylline except cyclopentyloxy
substituted xanthine 23. The chloroarylpiperazinyl substituted
xanthine 25 was the most effective compound of the series with no
jerks, least severity of bronchospasm and 100% survival of animals.
The 8-benzyl substituted xanthines 31e34 displayed decreased
bronchospasmolytic effects in comparison to theophylline and 8-
phenylxanthine congeners. The compounds 31 and 32 although
lack AR binding affinity but still produces bronchospasmolytic ef-
fects, with later displaying more prominence. Cyclopentyloxy
3. Biological evaluation
The 8-substituted xanthine derivatives 17e25 and 31e34 were
evaluated using in vitro radioligand binding studies at cloned
adenosine receptors [19,20]. In this study all human subtypes were
stably transfected into Chinese Hamster Ovary (CHO) cells to study
their pharmacological profile in an identical cellular background
utilizing radioligand binding studies (A1, A2A, A3) or adenylyl
cyclase activity assays (A2B). Affinities of various newly synthesized
xanthine derivatives for adenosine A1, A2A, A2B and A3 receptors
have been summarized in Table 1. The in vivo bronchospasmolytic
activity of compounds 17e25 and 31e34 was studied using a his-
tamine chamber model [21]. The data obtained has been arranged
in Table 2. The results have been compared with standard drug
theophylline.
4. Results and discussion
Table 1 summarizes the observed binding affinities of newly
synthesized 8-phenyl/benzylxanthine derivatives towards various
human adenosine receptor (A1, A2A, A2B and A3) subtypes. In

330
R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
O
O
O
HO
CH3OH , Reflux
OH
OH
Conc.
H2SO4
(27)
(26)
RCH2CH2CH2Br
Reflux
K2CO3
EMK
O
Cl
O
Cl
O
HO
NaOH
O
O
(29)
(28)
Cl
O
NH2
NH2
N
EDCI
MeOH
O
O
N
(2)
Cl
O
N
O
H
N
H
N
NaOH/Dioxane
N
N
O
O
NH2
N
O
N
O
(30)
(31)
Various amines
R
O
O
H
N
N
N
O
N
N
(32-34)
O
N
N
R
N
O
=
O
(32)
(33)
(34)
Scheme 3. Synthetic route to 8-(p-substituted benzyl)xanthine derivatives 31e34.
substituted 8-phenylxanthine 23 shows high affinity for A2A but
bronchospasmolytic effects are missing. Still the possibility of a
causal relationship between A2A antagonism and broncholysis
cannot be ruled out. Bronchospasmolytic effects of the compounds
may be attributed to combined antiinflammatory effects (resulting
from A2A binding) and bronchorelaxant effects (due to inherent
property of xanthine skeleton), therefore to some extent bron-
chospasmolytic effects are related to A2A binding but not
completely in this particular series of xanthine derivatives.
and broncholytic effects. The effect of varying substituents on the 8-
phenyl ring on biological properties in case of both in vitro and
in vivo assays is clearly visible. Suitable introduction of 8-phenyl/
benzyl substituents on the xanthine scaffold results in potent and
selective binding for A2A adenosine receptors along with potent
bronchospasmolytic effects. These dual action xanthine derivatives
may act as useful candidates for asthma therapy.
5. Experimental protocols
In conclusion, the newly synthesized 8-phenylxanthines dis-
played significant binding affinity for adenosine receptors with
highest potency for A2A, resulting in selectivity for A2A versus other
AR subtypes. These compounds also produced potent broncho-
spasmolytic effects, however replacement of phenyl ring with
benzyl moiety resulted in notable reduction in adenosine affinity
5.1. Chemistry
All melting points were obtained using glass capillary tubes on
a Veego melting point apparatus and are uncorrected. Infrared (IR)
spectra were recorded on a PerkineElmer RX1 Fourier Transfrom-

R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
331
Table 1
Binding affinities of xanthine derivatives at various adenosine receptor subtypes (A1, A2A, A2B and A3).
Comp. No. (CODE)
Ki (
m
M)
A2A selectivity
A1/A2A
(A1)a
(A2A
)
(A2B
)
(A3)d
A2B/A2A
A3/A2A
b
c
17 (RB-337)
18 (RB-396)
19 (RB-394)
20 (RB-393)
21 (RB-395)
22 (RB-338)
23 (RB-397)
24 (RB-339)
25 (RB-448)
31 (RB-452)
32 (RB-453)
33 (RB-454)
34 (RB-455)
1.82 (1.55e2.13)
1.59 (1.19e2.13)
1.33 (1.11e1.60)
2.58 (2.48e2.68)
1.20 (1.05e1.37)
>10
0.40 (0.21e0.77)
0.25 (0.16e0.38)
0.29 (0.14e0.58)
0.31 (0.21e0.46)
0.20 (0.12e0.34)
0.045 (0.036e0.054)
0.072 (0.05e0.12)
0.042 (0.03e0.07)
0.12 (0.076e0.19)
>10
4.95 (3.21e7.64)
2.59 (1.81e3.72)
2.9 (2.21e3.79)
1.34 (1.04e1.72)
1.97 (1.51e2.56)
>10
9.38 (6.12e14.40)
6.82 (5.14e9.05)
6.51 (4.26e9.95)
2.74 (1.51e4.97)
4.60 (3.97e5.32)
>30
4.6
6.4
4.6
83
12
10
10
43
9.9
>220
14
25
1.4
e
24
27
22
8.8
23
>670
>420
54
57
e
6.0
>220
>420
21
15
e
>30
1.0
>10
0.89 (0.68e1.18)
1.85 (1.70e2.01)
>30
>30
>30
1.05 (0.79e1.41)
0.17 (0.15e0.19)
>10
>10
>10
2.27 (1.91e2.7)
6.89 (4.04e11.8)
>30
>30
>30
>10
3.17 (2.56e3.92)
8.09 (4.39e14.9)
e
e
e
>9.5
>3.7
>3.2
>1.2
>9.5
>3.7
>30
>10
>30
Shown values are geometric means from 3 to 5 experiments in mM with 95% confidence intervals in parentheses.
a
Displacement of specific (3H) CCPA binding in CHO cells, stably transfected with human recombinant A1 adenosine receptor, expressed as Ki (nM).
Displacement of specific (3H) NECA binding in CHO cells, stably transfected with human recombinant A2A adenosine receptor, expressed as Ki (nM).
Antagonist affinities were determined by inhibition of NECA-stimulated adenylyl cyclase activity in membrane preparations.
b
c
d
Displacement of specific (3H)HEMADO binding in CHO cells, stably transfected with human recombinant A3 adenosine receptor, expressed as Ki (nM).
Infrared spectrophotometer using potassium bromide pellets
ymax in cmꢁ1). Proton (1H) and carbon (13C) nuclear magnetic
5.1.1. General procedure for the synthesis of various aldehydes 3e9
Requisite alkylaminoethyl chloride hydrochloride, chloro-
bromopropane and cyclopentyl bromide (6.0 mmol) were added to
a stirred and refluxing slurry of 4-hydroxybenzaldehyde (1.0 g,
8.19 mmol) in ethyl methyl ketone (C4H8O) (40 ml) in the presence
of anhydrous potassium carbonate (2.0 g,14.47 mmol). The reaction
mixture was further refluxed for 6 h with continuous stirring. The
completion of the reaction was monitored by TLC. On completion,
the reaction mixture was cooled, filtered and the solvent was
removed under reduced pressure to obtain an oily residue of cor-
responding aldehyde 3e9, which was used as such for further
reaction.
(
resonance spectroscopy were performed using a Bruker AC-400F,
400 MHz spectrometer for solutions in deuteriochloroform
(CDCl3) and deuterated dimethylsulfoxide (DMSO-d6) and are re-
ported in parts per million (ppm) downfield from tetramethylsi-
lane (Me4Si) as internal standard. The spin multiplicities are
indicated by the symbols, s (singlet), d (doublet), t (triplet), q
(quartet), p (pentet), m (multiplet), br (broad) and the coupling
constants (J) are given in Hertz (Hz). Elemental analyses were
carried out on a Thermo-Flash EA-1112 CHNSeO analyzer. The
results are within 0.4% of the theoretical values. Precoated plates
with silica gel G (E. Merck 60 F254, 0.25 mm) were used for thin
layer chromatography (TLC). Chromatographic spots were visual-
ized by ultra-violet light in the UV cabinet (Perfit, India). Anhy-
drous sodium sulfate was utilized as drying agent. All solvents
were freshly distilled and dried prior to use according to standard
procedures.
5.1.2. General procedure for the synthesis of various benzylidene
derivatives 10e16
To a stirred solution of 5,6-diamino-1,3-dimethyluracil (2) (1.0 g,
5.87 mmol) in MeOH-AcOH (4:1, 40 mL) was slowly added the
solution of the above obtained oily residue of the relevant aldehyde
3e9 in methanol (24 ml). The reaction mixture was further stirred
overnight at room temperature. The completion of the reaction was
monitored by TLC. The residue obtained after removal of solvent
under reduced pressure was dissolved in ice-cold water and made
alkaline with sodium hydroxide. The resultant turbid solution was
cooled in ice for complete precipitation. The precipitate obtained
was filtered off, washed with ice cold water and dried to obtain the
corresponding benzylidene derivatives 10e16, which were used as
such for further reaction.
Table 2
Protection by xanthine derivatives against bronchospasm induced by histamine
aerosol (5 ml of 1% w/v aerosoled in 1 min) in guinea pigs.
Comp. no.
(Code)
Mean time in
seconds for
onset of
Duration of
jerks in
seconds
Severity of
bronchospasm
Survival
(%)
bronchospasm
Mean ꢂ S.E.M
Mean ꢂ S.E.M
Control
67 ꢂ 2a
172 ꢂ 4a
89 ꢂ 7
39 ꢂ 1a
49 ꢂ 3a
22 ꢂ 2a
45 ꢂ 2a
32 ꢂ 1a
30 ꢂ 2a
35 ꢂ 2a
71 ꢂ 4
e
þþþ
þ
0
100
100
75
Theophylline
17 (RB-337)
18 (RB-396)
19 (RB-394)
20 (RB-393)
21 (RB-395)
22 (RB-338)
23 (RB-397)
24 (RB-339)
25b (RB-448)
31 (RB-452)
32b (RB-453)
33b (RB-454)
34 (RB-455)
92 ꢂ 3
5.1.3. General procedure for the synthesis of various 8-(substituted-
phenyl)xanthine derivatives 17e25
111 ꢂ 5
115 ꢂ 3
121 ꢂ 4a
104 ꢂ 5
111 ꢂ 6
114 ꢂ 5
72 ꢂ 3
þ
þþ
þ
Benzylidene derivatives 10e16 (2.5 mmol) thus obtained were
refluxed separately in thionyl chloride (20 ml) for 30e40 min to
affect cyclization. The excess thionyl chloride was removed under
reduced pressure to obtain a solid product. Ice cold water was
added to it and the resultant suspension was neutralized with
ammonium hydroxide solution. The precipitate obtained was
collected by filtration, dried and recrystallized from a mixture of
DMF and ethanol to afford the desired products 17e23,
respectively.
Further, the chloropropoxy derivative 22 was fused with imid-
azole and 1-(2-chlorobenzyl)piperazine at 160 ꢀC for 2 h to afford
the desired compounds 24 and 25, respectively. Imidazolyl
substituted xanthine 24 was crystallized from a mixture of DMF
75
þ
100
100
100
50
75
100
50
100
50
50
þ
þ
þþ
þþ
þ
108 ꢂ 4
109 ꢂ 5
75 ꢂ 4
44 ꢂ 3a
þþ
þ
108 ꢂ 4
83 ꢂ 3
e
e
þþ
þþ
73 ꢂ 3
23 ꢂ 3a
Number of animals in each group (N) ¼ 4.
Dose of standard and tested compounds ¼ 50 mg/kg.
a
Tukey’s test; p < 0.05.
No jerks were observed in animals after treatment with test compounds.
b

332
R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
and ethanol and compound 25 using a mixture of chloroform and
methanol.
121.53 (ArC), 128.03 (2xArCH), 148.47 (ArC), 149.75 (ArC), 151.13
(ArC), 154.04 (C]O) and 159.52 ppm (C]O). Anal. Calc. for
C19H23N5O3: C, 61.77; H, 6.27; N, 18.96. Found: C, 61.80; H, 6.27; N,
5.1.3.1. 8-[4-(2-Dimethylaminoethoxy)phenyl]-1,3-dimethylxanthine
18.71%.
(17). Yield: 54.3%; m.p. >280 ꢀC. FTIR: 3174, 1691, 1650, 1482, 1245,
1183; 1H NMR (CDCl3 þ DMSO-d6):
d
2.93 (s, 6H, eNe(CH3)2), 3.39 (s,
5.1.3.6. 8-[4-(3-Chloropropoxy)phenyl]-1,3-dimethylxanthine (22).
Yield: 114.7%; m.p. >220 ꢀC. FTIR: 3163, 2947, 1693, 1652, 1484,
3H, NeCH3), 3.56 (s(br), 2H, eCH2N<), 3.62 (s, 3H, NeCH3), 4.49 (t,
2H, eOCH2e), 7.04 (d, 2H, 2-CH and 6-CH, aromatic, Jo ¼ 8.92 Hz),
8.14 (d, 2H, 3-CH and 5-CH, aromatic, Jo ¼ 8.92 Hz) and 13.44 ppm (s,
1293,1026; 1H NMR (DMSO-d6):
d 2.27 (p, 2H, eCH2CH2CH2Cl), 3.43
(s, 3H, NeCH3), 3.65 (s, 3H, NeCH3), 3.77 (t, 2H, eCH2Cl,
J ¼ 6.32 Hz), 4.18 (t, 2H, eOCH2e, J ¼ 5.84 Hz), 6.98 (d, 2H, 2-CH and
6-CH, aromatic, Jo ¼ 8.84 Hz), 8.10 (d, 2H, 3-CH and 5-CH, aromatic,
Jo ¼ 8.84 Hz) and 13.26 ppm (s, 1H, NeH). 13C NMR (DMSO-d6):
1H, NeH). 13C NMR (DMSO-d6):
d
27.62 (2ꢃ NeCH3), 29.59 (2ꢃ Ne
CH3), 44.65 (NeCH2), 56.88 (OeCH2), 107.1 (ArC), 114.66 (2ꢃ ArCH),
121.51 (ArC)) 128.01 (2ꢃ ArCH), 148.2 (ArC), 149.95 (ArC), 151.16
(ArC),154.23 (C]O) and 160 ppm (C]O). Anal. Calc. for C17H21N5O3:
C, 59.46; H, 6.16; N, 20.39%. Found: C, 59.34; H, 6.27; N, 20.21%.
d
27.74 (NeCH3), 29.72 (NeCH3), 31.57 (CH2), 41.88 (CH2Cl), 64.48
(OeCH2), 107.27 (ArC), 114.82 (2xArCH), 121.39 (ArC), 128.06
(2xArCH), 148.54 (ArC), 149.87 (ArC), 151.17 (ArC), 154.08 (C]O)
and 159.9 ppm (C]O). Anal. Calc. for C16H17N4O3Cl: C, 55.15; H,
4.92; N, 16.08. Found: C, 55.33; H, 4.68; N, 16.36%.
5.1.3.2. 8-[4-(2-Diethylaminoethoxy)phenyl]-1,3-dimethylxanthine
(18). Yield: 89.6%; m.p. >280 ꢀC. FTIR: 3168, 1696, 1650, 1478,1245;
1H NMR (DMSO-d6):
d 1.32 (t, 6H, eN(CH2CH3)2), 3.16 (s, 4H, e
N(CH2CH3)2), 3.34 (s, 3H, NeCH3), 3.48 (s, 2H, eCH2N<), 3.57 (s, 3H,
NeCH3), 4.40 (s, 2H, eOCH2e), 7.05 (d, 2H, 2-CH and 6-CH, arom,
Jo ¼ 8.84 Hz), 8.14 (d, 2H, 3-CH and 5-CH, arom, Jo ¼ 8.76 Hz) and
5.1.3.7. 8-[4-(Cyclopentyloxy)phenyl]-1,3-dimethylxanthine
Yield: 60.4%; m.p. 208e210 ꢀC. FTIR: 3148, 2939, 1704, 1647, 1606,
1474, 1367, 1051; 1H NMR (CDCl3):
cyclopentyl), 3.42 (s, 3H, NeCH3), 3.65 (s, 3H, NeCH3), 4.80e4.91
(m, 1H, OeCH<, cyclopentyl), 6.93 (d, 2H, 2-CH and 6-CH, aromatic,
Jo ¼ 8.80 Hz) and 8.08 ppm (d, 2H, 3-CH and 5-CH, aromatic,
Jo ¼ 8.80 Hz). Anal. Calc. for C18H20N4O3: C, 63.52; H, 5.92; N, 16.46.
Found: C, 63.42; H, 5.80; N, 16.56%.
(23).
d
1.78e2.01 (m, 8H, 4ꢃ eCH2e,
13.54 (s, 1H, NeH). 13C NMR (DMSO-d6):
d
10.86 (2xCH3), 27.74 (Ne
CH3), 29.72 (NeCH3), 47.01 (Ne(CH2)2), 50.84 (NeCH2), 65.33 (Oe
CH2), 107.20 (ArC), 114.84 (2ꢃ ArCH), 121.30 (ArC), 128.02 (2ꢃ
ArCH), 148.49 (ArC), 149.81 (ArC), 151.15 (ArC), 154.04 (C]O) and
159.78 ppm (C]O). Anal. Calc. for C19H25N5O3: C, 61.48; H, 6.79; N,
18.87. Found: C, 61.38; H, 6.65; N, 18.82%.
5.1.3.8. 1,3-Dimethyl-8-[4-(3-imidazol-1-ylpropoxy)phenyl]xanthine
5.1.3.3. 1,3-Dimethyl-8-[4-(2-piperidin-4-ylethoxy)phenyl]xanthine
(24). Yield: 84.6%; m.p. 255 ꢀC (decomp.). FTIR: 3167, 2946, 1692,
(19). Yield: 25.2%; m.p. 258e260 ꢀC. FTIR: 3165, 2920, 1693, 1648,
1650, 1482, 1291, 1026; 1H NMR (DMSO-d6):
d 2.28 (p, 2H, e
1473, 1241, 1019; 1H NMR (DMSO-d6):
d
1.21e1.23 (m, 2H, eCH2e,
CH2CH2CH2N<), 3.42 (s, 3H, NeCH3), 3.65 (s, 3H, NeCH3), 3.99 (t,
2H, eCH2N<, J ¼ 5.16 Hz), 4.23 (t, 2H, eOCH2e, J ¼ 6.64 Hz), 6.96e
7.00 (m, 3H, 2-CH and 6-CH, aromatic and CH, imidazole), 7.03 (s,
1H, CH, imidazole), 7.52 (s, 1H, CH, imidazole), 8.09 (d, 2H, 3-CH and
5-CH, aromatic, Jo ¼ 7.40 Hz) and 13.25 ppm (s, 1H, NeH). 13C NMR
piperidine), 1.95e1.97 (m, 4H, 2ꢃ CH2, piperidine), 2.98e3.06 (m, 4H,
Ne(CH2)2, piperidine), 3.51e3.53 (m, 5H, NeCH3 and eCH2N<), 3.59
(s, 3H, NeCH3), 4.51 (t, 2H, eOCH2e, J ¼ 5.97 Hz), 7.02 (d, 2H, 2-CH and
6-CH, arom, Jo ¼ 8.84 Hz), 8.13 (d, 2H, 3-CH and 5-CH, arom,
Jo ¼ 8.64Hz)and10.62(s,1H, NeH).13CNMR(DMSO-d6):
d
21.19 (CH2),
(DMSO-d6): d 27.73 (NeCH3), 29.71 (NeCH3), 30.04 (CH2), 42.97
22.39 (2ꢃ CH2), 27.74 (NeCH3), 29.71 (NeCH3), 52.67 (Ne(CH2)2),
54.67 (NeCH2), 62.49 (OeCH2),107.22 (ArC),115.01 (2ꢃ ArCH),121.85
(ArC),128.03 (2xArCH),148.47 (ArC),149.63 (ArC),151.11 (ArC),154.02
(C]O) and 159.01 ppm (C]O). Anal. Calc. for C20H25N5O3: C, 62.65; H,
6.57; N, 18.26. Found: C, 62.80; H, 6.80; N, 18.46%.
(NeCH2), 64.70 (OeCH2), 107.20 (ArC), 114.80 (2xArCH), 119.45
(ArCH, imid), 121.38 (ArC), 122.67 (ArCH, imid), 128.04 (2xArCH),
137.27 (ArCH, imid), 148.49 (ArC), 149.83 (ArC), 151.15 (ArC), 154.04
(C]O) and 159.92 ppm (C]O). Anal. Calc. for C19H20N6O3: C, 59.99;
H, 5.30; N, 22.09. Found: C, 59.68; H, 5.17; N, 22.29%.
5.1.3.4. 1,3-Dimethyl-8-[4-(2-morpholin-4-ylethoxy)phenyl]xanthine
5.1.3.9. 8-[4-{3-(4-(2-Chlorobenzyl)piperazin-1-yl)propoxy}phenyl]-
(20). Yield: 52.3%; m.p. >280 ꢀC. FTIR: 3174, 2951, 1691, 1650, 1482,
1,3-dimethylxanthine (25). Yield: 33.5%; m.p. >220 ꢀC. FTIR: 3169,
1250, 1114; 1H NMR (DMSO-d6):
d
2.59 (s(br), 4H, eN(CH2)2, mor-
2942, 1694, 1650, 1478, 1291, 1048; 1H NMR (CDCl3):
d 2.02 (p, 2H, e
pholine), 2.82 (t, 2H, eCH2N<, J ¼ 5.64 Hz), 3.38 (s, 3H, NeCH3),
3.62 (s, 3H, NeCH3), 3.70 (t, 4H, O(CH2)2, morpholine), 4.17 (t, 2H, e
OCH2e, J ¼ 5.62 Hz), 6.99 (d, 2H, 2-CH and 6-CH, arom, Jo ¼ 8.88 Hz),
8.09 (d, 2H, 3-CH and 5-CH, arom, Jo ¼ 8.88 Hz) and 13.32 (s, 1H, Ne
OCH2CH2CH2-), 2.17 (s, 2H, eCH2eN<, J ¼ 6.10 Hz), 2.56e2.60 (m,
8H, 4ꢃ eCH2e, piperazine), 3.54 (s, 3H, NeCH3), 3.70 (s, 3H, Ne
CH3), 3.64 (s, 2H, eCH2e), 4.09 (t, 2H, eOCH2e, J ¼ 6.30 Hz), 6.98 (d,
2H, 2-CH and 6-CH, aromatic, Jo ¼ 8.76 Hz), 7.22e7.29 (m, 2H, 40-CH
and 50-CH, aromatic), 7.35 (dd, 1H, 60-CH, aromatic, Jm ¼ 1.36 Hz;
Jo ¼ 7.72 Hz), 7.47 (dd, 1H, 30-CH, aromatic, Jo ¼ 7.04 Hz), 8.15 (d, 2H,
3-CH and 5-CH, aromatic, Jo ¼ 8.76 Hz) and 13.38 ppm (s, 1H, NeH).
H). 13C NMR (DMSO-d6):
d 27.72 (NeCH3), 29.70 (NeCH3), 53.47
(Ne(CH2)2), 55.67 (NeCH2), 63.49 (OeCH2), 65.13 (O-(CH2)2), 107.11
(ArC), 114.01 (2xArCH), 123.85 (ArC), 129.03 (2xArCH), 148.17 (ArC),
149.61 (ArC), 153.11 (ArC), 154.12 (C]O) and 159.11 ppm (C]O).
Anal. Calc. for C19H23N5O4: C, 59.21; H, 6.01; N, 18.17. Found: C,
59.44; H, 6.14; N, 18.04%.
13C NMR (DMSO-d6):
d 27.58 (NeCH3), 29.01 (CH2), 29.55 (NeCH3),
52.52 (Ne(CH2)2), 52.70 (Ne(CH2)2), 54.21 (NeCH2), 58.53 (Ne
CH2eAr), 65.83 (OeCH2), 107.13 (ArC), 114.45 (2ꢃ ArCH), 121.12
(ArC)) 126.59 (ArCH), 127.96 (2ꢃ ArCH), 128.14 (ArCH), 128.99
(ArCH), 130.49 (ArCH), 133.33 (ArC), 135.48 (ArC),148.45 (ArC),
149.98 (ArC), 151.12 (ArC), 153.98 (C]O) and 160.11 ppm (C]O).
Anal. Calc. for C27H31N6O3Cl: C, 62.00; H, 5.97; N, 16.07. Found: C,
59.92; H, 5.88; N, 16.14%.
5.1.3.5. 1,3-Dimethyl-8-[4-(2-pyrrolidin-1-ylethoxy)phenyl]xanthine
(21). Yield: 35.7%; m.p. >220 ꢀC. FTIR: 3165, 2949, 1694, 1650, 1477,
1244, 1053; 1H NMR (DMSO-d6):
d
1.86 (s, 4H, 2ꢃ CH2, pyrrolidine),
2.88 (s, 4H, eN(CH2)2, pyrrolidine), 3.14 (s, 2H, eCH2N<), 3.33 (s,
3H, NeCH3), 3.61 (s, 3H, NeCH3), 4.24 (s, 2H, eOCH2e), 6.99 (d, 2H,
2-CH and 6-CH, arom, Jo ¼ 8.72 Hz), 8.11 (d, 2H, 3-CH and 5-CH
arom, Jo ¼ 8.68 Hz) and 13.46 (s, 1H, NH). 13C NMR (DMSO-d6):
5.1.4. 6-Amino-5-[{4-(3-chloropropoxy)carboxacetamido}phenyl]-
1,3-dimethyluracil (30)
A solution of 4-hydroxyphenylacetic acid (26) (1.0 g, 6.02 mmol)
in methanol (30 ml) was refluxed for 3 h in the presence of a
d
22.79 (2ꢃ CH2), 27.72 (NeCH3), 29.70 (NeCH3), 53.37 (NeCH2),
53.86 (Ne(CH2)2), 65.06 (OeCH2), 107.23 (AreC), 114.90 (2ꢃ ArCH),

R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
333
catalytic amount of sulfuric acid, the reaction being monitored by
thin layer chromatography. On completion, the excess solvent was
removed to afford an oily product of methyl-4-hydroxyphenyl ac-
etate (27), which was used as such for further reaction.
5.1.6.1. 1,3-Dimethyl-8-[4-(3-morpholinopropoxy)benzyl]xanthine
(32). Yield: 44.1%; m.p. 198e200 ꢀC. FT-IR: 3146, 3095, 3037, 2940,
2870, 2833, 1703, 1659, 1561, 1400, 1245, 1059, 984 and 798 cmꢁ1
.
1H NMR (CDCl3):
d
1.95 (p, 2H, eOCH2CH2CH2e, J ¼ 7.18 Hz), 2.46
The ester 27 so obtained was heated under reflux in ethyl
methyl ketone (10 ml) with 1-bromo-3-chloropropane (1.0 ml)
in the presence of anhydrous potassium carbonate (1.0 g). The
reaction mixture was further refluxed with stirring for 8 h and
the completion of the reaction was monitored by thin layer
chromatography. After cooling, the reaction mixture was filtered
off and the excess solvent was removed to dryness to give
methyl-[4-(3-chloropropoxy)phenyl]acetate [22] (28), which
was further treated with 1.0 N sodium hydroxide. The mixture
was stirred at room temperature for 8 h for completion of the
reaction. The reaction mixture was further acidified and on
continuous stirring some semisolid product separated out. The
precipitate so obtained was filtered, washed with water and
dried to obtain 4-(3-chloropropoxy)phenylacetic acid [22] (29,
0.35 g) mp 83e87 ꢀC.
(s(br), 4H, Ne(CH2)2, morpholine), 2.50 (t, 2H, eOCH2CH2CH2-N,
J ¼ 7.34 Hz), 3.43 (s, 3H, NeCH3), 3.62 (s, 3H, NeCH3), 3.71 (t, 4H, O-
(CH2)2, morpholine J ¼ 4.64 Hz), 3.99 (t, 2H, eOCH2e, J ¼ 6.32 Hz),
4.13 (s, 2H, eCH2e), 6.84 (d, 2H, 2-CH and 6-CH, aromatic,
Jo ¼ 6.62 Hz), 7.24 (d, 2H, aromatic, 3-CH and 5-CH, Jo ¼ 8.64 Hz) and
11.84 ppm (s, 1H, NeH). 13C NMR (DMSO-d6):
d 26.26 (NeCH3),
28.36 (NeCH3), 30.28 (CH2), 34.63 (CH2), 53.67 (Ne(CH2)2), 55.52
(NeCH2), 66.07 (O-CH2), 66.83 (O-(CH2)2), 106.79 (ArC), 114.91 (2ꢃ
ArCH), 127.74 (ArC), 129.85 (2xArCH), 149.39 (ArC), 151.54 (ArC),
154.29 (ArC), 155.63 (C]O) and 158.24 ppm (C]O). Anal. Calcd. for
C21H27N5O4: C, 61.0; H, 6.58; N, 16.94%. Found: C, 61.08; H, 6.60; N,
17.0%.
5.1.6.2. 1,3-Dimethyl-8-[4-{3-(1H-imidazol-1-yl)propoxy}benzyl]
xanthine (33). Yield: 87.4%; m.p. 210e212 ꢀC. FT-IR: 3382, 3143,
3100, 3035, 2987, 2941, 1706, 1662, 1560, 1401, 1241, 1060, 984 and
The compound 29 (1.0 g, 4.38 mmol) was stirred with 5,6-
diaminouracil (2) (1.0 g, 5.87 mmol) in methanol (40 ml) in pres-
ence of 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDCI)
[23] (1.0 g, 5.21 mmol) at room temperature. After 10 min the re-
action mixture turned turbid and was further stirred for 24 h. The
completion of reaction was monitored by thin layer chromatog-
raphy. The precipitate obtained was collected by filtration, washed
with methanol and dried to afford the desired product 30.
Yield: 135.7%; m.p. 218e220 ꢀC. FT-IR: 3321, 3200, 2955, 1705,
. d 2.21 (p, 2H, e
760 cmꢁ1 1H NMR (CDCl3-DMSO-d6):
OCH2CH2CH2e, J ¼ 8.12 Hz), 3.38 (s, 3H, NeCH3), 3.58 (s, 3H, Ne
CH3), 3.86 (t, 2H, eOCH2CH2CH2e, J ¼ 5.70 Hz), 4.04 (s, 2H, eCH2e),
4.19 (t, 2H, eOCH2e, J ¼ 6.70 Hz), 6.81 (d, 2H, 2-CH and 6-CH, ar-
omatic, Jo ¼ 8.64 Hz), 6.93 (s, 1H, CH, imidazole), 7.04 (s, 1H, CH,
imidazole), 7.24 (d, 2H, 3-CH and 5-CH, aromatic, Jo ¼ 8.64 Hz), 7.45
(s, 1H, CH, imidazole) and 12.99 ppm (s, 1H, NeH). 13C NMR (DMSO-
d6): d 27.61 (NeCH3), 29.71 (NeCH3), 30.13 (CH2), 33.46 (CH2), 42.87
1644, 1599, 1508, 1304, 1238, 1043, 930 and 756 cmꢁ1
.
1H NMR
2.23 (p, 2H, eOCH2CH2CH2Cl, J ¼ 6.04 Hz), 3.27
(NeCH2), 64.35 (O-CH2), 106.20 (ArC), 114.50 (2xArCH), 119.32
(ArCH, imid), 128.37 (ArCH, imid), 129.05 (ArC), 129.64 (2ꢃ ArCH),
137.29 (ArCH, imid), 148.10 (ArC), 151.12 (ArC), 152.78 (ArC), 154.03
(C]O) and 157.16 ppm (C]O). Anal. Calcd. for C20H22N6O3: C,
60.90; H, 5.62; N, 21.31%. Found: C, 60.85; H, 5.72; N, 21.20%.
(CDCl3-DMSO-d6):
d
(s, 3H, NeCH3), 3.41 (s, 3H, NeCH3), 3.67 (s, 2H, eCH2e), 3.75 (t, 2H,
eCH2Cl, J ¼ 6.30 Hz), 4.10 (t, 4H, eOCH2e, J ¼ 5.8 Hz), 5.87 (s, 2H, e
NH2), 6.88 (d, 2H, 2-CH and 6-CH, aromatic, Jo ¼ 8.52 Hz), 7.31 (d,
2H, 3-CH and 5-CH, aromatic, Jo ¼ 8.48 Hz) and 7.76 ppm (s, 1H, Ne
H). Anal. Calcd. for C17H21N4O3Cl: C, 55.66; H, 6.32; N, 15.27%.
Found: C, 55.80; H, 6.27; N, 15.17%.
5.1.6.3. 1,3-Dimethyl-8-[4-{3-(1,4-dioxa-8-azaspiro[4.5]decan-1-yl)-
propoxy}benzyl]-xanthine (34). Yield: 38.8%; m.p. 194e196 ꢀC. FT-
IR: 3147, 3096, 3037, 2955, 2820, 1706, 1661, 1561, 1403, 1244 and
797 cmꢁ1. 1H NMR (CDCl3):
d
1.75 (t, 4H, 2ꢃ eCH2e, J ¼ 5.70 Hz),
5.1.5. 8-[4-(3-Chloropropoxy)benzyl]-1,3-dimethylxanthine (31)
The compound 30 (1.0 g, 2.73 mmol) was refluxed in a mixture
of sodium hydroxide:dioxane (1:1) for 3 h to affect cyclization. The
mixture was cooled and acidified with dil. HCl (4 N). The precipitate
obtained was collected by filtration, dried and crystallized from a
mixture of chloroform and methanol to afford the desired product
31. Yield: 69.2%; m.p. 228e230 ꢀC. FT-IR: 3142, 3034, 1705, 1655,
1.95 (p, 2H, eOCH2CH2CH2e, J ¼ 7.18 Hz), 2.52e2.55 (m, 6H, e
OCH2CH2CH2-N and N(CH2)2), 3.41 (s, 3H, NeCH3), 3.62 (s, 3H, Ne
CH3), 3.95 (s, 4H, 2ꢃ eOCH2e), 4.0 (t, 2H, eOCH2e, J ¼ 6.30 Hz), 4.12
(s, 2H, eCH2e), 6.86 (d, 2H, 2-CH and 6-CH, aromatic, Jo ¼ 8.647 Hz),
7.21 (d, 2H, 3-CH and 5-CH, aromatic, Jo ¼ 8.60 Hz) and 11.84 ppm (s,
1H, NeH). 13C NMR (DMSO-d6):
d 26.48 (CH2), 27.63 (NeCH3), 29.72
(NeCH3), 33.46 (CH2), 34.41 (2ꢃ CH2), 50.89 (Ne(CH2)2), 53.98 (Ne
CH2), 63.50 (O-(CH2)2), 65.75 (O-CH2), 106.15 (ArC), 106.41 (C),
114.46 (2ꢃ ArCH), 128.73 (ArC), 129.61 (2ꢃ ArCH), 148.14 (ArC),
151.12 (ArC), 152.87 (ArC), 154.04 (C]O) and 157.40 ppm (C]O).
Anal. Calcd. for C24H31N5O5: C, 61.39; H, 6.65; N, 14.91%. Found: C,
61.29; H, 6.49; N, 15.02%.
1504, 1522, 1400, 1244, 938 and 750 cmꢁ1. 1H NMR (CDCl3):
d 2.22
(p, 2H, eOCH2CH2CH2Cl, J ¼ 6.04 Hz), 3.42 (s, 3H, NeCH3), 3.61 (s,
3H, NeCH3), 3.73 (t, 2H, eCH2Cl, J ¼ 6.32 Hz), 4.08e4.11 (m, 4H, e
OCH2e and eCH2e), 6.86 (d, 2H, 2-CH and 6-CH, aromatic,
Jo ¼ 8.68 Hz), 7.24 (d, 2H, 3-CH and 5-CH, aromatic, Jo ¼ 8.68 Hz) and
11.94 ppm (s, 1H, NeH). 13C NMR (DMSO-d6):
d 27.62 (NeCH3),
29.71 (NeCH3), 31.60 (CH2), 33.46 (CH2), 41.96 (CH2Cl), 64.13 (O-
CH2), 106.15 (ArC), 114.50 (2ꢃ ArCH), 129.04 (ArC), 129.66 (2ꢃ
ArCH), 148.14 (ArC), 151.11 (ArC), 152.82 (ArC), 154.03 (C]O) and
157.14 ppm (C]O). Anal. Calcd. for C17H19N4O3Cl: C, 56.28; H, 5.28;
N, 15.44%. Found: C, 56.18; H, 5.37; N, 15.55%.
5.2. Biological activity
5.2.1. Adenosine binding assays
The adenosine binding assays were performed as reported
earlier [19,20].
5.1.6. General procedure for the synthesis of various amine fused 8-
(substituted-benzyl)xanthine derivatives 32e34
5.2.1.1. Binding studies for A1 and A2A receptors
5.2.1.1.1. Materials. [3H]CCPA
(2-chloro-N6-cyclo-
A mixture of compound 31 (1.0 g, 2.76 mmol) and the desired
amine (in excess) was heated for 1 h at 80e100 ꢀC. The completion
of reaction was monitored with thin layer chromatography. The
excess amine was removed by washings with dry ether to obtain a
solid product, which was collected by filtration, dried and crystal-
lized from methanol to afford the corresponding products 32e34.
pentyladenosine) was from PerkinElmer, Rodgau, Germany; [3H]
NECA (50-N-ethylcarboxamidoadenosine) was obtained from
Amersham, Braunschweig, Germany; [3H]HEMADO (2-(hexyn-1-
yl)-6-methyladenosine) was from Tocris, Bristol, UK and [a-
32P]
ATP was from Hartmann Analytik, Braunschweig, Germany. The 96-
well microplate filtration system (Multiscreen MAFC) was obtained

334
R. Yadav et al. / European Journal of Medicinal Chemistry 75 (2014) 327e335
from Millipore, Eschborn, Germany. Cell culture media and fetal calf
serum were purchased from Pan Systems, Aidenbach, Germany.
Penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine and
G418 were from Gibco-Life Technologies, Eggenstein, Germany.
5.2.1.1.2. Cell culture. The cells were grown adherently and
maintained in Dulbecco’s Modified Eagles Medium with nutrient
mixture F12 (DMEM/F12) without nucleosides, containing 10% fetal
5.2.2.1. Animals. Male guinea-pigs (Dunkin Hartley) of 250 ꢂ 30 g,
bred in the disease free small animal house of Chaudhary Charan
Singh Haryana Agriculture University (Hisar, Haryana) were ob-
tained. The animals were housed under standard laboratory con-
ditions, maintained on a 12 h light and dark cycle and had free
access to food (carrots, cucumbers, leafy vegetables etc) and water.
The experimental protocols were approved by the Institutional
Animal Ethics Committee of the Panjab University, Chandigarh
(CAH/1476 dated 14.09.2009), and conducted according to the In-
dian National Science Academy Guidelines for the use and care of
experimental animals.
calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), L-
glutamine (2 mM) and Geneticin (G418, 0.2 mg/ml; A2B, 0.5 mg/ml)
at 37 ꢀC in 5% CO2/95% air. Cells were split 2 or 3 times weekly at a
ratio between 1:5 and 1:20. For binding assays the culture medium
was removed, cells were washed with PBS and frozen in the dishes
until preparation of membranes. The cells utilized for cAMP de-
terminations had a viability >95%, as assessed by the exclusion of
tryptan blue.
5.2.2.2. Drugs. Histamine hydrochloride (Himedia, India), theoph-
ylline, carboxymethyl cellulose (CMC) and test compounds.
5.2.1.1.3. Membrane preparation. Crude membranes for radio-
ligand binding experiments were prepared by thawing frozen cells
followed by scraping them off the petridishes in ice-cold hypotonic
buffer (5 mM Tris/HCl, 2 mM EDTA, pH 7.4). The cell suspension was
homogenized on ice (Ultra-Turrax, 2 ꢃ 15 s at full speed) and the
homogenate was spun for 10 min (4 ꢀC) at 1000 g. The supernatant
was then centrifuged for 30 min at 100,000 g. The membrane pellet
was resuspended in 50 mM Tris/HCl buffer pH 7.4 (for A3 adenosine
receptors: 50 mM Tris/HCl, 10 mM MgCl2, 1 mM EDTA, pH 8.25),
frozen in liquid nitrogen at a protein concentration of 1e3 mg/ml
and stored at ꢁ80 ꢀC. For the measurement of adenylyl cyclase
activity a slightly modified protocol with only one centrifugation
step was used. Fresh cells were homogenized and the homogenate
was sedimented for 30 min at 54,000 g. The resulting pellet was
resuspended in 50 mM Tris/HCl pH 7.4 and used for the adenylyl
cyclase assay immediately.
5.2.2.3. Experimental protocol. Three groups of animals (4 in each)
were made and designated as I, II and III for control (CMC þ distilled
water), standard (CMC þ theophylline þ distilled water) and test
drug (CMC þ test drug þ distilled water), respectively. The grouped
animals were kept for overnight fasting and were pretreated with
the test drug (50 mg/kg), theophylline (50 mg/kg), and CMC (con-
trol) per oral application 1 h before exposure to aerosol. Each group
of the animals was kept in the histamine chamber (M/s Inco,
Ambala) separately and exposed to histamine aerosol. Five ml of 1%
solution of histamine was aerosoled in 1 min to each animal of each
group. The onset of bronchospasm, duration of jerks, severity of
bronchospasm and death or survival of the animals was recorded
for each group. The animals remained in the chamber for 8 min
after which they were removed to fresh air and fed with proper
water and food.
5.2.1.1.4. Radioligand binding. Dissociation constants of unla-
beled compounds (Ki values) were determined in radioligand
competition experiments performed in a microplate format utiliz-
ing a 96-well microplate filtration system (Millipore Multiscreen
MAFC) [19,20]. For binding experiments at A1 adenosine receptors
[3H]CCPA (1 nM) was used as a radioligand, while [3H]NECA
(10 nM) and [3H]HEMADO (1 nM) were used for A2A and A3
Acknowledgments
The financial support provided by the Indian Council of Medical
Research, New Delhi, India is gratefully acknowledged.
Appendix A. Supplementary data
adenosine receptor binding. Membranes (10e50
mg of protein)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.045.
from CHO cells stably transfected with the respective adenosine
receptor subtype were incubated for 3 h at 25 ꢀC, filtered through
the built in filter at the bottom of the wells of the microplates and
washed three times with 200 ml of ice-cold binding buffer. After
addition of 20 ml of scintillator to the dried filter plates samples
were counted in a Wallac Micro-Beta counter. Nonspecific binding
was determined in the presence of 1 mM theophylline (A1) or
100 mM R-PIA (N6-phenylisopropyladenosine) (A2A, A3). All binding
data were calculated by non-linear curve fitting with the program
SCTFIT.
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