Bronchospasmolytic activity and adenosine receptor binding of some newer 1,3-dipropyl-8-phenyl substituted xanthine derivatives

Article abstract of DOI:10.1111/cbdd.13673

The aldehyde derivatives of 1,3-dipropyl xanthines as described in this paper, constitutes a new series of selective adenosine ligands displaying bronchospasmolytic activity. The effect of substitution at third- and fourth-position of 8-phenyl xanthine ha

Full text of DOI:10.1111/cbdd.13673

DR DIVYA DHAWAL BHANDARI (Orcid ID : 0000-0002-2813-0267)
Article type : Research Article
BRONCHOSPASMOLYTIC ACTIVITY AND ADENOSINE RECEPTOR
BINDING OF SOME NEWER 1,3-DIPROPYL-8-PHENYL SUBSTITUTED
XANTHINE DERIVATIVES
Divya Gumber1, Divya Yadav1, Rakesh Yadav*1, Sonja Kachler2, K.N. Klotz2
1Department of Pharmacy, Banasthali University, Banasthali-304022, Rajasthan, India
2Institut für Pharmakologie und Toxikologie, Universität Würzburg, Germany
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13673
This article is protected by copyright. All rights reserved

*Correspondence:
Dr Rakesh Yadav
Associate Professor
Department of Pharmacy, Banasthali University,
Banasthali-304022, Rajasthan, India.
Email:rakesh_pu@yahoo.co.in
Mob. No.: 9694891228
ABSTRACT
The aldehyde derivatives of 1,3-dipropyl xanthines as described in this paper, constitutes a new series
of selective adenosine ligands displaying bronchospasmolytic activity. The effect of substitution at
3rd- and 4th-position of 8-phenyl xanthine has also been taken into consideration. The synthesized
compounds showed varying binding affinities at different adenosine receptor subtypes (A1, A2A, A2B
and A3) and also good in vivo bronchospasmolytic activity against histamine aerosol induced asthma
in guinea pigs. Most of the compounds showed maximum affinity towards the A2A receptor subtype.
The monosubstituted 3-aminoalkoxyl 8-phenyl xanthine with a diethylmoiety (compound 12e) was
found to be most potent A2Aadenosine receptor ligand (Ki = 0.036 µM) followed by disubstituted 4-
aminoalkoxyl-3-methoxy-8-phenyl xanthine (Ki= 0.050µM) (compound 10a).
KEYWORDS: Xanthines, Adenosine, Radioligand binding, Bronchospasmolytic, Antiasthmatics.
1. Introduction
This article is protected by copyright. All rights reserved

The therapeutic ability of adenosine was first evaluated in 1930’s (Kaiser, 1999). Adenosine is a
universal signaling nucleoside molecule containing an adenine group attached to ribose sugar.
Adenosine and its receptors are used clinically as targets for drug development since 1940’s (Trevor,
2014). There are four subtypes of adenosine receptors designated as A1, A2A, A2B, A3 and all belong to
the GPCRs (G Protein-coupled receptors). All the adenosine receptors play therapeutic roles in
treating various cardiovascular diseases (Fredholm, 2001), nervous and renal system disorders
(Linden, 2005; Ascherio, 2001), pulmonary diseases (Fozard, 2002), inflammatory
disorders(Erdmann, 2005; Antonioli, 2013), endocrine disorders, and visual disorders and also as
targets in treating cancer (Neary, 1998; Gessi, 2011; Sousa, 2018). Adenosine receptors were
categorized as A1 and A2 intially by Brunstock in 1978 depending on their ability to inhibit or
stimulate adenyl cyclase (Polosa, 2002). The A2 receptors were further classified as A2A and A2B
based on their high affinity and low affinity to stimulate adenyl cyclase (Aherne, 2011). The
activation of the A2A AR produces broad-spectrum anti-inflammatory activity in various allergic
models of asthma thus making them useful alternatives to glucocorticosteroids in the treatment of
asthma (Fozard, 2002). The elucidation of a A2B low-affinity subtype of the A2 adenosine receptorsled
to the possibility that there might be a physiological receptor to counter balance the high-affinity A2A
AR models but lack of pharmacologically specific targets reduced their physiological relevance
(Aherne, 2011).
The high affinity adenosine A2A receptors are expressed on mast cells and A2A agonists increase
intracellular levels of cAMP which in turn inhibits the release of histamine and other inflammatory
stimuli from mast cells thus proven effective in asthmatic patients (Spicuzza, 2003; Baraldi, 2008).
The low affinity A2B receptor antagonists are under investigation based on their inhibiting action of
adenosine on mast cells in addition to pro-inflammatory effects (Brown, 2008).
Theophylline is a xanthine derivative that shows a low affinity (in the micromolar range) for
adenosine receptor, A2B as an antagonist and does not discriminate between the A1 and A2 receptor
subtypes while at relatively high concentrations, it inhibits proliferation of human peripheral blood
mononuclear cells showing anti-proliferative action. (Landells, 2000). The binding affinity of
theophylline is more towards A2A receptors in low concentrations as compared to A1 and A2B (Yasui,
2000) and some of the Antiasthmatic effect observed with theophylline is also due to A2B receptor
This article is protected by copyright. All rights reserved

antagonism. The anti-proliferative effect of theophylline is seen only at higher concentrations then
required to antagonize A2B receptors while A2A agonist action can be seen in micromolar range.
(Landells, 2000).
The affinity for adenosine receptors is increased when various substitutions in the xanthine moiety are
introduced. Most of the A2A agonists are directly related to adenosine itself so various synthetic
modifications were done to the naturally occurring xanthines which are structural analogs of
adenosine, to produce selective A2A receptor ligands (Müller, 2011).
The various substituted xanthines retain almost 10,000 times higher affinity towards adenosine
receptors as compared to theophylline and caffeine which are present naturally. In spite of broader
pharmaceutical applications and similarity to purine family, xanthine-based research has not taken up
full measure thus limiting synthesis of xanthine-based molecules (Monteiro, 2016). The most
noticeable reason for this is adverse synthetic procedures such as ring closure and condensation
method for the production of new products (Allwood, 2007; Hayallah, 2002; Sakai, 1992; Kim, 2010;
Bandyopadhyay, 2012; Chen, 2014; Lee, 2016). In terms of xanthine-based drug development, large
scale synthesis is not supported thus there is crucial need for the development of feasible synthetic
methods so that new xanthine-based drug candidates can be introduced. Xanthine provides maximum
possible sites of substitutions. There are five sites for mono substitution [1-, 3-, 7-, 8- and 9-], for di-
substitutions there are eight sites [1,3-, 1,7-, 1,8-, 1,9-, 3,7-, 3,8-, 3,9- and 7, 8-] and there are three
sites for tri- substitutions [1,3,7-,1,3,8-, 1,3,9-] (Miyamoto, 1993; Muller, 1998; Bansal, 2010). The
structure of xanthine gives extreme possibility for variation in derivatization because of the presence
of N1, N3, N7 and C8 positions for substitution. It was also observed that the increase in the length of
carbon chain in 1- and 3-position from methyl to propyl increases the affinity of xanthine derivatives
towards adenosine receptors.
Theophylline and other substituted xanthine derivatives possess inhibitory effect at adenosine
receptors especially A1 and A2 where A1 is inhibitory and A2 is stimulatory to adenylate cyclase
(Persson, 1981). Enprofylline (A), a 3-propyl xanthine proved more potent than theophylline in
regulating adenosine induced contraction of airways (Belardinelli, 1981).
This article is protected by copyright. All rights reserved

O
N
H
N
NH
N
O
C3H7
A
( )
Considering all the literature reports into account, 1,3-dipropyl substituted xanthine derivatives were
synthesized by substituting the 3rd- and 4th-position of the 8-phenyl ring introduced into the xanthine
scaffold. Studies indicated that suitable substitution and position of substituents results in high affinity
compounds (Bansal, 2009). The para and meta shifting of substituent play an important role towards
binding affinity at adenosine receptors. The synthesized compounds were evaluated biologically using
in vitro radioligand binding and in vivo histamine aerosol induced asthma in guinea pigs (Zabeer,
2006).
2. Results and discussion
2.1 Chemistry
The key intermediate 5,6-Diamino-1,3-dipropyluracil (3) was synthesized by the condensation of
N,N’-dipropyluracil (1) and cyanoacetic acid (2)followed by nitrosation and reduction as depicted in
Scheme 1 (Papesch, 1951; Blicke, 1954). The synthesis of substituted aldehydes 4-6 (a-f), Schiff
bases 7-9 (a-f) and substituted xannthines10-12 (a-f) is depicted in Scheme 2. The starting aldehydes
vanillin (4), isovanillin (5), and 3-hydroxybenzaldehyde (6) possesses a hydroxy group at para and
meta positions where different amines (morpholine, piperidine, dimethylamine, pyridine,
diethylamine and phthalimide derivatives of 2-chloroethyl aminohydrochlorides) were substituted to
obtain substituted aldehydes4-6(a-f).
This article is protected by copyright. All rights reserved

O
N
O
CNCH2COOH
NaOH
N
N
N
H
H
O
NH2
N, N'-Dipropylurea
(1)
6-Amino-1,3-dipropyluracil
NaNO2
CH3COOH
O
O
NH2
NO
N
N
Na2S2O4
NH4OH
O
N
NH2
O
N
NH2
(3)
(2)
5-Nitroso-6-amino-1,3-dipropyluracil
5, 6-Diamino-1,3-dipropyluracil
Scheme 1
These aldehydes 4-6 (a-f) were condensed with key intermediate dipropyluracil (3) at reflux in the
presence of ethanol and acetic acid to synthesize Schiff bases 7-9 (a-f) which in turn were cyclized in
refluxing thionyl chloride to synthesize resultant xanthine derivatives 10-12 (a-f) as depicted in
Scheme 2. The structures of the synthesized compounds were characterized by various spectral and
elemental analyses. A singlet for one proton at ~11.3 ppm for N=CH was observed in all theSchiff
bases 7-9 (a-f) while disappearance of this peak on treatment with thionyl chloride confirmed
cyclization of xanthine derivatives 10-12(a-f).
This article is protected by copyright. All rights reserved

R
O
N
O
N
R
NH2
NH2
N
O
EtOH/AcOH
Reflux
N
N
R1
+
R1
O
O
NH2
(3)
4-6 (a-f)
7-9 (a-f)
SOCl2
R
R1
O
R
O-CH2-CH2-R2
O-CH3
O-CH3
4,7,10
5,8,11
N
N
R1
O-CH2-CH2-R2
O-CH2-CH2-R2
N
H
O
N
H
6,9,12
10-12 (a-f)
(c)
(f)
(a)
(d)
(b)
N
N
N
O
N
R2=
O
N
(e)
N
O
Scheme 2:
Synthetic route for the synthesis of various 8-phenyl substituted xanthines,
10 a-f; 11a-f and 12 a-f.
2.2 Radioligand binding assays
Adenosine receptor binding affinity of various xanthine derivatives at adenosine receptors (A1,
A2A, A2B and A3) was also determined and summarized in Table 1. The substituted xanthines 10-
12 (a-f) showed potent binding affinities towards various adenosine receptor subtypes whereas
they showed high selectivity towards A2A subtype.Monosubstituted8-phenyl xanthines 12b, 12d
and 12e with a polar side chain at meta position of the 8-phenyl ring showed higher affinity and
selectivity towards A2A receptor subtypes followed by10a-f and 11 a-f derivatives with an
additional methoxy group at ortho position to polar side chain.. Among all the series the
monosubstituted derivatives, 8-[3-{2-(phthalimido)-ethoxy}-phenyl]-1,3-dipropylxanthine, 12e
This article is protected by copyright. All rights reserved

with Ki=0.036 µm found as the compound with the highest affinity of the series. On comparing
disubstituted derivatives, the derivatives with polar side chain at para position of 3,4-disubstituted
8-phenyl xanthines (compounds 10a) was found to have higher affinity than the derivatives with
polar side chain at meta position (compounds11f). Thus, the newly synthesized derivatives
displayed higher affinity and selectivity towards A2A receptor subtypes then the previously
reported xanthine derivatives.
Table 1: Binding Affinities of Various Xanthine derivatives at adenosine receptors (A1, A2A, A2B and A3)
S.
No.
1
Comp.
(Code)
A1
A2A
A2B
(Ki µm)
> 100
A3
(Ki µm)
(Ki µm)
(Ki µm)
10a (RY-67)
10b (RY-69)
0.248 (0.209-0.296)
1.50 (1.39-1.63)
0.050 (0.037-0.068)
0.622 (0.465-0.833)
0.593 (0.503-0.698)
1.12 (0.941-1.34)
2
> 100
3
4
10c (RY-71)
10d (RY-73)
10e (RY-75)
10f (RY-77)
11a (RY-79)
11b (RY-81)
11c (RY-83)
11d (RY-85)
11e (RY-87)
11f (RY-89)
1.20 (1.01-1.42)
1.19 (0.933-1.51)
0.813 (0.620-1.07)
2.05 (1.76-2.40)
0.481 (0.338-0.685)
0.364 (0.323-0.409)
0.222 (0.127-0.390)
0.543 (0.293-1.04)
0.268 (0.156-0.461)
0.818 (0.551-1.210)
1.71 (1.55-1.89)
> 100
> 100
1.20 (0.882-1.62)
0.861 (0.584-1.27)
0.622 (0.484-0.799)
4.62 (3.71-5.74)
5
> 100
6
> 100
7
0.757 (0.720-0.796)
0.896 (0.700-1.15)
2.96 (2.64-3.33)
> 100
1.05 (0.713-1.54)
1.780 (1.250-2.530)
2.47 (1.81-3.36)
8
> 100
9
> 100
10
11
12
0.891 (0.662-1.20)
0.749 (0.436-1.29)
0.189 (0.168-0.214)
2.59 (2.16-3.10)
0.655 (0.578-0.743)
0.870 (0.832-0.909)
0.055 (0.046-0.065)
1.27 (1.18-1.37)
> 100
1.33 (1.01-1.77)
> 100
1.39 (1.05-1.83)
> 100
0.255 (0.158-0.412)
2.43 (2.03-2.92)
13 12a (RY-103)
14 12b (RY-105)
15 12c (RY-107)
16 12d (RY-109)
17 12e (RY-111)
18 12f (RY-113)
> 10
0.110 (0.062-0.195)
3.26 (2.26-4.69)
0.054 (0.049-0.058)
2.12 (1.58-2.86)
1.26 (1.13-1.40)
> 10
0.639 (0.411-0.993)
2.86 (2.02-4.06)
0.196 (0.116-0.334)
0.107 (0.080-0.143)
0.119 (0.101-0.141)
0.116 (0.096-0.141)
0.036 (0.032-0.041)
0.058 (0.044-0.077)
3.430 (2.730-4.320)
1.88 (1.33-2.67)
2.86 (1.87-4.39)
2.020 (1.89-2.16)
0.542 (0.408-0.721)
0.088 (0.080-0.098)
Shown values are geometric means from 3 to 5 experiments in µM with 95% confidence intervals in parentheses, where:
This article is protected by copyright. All rights reserved

a. Displacement of specific (3H)CCPA binding in CHO cells, stably transfected with human recombinant A1 adenosine receptor,
expressed as Ki (µM).
b. Displacement of specific (3H)NECA binding in CHO cells, stably transfected with human recombinant A2A adenosine
receptor, expressed as Ki (µM).
c. Antagonist affinities were determined by inhibition of NECA-stimulated adenylyl cyclase activity in membrane preparations.
d. Displacement of specific (3H)HEMADO binding in CHO cells, stably transfected with human recombinant A3 adenosine
receptor, expressed as Ki (µM).
2.3 Bronchospasmolytic activity
The newly synthesized 1,3-dipropyl-8-substituted xanthines 10a, 10b, 10c, 10f, 11a, 11b and 12a-
fwere evaluated against histamine aerosol-induced asthma in guinea pigs. The compounds showed a
significant protection profile against the standard drug theophylline as evident from increased time for
the onset of bronchospasm as compared to standard drug. In the vanillin substituted series, among10a,
10b, 10c and 10f, derivative 10f with a phthalimide substitution was found to be most effective as it
took more time for onset of spasm as compared to standard drug with 100% survival rate. The
isovanillin substituted series i.e. 11a and 11bshowed lower values for onset of spasm as compared to
standard but with 100% survival rate. The 3-hydroxybenzaldehyde substituted xanthine derivatives
series, 12 a-f proved to be most effective among all as their onset of spasm time is much higher in
comparison to standard drug. Among this series, derivatives 12e and 12f with a diethyl and
phthalimide substitution showed maximum time for the onset of bronchospasm followed by no jerks
and 100% survival rate, thus, considered as most effective in vivo (Table 2).
All the newly synthesized derivatives exhibited prominent binding affinity towards A2A receptors and
also exhibited prominent bronchospasmolytic activity. The compound 12e from the 3-
monosubstituted series found as most potent towards A2A receptor ligand and also took maximum
time for the onset of bronchospasm followed by 12f compound of this series. Among the other two
series, compound 10f of vanillin series exhibited higher activity then the standard drug.
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
Mean time in seconds for
onset of bronchospasm
Mean ± S.E.M
Duration of jerks in
seconds
Compound
No.
Severity of
Survival
(%)
Bronchospasm
Mean ± S.E.M
This article is protected by copyright. All rights reserved

Mean time in seconds for
onset of bronchospasm
Mean ± S.E.M
51.8 ± 3.49
Duration of jerks in
seconds
Compound
No.
Severity of
Survival
(%)
Bronchospasm
Mean ± S.E.M
183.4 ± 10.59
94.8 ± 6.72*#
32.6 ± 4.11*#
42.8 ± 4.02*#
56.2 ± 7.57#
36.2 ± 3.56*#
39.6 ± 4.19*#
50.2 ± 1.93*
52.6 ± 7.58*#
58.8 ± 8.28*
60 ± 4.45*
Control
+++
+
0
Theophylline
10a(RY-067)
10b(RY-069)
10c(RY-071)
10f(RY-077)
11a(RY-079)
11b(RY-081)
12a(RY-103)
12b(RY-105)
12c(RY-107)
12d(RY-109)
12e(RY-111)
12f(RY-113)
105.6 ± 7.73*
98.26 ± 9,54*
86.33 ± 5.43*
97.87 ± 3,11*
115.34 ± 2.16*
101.18 ± 6.23*
50.19 ± 8.19*#
125.65 ± 5.76*
120.13 ± 3.53*
172.19 ± 4.31*#
126.21 ± 4.37*
480 ± 0*#
100
100
100
100
100
100
100
100
100
100
100
100
100
++
++
+++
+
+
+++
++
+++
++
++
-
36.6 ± 6.57*#
-
480 ± 0*#
-
-
Number of animals in each group (N) = 5
(-) means= not observed
*Newman-Keuls Multiple Comparison Test; p<0.05 as compare to normal control
#Newman-Keuls Multiple Comparison Test; p<0.05 as compare to theophylline
3. Conclusion
The newly synthesized 1,3-dipropyl-8-substituted xanthines showed prominent binding affinity for
adenosine receptors subtypes except the A2B subtype with maximum affinity towards A2A receptors.
Thus, a conclusion can be drawn regarding A2A receptor agonist action and A2B receptor antagonist
action of xanthines as synthesized derivatives showed maximum binding affinity at A2A and minimum
at A2B. The suitable substitution on para and meta position of 8-phenyl ring resulted in high affinity
compounds with potent bronchospasmolytic activity. The monosubstituted xanthine derivative 12e
found as highly potent bronchospasmolytic agent along with highest affinity towards A2A receptors
followed by para-substituted derivative 10a and then meta-substituted 11a. Thus, the number of
substitutions and their position plays an important role in defining the chemistry of xanthines as
This article is protected by copyright. All rights reserved

bronchospasmolytic agents and also provides specific binding affinities towards various adenosine
receptors.
4. Experimental section
4.1 Chemistry
All melting points were obtained using glass capillary tubes on Veego melting point
apparatus and are uncorrected. The purity of the compounds was confirmed by thin layer
chromatography using precoated plates with silica gel G (Merck 60 F254) and the spots were visualized
in iodine chamber. Infrared (IR) spectra were recorded on Agilent Technology cary 600 series
Fourier Transform-Infrared spectrophotometer using potassium bromide pellets (max in
1
cm-1). 13C and H-NMR spectroscopy were performed using a Bruker model 400 MHz
spectrometer in deuterated dimethylsulfoxide (DMSO-d6) and are reported in parts per
million (ppm) downfield from tetramethylsilane (Me4Si) as internal standard. The spin
multiplicities are indicated as symbols, s (singlet), d (doublet), t (triplet), q (quartet), p
(pentet), m (multiplet) and the coupling constants (J) are given in Hertz (Hz). Anhydrous
sodium sulfate was used as drying agent and all the solvents were freshly distilled off and
dried prior to use according to the standard procedures. Elemental Analysis was carried out
on Perkin Elmer series II-2400 analyzer. The results so observed were within the limits.
4.1.1 General method for the synthesis of compound 3
A mixture of N, N’-dipropylurea (1.0 g, 6.94 mmol), cyanoacetic acid (1.5 g, 17.64 mmol) and acetic
anhydride (2.5 ml) was refluxed at 70-80 °C (3 h) excluding moisture. The excess anhydride and
acetic acid formed during the reaction were removed under vacuum. The residue was then cooled (0-
5°C) and a solution of 5 % sodium hydroxide (40 ml) was added with stirring resulting in
precipitation of 6-amino-1, 3-dipropyluracil. A sodium nitrite solution (1.0 g, 14.49 mmol in 8 ml of
water) was added to cool, stirred mixture and acidified with acetic acid dropwise (2 ml) over a period
of one hour resulting in red-violet precipitates. The mixture was then kept on stirring at room
temperature for a period of overnight. The mixture so obtained was then cooled, filtered off, washing
This article is protected by copyright. All rights reserved

was done with water and diethyl ether followed by drying to get 6-amino-1, 3-dipropyl-5-nitrosouracil
2 (1.33 g, 63.63 %), mp 215-220 °C.
The sodium dithionite (2.74 g, 15.73 mmol) was then added slowly with stirring. The salt dissolved
and underwent a series of color changes. The solution was kept on stirring for further two hours at
room temperature followed by cooling in an ice bath. The precipitates so obtained were filtered off,
washing was done with few drops of cool water followed by drying to get 5, 6-diamino-1, 3-
dipropyluracil (3, 0.76 g, 80.85 %), mp 128-132 °C.
4.1.2. General method for the synthesis of substituted aldehydes 4-6 (a-f)
Various amines such as 4-(2-chloroethyl)-amino hydrochlorides (1 g), [4-(2-chloroethyl)-morpholine
hydrochloride (a, 5.36 mmol), 4-(2-chloroethyl)-piperidine hydrochloride (b, 5.42 mmol), 2-chloro-
N,N’-dimethyl ethanamine (c, 6.93 mmol), 1-(2-chloroethyl)-pyrrolidine (d, 5.88 mmol), 2-chloro-
N,N’-diethyl ethanamine (e, 5.82 mmol) and 1-(2-chloroethyl)-phthalimide (f, 4.77 mmol)] were
added to the refluxing suspension of 4-Hydroxy-3-methoxy-benzaldehyde (4, 1 g, 6.58 mmol), 3-
Hydroxy-4-methoxy-benzaldehyde (5, 1 g, 6.56 mmol) and 3-Hydroxybenzaldehyde (6, 1g, 6.80
mmol) in ethyl methyl ketone (20 ml). Dried potassium carbonate (3.0 g) was added to avoid
incorporation of any moisture. After completion, suspension was filtered and reduced on rotary
evaporator to afford the targeted compounds 4-6 (a-f).
4.1.3. General procedure for the synthesis of compounds 7-9 (a-f)
To the stirred solution of 4-6 (a-f) in ethanol (5 ml), added 5, 6-diamino-1,3-dipropyluracil (3, 1 g,
4.42 mmol) with ethanol and acetic acid (8:2, 10 ml) and refluxed. After completion, excess amount
of the solvent was evaporated at rotary evaporator. Added ice cold water to the residue and filtered to
afford 7-9 (a-f).
4.1.4 General procedure for the synthesis of compounds 10-12 (a-f)
The Schiff bases 7-9 (a-f) (1 g) were refluxed in thionyl chloride (15 ml) for 1 h at 70-80°C. Excess
solvent was reduced at rotary evaporator and residue so obtained was neutralized to result in
precipitation. The reaction mixture was cooled, filtered off and washed with cold water to obtain
desired derivatives 10-12 (a-f).
This article is protected by copyright. All rights reserved

4.1.4.1. 8-[4-(2-Morpholinoethoxy]-3-methoxyphenyl)-1,3-dipropyl xanthine (RY-067, 10a)1.46 g,
69.56 %, mp 234-238 °C; ¹H-NMR (400 MHz, CDCl3): δ 11.06 (s, 1H, -NH), 7.75 (s, 1H, Ar-H),
7.26 (s, 1H, Ar-H), 6.96 (s, 1H, Ar-H), 4.31 (s, 3H, -OCH3), 3.95 (s, 4H, 2 x -OCH2-), 3.75 (s, 2H, -
OCH2-), 2.89 (s, 4H, 2 x -NCH2-), 2.63 (s, 6H, 3 x -NCH2-),1.47 (s, 4H, 2 x -CH2-) and 0.99 ppm (s,
6H,2 x -CH3); 13C-NMR (100 MHz, CDCl3): δ 10.08-11.10 (2 CH3), 20.01 (2 CH2), 44.72 (2 N-CH2),
53.37-54.01 (3 N-CH2), 56.20-62.01 (3 O-CH2), 65.57 (O-CH3), 112.51 (Ar-CH), 114.41-115.56 (2
Ar-C), 119.54 (2 Ar-CH), 123.47 (Ar-C), 146.98-151.08 (3 Ar-C), 149.94 (C=O) and 152.61 ppm
(C=O); FT-IR νmax(KBr)cm-1: 3410, 3202, 2959, 1691, 1647, 1556, 1478, 1271, 1168; Anal. Cal. for
C24H33N5O5: C, 61.13%; H, 7.06%; N, 14.85% Found: C,63.15%; H, 6.37%; N, 16.38%.
4.1.4.2 8-[4-(2-Piperidinoethoxy]-3-methoxyphenyl)-1,3-dipropyl-xanthine (RY-069, 10b) 0.97 g, 99
%, mp 137-141 °C; ¹H-NMR (400 MHz, CDCl3): δ 7.66 (s, 1H, Ar-H), 7.22 (s, 1H, Ar-H), 6.93 (s,
1H, Ar-H), 4.07 (s, 1H, -NH), 3.90 (s, 4H, 2 x -NCH2-), 3.73 (s, 6H, 3 x -NCH2-), 3.01 (s, 2H, -OCH2-
), 2.75 (s, 3H, -OCH3), 1.68 (s, 10H, 5 x -CH2-) and 1.18 ppm (s, 6H,2 x -CH3); 13C-NMR (100MHz,
CDCl3):δ10.08-11.10 (2 CH3), 20.01 (2 CH2), 24.73 (3 CH2), 43.72 (2 N-CH2), 54.01 (3 N-CH2),
56.07 (O-CH2), 67.57 (O-CH3), 112.51 (Ar-CH), 115.41 (Ar-C), 119.54-120.02 (2 Ar-CH), 123.47
(Ar-C), 146.98 (Ar-C), 150.08 (Ar-C), 151.08 (ArC), 151.94 (C=O), 153.61 (C=O) and 154.08 ppm
(ArC); FT-IRνmax(KBr) cm-1:3565, 2920, 1698, 1650, 1516, 1460, 1258, 1209; Anal. Cal. for
C25H35N5O4:C,63.94%; H,7.51%; N,14.91%, Found: C,64.18%; H, 6.28%;N, 15.34%.
4.1.4.3.8-[4-{2-(Dimethylamino)-ethoxy}-3-methoxyphenyl]-1,3-dipropyl-xanthine (RY-071,10c) 0.71
g, 71.42 %, mp 169-171 °C; ¹H-NMR (400MHz, CDCl3):δ 7.69 (s, 1H,-NH), 6.94 (s, 3H, Ar-H), 3.91
(s, 6H, -3 x -NCH2-), 3.00 (s, 6H, 2 x -NCH3-), 2.49 (s, 3H, -OCH3), 2.46 (s, 2H, -OCH2-), 1.62 (s,
13
4H, 2 x -CH2-) and 0.85 ppm (s, 6H, 2 x -CH3); C-NMR (100 MHz, CDCl3):δ 10.08 (CH3), 11.10
(CH3), 21.07(CH2), 23.01 (CH2), 43.72 (N-CH2), 45.05 (N-CH2), 47.09 (2 N-CH3), 54.01 (N-CH2),
56.07 (O-CH2), 66.57 (O-CH3), 107.51-153.85 (Ar-C), 154.94 (C=O) and 157.61 ppm (C=O); FT-IR
νmax(KBr) cm-1:3565, 2920, 1699, 1650, 1517, 1460, 1259, 1179; Anal. Cal. for C22H31N5O4:C,
61.52%; H, 7.27%; N, 16.31%; O, 14.90%; Found: C, 61.54%; H, 6.75%; N, 16.65%.
4.1.4.4. 8-[4-{2-(Pyrrolidin-1-yl)-ethoxy}-3-methoxyphenyl]-1,3-dipropyl-xanthine(RY-073, 10d)0.95
g, 99 %, mp 139-141 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 7.70 (s, 1H, Ar-H), 6.94 (s, 2H,
Ar-H), 4.13 (s, 1H, -NH), 3.70 (s, 10H, 5 x -NCH2-), 3.38 (s, 2H, -OCH2), 3.14 (s, 3H, -OCH3-), 1.67
This article is protected by copyright. All rights reserved

(s, 8H, 4 x -CH2-) and 0.96 ppm (s, 6H, 2x-CH3); 13C-NMR (100MHz, CDCl3):δ 11.01-12.08 (2 CH3),
21.06 (2 CH2), 23.97 (2 CH2), 44.78-55.67 (5 N-CH2), 56.62 (O-CH3), 62.06 (O-CH2), 107.86-
154.45 (Ar-C) and 157.98 ppm (C=O); FT-IR νmax(KBr) cm-1:3565, 2918, 1698, 1649, 1516, 1460,
1259, 1038; Anal. Cal. for C24H33N5O4:C, 63.29%; H,7.31%; N,15.38%; O,14.05%;Found:C,
64.16%; H, 7.50%; N, 14.32%.
4.1.4.5. 8-[4-{2-(Diethylamino)-ethoxy}-3-methoxyphenyl]-1,3-dipropyl xanthine (RY-075, 10e)0.90
g, 90.45 %, mp 117-120 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 8.04 (s, 1H, -NH), 7.31 (s, 3H,
Ar-H), 4.34 (s, 4H, -NCH2-), 4.06 (t, 2H, -NCH2-), 3.92 (t, 2H, -OCH2), 3.88 (s, 3H, -OCH3-), 3.47
(s, 2H, -CH2-), 1.82-1.77 (q, 2H, -NCH2-), 1.67-1.61 (q, 2H, -NCH2-) and 0.97-0.91 ppm (q, 10H, 2 x
13
-CH2-, 2 x -CH3); C-NMR (100 MHz, CDCl3):δ 10.91 (CH3), 11.04 (CH3), 14.04 (2 CH3), 20.80
(CH2), 20.85 (CH2), 42.13 (2 N-CH2), 44.41-54.08 (3 N-CH2), 47.40 (O-CH2), 55.80 (O-CH3),
113.81-151.74 (C=O) and 154.22 ppm (C=O); FTIR νmax(KBr) cm-1:3565, 2920, 1698, 1649, 1516,
1460, 1259, 1021; Anal. Cal. for C24H35N5O4:C, 63.00%; H, 7.71%; N, 15.31%; O, 13.99%;Found:C,
64.52%; H, 6.67%; N, 13.32%.
4.1.4.6. 8-[4-{2-(Phthalimido)-ethoxy}-3-methoxyphenyl]-1,3-dipropyl-xanthine (RY-077, 10f)0.60 g,
1
60.24 %, mp 86-90 °C; H-NMR (400 MHz, CDCl3):δ 7.79 (s, 4H, Ar-H), 7.67 (s, 3H, Ar-H, 1H, -
NH), 3.97 (s, 6H, 3 x -NCH2-), 3.70 (s, 5H, -OCH2-, -OCH3), 1.62 (s, 4H, 2 x -CH2-) and 0.92 ppm (s,
6H, 2x-CH3); 13C-NMR (100MHz, CDCl3):δ 10.08 (CH3), 11.01 (CH3), 42.21 (CH2), 39.45 (N-CH2),
40.85 (N-CH2), 56.62 (O-CH3), 66.45 (O-CH2), 107.07-156.62 (Ar-C), 158.04 (C=O) and 167.99
ppm (C=O); FT-IR νmax(KBr) cm-1:3028, 2961, 1768, 1704, 1648, 1542, 1462, 1260, 1088; Anal. Cal.
for C28H29N5O6:C, 63.28%; H, 5.51%; N, 13.19%; O, 18.06%;Found:C, 64.46%; H, 6.03%; N,
14.51%.
4.1.4.7. 8-[3-(2-Morpholinoethoxy)-4-methoxyphenyl]-1,3-dipropyl-xanthine (RY-079, 11a)0.99 g, 99
%, mp192-195 °C (decomp.); ¹H-NMR (400 MHz, CDCl3): δ 11.43 (s, 1H, -NH), 7.59 (s, 1H, Ar-H),
6.84 (s, 2H, Ar-H), 4.28 (s, 3H, -OCH3), 4.04 (s, 6H, 3 x -OCH2-), 3.81 (s, 4H, 2 x -NCH2-), 3.03 (m,
13
6H, 3 x -NCH2-),1.76 (s, 2H, -CH2-), 1.60 (s, 2H, -CH2-) and 0.89 ppm (s, 6H, 2 x CH3); C-NMR
(100MHz, CDCl3):δ 10.08-11.10 (2 CH3), 20.01 (2 CH2), 44.72 (2 N-CH2), 53.37-54.01 (3 N-CH2),
56.20-62.01 (3 O-CH2), 65.57 (O-CH3), 112.51 (Ar-CH), 114.41-115.56 (2 Ar-C), 119.54 (2 Ar-CH),
123.47 (Ar-C), 146.98-151.08 (3 Ar-C), 149.94 (C=O) and 152.61 ppm (C=O); FT-IR νmax(KBr)cm-
This article is protected by copyright. All rights reserved

1: 3564, 3179, 2961, 1699, 1654, 1543, 1488, 1262, 1210; Anal. Cal. for C24H33N5O5: C, 61.13%; H,
7.05%; N, 14.85%; O, 16.96%; Found: C, 32.95%; H, 4.58%; N, 7.66%.
4.1.4.8. 8-[3-(2-Piperidinoethoxy)-4-methoxyphenyl]-1,3-dipropyl xanthine (RY-081; 11b) 0.99 g,
99.2 %, mp 199-202 °C (decomp); ¹H-NMR (400 MHz, CDCl3):δ 7.64 (s, 1H, Ar-H), 7.19 (s, 1H, Ar-
H), 6.83 (s, 1H, Ar-H), 4.20 (s, 1H, -NH), 4.07 (s, 4H, 2 x -NCH2-), 3.83 (s, 6H, 3 x -NCH2-), 2.83 (s,
13
2H, -OCH2-), 2.54 (s, 3H, -OCH3), 1.58 (s, 10H, 5 x -CH2-) and 0.88 ppm (s, 6H, 2 x -CH3); C-
NMR (100 MHz, CDCl3):δ 11.25 (2 CH3), 21.30 (2 CH2), 23.97 (3 CH2), 43.16 (2 N-CH2), 45.20-
55.03 (3 N-CH2), 56.31 (O-CH3), 66.80 (O-CH2), 107.86 - 149.99 (Ar-C),150.90 (C=O), and 157.98
ppm (C=O); FT-IRνmax(KBr)cm-1:3447, 3123, 2959, 1697, 1652, 1541, 1486, 1260, 1210; Anal. Cal.
for C25H35N5O4:C, 63.95%; H, 7.52%; N 14.81%; O 13.63% Found: C, 64.42%; H, 8.11%; N,
13.76%.
4.1.4.9. 8-[3-{2-(Dimethylamino)-ethoxy}-4-methoxyphenyl]-1, 3-dipropyl xanthine (RY-083;
11c)0.40 g, 40.81 %, mp 205-208 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 6.94 (s, 3H, Ar-H),
3.98 (m, 13H, -NH, 3 x -NCH2-, 2 x -NCH3), 1.81 (m, 3H, -OCH3), 1.67 (m, 2H, -OCH2-) and 0.96
ppm (m, 10H, 2 x -CH2, 2x-CH3); 13C-NMR (100MHz, CDCl3):δ 10.08 (CH3), 11.10 (CH3),
21.07(CH2), 23.01 (CH2), 43.72 (N-CH2), 45.05 (N-CH2), 47.09 (2 N-CH3), 54.01 (N-CH2), 56.07
(O-CH2), 66.57 (O-CH3), 107.51-153.85 (Ar-C), 154.94 (C=O) and 157.61 ppm (C=O); FT-
IRνmax(KBr)cm-1:3421, 3199, 2964, 1663, 1650, 1542, 1462, 1259, 1179; Anal.Cal. for C22H31N5O4:
C, 61.52%; H, 7.27%; N, 16.31%; O, 14.90%; Found: C, 61.54%; H, 6.75%; N, 16.65%.
4.1.4.10. 8-[3-{2-(Pyrrolidin-1-yl)-ethoxy}-4-methoxyphenyl]-1, 3-dipropyl xanthine (RY-085;
11d)0.84 g, 84.03 %, mp 145-148 °C (decomp.); ¹H-NMR (400MHz, CDCl3):δ 7.62 (s, 1H, Ar-H),
6.84 (s, 2H, Ar-H), 4.36 (s, 1H, -NH), 3.88 (s, 4H, 2 x -NCH2-), 3.82 (s, 6H, 3 x -NCH2-), 3.32 (s, 2H,
-OCH2-), 3.17 (s, 3H, -OCH3), 1.76 (s, 4H, 2 x -CH2-), 1.60 (s, 4H, 2 x -CH2-) and 0.88 ppm (s, 6H,
13
2xCH3); C-NMR (100MHz, CDCl3):δ 11.01-12.08 (CH3), 21.06 (CH2), 23.97 (CH2), 44.78-55.67
(N-CH2), 56.62 (O-CH3), 62.06 (O-CH2), 107.86 -154.45 (Ar-C) and 157.98 ppm (C=O);FT-IR
νmax(KBr) cm-1:3744, 2961, 1698, 1655, 1552, 1519, 1258, 1181; Anal. Cal. for C24H33N5O4:C,
63.29%; H, 7.31%; N, 15.38%; O, 14.05%, Found: C, 64.02%; H, 6.15%; N, 14.67%.
This article is protected by copyright. All rights reserved

4.1.4.11. 8-(3-(2-(Diethylamino)-ethoxy)-4-methoxyphenyl)-1,3-dipropyl xanthine (RY-087, 11e) 0.99
g, 99 %, mp 185-188 °C (decomp.);¹H-NMR (400 MHz, CDCl3):δ 11.26 (s, 1H, -NH), 6.87 (s, 3H,
Ar-H), 3.87 (m, 10H, 5 x -NCH2-), 1.70 (s, 2H, -OCH2-), 1.48 (s, 3H, -OCH3), 1.09 (s, 4H, 2 x -CH2-)
and 0.88 ppm (s, 12H, 4x-CH3); 13C-NMR (100MHz, CDCl3):δ 10.91 (CH3), 11.04 (CH3), 14.04
(CH3), 20.80 (CH2), 20.85 (CH2), 42.13 (N-CH2), 44.41-54.08 (3 N-CH2), 47.40 (O-CH2), 55.80 (O-
CH3), 113.81-151.55 (Ar-C), 151.74 (C=O) and 154.22 ppm (C=O); FT-IR νmax(KBr)cm-1:3615,
2959, 1699, 1651, 1547, 1463, 1257, 1175;Anal. Cal. for C24H35N5O4: C, 63.00%; H, 7.71%; N,
15.31%; O, 13.99%, Found: C, 64.13%; H, 7.20%; N, 16.93%.
4.1.4.12. 8-[3-{2-(Phthalimido)-ethoxy}-4-methoxyphenyl]-1, 3-dipropyl xanthine (RY-089; 11f)0.80
g, 80.54 %, mp 266-270 °C (decomp.); ¹H-NMR (400MHz, CDCl3):δ 13.25 (s, 1H, -NH), 8.99 (s, 1H,
Ar-H), 7.92 (s, 1H, Ar-H), 7.55 (d, 2H, Ar-H, Jortho= 7.48 Hz), 6.88 (d, 2H, Ar-H, Jortho= 8.32 Hz),
4.01 (t, 2H, -NCH2-,J1=7.00, J2=6.92), 3.87 (s, 2H, -NCH2-), 3.85 (s, 2H, -NCH2-), 3.82 (s, 3H, -
OCH3), 1.74 (m, 2H, -OCH2-),1.58 (m, 2H, -CH2-) and 0.88 ppm (m, 8H, -CH2-, 2 x -CH3);13C-NMR
(100 MHz, CDCl3):δ 10.92 (CH3), 11.03 (CH3), 42.98 (2 CH2), 39.45 (N-CH2), 44.33 (2 N-CH2),
55.45 (O-CH3), 64.37 (O-CH2), 107.07-119.91 (3 Ar-CH), 114.41 (Ar-C), 123.55 (Ar-C), 127.27 (2
Ar-CH), 132.33 (2 ArCH, 2 ArC), 146.63 (Ar-C), 149.98 (2 Ar-C), 151.02 (C=O), 156.62 (Ar-C),
158.04 (C=O) and 167.99 ppm (2 C=O); FT-IR νmax(KBr)cm-1: 3186, 2960, 1742, 1694, 1651, 1525,
1490, 1264, 1145; Anal.Cal. for C28H29N5O6: C, 63.27%; H, 5.50%; N, 13.18%; O, 18.06%, Found:
C, 62.88%; H, 5.33%; N, 14.63%.
4.1.4.13. 8-[3-(2-Morpholinoethoxy)-phenyl]-1,3-dipropylxanthine (RY-103, 12a)0.50 g, 50.42 %, mp
150-155 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 7.45 (s, 2H, Ar-H), 7.03 (m, 2H, Ar-H), 4.06
(m, 4H, -2OCH2-), 2.09 (m, 1H, NH), 1.76 (m, 2H, -OCH2-), 1.69 (m,4H, 2 x -NCH2-), 1.41 (m, 2H, -
13
NH2), 1.25 (m, 2H, -CH2-), 0.95 (m, 6H, 3 x -CH2), 0.07 (m, 6H, -2 x -CH3); C-NMR (100 MHz,
CDCl3):δ 10.08-11.01 (2 CH3), 20.01 (2 CH2), 44.39 (2 N-CH2), 54.02 (3 N-CH2), 65.64 (2 O-CH2),
66.08 (O-CH2), 111.10 -156.41 (ArC) and 162.75 (C=O) and 168.05 ppm (C=O); FT-IR νmax. (KBr)
cm-1:2962, 1701, 1655, 1548, 1258; Anal.Cal. for C23H31N5O4: C, 62.57%; H, 7.08%; N, 15.86%; O,
14.49%, Found:C, 60.57%; H, 6.18%; N, 14.16%.
4.1.4.14. 8-[3-(2-Piperidinoethoxy)-phenyl]-1,3-dipropylxanthine (RY-105, 12b)0.40 g, 40.27 %, mp
180-185 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 7.83 (d, 2H, Ar-H), 7.37 (s, 1H, Ar-H), 7,00 (s,
This article is protected by copyright. All rights reserved

1H, Ar-H), 4.17 (m, 6H, 3 x -NCH2-), 2.92 (s, 2H, -OCH2-), 2.65 (s, 4H, 2 x -NCH2-), 1.86 (s, 2H, -
CH2), 1.68 (s, 6H, 3 x -CH2-), 1.49 (s,2H, -CH2), 1.01 (m, 6H,2xCH3); 13C-NMR (100MHz, CDCl3):δ
11.37-11.53 (2 CH3), 21.24-25.56 (5 CH2), 47.28-57.70 (5 N-CH2), 65.64 (O-CH2), 108.23-151.41 (5
Ar-C), 119.10 (2 Ar-CH), 129.90 (2 Ar-CH), 155.75 (C=O) and 159.05 ppm (C=O); FT-IR
νmax(KBr) cm-1:3196 (-NH), 2933 (Ar-CH), 2787 (Ali-CH), 1698 (carbonyl), 1655 (CN), 1553 (H-
C=C-H), 1259 (CO), 1186 (C-N); Anal. Cal. for C24H33N5O3:C, 65.58%; H, 7.57%; N, 15.93%; O,
10.92%, Found:C, 64.98%; H, 7.01%; N, 14.97%.
4.1.4.15. 8-[3-{2-(Dimethylamino)-ethoxy}-phenyl]-1,3-dipropylxanthine (RY-107, 12c)0.86 g, 86.33
%, mp>280 °C (decomp.); ¹H-NMR (400 MHz, DMSO-d6):δ 7.30-6.91 (m, 4H, Ar-H), 4.01-3.83 (m,
4H, -OCH2, -NCH2), 1.93-1.55 (m, 4H, 2 x -NCH2-), 1.80 (s, 4H, 2 x -CH2-), 0.85 (s, 12H, 2 x -
13
NCH3, 2 x -CH3); C-NMR (400 MHz, DMSO-d6):δ 10.37-11.53 (2 CH3), 21.24 (2 CH2), 43.66 (2
N-CH2), 45.80 (2 N-CH3), 57.70 (N-CH2), 65.64 (O-CH2), 115.23-152.41 (5 Ar-C), 119.10-132.90 (4
Ar-CH) and 151.75-156.05 ppm (2 C=O); FT-IR νmax. (KBr) cm-1:3177 (-NH), 2960 (Ar-CH), 1695
(C=O), 1653 (C=N), 1550 (Ar-C=C), 1256 (C-O), 1175 (C-N); Anal. Cal. for C21H29N5O3:C, 63.14%;
H, 7.32%; N, 17.53%; O, 12.01%, Found:C, 60.14%; H, 6.82%; N, 16.83%.
4.1.4.16. 8-[3-(2-Pyrrolidinoethoxy)-phenyl]-1,3-dipropylxanthine (RY-109, 12d)0.99 g, 99 %,
mp230-235 °C (decomp.); ¹H-NMR (400MHz, DMSO-d6):δ 13.76 (s, 1H, -NH), 7.31 (m, 4H, Ar-H),
4.42 (s, 2H, -OCH2-), 3.96 (s, 6H, 3 x -NCH2-), 3.89 (s, 4H, 2 x NCH2-), 2.08 (d, 4H, 2 x -CH2), 1.77
(d, 4H, 2 x -CH2-) and 0.91 ppm (s, 6H, 2 x -CH3);13C-NMR (100 MHz, DMSO-d6):δ 10.08-11.01 (2
CH3), 20.08-25.56 (4 CH2), 44.05 (2 N-CH2), 54.08-56.67 (3 N-CH2), 66.07 (O-CH2), 114.67-128.89
(4 Ar-CH, Ar-C), 121.45 (Ar-C), 150.04 (Ar-C), 152.87 (C=O), 156.08 (2 Ar-C) and 156.78 ppm
(C=O); FTIR νmax(KBr) cm-1:3223 (-NH), 2962 (Ar-CH), 1694 (C=O), 1648 (C=N), 1451 (Ar-C=C),
1254 (C-N); Anal. Cal. for C23H31N5O3:C, 64.93%; H, 7.35%; N, 16.47%; O, 11.28%; Found: C,
65.34%; H, 8.31%; N, 15.12%.
4.1.4.17. 8-[3-{2-(Diethylamino)-ethoxy}-phenyl]-1,3-dipropylxanthine (RY-111, 12e)0.76 g,76.92 %,
mp 100-105 °C (decomp.); ¹H-NMR (400MHz, DMSO-d6):δ 13.72 (s, 1H, -NH), 7.8(m, 1H, Ar-H),
7.40 (m, 2H, Ar-H), 7.05 (m, 1H, Ar-H), 4.40 (m, 2H, -OCH2-), 4.07 (t, 2H, 2 x -NCH2-), 3.92 (m,
4H, 2 x -NCH2-), 1.79 (q, 4H, 2 x -N-CH2), 1.63 (q, 2H, -CH2-), 1.30 (m, 6H, 2 x -CH3) and 0.93 ppm
(m, 8H, 2xCH3, -CH2-); 13C-NMR (100MHz, DMSO-d6):δ 10.08-11.01 (2 CH3), 13.08 (2 CH3), 20.08
This article is protected by copyright. All rights reserved

(2 CH2), 44.03-53.78 (5 N-CH2), 66.07 (O-CH2), 114.67-150.04 (Ar-C), and 156.78 ppm (C=O);
FTIR νmax(KBr) cm-1:3181 (NH), 2961 (CH), 1743 (carbonyl), 1696 (C=O), 1651 (cyano), 1549 (H-
C=C-H), 1257 (C-N); Anal. Cal. for C23H33N5O3:C, 64.61%; H, 7.78%; N, 16.38%; O, 11.23%,
Found:C, 62.61%; H, 8.08%; N, 16.38%.
4.1.4.18. 8-[3-{2-(Phthalimido)-ethoxy}-phenyl]-1,3-dipropylxanthine (RY-113, 12f) 0.80 g, 80.69 %,
mp 274-280 °C (decomp.); ¹H-NMR (400 MHz, CDCl3):δ 13.66 (s, 1H, -NH), 7.56(m, 2H, Ar-H),
7.25 (t, 3H, Ar-H, Jortho= 7.88Hz, Jortho= 7.84Hz),6.87 (d, 2H, Ar-H, Jpara= 1.96Hz), 6.85 (d, 1H, Ar-
H, Jpara= 1.59Hz), 4.05 (t, 2H, -OCH2-, J1=7.00, J2=7.48), 3.90 (t, 2H, -NCH2-, J1=7.32, J2=7.52),
1.77 (m, 4H, 2 x NH2-), 1.61 (m, 4H, 2 x -CH2-) and 0.93 ppm (m, 6H, 2x-CH3);13C-NMR
(100MHz, CDCl3): δ 10.8-11.01 (2 CH3), 20.02 (2 CH2), 39.04-44.89 (3 N-CH2), 66.04 (O-CH2),
114.32 (2 Ar-CH, Ar-C), 123.76 (Ar-C), 128.05 (4 Ar-CH), 132.44 (2 Ar-CH, 2 Ar-C), 150.78 (Ar-
C), 152.04 (C=O), 156.04 (2 Ar-C), 156.87 (C=O) and 167.09 ppm (2 C=O); FT-IR νmax. (KBr) cm-1:
3192 (-NH-), 2963 (Ar-CH-), 2876 (Ali-CH-), 1695 (C=O), 1644 (C=N), 1464 (Ar-C=C), 1262 (C-
N), 1186 (C-O); Anal. Cal. for C27H27N5O5:C, 64.66%; H, 5.43%; N, 13.96%; O, 15.95%,Found:C,
62.15%; H, 6.13%; N, 14.16%.
4.2 Biological methods
4.2.1Radioligand binding assays
The binding affinities at A1, A2A and A3 receptors (Ki-values) were determined in radioligand
competition experiments. The binding affinities were tested using a 96 well microplate filtration
system. The radioligands used were the agonists [3H] CCPA (1 nM), [3H]NECA (10 nM) and
[3H]HEMADO (1 nM) for A1, A2A and A3, respectively. The samples were first incubated with
membrane protein at 25ºC for 3 hours and then filtered followed by washing with ice cold binding
buffer. After addition of scintillator to the dried filter plates, sample were counted. The binding data
was calculated by nonlinear curve fitting using the program Prism [GraphPad Software](Klotz, 1998;
Klotz, 2007).
4.2.2 Bronchospasmolytic activity
This article is protected by copyright. All rights reserved

The newly synthesized derivatives 10a, 10b, 10c, 10f, 11a, 11b, 12(a-f)were evaluated for their in
vivo bronchospasmolytic activity against the standard drug theophylline by histamine aerosol induced
asthma in guinea pigs according to the method of Zabeer (Zabeer, 2006).
4.2.2.1 Experimental animals Guinea-pigs (Male; Dunkin Hartley) of 220-280 g, were obtained from
LLRUAS, Hisar after the approval of IAEC, Banasthali University (BV/IAEC/2016/I dated
08.10.2016, Ref. No. BV/3421/16-17). The experimental animals so procured were kept in standard
conditions as prescribed with proper food and water. They were monitored for 12 h day and night
cycle for experimentation.
4.2.2.2 Drugs used. Histamine hydrochloride (bronchospasm agent), theophylline (standard drug),
carboxymethyl cellulose (suspending agents) and test compounds (synthesized compound).
4.2.2.3. Experimental protocol. Guinea pigs (n =5) were designated as I for control animals (fed with
carboxymethyl cellulose and water); II for positive animals (carboxymethyl cellulose, theophylline
and water) and III for test animals (carboxymethyl cellulose, test drug and water). The assigned
animal groups were allowed for fasting before treatment. The experimental animals were exposed in
histamine chamber with histamine aerosol for 5 min after dosing of 1 h. Prior to exposure; animals
received test drug (50 mg/kg), theophylline (50 mg/kg) and carboxymethyl cellulose orally,
respectively. Different in vivo pharmacological behaviors of each animal was observed such as
bronchospasm, jerks, death or survival. The onset of bronchospasm, duration of jerks, severity of
bronchospasm and death or survival of the animal was recorded for each group. The results were
expressed as Mean ± SEM. (Table 2). The animals were allowed to remain in the chamber for 15
min, if survived, animals were removed from the chamber and placed in fresh atmosphere with proper
diet (Zabeer, 2006).
Acknowledgements
Authors are thankful to UGC, New Delhi for providing financial assistance and to the Vice
Chancellor, Banasthali University for providing necessary facilities. The NMR facility provided by
SAIF, Panjab University, Chandigarh is also highly acknowledged.
Conflict of interest
This article is protected by copyright. All rights reserved

The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
References
Aherne, C. M., Kewly, E. M.,Eltzschig, H. K. (2011).The resurgence of A2B adenosine receptor
signaling, Biochimica et Biophysica Acta, 1808, 1329–1339
Antonioli, L. ,Blandizzi, C. , Pacher, P. , &Hasko, G. (2013) Immunity, Inflammation and Cancer
: A leading role for adenosine. Nature, 13, 842-857.
Ascherio, A. (2001)Prospective study of caffeine consumption and risk of Parkinson’s disease in
men and women. Annals of Neurology, 50, 56–63.
Allwood, M. B. ,Cannan, B. , Van Aalten, D. M. F. , &Eggleston., I. M. (2007)Efficient synthesis
of 1,3,7-substituted xanthines by a safety-catch protection strategy. Tetrahedron, 63, 12294-
12302.
Bansal, R. ,Kumar, G. , Gandhi, D. , Yadav, R. ,Young, L. C. , &Harvey, A. L.(2010) Synthesis of
8-(cyclopentyloxy)phenyl substituted xanthine derivatives as adenosine A2A ligands,
Arzneimittelforschung, 60, 131-136.
Bansal, R. ,Kumar, G. , Gandhi, D. , Young, L. C. ,&Harvey, A. L. (2009) Synthesis of a series of
8-(substituted-phenyl)xanthines and a study on the effects of substitution pattern of phenyl
substituents on affinity for adenosine A1 and A2A receptors. European Journal of Medicinal
Chemistry, 44, 2122-2127.
Bandyopadhyay, P., Agrawal, S. K. ,Sathe, M. , Sharma, P. , &Kaushik, M. P. (2012)A facile and
rapid one-step synthesis of 8-substituted xanthine derivatives via tandem ring closure at room
temperature. Tetrahedron, 68, 3822-3827.
Baraldi, P. G. ,Tabrizi, M. A. , Gessi, S. , &Borea, P. A. (2008)Adenosine receptor antagonists:
Translating medicinal chemistry and pharmacology into clinical utility.Chemical Reviews, 108,
238-263.
This article is protected by copyright. All rights reserved

Belardinelli, L. ,Mattos, E. C. , &Beme, R. M. (1981)Evidence for adenosine mediation of
atrioventricular block in the ischaemicmyocardium.Journal of Clinical Investigation, 68, 195-205.
Blicke, F. F. ,&Godt, H. C. (1954)Reactions of 1, 3-Dimethyl-5, 6-diaminouracil.Journal of
American Chemical Society, 76, 2798-2800.
Brown, R. A. , Spina, D. , & Page, C. P. (2008) British Journal of Pharmacology, 153, 5446-
5456.
Chen, Y. ,Wang, B. , Guo, Y. , Zhou, Y. , Pan, L. , Xiong, L., Yu, S. , & Li, Z.(2014) Synthesis
and biological activities of novel methyl xanthine derivatives. Chemical Research in Chinese
Universities, 30,98-102.
Erdmann, A. A.(2005) Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits
IL-2 secretion in vitro and IL-2 driven expansion in vivo.Blood, 105, 4707–4714.
Fozard, J. R. ,Ellis, K. M. , VillelaDantas, M. F. , Tigani, B. ,&Mazzoni, L.(2002) Effects of CGS
21680, a selective adenosine A2A receptor agonist, on allergic airways inflammation in the rat.
European Journal of Pharmacology, 438, 183–188.
Fredholm, B. B. ,IJzerman, A. P. , Jacobson, K. A. , Klotz, K. A. , &Linden, J. (2001)International
Union
of
Pharmacology.
XXV.
Nomenclature
and
classificationofadenosinereceptors.Pharmacological Reviews, 53, 527–552.
Gessi, S. ,Merighi, S. ,Sacchetto, V. , Simioni, C. , &Borea, P. A.(2011) Adenosine Receptors and
Cancer. Biochimica et Biophysica Acta, 1801, 1400-1412.
Hayallah, A. M. ,Sandoval-Ramírez, J. , Reith, U. , Schobert, U. , Preiss, B. , Schumacher, B. ,
Daly, J. , & Muller, C. E. (2002) 1,8-Disubstituted Xanthine Derivatives:ꢀ Synthesis of Potent
A2B-Selective Adenosine Receptor Antagonists. Journal of Medicinal Chemistry, 45, 1500-1510.
Kaiser, S. M. ,&Quinn., R. J. (1999) Adenosine receptors as potential therapeutic targets. Drug
Discovery Today,12, 542-551.
Kim, D. ,Jun, H. , Lee, H. , Hong, S. S. , &Hong, S.(2010) Development of New Fluorescent
Xanthines as Kinase Inhibitors. Organic Letters, 12, 1212-1215.
Klotz, K. N. ,Falgner, N. , Kachler, S. , Lambertucci, C. , Vittori, S. , Volpini, R. ,&
Cristalli, G.(2007) [3H]HEMADO— a novel tritiated agonist selective for the human adenosine
A3 receptor. European Journal of Pharmacology, 556,14-18.
This article is protected by copyright. All rights reserved

Klotz, K. N. ,Hessling, J. , Hegler, J. , Owman, C. , Kull, B. , Fredholm, B. B. , &Lohse, M.
J.(1998) Comparative pharmacology of human adenosine receptor subtypes – characterization of
stably transfected receptors in CHO cells. Naunyn-Schmiedeberg’s Archives of Pharmacology,
357, 1-9.
Landells, L. J. , Jensen, M. W. , Orr, L. M. , Spina, D. , O’Connor, B. J. , & Page, C. P. (2000)
The role of adenosine receptors in the action of theophylline on human peripheral blood
mononuclear cells from healthy and asthmatic subjects. British Journal of Pharmacology, 129,
1140-1144.
Lee, D. , Lee, S. , Liu, K. H. , Bae, J. S. , Baek, D. J. , & Lee., T.(2016) Solid-Phase Synthesis of
1,3,7,8-Tetrasubstituted Xanthine Derivatives on Traceless Solid Support. ACS Combinatorial
Science, 18,70-74.
Linden, J. (2005)Adenosine in tissue protection and tissue regeneration. Molecular
Pharmacology, 67, 1385–1387.
Miyamoto, K. ,Yamamoto, Y. , Kurita, M. , Sakai, R. , Konno, K. , Sanae, F. , Ohshima, T. ,
Takagi, K. , & Hasegawa, T. (1993) Bronchodilator activity of xanthine derivatives substituted
with functional groups at the 1- or 7-position. Journal of Medicinal Chemistry, 36,1380-1386.
Monteiro, J. P. , Alves, M. G. , Oliveira, P. F. & Silva, B. M. (2016) Structure-Bioactivity
Relationships of Methylxanthines: Trying to Make Sense of All the Promises and the Drawbacks.
Molecules, 21, 1-32.
Müller, C. , & Jacobson, K. A. (2011) Xanthines as adenosine receptor antagonists. Handbook of
Experimental Pharmacology, 200, 1-59.
Muller, C. E. , Deters, D. , Dominik, A. , & Pawlowski, M. (1998) Synthesis of Paraxanthine and
Isoparaxanthine Analogs (1,7- and 1,9-Substituted Xanthine Derivatives). Synthesis,1428-1436.
Neary, J. T. ,McCarthy, M. , Kang, Y. , & Zuniga, S. (1998)Mitogenic signalling from P1 and P2
purinergic receptors to mitogen-activated protein kinase in human fetal astrocyte
cultures.Neuroscience Letters, 242,159–162.
Papesch, V. ,&Schroeder, E. F. (1951)Synthesis of 1-Mono- and 1, 3-Di-Substituted 6-
Aminouracils. DiureticActivity.Journal of Organic Chemistry, 16, 1879-1890.
This article is protected by copyright. All rights reserved

Persson,
C.
G.
A.
,&Kjellin,
G.
(1981)Enprofylline,
a
principally
new
antiasthmaticxanthine.ActaPharmacologicaetToxicologica, 49, 313-316.
Polosa, R. (2002) Adenosine receptor subtypes: their relevance to adenosine mediated responses
in asthma and chronic obstructive pulmonary disease. European Respiratory Journal, 20, 488-
496.
Sakai, R. ,Konno, K. , Yamamoto, Y. , Sanae, F. , Takagi, K. , &Hasegawa, T. (1992) Effects of
alkyl substitutions of xanthine skeleton on bronchodilation. Journal of Medicinal Chemistry, 35,
4039-4044.
Spicuzza, L. ,Bonfiglio, C. , &Polosa, R. (2003) Research applications and implications
ofadenosine in diseased airways. Trends in Pharmacological Sciences, 24,409–413.
Sousa, J. B. ,Fresco, P. ,Diniz, C. , &Goncalves, J. (2018) Adenosine Receptor Ligands on Cancer
Therapy : A review of Patent Literature. Recent Patents on Anti-Cancer Drug Discovery, 13, 40-
69.
Trevor, J. L. ,&Deshane, L. S. (2014) Refractory asthma: mechanisms, targets, and
therapy.Allergy,69, 817–827.
Yasui, K. ,Agematsu, K. , Shinozaki, K. , Hokibara, S. , Nagumo, H. , Nakazawa, T. , &
Komiyama, A. (2000) Theophylline induces neutrophil apoptosis through adenosineA2A
receptor antagonism. Journal of Leukocyte Biology, 67, 529-535.
Zabeer, A. , Bhagat, A. , Gupta, O. P. , Singh, G. D. , Youssouf, M. S. , Dhar, K. L. , Suri, O.
P. , Suri, K. A. , Satti, N. K. , Gupta, B. D. , &Qazi, G. N.(2006) Synthesis and bronchodilator
activity of new quinazolin derivative. European Journal of Medicinal Chemistry, 41, 429-434.
This article is protected by copyright. All rights reserved