Synthesis and Evaluation of a New Series of 8-(2-Nitroaryl)Xanthines as Adenosine Receptor Ligands

Article abstract of DOI:10.1002/ddr.21317

(Table presented.). A new series of 1,3-dimethylxanthine derivatives bearing 8-(2-nitroaryl) residue was synthesized and evaluated for affinity for recombinant human adenosine receptors subtypes. Nitrate esters of 7-substituted-1,3-dimethyl-8-phenylxanthines were also synthesized and tested. Introducing a nitro substituent at the 2-position of the 8-substituted phenyl ring resulted in generally low affinity for adenosine receptors (ARs), selectivity toward the A_2A subtype was enhanced in some of the compounds. 8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,3-dimethylxanthine (9e) proved to be a potent compound among the 2-nitrophenyl substituted xanthines exhibiting a K_i = 1 μM at human A_2A ARs with at least 30 fold selectivity versus human A₁ and A_2B ARs. Replacement of 8-chloropropoxy phenyl with 8-nitrooxypropoxy phenyl resulted in a negligible change in binding affinity of the 8-substituted xanthines for various AR subtypes. Drug Dev Res 77 : 241–250, 2016.

Full text of DOI:10.1002/ddr.21317

DRUG DEVELOPMENT RESEARCH 00 : 00–00 (2016)
Research Article
Synthesis and Evaluation of a New Series of 8-(2-
Nitroaryl)Xanthines as Adenosine Receptor Ligands
Ranju Bansal,1* Gulshan Kumar,1 Suman Rohilla,1 Karl-Norbert Klotz,2 Sonja Kachler,2
Louise C. Young,3 and Alan L. Harvey3
1University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
2
€
€
€
Institut fur Pharmakologie und Toxikologie, Universitat Wurzburg, Wurzburg, Germany
3Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161
Cathedral Street, Glasgow G4 0RE, United Kingdom
Strategy, Management and Health Policy
Enabling
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Preclinical
Research
Postmarketing
Phase IV
Technology,
Genomics,
Proteomics
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
A new series of 1,3-dimethylxanthine derivatives bearing 8-(2-nitroaryl) residue was
synthesized and evaluated for affinity for recombinant human adenosine receptors subtypes. Nitrate
esters of 7-substituted-1,3-dimethyl-8-phenylxanthines were also synthesized and tested. Introducing a
nitro substituent at the 2-position of the 8-substituted phenyl ring resulted in generally low affinity for
adenosine receptors (ARs), selectivity toward the A2A subtype was enhanced in some of the com-
pounds. 8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,3-dimethylxanthine (9e) proved to be a potent
compound among the 2-nitrophenyl substituted xanthines exhibiting a Ki 5 1 lM at human A2A ARs
with at least 30 fold selectivity versus human A1 and A2B ARs. Replacement of 8-chloropropoxy phenyl
with 8-nitrooxypropoxy phenyl resulted in a negligible change in binding affinity of the 8-substituted
C
V
xanthines for various AR subtypes. Drug Dev Res 00 : 000–000, 2016.
2016 Wiley Periodicals, Inc.
Key words: nitroxanthines; adenosine receptors; nitrate esters
well-known binding affinity for ARs. Xanthines have
been widely explored in terms of affinity and selectiv-
ity for ARs [Daly, 2000; Bansal et al., 2009; Yadav
et al., 2014]. Introduction of 8-aryl/heteroaryl sub-
stituents in the xanthine scaffold results in spectacu-
lar alterations in their affinity for ARs [Baziard-
INTRODUCTION
Adenosine, an endogeneous nucleoside present
ubiquitously in all mammalian cells, modulates a vari-
ety of physiological functions via G protein-coupled
adenosine receptors, classified as A1, A2A, A2B, and
A3 subtypes. For each subtype, selective agonists, and
antagonists have been developed as potential new
drug candidates for the treatment of various ailments
such as neurodegenerative, respiratory, cardiac, imm-
une, and inflammatory disorders [Muller and Jacob-
son, 2011; Chen et al., 2013; Layland et al., 2014].
The development of potent and selective ligands for
adenosine receptors (ARs) has been a dynamic area
of research for several decades.
*Correspondence to: Ranju Bansal, University Institute of
Pharmaceutical Sciences, Panjab University, Chandigarh
160014, India. E-mail: ranju29in@yahoo.co.in
Contract grant sponsor: HSCST, Haryana, India and Common-
wealth Fellowship from the Association of Commonwealth
Universities.
Received 9 May 2016; Accepted 29 May 2016
Substituted xanthines represent a privileged
Published online in Wiley Online Library (wileyonlinelibrary.
class of pharmacologically active compounds with com). DOI: 10.1002/ddr.21317
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V
2016 Wiley Periodicals, Inc.

2
BANSAL ET AL.
Mouysset et al., 1995]. Several 8-(substituted phenyl)
derivatives are potent AR ligands [Daly et al., 1986].
It has been observed that the substitution pattern of
8-phenyl substituents dramatically affects the AR
binding affinity. 8-(4-Nitrophenyl)-1,3-dimethylxan-
thine (1) (Fig. 1) has been reported as a potent A1
AR antagonist with an IC50 value of 8 nM in com-
petition for N6-[3H] cyclohexyladenosine binding at
bovine brain membranes [Hamilton et al., 1985].
We decided to synthesize a new series of 8-
nitroarylxanthines and study their binding affinity for
ARs to gain further insight into the structure-activity
relationship of xanthines at ARs. In view of the signif-
icance of nitrate esters (2) to display a variety of bio-
logical actions due to their ability to generate nitric
oxide [Lagas and Duchateau, 1988; Kerwin et al.,
1995], it was further planned to synthesize and study
the effect of introducing nitrate esters on adenosine
binding affinity of xanthines.
Fig. 1. Structures of nitroxanthine (1) and nitrate ester (2).
benzaldehyde, 4-cyclopentyloxy-3methoxybenzaldehyde
(1 g) was added in small portions to the stirred ice cold
mixture of concentrated nitric acid (3 ml) and concen-
trated sulfuric acid (2 ml). The reaction mixture was fur-
ther stirred in ice for 20–25 min and then at room
temperature for 15 min. The reaction mixture was
poured onto crushed ice. The yellow colored precipitate
obtained was filtered off, washed with ice cold water,
dried, and crystallized from ethanol to obtain the corre-
sponding nitroaldehydes 7b–e.
4,5-Dimethoxy-2-nitrobenzaldehyde (7b). Yield:
1
(0.9 g, 70.86%), m.p. 126–1288C. H NMR (CDCl3):
d 4.15 (s, 6H, 2 X AOCH3), 7.45 (s, 1H, CH, aro-
matic), 7.65 (s, 1H, CH, aromatic), and 10.25 ppm (s,
1H, ACHO).
METHODS AND MATERIALS
Chemistry
4-Methoxy-2-nitrobenzaldehyde (7c). Yield:
All melting points were taken on a Veego melt-
ing point apparatus and are uncorrected. Infrared
(IR) spectra were recorded on Perkin-Elmer 882 and
RX1 FT-IR spectrophotometers as potassium bro-
mide pellets (tmax in cm21). Proton (1H) nuclear
magnetic resonance spectroscopy was performed
using a Brucker AC-300F, 300 MHz spectrometer
for solutions in deuteriochloroform, deuterated dime-
thylsulfoxide and are reported in parts per million
(ppm). Tetramethylsilane (Me4Si) was used as an
internal reference. The spin multiplicities are desig-
nated by the symbols, s (singlet), d (doublet), t (tri-
plet), q (quartet), p (pentet), m (multiplet), and br
(broad). Elemental analyses were carried out on a
Perkin-Elmer-2400 CHN elemental analyzer. Plates
for thin layer chromatography (TLC) were prepared
with silica gel G according to method of Stahl (E.
Merck) using ethyl acetate as solvent and activated at
1108C for 30 min. Iodine was used to develop the
TLC plates. Anhydrous sodium sulfate was utilized as
drying agent. All solvents were freshly distilled and
dried prior to use according to standard procedures.
Nitroaldehyde 7a was prepared by a reported proce-
dure [Bogert and Elder, 1929]. 7-Substituted xan-
thine derivatives 17, 18, and 22 were also
synthesized as described [Bansal et al., 2011].
1
(0.98 g, 74.0%), m.p. 76–808C. H NMR (CDCl3): d
4.05 (s, 3H, AOCH3), 7.30 (d, 1H, CH, aromatic,
Jo 5 7.80 Hz), 8.15 (dd, 1H, CH, aromatic, Jo 5 8.0,
Jm 5 2.0 Hz), 8.35 (d, 1H, CH, aromatic, Jm 5 2.0
Hz), and 10.35 ppm (s, 3H, ACHO).
3,4,5-Trimethoxy-2-nitrobenzaldehyde
(7d).
Yield: (0.6 g, 48.78%), m.p. 110–1128C. 1H NMR
(CDCl3): d 4.05 (s, 9H, 3 X AOCH3), 7.30 (s, 1H,
CH, aromatic), and 9.90 ppm (s, 1H, ACHO).
4-Cyclopentyloxy-5-methoxy-2-nitrobenzaldehyde
1
(7e). Yield: (0.67 g, 55.83%), m.p. 80–848C. H NMR
(CDCl3): d 1.80 (br s, 8H, 4 X CH2, cyclopentyl), 4.05
(s, 3H, AOCH3), 4.95 (s, 1H, OACH<, cyclopentyl),
7.40 (m, 1H, CH, aromatic), 7.66 (s, 1H, CH, aromatic),
and 10.55 ppm (s, 1H, ACHO).
General Method for the Synthesis of Substituted
Aldehydes 10a–g
The respective hydrochlorides of b-dialkylami-
noethyl chloride (6.57 mmol), 1-bromo-3-chloro-
propane (1.5 ml, in excess)/1-bromo-2-chloroethane
(1.5 ml, in excess) were added to a stirred and reflux-
ing slurry of vanillin (1.0 g, 6.57 mmol) in ethyl
methyl ketone (40 ml) in the presence of anhydrous
potassium carbonate (2.0 g, 14.47 mmol) as reported
earlier [Bansal et al., 2009, 2011]. The reaction mix-
ture was further refluxed for 6 h with continuous stir-
ring. The completion of the reaction was monitored
by TLC. On completion, the reaction mixture was
General Method for the Synthesis
of Nitroaldehydes 7b–e
Requisite aromatic aldehyde 3,4-dimethoxyben-
zaldehyde, 4-methoxybenzaldehyde, 3,4,5-trimethoxy- cooled, filtered and the solvent was removed under
Drug Dev. Res.

SYNTHESIS AND EVALUATION OF A NEW SERIES OF 8-(2-NITROARYL)XANTHINES
3
reduced pressure to obtain an oily residue of the cor- AOCH3), 4.29 (t, 2H, AOCH2A, J 5 5.91 Hz), 7.40
responding substituted aldehyde 10a–g, which was (s, 1H, CH, aromatic), 7.65 (s, 1H, CH, aromatic),
and 10.44 ppm (s, 1H, ACHO).
used as such for further reaction.
4-(2-Chloropropoxy)-3-methoxy-2-nitrobenzalde-
1
General Method for the Synthesis
hyde (11f). Yield: (1.1 g, 92.43%), m.p 76–808C. H
NMR (CDCl3): d 2.35 (m, 2H, ACH2CH2A), 3.78 (t,
2H, ACH2Cl, J 5 6.0 Hz), 4.05 (s, 3H, AOCH3), 4.25
(t, 2H, AOCH2A, J 5 6.0 Hz), 7.45 (s, 1H, CH, aro-
matic), 7.55 (s, 1H, CH, aromatic), and 10.25 ppm (s,
1H, ACHO).
4-(2-Chloroethoxy)-3-methoxy-2-nitrobenzaldehyde
(11g). Yield: (0.58 g, 48.33%), m.p 158–1608C. 1H NMR
(CDCl3): d 3.93 (t, 2H, ACH2Cl, J5 6.36 Hz), 4.02 (s,
3H, AOCH3), 4.42 (t, 2H, AOCH2A, J5 5.71 Hz), 7.43
(s, 1H, CH, aromatic), 7.63 (s, 1H, CH, aromatic), and
10.45 ppm (s, 1H, ACHO).
of Nitroaldehydes 11a–g
The above obtained substituted aldehyde 10a–g
(oily residue) was added in small portions to the
stirred ice cold mixture of concentrated nitric acid
(3 ml) and concentrated sulfuric acid (2 ml). The
reaction mixture was further stirred in ice for 20–25
min and then at room temperature for 15 min and
then poured onto crushed ice. The yellow colored
precipitate obtained was filtered off, washed with ice
cold water, dried and crystallized from ethanol to
obtain corresponding compound 11a–g.
4-(2-Dimethylaminoethoxy)-5-methoxy-2-nitro-
benzaldehyde (11a). Yield: (0.35 g, 29.16%), m.p. 60–
648C. H NMR (CDCl3): d 2.43 (t, 6H, AN(CH3)2),
General Method for the Synthesis of Benzylidene
Derivatives 8a–e, 12a–g
1
To a stirred solution of 5,6-diamino-1,3-dime-
thyluracil (6, 1.0 g, 5.87 mmol) in MeOH–AcOH
(4:1, 40 ml) was slowly added the requisite nitroalde-
hyde 7a–e and 11a–g (1.0 g) in methanol (24 ml).
Yellow colored precipitate appeared during addition.
The reaction mixture was further stirred overnight at
room temperature and the completion of reaction
was monitored by TLC. The precipitate obtained was
filtered off, washed with methanol and dried to
obtain corresponding quite unstable benzylidene
derivatives 8a–e and 12a–g, which were used as
such for further cyclization. The physicochemical
data of the intermediates 8a–e and 12a–g are pre-
sented in Table 1.
2.84 (t, 2H, ACH2N<, J 5 5.67 Hz), 3.99 (s, 3H,
AOCH3), 4.24 (t, 2H, AOCH2A, J 5 5.74 Hz), 7.40
(s, 1H, CH, aromatic), 7.64 (s, 1H, CH, aromatic),
and 10.44 ppm (s, 1H, ACHO).
4-(2-Diethylaminoethoxy)-5-methoxy-2-nitroben-
zaldehyde (11b). Yield: (0.38 g, 32.47%), m.p 72–
76ꢀC. 1H NMR (CDCl3):
d
1.09 (t, 6H,
AN(CH2CH3)2, J 5 7.03 Hz), 2.68 (m, 4H,
AN(CH2CH3)2), 2.99 (t, 2H, ACH2N<, J 5 5.7 Hz),
4.00 (s, 3H, AOCH3), 4.21 (t, 2H, AOCH2A, J 5 5.6
Hz), 7.01 (s, 1H, CH, aromatic), 7.66 (s, 1H, CH,
aromatic), and 9.84 ppm (s, 1H, ACHO).
3-Methoxy-2-nitro-4-(2-piperidin-1-ylethoxy)ben-
zaldehyde (11c). Yield: (0.25 g, 21.36%), m.p 110–
1148C. 1H NMR (CDCl3): d 1.47 (m, 2H, ACH2,
piperidine), 1.62 (m, 4H, 2 X CH2, piperidine), 2.54
(s, 4H, AN(CH2)2, piperidine), 2.86 (t, 2H,
ACH2N<, J 5 6.10 Hz), 4.00 (s, 3H, AOCH3), 4.27
(t, 2H, AOCH2A, J 5 6.12 Hz), 7.40 (s, 1H, CH, aro-
matic), 7.68 (s, 1H, CH, aromatic), and 10.44 ppm
(s, 1H, ACHO).
General Method for Synthesis of Various Xanthine
Derivatives 9a–e and 13a–g
Benzylidene derivatives 8a–e and 12a–g (1.0 g,
2 mmol) thus obtained were cyclized individually by
refluxing in thionyl chloride (20 ml) for 30–40 min.
The excess thionyl chloride was removed under
reduced pressure to get a solid product. Ice cold
water was added to it and resulting suspension was
neutralized with ammonium hydroxide solution. The
precipitate obtained was collected by filtration, dried
and recrystallized from ethanol to afford the desired
products 9a–e and 13a–g, respectively.
5-Methoxy-4-(2-morpholin-4-ylethoxy)-2-nitrobe-
nzaldehyde (11d). Yield: (0.4 g, 34.48%), m.p 98–
1028C. 1H NMR (CDCl3):
d
2.61 (m, 4H,
AN(CH2)2, morpholine), 2.90 (t, 2H, ACH2N<,
J 5 5.77 Hz), 3.74 (m, 4H, O(CH2)2, morpholine),
4.00 (s, 3H, AOCH3), 4.29 (t, 2H, AOCH2A,
J 5 5.83 Hz), 7.42 (s, 1H, CH, aromatic), 7.67 (s, 1H,
CH, aromatic), and 10.45 ppm (s,1H, ACHO).
1,3-Dimethyl-8-(6-nitrobenzo[1,3]dioxol-5-yl)xant-
hine (9a). Yield: (0.94 g, 94.56%), m.p. 318–3228C.
FTIRtmax (KBr): 3040, 2920, 1695, 1640, 1490, 1360,
5-Methoxy-2-nitro-4-(2-pyrrolidin-1-ylethoxy)be-
nzaldehyde (11e).Yield: (0.38 g, 32.20%), m.p 122–
1
1240, and 1005 cm21. H NMR (CDCl3-DMSO-d6): d
1
1268C. H NMR (CDCl3): d 1.83 (m, 4H, 2 X CH2,
3.35 (s, 3H, NACH3), 3.54 (s, 3H, NACH3), 6.23 (s, 2H,
pyrrolidine), 2.68 (m, 4H, AN(CH2)2, pyrrolidine), AOACH2AOA), 7.18 (s, 1H, CH, aromatic), 7.94 (s,
3.02 (t, 2H, ACH2N<, J 5 5.94 Hz), 4.00 (s, 3H, 1H, CH, aromatic), and 13.74 ppm (br s, 1H, NAH).
Drug Dev. Res.

4
BANSAL ET AL.
7.19 (s, 1H, CH, aromatic), 7.70 (s, 1H, CH, aromatic),
and 13.30 ppm (br s, 1H, NAH). Calcd. for
C19H21N5O6: C, 54.94; H, 5.09; N, 16.86%. Found: C,
54.76; H, 4.91; N, 16.44%.
TABLE 1. Physicochemical Data of Benzylidene Derivatives 8a–e
and 12a–g
Comp no
Yield (%)
m.p. (8C)
Comp no
Yield
m.p. (8C)
8-[4-(2-Dimethylaminoethoxy)-5-methoxy-2-nitro-
phenyl]-1,3-dimethylxanthine (13a). Yield: (0.1 g,
25.12%), m.p. 148–152ꢀC. FTIRtmax (KBr): 2920, 1710,
8a
8b
8c
8d
8e
12a
44.11
58.68
63.07
26.83
34.69
52.8
260–264
258–262
>300
160–170
238–242
200–204
12b
12c
12d
12e
12f
33.33
34.35
35.46
27.57
36.92
47.61
208–214
214–218
160–164
202–208
200–204
234–238
1
1650, 1510, 1340, 1220, and 1050 cm21. H NMR
(CDCl3): d 2.43 (t, 6H, AN(CH3)2), 2.91 (t, 2H,
ACH2N<, J 5 5.45 Hz), 3.33 (s, 3H, NACH3), 3.66 (s,
3H, NACH3), 3.98 (s, 3H, AOCH3), 4.26 (t, 2H,
AOCH2A, J 5 5.54 Hz), 7.24 (s, 1H, CH, aromatic),
and 7.71 ppm (s, 1H, CH, aromatic). Calcd. for
C18H22N6O6: C, 51.67; H, 5.30; N, 20.09%. Found: C,
50.91; H, 4.98; N, 19.81%.
12g
Calcd. for C14H11N5O6: C, 48.70; H, 3.21; N, 20.28%.
Found: C, 48.31; H, 2.92; N, 20.32%.
8-[4-(2-Diethylaminoethoxy)-5-methoxy-2-nitro-
phenyl]-1,3-dimethylxanthine (13b). Yield: (0.12 g,
20.33%), m.p. 132–136ꢀC. FTIRtmax (KBr): 2974,
8-(4,5-Dimethoxy-2-nitrophenyl)-1,3-dimethyl-
xanthine (9b). Yield: (0.73 g, 72.94%), m.p. 304–
3088C. FTIRtmax (KBr): 3158, 2920, 1704, 1652,
1708,1655, 1509, 1335, 1220, and 1051 cm21 1H
.
1546, 1475, 1351, 1281, and 1011 cm21. H NMR
1
NMR (CDCl3): d 1.09 (t, 6H, AN(CH2CH3)2,
J 5 7.12 Hz), 2.67 (m, 4H, AN(CH2CH3)2), 2.98 (t,
2H, ACH2N<, J 5 6.50 Hz), 3.32 (s, 3H, NACH3),
3.66 (s, 3H, NACH3), 3.99 (s, 3H, AOCH3), 4.21
(t,2H, AOCH2A, J 5 6.25 Hz), 7.21 (s, 1H, CH, aro-
matic), and 7.74 ppm (s, 1H, CH, aromatic). Calcd.
for C20H26N6O6: C, 53.80; H, 5.87; N, 18.82%.
Found: C, 53.67; H, 5.66; N, 18.21%.
(CDCl3): d 3.33 (s, 3H, NACH3), 3.67 (s, 3H,
NACH3), 4.03 (s, 6H, 2 X AOCH3), 7.25 (s, 1H, CH,
aromatic), 7.70 (s, 1H, CH, aromatic), and 13.10 ppm
(br s, 1H, NAH). Calcd. for C15H15N5O6: C, 49.86;
H, 4.18; N, 19.38%. Found: C, 49.34; H, 3.90; N,
18.88%.
1,3-Dimethyl-8-(4-methoxy-2-nitrophenyl)xanthine
(9c). Yield: (0.7g, 70.49%), m.p. >3008C. FTIRtmax
(KBr): 2941, 1709, 1662, 1522, 1333, 1223, and
1052 cm21. 1H NMR (CDCl3): d 3.41 (s, 3H, NACH3),
3.63 (s, 3H, NACH3), 4.04 (s, 3H, AOCH3), 7.23 (d, 1H,
CH, aromatic, Jo 5 8.93 Hz), 8.41 (dd, 1H, CH, aromatic,
Jo 5 8.73, Jm 5 2.14 Hz), and 8.75 ppm (d, 1H, CH, aro-
matic, Jm 5 2.12 Hz). Calcd. for C14H13N5O5: C, 50.76;
H, 3.95; N, 21.14%. Found: C, 50.45; H, 3.85; N,
20.81%.
1,3-Dimethyl-8-[5-methoxy-2-nitro-4-(2-piperidin-
1-ylethoxy)phenyl]xanthine (13c). Yield: (0.3 g, 43.10%),
m.p. 142–1468C. FTIRtmax (KBr): 2941, 2793, 1709,
1652, 1503, 1334, 1221, and 1048 cm21 1H NMR
.
(CDCl3): d 1.48 (m, 2H, ACH2, piperidine), 1.63 (p, 4H,
2 X CH2, piperidine), 2.55 (s, 4H, AN(CH2)2, piperi-
dine), 2.87 (t, 2H, ACH2N<, J5 6.01 Hz), 3.34 (s, 3H,
NACH3), 3.66 (s, 3H, NACH3), 3.99 (s, 3H, AOCH3),
4.27 (t, 2H, AOCH2A, J5 5.96 Hz), 7.23 (s, 1H, CH,
aromatic) and 7.74 ppm (s, 1H, CH, aromatic). Calcd. for
C21H26N6O6: C, 55.01; H, 5.72; N, 18.33%. Found: C,
54.78; H, 5.37; N, 18.13%.
1,3-Dimethyl-8-(3,4,5-trimethoxy-2-nitrophenyl)
xanthine (9d). Yield: (0.33 g, 47.4%), m.p. 256–
2608C. FTIRtmax (KBr): 3107, 2949, 1699, 1642,
1606, 1577, 1542, 1476, 1375, 1197, and 1027 cm21
.
1,3-Dimethyl-8-[5-methoxy-4-(2-morpholin-4-yle-
thoxy)-2-nitrophenyl]xanthine (13d). Yield: (0.08 g,
8.08%), m.p. 198–2028C. FTIRtmax (KBr): 2920, 1710,
1H NMR (CDCl3): d 3.39 (s, 3H, NACH3), 3.62 (s,
3H, NACH3), 3.98 (s, 3H, AOCH3), 3.99 (s, 3H,
AOCH3), 4.02 (s, 3H, AOCH3), 7.22 (s, 1H, CH,
aromatic), and 12.55 ppm (br s, 1H, NAH). Calcd.
for C16H17N5O7: C, 49.11; H, 4.38; N, 17.89%.
Found: C, 48.78; H, 3.87; N, 17.39%.
1
1655, 1510, 1335, 1270, 1220, and 1115 cm1. H NMR
(CDCl3): d 2.62 (t, 4H, AN(CH2)2, morpholine, J5 4.61
Hz), 2.91 (t, 2H, ACH2N<, J5 5.72 Hz), 3.33 (s, 3H,
NACH3), 3.66 (s, 3H, NACH3), 3.74 (t, 4H, O(CH2)2,
morpholine, J5 4.65 Hz), 4.00 (s, 3H, AOCH3), 4.26 (t,
2H, AOCH2A, J5 5.79 Hz), 7.25 (s, 1H, CH, aromatic),
and 7.74 ppm (s, 1H, CH, aromatic). Calcd. for
C20H24N6O7: C, 52.17; H, 5.25; N, 18.25%. Found: C,
51.88; H, 4.98; N, 17.93%.
8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,
3-dimethylxanthine (9e). Yield: (0.17 g, 24.13%), m.p.
220–2248C. FTIRtmax (KBr): 2940, 1710, 1640, 1580,
1
1520, 1335, 1280, 1220, and 1050 cm21. H NMR
(CDCl3): d 1.71 (m, 2H, CH2, cyclopentyl), 1.88 (m,
4H, 2 X CH2, cyclopentyl), 2.06 (m, 2H, CH2, cyclopen-
tyl), 3.31 (s, 3H, NACH3), 3.68 (s, 3H, NACH3), 3.99
1,3-Dimethyl-8-[5-methoxy-2-nitro-4-(2-pyrrolidin-
1-ylethoxy)phenyl]xanthine (13e). Yield: (0.26 g,
(s, 2H, AOCH3), 4.92 (m, 1H, OACH<, cyclopentyl), 43.55%), m.p. 118–1228C. FTIRtmax (KBr): 2925,
Drug Dev. Res.

SYNTHESIS AND EVALUATION OF A NEW SERIES OF 8-(2-NITROARYL)XANTHINES
5
1677, 1630, 1522, 1349, 1211, and 1052 cm21.1H matic), and 7.56 ppm (s, 1H, CH, imidazole). Calcd.
NMR (CDCl3): d 1.63 (p, 4H, 2 X CH2, pyrrolidine), for C20H21N7O6: C, 52.74; H, 4.65; N, 21.53%. Found:
2.67 (m, 4H, AN(CH2)2, pyrrolidine), 3.04 (t, 2H, C, 52.60; H, 4.35; N, 21.23%.
1,3-Dimethyl-8-[4-(2-imidazol-1-ylethoxy)-5-
methoxy-2-nitrophenyl]xanthine (15). Yield: (0.34 g,
45.09%), m.p. 260–264ꢀC. FTIRtmax (KBr): 2946, 1703,
CH2N<, J 5 5.83Hz), 3.34 (s, 3H, NACH3), 3.66 (s,
3H, NACH3), 3.96 (s, 3H, AOCH3), 4.28 (t, 2H,
AOCH2A, J 5 5.84 Hz), 7.23 (s, 1H, CH, aromatic),
and 7.71 ppm (s, 1H, CH, aromatic). Calcd. for
C20H24N6O6: C, 54.05; H, 5.44; N, 18.91%. Found:
C, 53.98; H, 5.25; N, 18.71%.
1661, 1580, 1500, 1443, 1332, 1217, and 1050 cm21
.
1H NMR (CDCl3-DMSO-d6): d 3.39 (s, 3H, NACH3),
3.57 (s, 3H, NACH3), 3.99 (s, 3H, AOCH3), 4.41 (t,
8-[4-(3-Chloropropoxy)-5-methoxy-2-nitrophenyl]- 2H, ACH2N<, J5 4.72 Hz), 4.48 (t, 2H, AOCH2A,
1,3-dimethylxanthine (13f). Yield: (0.75 g, 75.75%), J5 4.72 Hz), 7.02 (s, 1H, CH, imidazole), 7.21 (s, 1H,
m.p. 232–2368C. FTIRtmax (KBr): 2930, 1695, 1660, CH, imidazole), 7.29 (s, 1H, CH, aromatic), 7.55 (s,
1
1550, 1335, 1270, 1220, and 1050 cm21. H NMR 1H, CH, aromatic), and 7.77 ppm (s, 1H, CH, imidaz-
(CDCl3): d 2.36 (p, 2H, ACH2CH2A), 3.32 (s, 3H, ole). Calcd. for C19H19N7O6: C, 51.70; H, 4.34; N,
NACH3), 3.67 (s, 3H, NACH3), 3.80 (t, 2H, 22.21%. Found: C, 51.63; H, 4.14; N, 22.18%.
ACH2Cl, J 5 6.16 Hz), 4.01 (s, 3H, AOCH3), 4.31 (t,
General Procedure for the Synthesis
of 8-Nitrooxypropoxyphenylxanthines
18, 19, and 21
2H, AOCH2A, J 5 5.91 Hz), 7.23 (s, 1H, CH, aro-
matic), 7.73 (s, 1H, CH, aromatic), and 13.13 ppm
(br s, 1H, NAH). Calcd. for C17H18N5O6Cl: C,
48.18; H, 4.28; N, 16.52%. Found: C, 47.97; H, 3.90;
N, 16.20%.
A solution of silver nitrate (0.38 g, 2.23 mmol)
in acetonitrile (4 ml) was added to stirred solution of
desired xanthine derivative 16, 17, and 20 (0.50 g,
1.19 mmol) in sufficient amount of dry acetonitrile at
60ꢀC. The reaction mixture was further stirred for
10 h while keeping the temperature constant. The
solution was filtered off, filtrate was concentrated and
water was added to it. The precipitate obtained was
filtered off, washed with water, dried, and recrystal-
lized from acetone to afford corresponding xanthine
nitrate esters 18, 19, and 21.
8-[4-(2-Chloroethoxy)-5-methoxy-2-nitrophenyl]-
1,3-dimethylxanthine (13g). Yield: (0.56 g, 56.28%),
m.p. 286–290ꢀC. FTIRtmax (KBr): 3116, 1701, 1658,
1571, 1531, 1485, 1379, 1282, 1215, and 1023 cm21
.
1H NMR (CDCl3): d 3.35 (s, 3H, NACH3), 3.67 (s,
3H, NACH3), 3.92 (t, 2H, ACH2Cl, J 5 5.63 Hz),
4.03 (s, 3H, AOCH3), 4.41 (t, 2H, AOCH2A,
J 5 5.77 Hz), 7.32 (s, 1H, CH, aromatic), and 7.72
ppm (s, 1H, CH, aromatic). Calcd. for
C16H16N5O6Cl: C, 46.90; H, 3.94; N, 17.09%. Found:
C, 46.77; H, 3.76; N, 16.94%.
8-[3-Methoxy-4-(3-nitrooxypropoxy)phenyl-1,3,7-
trimethylxanthine (18) (0.2 g, 37.45%), m.p. 122–
124ꢀC. FTIRtmax (KBr): 2947, 1695, 1658, 1539, 1279,
1
1237, and 1028 cm21. H NMR (CDCl3): d 2.31 (m,
General Procedure for the Synthesis
of Xanthine Derivatives 14 and 15
2H, ACH2CH2A), 3.43 (s, 3H, NACH3), 3.63 (s, 3H,
A mixture of 8-[4-(3-chloropropoxy)]xanthine NACH3), 3.93 (s, 3H, NACH3), 4.05 (s, 3H, AOCH3),
derivatives 13f or 13g (1.0 g) and imidazole (1.0 g, 4.18 (t, 2H, AOCH2A, J 5 5.93 Hz), 4.72 (t, 2H,
14.68 mmol) was fused together at 1208C for 3 h. ACH2ONO2, J 5 6.15 Hz), 6.99 (d, 1H, CH, aromatic,
The fused reaction mixture was cooled, water was Jo 5 8.25 Hz), 7.15 (dd, 1H, CH, aromatic, Jo 5 8.13,
added to remove the unreacted imidazole and cooled Jm 5 2.12 Hz), and 7.25 ppm (m, 1H, CH, aromatic).
in ice. The solid obtained was filtered off, washed Calcd. for C18H21N5O7: C, 51.55; H, 5.05; N, 16.70%.
with water, dried and recrystallized from a mixture of Found: C, 51.21; H, 4.68; N, 16.23%.
1,3-Dimethyl-8-[3-methoxy-4-(3-nitrooxypropoxy)
methanol and chloroform to obtain the corresponding
phenyl]-7-propylxanthine (19) (0.4 g, 75.47%), m.p.
imidazole derivatives 14 and 15.
1,3-Dimethyl-8-[4-(3-imidazol-1-ylpropoxy)-5- 150–154ꢀC. FTIRtmax (KBr): 2960, 1697, 1655, 1537,
methoxy-2-nitrophenyl]xanthine (14). Yield: (0.73 g, 1475, 1438, 1260, and 1031 cm21. 1H NMR (CDCl3): d
67.91%), m.p. 238–242ꢀC. FTIRtmax (KBr): 2900, 0.88 (t, 3H, ACH2CH3, J 5 7.37 Hz), 1.86 (m, 2H,
1700, 1660, 1580, 1510, 1325, 1210, and 1040 cm21
.
ACH2CH3), 2.32 (m, 2H, ACH2CH2A), 3.44 (s, 3H,
1H NMR (CDCl3): d 2.33 (p, 2H, ACH2CH2A), 3.40 NACH3), 3.63 (s, 3H, NACH3), 3.92 (s, 3H, AOCH3),
(s, 3H, NACH3), 3.59 (s, 3H, NACH3), 4.01 (s, 3H, 4.19 (t, 2H, AOCH2A, J 5 5.89 Hz), 4.30 (t, 2H,
AOCH3), 4.07 (t, 2H, ACH2N<, J 5 5.82 Hz), 4.26 (t, NACH2, J 5 7.63 Hz), 4.72 (t, 2H, ACH2ONO2,
2H, AOCH2A,J 5 6.69 Hz), 7.02 (s, 2H, CH, imidaz- J 5 6.14 Hz), 6.98 (d, 1H, CH, aromatic, Jo 5 8.06 Hz),
ole), 7.29 (s, 1H, CH, aromatic), 7.51 (s, 1H, CH, aro- and 7.15 ppm (m, 2H, CH, aromatic). Calcd. for
Drug Dev. Res.

6
BANSAL ET AL.
200 ll. Nonradioactive DPCPX (Tocris Cookson,
Bristol, UK), in the concentration range of 1 nM to 3
lM, in half log units, was used as a standard to assess
assay integrity.
A2A Receptor Binding Assay
Twenty nanomolar [3H]ZM241385 (Tocris
Cookson, Bristol, UK) was incubated with 10 mg A2A
receptors in the presence or absence of compound in
50 mM Tris HCl buffer supplemented with 0.5mM
EDTA and 10 mM MgCl2 (Sigma Aldrich, Poole,
UK). ZM241385 (Tocris Cookson, Bristol, UK), over
the concentration range 0.1 pM-300 nM, was used as
a standard compound in this assay system. Total assay
volume was 0.2 ml. Reactants were incubated in a
Multiscreen MHAF B3 H60 filter plates (as above)
for 60 min at 25ꢀC.
Subsequently, both assays were treated a similar
manner: The reaction was terminated by rapid filtra-
tion of the multiscreen assay plates using a Millipore
manifold at a pressure of 700 mbar. The filters were
washed three times with 200 ll of the relevant assay
buffer. Scintillation fluid (Perkin Elmer, Beaconsfield,
UK) was added (100 ll/well), and bound radioactivity
determined using a Wallac Microbeta scintillation
counter (Beaconsfield, UK).
All results were expressed as a percentage of
the mean CPMs obtained as a result of the receptor
ligand reaction in the absence of any compound.
Data was plotted using GraphPad Prism, Version 2.0
(GraphPad, San Diego, CA). The apparent Ki of the
bound ligand was calculated for each active com-
pound using the Cheng–Prusoff Equation. KD values
of 1.6 nM (human A1 receptors) for [3H]DPCPX and
1 nM (human A2A receptors) for [3H]ZM241385
were previously determined (data not shown).
Fig. 2. Synthesis of 5,6-diamino-1,3-dimethyluracil (6). Reagents
and reaction conditions: (i) cyanoacetic acid, acetic anhydride,
reflux, sodium hydroxide; (ii) glacial acetic acid, sodium nitrite, ice
bath; (iii) concentrated NH4OH, sodium dithionite.
C20H25N5O7: C, 53.69; H, 5.63; N, 15.65%. Found: C,
53.43; H, 4.95; N, 15.39%.
1,3-Dimethyl-8-[3-(3-nitrooxypropoxy)phenyl]-7-
propylxanthine (21) (0.23 g, 43.07%), m.p. 120–
122ꢀC. FTIRtmax (KBr): 2961, 1697, 1660, 1540,
1
1281, 1224, and 1047 cm21. H NMR (CDCl3): d
0.87 (t, 3H, ACH2CH3, J 5 7.36 Hz), 1.84 (m, 2H,
ACH2CH3), 2.26 (m, 2H, ACH2CH2A), 3.44 (s, 3H,
NACH3), 3.63 (s, 3H, NACH3), 4.14 (t, 2H,
AOCH2A, J 5 5.85 Hz), 4.32 (t, 2H, NACH2,
J 5 7.52 Hz), 4.69 (t, 2H, ACH2ONO2, J 5 6.21 Hz),
7.05 (dd, 1H, CH, aromatic, Jo 5 8.42, Jm 5 2.31 Hz),
7.15 (m, 2H, CH, aromatic), and 7.43 ppm (t, 1H,
CH, aromatic, Jo 5 7.96 Hz). Calcd. for C19H23N5O6:
C, 54.67; H, 5.55; N, 16.78%. Found: C, 53.81; H,
5.26; N, 16.36%.
Adenosine Receptor Binding Assays
The synthesized compounds were tested in
radioligand binding assays using membranes from
cells expressing cloned human adenosine A1 (Euro-
screen, Belgium) and A2A receptors (Perkin Elmer,
Beaconsfield, UK) based on the methods previously
described [Bansal et al., 2009, 2011].
A2B Receptor Affinity
Due to the lack of a suitable radioligand the
affinity of ligands at A2B ARs was determined in
adenylyl cyclase experiments [Klotz et al., 1998].
Concentration-dependent inhibition of NECA-
stimulated adenylyl cyclase caused by antagonists was
measured in membranes from CHO cells stably
transfected with the human A2B AR. Membranes
were incubated with approximately 150,000 cpm of
[a-32P]ATP for 20 min and Ki-values were then cal-
culated from the measured IC50-values using the
A1 Receptor Binding
Basically, 10 mg human recombinant A1 recep-
tors were incubated with 20 nM [3H]DPCPX (Tocris
Cookson, Bristol, UK) in the presence or absence of
compound in a Hepes (20 mM) buffer containing
100 mM NaCl and 100 mM MgCl2 (Sigma Aldrich,
Poole, UK). The assay was incubated at 25ꢀC for 60
min in 96 well Multiscreen MHAF B3 H60 filter
plates presoaked in 0.3% polyethyleneimine (PEI)
(Millipore (Watford, UK). Total assay volume was Cheng and Prusoff equation [Klotz et al., 1998].
Drug Dev. Res.

SYNTHESIS AND EVALUATION OF A NEW SERIES OF 8-(2-NITROARYL)XANTHINES
7
Fig. 3. Synthesis of 8-(2-nitroaryl)substituted xanthine derivatives 9a–e. Reagents and reaction conditions: (i) MeOH/CH3COOH, room
temperature, 18 h; (ii) SOCl2, reflux, 30–40 min; NH4OH.
Fig. 4. Synthesis of 8-(2-nitroaryl)substituted xanthine derivatives 13a–g, 14, 15. Reagents and reaction conditions: (i) MeOH/CH3COOH,
room temperature, 18 h; (ii) SOCl2, reflux, 30–40 min; NH4OH.
Drug Dev. Res.

8
BANSAL ET AL.
Fig. 5. Synthesis of 8-nitrooxypropoxyphenylxanthines 18, 19, and 21. Reagents and reaction conditions: (i) AgNO3, CH3CN, 60ꢀC, 18 h.
RESULTS AND DISCUSSION
Chemistry
the compounds. A downfield shift (d 4.72) of
CH2AONO2 protons was observed as compared to
CH2ACl (d 3.90) of precursor chloro derivatives 16
and 17 in the NMR spectra. To comprehend the
effect of the substitution pattern of the phenyl ring,
nitrooxyaryl derivative 21 of 7-alkyl-8-(m-substituted)-
phenylxanthine (20) was also synthesized.
The synthetic pathways for the synthesis of 8-
(substituted phenyl)xanthines are depicted in Figures
2–5. 5,6-Diamino-1,3-dimethyluracil (6), the key
intermediate for the synthesis of all 8-(substituted-
phenyl)xanthines was synthesized according to
reported methods [Papesch and Schroeder, 1951;
Blicke and Godt, 1954] summarized in Figure 2.
Variously substituted nitroaldehydes (7a–e,
11a–g) were treated with compound 6 to afford cor-
responding benzylidene derivatives 8a–e and 12a–g.
A singlet integrating for one proton appeared at ꢁd
Adenosine Receptor Binding
The synthesized compounds were tested in
binding assays using cloned human adenosine A1 and
A2A receptors using [3H]DPCPX and [3H]ZM241385,
respectively. Affinities of synthesized xanthine deriva-
tives in radioligand binding assays at A1 and A2A
receptors are summarized in Table 2.
1
9.75 for N@CH in H NMR spectra of all the benzy-
lidenes. Subsequent cyclization [Senga et al., 1978] of
these compounds using thionyl chloride yielded the
desired corresponding 8-substituted 1,3-dimethylxan-
thines 9a–e and 13a–g (Figs. 3 and 4).
Imidazole derivatives 14 and 15 were also pre-
pared by fusing 13f and 13g with imidazole at 1208C
for 3 h [Bansal et al., 2011]. Compound structures
were confirmed using various spectral analyses, which
were consistent with the proposed structures as
detailed under experimental section.
7-Alkyl-8-[4-(3-chloropropoxy]phenyl derivatives
16 and 17, previously synthesized and described in
earlier reports [Bansal et al., 2011], were treated with
silver nitrate in acetonitrile at 60ꢀC to obtain nitrate
esters 18 and 19, respectively (Fig. 5). The structures
of the compounds were confirmed using various
spectral analyses. Asymmetric and symmetric stretch-
The majority of the newly synthesized 8-(2-
nitroaryl)xanthines displayed low affinity for ARs in
comparison to their complementary nitro group un-
substituted xanthine derivatives [Bansal et al., 2009],
however good selectivity for the A2A subtype was
observed with some compounds. 8-Nitroarylxanthine
9e bearing a cyclopentyloxy moiety, showed 30 times
higher binding affinity for adenosine A2A receptors
than A1 and A2B receptor subtypes. 8-(2-Nitrophenyl)
xanthines substituted with pyrrolidinylethoxy (13e),
chloroethoxy (13g) and imidazolylpropoxy (14) moi-
eties were also selective for A2A ARs. Introduction of
nitro group at 2- position of 8-aryl ring of xanthines
results in overall decreased affinity for ARs, however
selection of a suitably substituted 8-nitrophenyl ring
may result in improved selectivity for A2A receptors a
ing bands for ONO2 were present near 1540 and finding that may be useful for development of xan-
1280 cm21, respectively, in the IR spectra of both thine based A2A selective ligands. Introduction of an
Drug Dev. Res.

SYNTHESIS AND EVALUATION OF A NEW SERIES OF 8-(2-NITROARYL)XANTHINES
9
not appear to bring much change in adenosine bind-
ing affinity when compared with their chloropropoxy
counterparts. The current study provides additional
knowledge in the pursuit of substituted xanthines as
selective A2A AR ligands.
TABLE 2. AR Binding Affinities of Synthesized Xanthine Derivatives
Compound no.
A1 Ki (lM)
>30
A2A Ki (lM)
A2B Ki (lM)
>10
(9a)
5
(1.9–10)
>30
>30
>30
(9b)
(9c)
(9d)
(9e)
>30
>30
>30
>30
>30
>10
>30
>30
ACKNOWLEDGMENT
1
The financial support provided by the HSCST,
Haryana, India and Commonwealth Fellowship from
the Association of Commonwealth Universities,
United Kingdom is gratefully acknowledged.
(0.69–1.6)
>30
>30
>30
>30
(13a)
(13b)
(13c)
(13d)
(13e)
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
CONFLICT OF INTEREST
4
(1.4–13)
>30
Authors declare no conflict of interest.
(13f)
(13g)
>30
>30
>10
>30
2
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