Synthesis and theoretical studies on energetics of novel N- and O- perfluoroalkyl triazole tagged thienopyrimidines - Their potential as adenosine receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2009.12.075

A series of novel N- and O- perfluoroalkyl triazole tagged thienopyrimidines 6a-c and 7a-d was synthesized in two steps from thienopyrimidin-4-ones 2 through O- and N-propargylated regioisomers 3a-i and 4a-i respectively. Compound 2 was reacted with propargyl bromide to form O- and N-propargylated regioisomers 3 and 4 in definite proportions. Each regioisomer was separated and independently subjected to [3?+?2] cycloaddition using perfluoroalkyl azides through Click reaction under Sharpless conditions and obtained exclusively anti product in each case. The formation of two regioisomers in the first step and single anti addition product in the next step could be explained based on computational studies carried out at B3LYP/6-31G(d) level of theory. Results of Fukui function indices at the reactive centers are in accordance with the observations. On evaluation of the synthesized molecules for their binding affinities towards adenosine receptors, 4d and 4f were found to be selective to A₁ over A_2A receptors.

Full text of DOI:10.1016/j.ejmech.2009.12.075

European Journal of Medicinal Chemistry 45 (2010) 1739–1745
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and theoretical studies on energetics of novel N- and O- perfluoroalkyl
triazole tagged thienopyrimidines – Their potential as adenosine receptor ligands
,
a
b
b
c
B. Sirisha a, B. Narsaiah a , T. Yakaiah , G. Gayatri , G. Narahari Sastry , M. Raghu Prasad ,
*
A. Raghuram Rao d
a Fluoroorganic Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
b Molecular Modeling Division, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
c Vishnu College of Pharmacy, Bhimavaram, India
d University Institute of Pharmaceutical Sciences and UGC-CAS, Punjab University, Chandigarh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N- and O- perfluoroalkyl triazole tagged thienopyrimidines 6a–c and 7a–d was
synthesized in two steps from thienopyrimidin-4-ones 2 through O- and N-propargylated regioisomers
3a–i and 4a–i respectively. Compound 2 was reacted with propargyl bromide to form O- and N-prop-
argylated regioisomers 3 and 4 in definite proportions. Each regioisomer was separated and indepen-
dently subjected to [3 þ 2] cycloaddition using perfluoroalkyl azides through Click reaction under
Sharpless conditions and obtained exclusively anti product in each case. The formation of two
regioisomers in the first step and single anti addition product in the next step could be explained based
on computational studies carried out at B3LYP/6-31G(d) level of theory. Results of Fukui function indices
at the reactive centers are in accordance with the observations. On evaluation of the synthesized
molecules for their binding affinities towards adenosine receptors, 4d and 4f were found to be selective
to A1 over A2A receptors.
Received 24 September 2009
Received in revised form
1 December 2009
Accepted 31 December 2009
Available online 14 January 2010
Keywords:
Propargyl bromide
Thienopyrimidines
Perfluoroalkyl triazole
Sharpless conditions
Cycloaddition
Ó 2010 Elsevier Masson SAS. All rights reserved.
Regioisomers
1. Introduction
classic isosteric modification of triazolo thienopyrimidines is done
to evaluate them for adenosine binding activity. Perfluorinated alkyl
The bioisosteric replacement of benzene ring with
p
-excess
chain was selected to increase the lipophilicity of molecules in order
to cross the blood brain barrier. The synthesis of title compounds
was performed using Cu(I)-catalyzed Huisgen [3 þ 2] dipolar
cycloaddition reaction between an organic azide and an alkyne
commonly known as click chemistry. This reaction has several
applications in chemistry, biology and materials science. It has
enabled demanding bio-conjugations involving more number of
steps and has been used in activity-based protein profiling (ABPP) of
crude proteome homogenates for selective labeling of modified
bacterial cell walls and in the synthesis of novel biologically active
compounds [13–15]. Initially the synthesis of O-/N-propargylated
thienopyrimidines was carried out and the variation in the product
yield is correlated with the substituent at the 2nd position. Position
and orientation of attack of reagent on the transition state of the
staring material was predicted with the help of chemoinformatics to
elucidate the observed synthetic trends. Further, existence of two
reactive nitrogens inperfluoroalkyl azide in reactionwith alkyne can
lead to the formation of 1, 4 and 1, 5-disubstituted alkyl [1–3]
triazoles through their syn and anti additions. But, formation of
only 1,4-disubstituted triazole in considerable yields along with
thiophene has been an established ligand-based drug design
strategy to optimize various lead structures. Diverse functionalized
thiophene derivatives were reported as adenosine receptor ligands
for the past twenty years [1–3]. Thiophene fused pyrimidines called
thienopyrimidines exhibit DHFR inhibitory [4] CDK4 inhibitory [5],
anti-inflammatory [6] activities. Several quinazolines and thieno-
pyrimidines were reported from our laboratory as antihistaminic
[7] antibacterial [8] bronchodilatory [9], anti-inflammatory agents
[10] and as adenosine receptor ligands [11].
Based on the importance and in continuation to our investiga-
tions, we report the synthesis of novel derivatives with thienopyr-
imidine nucleus. Since five-membered rings with more number of
nitrogenslike tetrazole, triazole and imidazole is found to be the best
ranked substitutions in antagonizing a wide variety of macromo-
lecular targets [12], we attempted to synthesize O, N- propargylated
thienopyrimidines and 3/4-triazolyl thienopyrimidines. Such a non-
* Corresponding author. Tel.: þ91 40 27193630; fax: þ91 40 27193185.
E-mail address: narsaiah_1984@yahoo.co.in (B. Narsaiah).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.075

1740
B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
remaining amount of starting material in the experimental studies
led us to use density functional theory to unravel the experimental
observations. Molecular modeling studies were carried out and the
reactants, products and transition states were optimized at B3LYP/
6–31G* level of theoryas B3LYP with 6–31G* basis set is proved to be
better in modeling the reactions [16]. IRC calculations were per-
formed to characterize the transition states. Fukui function indices
were calculated using the following equations
I
Acetone
N3
F
17C8
F17C8
+
NaN3
H2O, reflux
Scheme 2. Synthesis of perfluorinated alkylazides 5.
1,4-disubstituted-1,2,3-triazole derivatives 6 and 7, respectively
according to Schemes 3 and 4. The reaction is considered to take
place via 1,3-dipolar cycloaddition of perfluoroalkyl azide to alkyne
through a preformed copper acetylide complex formation [24,25].
The percentage yields of products tabulated in Tables 1 and 2. The
cycloaddition reaction was also performed in DMSO in order to see
the influence of solvent on the formation of products and found to
give better yields.
fkþ ¼ ½qkðN þ 1Þ ꢀ qkðNÞꢁ for nucleophilic attack
(1)
(2)
(3)
h
i
fkꢀ
¼
qkðNÞ ꢀ qkðN ꢀ 1Þ for electrophilic attack
fkꢂ ¼ ½qkðN þ 1Þ ꢀ qkðN ꢀ 1Þꢁ for free radical attack
All the calculations were performed using G03W program [17].
The charge densities were obtained using AIM2000 program [18].
2.2. Modeling studies
Molecular modeling studies were carried out in order to
understand the observed synthetic trends. As the nature of
substituent (–R1 and –R2 groups) seems to be inconsequential with
respect to the observed yields of 3a–i and 4a–i, it was felt logical to
replace them with H in all computational calculations.
2. Results and discussion
2.1. Chemistry
From the experimental trends, the R group is mainly found to
influence the site of attack of nucleophile which would result in
formation of O- and N- propargylated thienopyrimidines 3 and 4
from thienopyrimidines 2. Computational calculations were per-
formed on thienopyrimidine 2 by taking R as H, –CH3 or –C6H5, as
representatives of unsubstituted, aliphatic (alkyl) and aromatic
(aryl) groups respectively. Both gas phase and solvent calculations
have been carried out on thienopyrimidine in order to examine the
solvent dependency. The activation and reaction energies (in kcal/
mol) for both gas and solvent phases are illustrated in Table 3. IRC
calculations were performed to characterize the transition states
which further confirmed the authenticity of transition states.
Initially the solvent effects on the reactivity were studied,
followed by the comparison of reactivity of CH3 and C6H5
substituted compounds with that of unsubstituted (H as substit-
uent) compounds which is followed by the explanation for the site
selective addition at N and O sites. From the data (Table 3) it can be
understood that the addition of solvent decreases the activation
The initial step involves the synthesis of thiophene 2-amino-
3-carboxylic acid ethyl ester 1 from cyanoethylacetate, ketone and
sulfur in ethanol using morpholine as base [19]. Then preparation of
thieno [2,3-d] pyrimidin-4(3H)-ones 2 from compound 1 and alkyl/
aryl nitrile in dioxane using dry HCl [20]. The thieno [2,3-d] pyr-
imidin-4(3H)-ones 2 were reacted with propargyl bromide in
acetone using potassium carbonate as a base to yield two products
namely O- and N-propargylated thieno pyrimidines 3 and 4 in
definite proportions [21] as outlined in Scheme 1. The formation of
each regioisomer depends on the bulkiness/electron-withdrawing
or donating nature of substituents present. The O-propargylated
product 3 is formed in major when R is aromatic, while N-prop-
argylated compound 4 is major when –R is aliphatic. In the second
step, synthesis of various perfluoroalkyl azides 5 was performed as
outlined in Scheme 2 [22]. All the products were separated through
column chromatography and characterized based on their differ-
ence in polarity.
Further the O- and N-propargylated thienopyrimidines 3 and 4
were independently reacted with perfluoroalkyl azide 5 in THF,
using copper (I) iodide as catalyst [23] and resulted in exclusively
energies and reaction energies (except for
H substituted
compounds). The H substituted compounds show increase in both
activation and reaction energies compared to the CH3 and C6H5
substituted compounds. Since variation in the yields seems to be
mainly due to the aliphatic and aromatic groups, further studies
were carried out based on the site selectivity at N and O positions.
Also from the table, it can be inferred that the N-propargylated
thienopyrimidine (4) with methyl substitution is found to be stable,
both kinetically and thermodynamically. Replacement of –R with
–C6H5 suggested the formation of kinetically stable O-propargy-
lated thienopyrimidine and thermodynamically stable N-prop-
argylated thienopyrimidines. However the difference between the
activation energies for 2-A–Me and 2-B–Me, and 2-A–Ph and 2-B–
Ph regioisomers in gas/solvent phases are 0.0/0.4 and 3.8/3.0
COOC2H5
NH2
R1
Morpholine
Ethanol
C2H5OOC
NC
+ R -CO-CH -R
+ S
1
2
2
R2
S
1a-c
O
R1
Dioxane
Dry HCl
RCN
NH
R2
Br
Acetone,
K2CO3
R
S
N
2a-i
R = -CH3, -C6H5, -CH2C6H4(4-F)
R1 = R2 = -CH3, -(CH2)4, -(CH2)3
C8F17
N
N
O
R1
O
O
O
N
R1
R1
N
R1
C8F17
R2
N
N3
R2
N
N
+
R2
CuI
N
R2
S
N
R
3
S
R
R = -C6H5
R1 = R2 = -CH3, -(CH2)4, -(CH2)3
R
S
R
S
N
6
N
3a-i
4a-i
Scheme 1. Synthesis of O- and N- propargylated thienopyrimidines 3 and 4.
Scheme 3. Synthesis of O- perfluoroalkyl triazole tagged thienopyrimidines 6.

B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
1741
O
R1
O
N
R1
C8F17
N
N
C8F17
R2
N
N3
R2
N
N
CuI
S
N
4
R
S
R
7
R = -CH3, -CH2C6H4(4-F)
R1 = R2 = -CH3, -(CH2)4, -(CH2)3
Scheme 4. Synthesis of N- perfluoroalkyl triazole tagged thienopyrimidines 7.
respectively. So, further studies are carried out in order to look at
the regioselectivity. The next section discusses the AIM analysis
which is followed by explaining the reactivity based on Fukui
function indices. These studies give a better understanding about
Further [3
þ
2] cycloaddition reaction between N- and
O-propargylated thienopyrimidines, and alkyl azides was analyzed.
Theoretical studies on such molecules using Cu as catalyst along
with the mechanism is well established [25]. The ‘C2H4C8F17’ group
on the azide nitrogen is replaced with ‘CH3’ and the results
obtained are illustrated in Table 3. The theoretical results clearly
suggested the formation of anti product over syn, both kinetically
and theromodynamically which were in accordance with experi-
mental results. This is assumed to be mainly due to the steric
repulsions arising from the bulky alkyl group attached to the azide.
the factors responsible for the formation of
regioisomer.
a particular
The electronic charge densities (r) and hydrogen bond lengths
(in Å) between ‘N–H’ and ‘O–H’ for 3 and 4 respectively are depicted
in Fig. 1. The hydrogen bond lengths and charge density values for
‘O–H’ and ‘N–H’ bonds are considered in order to see whether the
stabilizing factors in such cases can be attributed to hydrogen
bonding. From the Fig. 1, it can be inferred that, the hydrogen bond
‘N–H’ stabilizes 2-A–Ph–TS compared to 2-B–Ph–TS, while the
hydrogen bond between "O–H" stabilizes 2-B–Me–TS compared to
2-A–Me–TS.
The Fukui function indices for the reacting sites were calculated
as they have been proved to be better in explaining the regiose-
lectivity [26,27]. Fukui function indices are the local reactivity
descriptors introduced by Parr and coworkers based on the frontier
orbital concept given by Fukui [28–31]. Since the thienopyrimidine
exists in lactim and lactam forms, the reactant was considered
based on the product formation. In the case of O-propargylated,
lactim form and N-propargylated product, lactam form was
considered. Also as the thienopyrimidine acts as a nucleophile, fk–
values on the reactive centers were considered. The reactants along
with the Fukui function indices values are depicted in Fig. 2. From
the figure, it can be observed that in the case of ‘CH3’ substitution,
the reactive site ‘N’ (0.112) is preferred to ‘O’ (0.106) while in the
case of ‘C6H5’ substitution, ‘O’ is preferred (0.458) to ‘N’ (0.044).
From the above computational studies, it can be clearly understood
that when the –R is aromatic, O-propargylated product is feasible
while when R group is aliphatic, N-propargylated product is
feasible.
2.3. Pharmacological studies
Representative compounds were selected and tested for their
ability to displace [3H] 2-chloro-N6-cyclohexyladenosine
([3H]CCPA) from A1ARs and [3H]MSX-2 from A2AAR in rat cortical
and striatal membranes, respectively [11]. The A1 and A2A receptor
binding affinities of compounds are expressed as Ki values of
radioligand binding (at 1.0
Table 4.
mM concentration) and presented in
All the compounds evaluated were found to bind with adeno-
sine A1 and A2A receptors with moderate efficiency. The compounds
4d, 4f, 4h were selectively bind to A1 receptor. The compound 4a
was found to be potent in binding with A1 and A2A receptor but
with lack of selectivity. The substitution at second position was
found to influence the binding, where selectivity towards A2A
receptor was increased with aryl group (4e) and reduced with alkyl
and aralkyl substitutions (4d, 4f). Except 4d, 4f, 4h, all other
molecules evaluated were showed better binding profiles when
compared to standard DPCPX.
3. Conclusion
The formation of O- and N-propargylated thienopyrimidines in
definite proportions was established based on substitution present
in 2nd position. Further [3 þ 2] cycloaddition of 3 or 4 with
perfluorinated azides resulted in the exclusive formation of ‘anti’
product. The formation of products was analyzed both kinetically
and thermodynamically at B3LYP/6-31G* level of theory. Fukui
function indices values and charge critical densities were in
accordance with the observed experimental trends. Representative
compounds were found to be selective for A1 over A2A receptors on
Table 1
Percentage yields of propargylated thienopyrimidines 3 and 4.
Cmpd
R1
R2
R
% Yield
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
p-F–C6H4–CH2
p-F–C6H4–CH2
CH3
CH3
C6H5
8.5
76.7
88.18
4.4
15.17
75.89
7.11
80.16
82.31
6.09
4.0
CH3
CH3
CH3
CH3
CH3
CH3
Table 2
Percentage yields of perfluoroalkyl triazole tagged thienopyrimidines 6 and 7.
C6H5
Cmpd
R1
R2
R
% Yield
p-F–C6H4–CH2
p-F–C6H4–CH2
CH3
CH3
C6H5
C6H5
p-F–C6H4–CH2
p-F–C6H4–CH2
6a
6b
6c
7a
7b
7c
7d
–(CH2)4–
CH3
–(CH2)3–
–(CH2)4–
CH3
C6H5
C6H5
C6H5
CH3
CH3
p-F–C6H4–CH2
CH3
59.33
42.45
52.93
69.44
40.22
45.00
41.66
4f
CH3
70.79
7.6
CH3
3g
4g
3h
4h
3i
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
80.45
92.02
4.38
8.88
71.04
CH3
CH3
CH3
–(CH2)3–
4i

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B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
Table 3
H
H
Activation and reaction energies of the compounds studied. Values in parenthesis
are obtained using PCM model, acetone is used as solvent.
O
O
N
0.106
0.458
H
O
O
N
N
H
N
N
S
S
S
N
O
Me
Ph
R
S
R
S
O
N
2-A
N
2-B
H
R=H, Me, Ph
H
N
N
0.044
0.112
DE
z (K. cal)
DEr (K. cal)
2-A–H
23.7(21.7)
23.6 (5.5)
23.1 (6.1)
22.5(17.1)
23.6(5.1)
26.9(9.1)
16.8
16.5
17.1
16.7
ꢀ64.7 (ꢀ129.3)
ꢀ63.49 (ꢀ20.4)
ꢀ63.09 (ꢀ19.9)
ꢀ74.0 (ꢀ140.0)
ꢀ71.81 (ꢀ30. 6)
ꢀ65.52 (ꢀ23.3)
ꢀ71.1
S
N
Ph
N
Me
2-A–Me
2-A–Ph
2-B–H
2-B–Me
2-B–Ph
Fig. 2. Reactants along with their fukui function indices
a VG 7070 H instrument at 70 eV. All reactions were monitored by
thin layer chromatography (TLC) on precoated silica gel 60
F254 (mesh); Spots were visualized with UV light. Merck silica gel
(100–200 mesh) was used for chromatography. CHN analyses were
recorded on a Vario EL analyser.
3 þ RN3-syn
3 þ RN3-anti
4 þ RN3-syn
4 þ RN3-anti
ꢀ73.3
ꢀ70.7
ꢀ75.9
4.2. Synthesis
evaluation of their adenosine binding affinities which offers a clue
to design more potent ligands with improved selectivity.
Preparation of 2-amino-4,5-substituted thiophene-3-carboxylic
acid ethyl ester (1) [19]
Preparatin of 2, 5, 6 substituted thienopyrimidin-4-one (2) [20].
Preparation of propargylated thieno [2,3-d] pyrimidines (3) and
thieno [2,3-d] pyrimidin-4(3H)ones (4).
4. Experimental section
4.1. General
Melting points were recorded on Casia-siamia (VMP-AM)
melting point apparatus and are uncorrected. IR spectra were
recorded on a Perkin–Elmer FT-IR 240-C spectro photometer using
KBr optics. 1H NMR spectra were recorded on Gemini varian
200 MHz, Bruker AV 300 MHz and Unity 400 MHz spectrometer in
DMSO-d6 or CDCl3 using TMS as an internal standard. Electron
impact (EI) and chemical ionization mass spectra were recorded on
4.2.1. General procedure
Compound 2 (3.0 mmol) and potassium carbonate (6.0 mmol)
was taken in acetone (20 mL) and propargylbromide (6.0 mmol) was
added drop-wise. The reaction mixture was heated under reflux for
5 h, concentrated in vacuo and the residue was washed with n-
hexane to remove excess propargyl bromide followed by treated
with ice-cold water. The separated solid product was collected by
filtration. The O- and N- propargylated products 3 and 4 were
separated by column chromatography using n-hexane:ethylacetate.
4.2.2. 2-Methyl-4-(prop-2-yn-1-yloxy)-5,6,7,8-tetrahydro[1] benzo-
thieno[2,3-d]pyrimidine (3a)
White solid, yield: 8.5% (65 mg), mp:135 ꢃC, IR (KBr): 2116
(–C^C–) cmꢀ1 1H NMR (CDCl3): 1.86–1.96 (m, 4H, 2CH2), 2.42–
;
2.46 (s, 1H, –C^CH), 2.66 (s, 3H, CH3), 2.78–2.86 (m, 2H, CH2), 2.92–
Table 4
Radioligand binding data of the molecules with A1 receptor (rat brain cortical
membranes) and A2A receptor (rat brain striaital membranes).
Cmpd
A1[3H]CCPA
A2A[3H] MSX-2
Ki ꢄ SEM [nM]
3b
3h
4a
4c
4d
4e
4f
4g
4h
7a
(ꢀ10 ꢄ 4) (n ¼ 4)
(ꢀ3 ꢄ 7) (n ¼ 3)
(0 ꢄ 3) (n ¼ 4)
(21 ꢄ 5) (n ¼ 4)
(6 ꢄ 4) (n ¼ 4)
(ꢀ1 ꢄ 2) (n ¼ 3)
(4 ꢄ 8) (n ¼ 4)
(6 ꢄ 5) (n ¼ 4)
(5 ꢄ 3) (n ¼ 4)
(ꢀ14 ꢄ 7) (n ¼ 4)
0.5 þ 0.2
(3 ꢄ 6) (n ¼ 3)
(ꢀ10 ꢄ 9) (n ¼ 4)
(7 ꢄ 10) (n ¼ 3)
(14 ꢄ 6) (n ¼ 3)
3790 ꢄ 370 (n ¼ 3)
(29 ꢄ 6) (n ¼ 4)
7620 ꢄ 2930 (n ¼ 3)
(18 ꢄ 13) (n ¼ 3)
9700 ꢄ 640 (n ¼ 3)
(ꢀ8 ꢄ 3) (n ¼ 4)
157 þ 6.0
Fig. 1. Bond distances (normal) in Å and electronic charge densities in amu on the ‘N/
H’ and ‘O/H’; bonds for the alkynyl thienopyrimidine transition states Transition state
is represented as ‘TS’.
DPCPX

B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
1743
3.02 (m, 2H, CH2), 5.10 (s, 2H, OCH2); EIMS, m/z: 259 (Mþ þ 1); Anal.
Calcd. for C14H14N2OS: C, 65.09; H, 5.46; N, 10.84%. Found: C, 64.84;
H, 5.68; N, 11.02%.
(s, 2H, NCH2); EIMS, m/z: 233(Mþ
C12H12N2OS: C, 62.04; H, 5.21; N, 12.06%. Found: C, 61.88; H, 5.03;
N, 12.24%.
þ
1); Anal. Calcd. for
4.2.3. 2-Methyl-3-prop-2-yn-1-yl-5, 6, 7, 8-tetrahydro [1]
benzothieno[2,3-d]pyrimidin-4(3H)-one (4a)
4.2.10. 5,6-Dimethyl-2-phenyl-4-(prop-2-yn-1-yloxy) thieno
[2,3-d]pyrimidine (3e)
White solid, yield: 76.7% (595 mg), mp: 195 ꢃC; IR (KBr): 1679
White solid, yield: 82.31% (720 mg), mp: 153 ꢃC, IR(KBr): 2119
(–C^C–) cmꢀ1; 1H NMR (CDCl3): 2.42–2.51 (m, 7H, –C^CH, 2CH3),
5.24 (m, 2H, OCH2), 7.38–7.47 (m, 3H, Ar–H), 8.42–8.48 (m, 2H, Ar–
H); EIMS, m/z: 295 (Mþ þ 1); Anal. Calcd. for C17H14N2OS: C, 69.36;
H, 4.7; N, 9.52%. Found: C, 69.54; H, 5.01; N, 9.27%.
(C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 1.78–1.92(m, 4H,
;
2CH2), 2.22–2.26 (s, 1H, –C^CH), 2.68(s, 3H, CH3), 2.72–2.78(m, 2H,
CH2), 2.91–3.2 (m, 2H, CH2), 4.85 (s, 2H, NCH2); EIMS, m/z: 259
(Mþ þ 1); Anal. Calcd. for C14H14N2OS: C, 65.09; H, 5.46; N, 10.84%.
Found: C, 65.27; H, 5.21; N, 11.06%.
4.2.11. 5, 6-Dimethyl-2-phenyl-3-prop-2-yn-1-ylthieno [2, 3-d]
pyrimidin-4 (3H)-one (4e)
4.2.4. 2-Phenyl-4- (prop-2-yn-1-yloxy)-5,6,7,8-tetrahydro
[1]benzothieno [2,3-d]pyrimidine (3b)
White solid, yield: 6.1% (55 mg), m.p. 185 ꢃC, IR (KBr):1643
White solid, yield: 88.18% (845 mg), mp:121 ꢃC, IR (KBr): 2118
(–C^C–) cmꢀ1; 1H NMR (CDCl3): 1.84–1.98 (m, 4H, 2CH2), 2.42–2.46
(s, 1H, –C^CH), 2.82–2.90 (m, 2H, CH2), 2.84–3.3 (m, 2H, CH2), 5.24
(s, 2H, OCH2), 7.40–7.49 (m, 3H, Ar–H), 8.41–8.49 (m, 2H, Ar–H);
EIMS, m/z: 321 (Mþ þ 1); Anal. Calcd. for C19H16N2OS: C, 71.22; H,
5.03; N, 8.74%. Found: C, 71.39; H, 4.87; N, 8.99%.
(C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 2.31–2.36 (s, 1H,
;
–C^CH), 2.46 (s, 3H, CH3), 2.56 (s, 3H, CH3), 4.68 (m, 2H, NCH2),
7.51–7.59 (m, 3H, Ar–H), 7.72–7.78 (m, 2H, Ar–H); EIMS, m/z: 295
(Mþ þ 1); Anal. Calcd. for C17H14N2OS: C, 69.36; H, 4.79; N, 9.52%.
Found: C, 69.20; H, 5.02; N, 9.68%.
4.2.12. 2-(4-Fluorobenzyl) -5,6-dimethyl-4- (prop-2-yn-1-yloxy)
thieno [2,3-d] pyrimidine (3f)
4.2.5. 2-Phenyl-3-prop-2-yn-1-yl-5,6,7,8-tetrahydro [1]
benzothieno [2,3-d] pyrimidin-4(3H)-one (4b)
White solid, yield: 4% (30 mg), mp: 105 ꢃC, IR (KBr): 2119
White solid, yield: 4.4% (45 mg), mp:175 ꢃC, IR (KBr):1672
(–C^C–) cmꢀ1 1H NMR (CDCl3):), 2.44–2.56 (m, 7H, -ChCH,
;
(C]O), 2116 (–C^C–) cmꢀ1
;
1H NMR (CDCl3): 1.78–1.90 (m, 4H,
2CH3), 4.10 (s, 2H, CH2), 5.34 (s, 2H, OCH2), 7.02 (m, 2H, Ar–H), 7.28
2CH2), 2.22–2.26 (s, 1H, –C^CH), 2.72–2.78 (m, 2H, CH2), 2.91–3.2
(m, 2H, CH2), 4.74 (s, 2H, NCH2), 7.51–7.59 (m, 3H, Ar–H), 7.72–7.78
(m, 2H, Ar–H); EIMS, m/z: 321 (Mþ þ 1); Anal. Calcd. for
C19H16N2OS: C, 71.22; H, 5.03; N, 8.74%. Found: C, 71.05; H, 4.73; N,
9.0%.
(m, 2H, Ar–H) EIMS, m/z: 327 (Mþ
C18H15FN2OS: C, 66.24; H, 4.63; N, (8.58) %. Found: C, 66.49; H, 4.47;
N, 8.75%.
þ
1); Anal. Calcd. for
4.2.13. 2-(4-Fluorobenzyl)-5,6-dimethyl-3-prop-2-yn-
1-ylthieno[2,3-d]pyrimi din-4(3H)-one (4f)
4.2.6. 2-(4-Fluorobenzyl)-4-(prop-2-yn-1-yloxy)-5, 6, 7,
8-tetrahydro [1]benzothieno[2,3-d] pyrimidine (3c)
White solid, yield: 70.8% (695 mg), mp: 128 ꢃC, IR
(KBr):1670 (C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 2.28–
;
White solid, yield: 15.17% (160 mg), mp: 146 ꢃC, IR (KBr): 2116
2.32 (s, 1H, –C^CH), 2.42 (s, 3H, CH3), 2.47 (s, 3H, CH3), 4.38 (s,
2H, CH2), 4.75 (s, 2H, NCH2), 7.02–7.10 (m, 2H, Ar–H), 7.25–7.35
(m, 2H, Ar–H); EIMS, m/z: 327 (Mþ þ 1); Anal. Calcd. for
C18H15FN2OS: C, 66.24; H, 4.63; N, 8.58%. Found: C, 65.99; H,
4.85; N, 8.76%.
(–C^C–) cmꢀ1 1H NMR (CDCl3): 1.32–1.38(m, 2H, CH2), 1.38–
;
1.41(s, 1H, –C^CH), 1.81–1.93 (m, 4H, 2CH2), 2.78–2.85 (m, 2H,
CH2), 4.10 (s, 2H, CH2), 5.04 (s, 2H, OCH2), 6.85–6.98 (m, 2H, Ar–H),
7.22 (s, 1H, Ar–H), 7.28–7.36 (m, 1H, Ar–H); EIMS, m/z: 353
(Mþ þ 1); Anal. Calcd. for C20H17FN2OS: C, 68.16; H, 4.86; N, 7.85%.
Found: C, 68.38; H, 5.04; N, 7.7%.
4.2.14. 2-Methyl-4-(prop-2-yn-1-yloxy)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d] pyrimidine (3g)
4.2.7. 2-(4-Fluorobenzyl)-3-prop-2-yn-1-yl-5,6,7,
White solid, yield: 7.6% (55 mg), mp: 118 ꢃC, IR (KBr): 2116
(–C^C–) cmꢀ1;1H NMR (CDCl3): 2.40–2.44 (s, 1H, –C^CH), 2.46–
2.51 (m, 2H, CH2), 2.66(s, 3H, CH3), 2.98–3.08 (m, 4H, 2CH2), 5.12 (s,
2H, OCH2); EIMS, m/z: 245 (Mþ þ 1); Anal. Calcd. for C13H12N2OS: C,
63.91; H, 4.95; N, 11.47%. Found: C, 64.14; H, 4.79; N, 11.68%.
8-tetrahydro[1]benzothieno [2,3-d] pyrimidin -4(3H) – one (4c)
White solid, yield: 75.89% (800 mg), mp: 167 ꢃC, IR (KBr): 1620
(C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 1.78–1.92 (m, 4H,
;
2CH2), 2.21–2.28 (s, 1H, –C^CH), 2.72–2.86 (m, 2H, CH2), 2.91–3.06
(m, 2H, CH2), 4.30 (s, 2H, CH2), 4.76 (s, 2H, NCH2), 6.92–7.08 (m, 2H,
Ar–H), 7.18–7.31 (m, 2H, Ar–H); EIMS, m/z: 353 (Mþ þ 1); Anal.
Calcd. for C20H17FN2OS: C, 68.16; H, 4.86; N, 7.85%. Found: C, 68.05;
H, 5.11; N, 8.12%.
4.2.15. 2-Methyl-3-prop-2-yn-1-yl-3,5,6,7-tetrahydro-
4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-one (4g)
White solid, yield: 84.45% (620 mg), mp: 210 ꢃC, IR (KBr):1670
(C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 2.28–2.32 (s, 1H,
;
4.2.8. 2,5,6-Trimethyl-4- (prop-2-yn-1-yloxy)thieno[2,3-d]pyrimidine
(3d)
–C^CH), 2.42–2.56 (m, 2H, CH2), 2.47 (s, 2H, CH2), 2.72 (s, 3H, CH3),
2.78–2.98 (m, 2H, CH2), 4.88 (s, 2H, NCH2); EIMS, m/z: 245
(Mþ þ 1); Anal. Calcd. for C13H12N2OS: C, 63.91; H, 4.95; N, 11.47%.
Found: C, 63.75; H, 5.18; N, 11.65%.
White solid, yield: 7.11% (50 mg), mp: 129 ꢃC, IR(KBr):2116
(–C^C–) cmꢀ1; 1H NMR (CDCl3): 2.38–2.48 (m, 7H, –C^CH, 2CH3),
2.62 (s, 3H, CH3), 5.10 (s, 2H, OCH2); EIMS, m/z: 233 (Mþ þ 1); Anal.
Calcd. for C12H12N2OS: C, 62.04; H, 5.21; N, 12.06%. Found: C, 62.3;
H, 5.04; N, 11.76%.
4.2.16. 2-Phenyl-4-(prop-2-yn-1-yloxy)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (3h)
White solid, yield: 92.02% (845 mg), mp 159 ꢃC, IR (KBr):2116
(–C^C–); 1H NMR (CDCl3): 2.44–2.46 (s, 1H, –C^CH), 2.46–2.52
(m, 2H, CH2), 2.98–3.11 (m, 4H, 2CH2), 5.25 (s, 2H, OCH2), 7.38–7.48
(m, 3H, Ar–H), 8.43–8.52 (m, 2H, Ar–H); EIMS, m/z: 307 (Mþ þ 1);
Anal. Calcd. for C18H14N2OS: C, 70.56; H, 4.61; N, 9.14%. Found: C,
70.26; H, 4.87; N, 8.97%.
4.2.9. 2,5,6-Trimethyl-3-prop-2-yn-1-ylthieno[2,3-d]pyrimidin-
4(3H)-one (4d)
White solid, yield: 80.16% (560 mg), mp: 153 ꢃC, IR (KBr): 1620
(C]O), 2116 (–C^C–) cmꢀ1 1H NMR (CDCl3): 2.21–2.26 (s, 1H,
;
–C^CH), 2.36 (s, 3H, CH3), 2.44 (s, 3H, CH3), 2.68 (s, 3H, CH3), 4.86

1744
B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
4.2.17. 2-Phenyl-3-prop-2-yn-1-yl-3,5,6,7-tetrahydro-
4H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-4-one (4h)
White solid, yield: 4.38% (40 mg), mp: 205 ꢃC, IR (KBr): 1672
(C]O), 2116 (–C^C–); 1H NMR (CDCl3): 2.28–2.33 (s, 1H,
–C^CH), 2.47–2.58 (m, 2H, CH2), 2.82–3.0 (m, 2H, CH2), 3.08–
3.18 (m, 2H, CH2) 4.68 (s, 2H, NCH2), 7.49–7.56 (m, 3H, Ar–H),
7.69–7.77 (m, 2H, Ar–H); EIMS, m/z: 307 (Mþ þ 1); Anal. Calcd.
for C18H14N2OS: C, 70.56; H, 4.61; N, 9.14%. Found: C, 70.74; H,
4.36; N, 9.36%.
2.42–2.51 (m, 6H, 2CH3), 2.73–2.9 (m, 2H, CH2), 4.60–4.65 (t, 2H,
NCH2), 5.82 (s, 2H, OCH2), 7.40–7.50 (m, 3H, Ar–H), 7.65 (s, 1H,
–C]CH), 8.45–8.52 (m, 2H, Ar–H); EIMS, m/z: 784 (Mþ þ 1); Anal.
Calcd. for C27H18F17N5OS: C, 41.39; H, 2.32; N, 8.94%. Found: C,
41.22; H, 2.16; N, 9.19%.
4.2.23. 2-Phenyl-4{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadeca
fluoro decyl)-1H-1, 2, 3-triazol-4-yl)methyl}oxy)-6,7-dihydro-
5H-cyclopenta[4,5] thieno[2,3-d]pyrimidine (6c)
White solid, yield: 52.93% (420 mg), mp: 198.6 ꢃC; IR (KBr): 1572
4.2.18. 2-(4-Fluorobenzyl)-4-(prop-2-yn-1-yloxy)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d] pyrimidine (3i)
(C]N), 1501 (C]C), 1255 (C–O–C) cmꢀ1 1H NMR (CDCl3): 2.47–
:
2.58 (m, 2H, CH2), 2.75–2.98(m, 2H, CH2), 3.02–3.12 (m, 4H, 2CH2),
4.60–4.69(t, 2H, NCH2), 5.85(s, 2H, OCH2), 7.45–7.56 (m, 3H, Ar–H),
7.70(s, 1H, –C]CH), 8.52–8.58(m, 2H, Ar–H); EIMS, m/z: 796
(Mþ þ 1); Anal. Calcd. for C28H18 F17N5OS: C, 42.27; H, 2.28; N,
8.80%. Found: C, 42.45; H, 2.50; N, 8.55%.
White solid, yield: 8.88%, (90 mg), mp: 120 ꢃC, IR (KBr):2119
(–C^C–) cmꢀ1; 1H NMR (CDCl3): 2.30–2.32 (s, 1H, –C^CH), 2.50–
2.54 (m, 2H, CH2), 2.80–3.0 (m, 2H, CH2), 3.06–3.14(m, 2H, CH2)
4.10(s, 2H, CH2), 5.4(s, 2H, OCH2), 7.04–7.2(m, 2H, Ar–H), 7.28 (m,
2H, Ar–H); EIMS, m/z: 339 (Mþ
C19H15FN2OS: C, 67.44; H, 4.47; N, 8.28%. Found: C, 67.16; H, 4.72;
N, 8.46%.
þ
1); Anal. Calcd. for
4.2.24. 2-Methyl-3-{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
10-heptadeca fluoro decyl)-1H-1,2,3-triazol-4-yl)methyl}-5,6,7,
8-tetrahydro [1]benzothieno [2,3-d]pyrimidin-4(3H)-one (7a)
White solid, yield: 69.44% (518 mg), mp: 189 ꢃC; IR(KBr):1620
4.2.19. 2-(4-Fluorobenzyl)-3-prop-2-yn-1-yl-3,5,6,7-tetrahydro-
4H-cyclopenta [4,5] thieno [2,3-d] pyrimidin-4-one (4i)
(C]O), 1551 (C]N), 1456 (C]C) cmꢀ1 1H NMR (CDCl3): 1.8–1.95
;
White solid, yield: 71.04% (720mg), mp: 140 ꢃC, IR (KBr):1670
(m, 4H, 2CH2), 2.8 (s, 3H, CH3), 2.85–2.90 (m, 4H, 2CH2), 2.95 (s,
2H, CH2), 4.57–4.68 (m, 2H, NCH2), 5.30 (s, 2H, NCH2), 7.8(s, 1H,
(C]O), 2116 (–C^C–) cmꢀ1
;
1H NMR (CDCl3): 2.28–2.32 (s,
1H, –C^CH), 2.50–2.56(m, 2H, CH2), 2.82–3.0(m, 2H, CH2), 3.06–
3.14(m, 2H, CH2), 4.10(s, 2H, CH2), 4.52(s, 2H, NCH2), 7.04–7.2(m,
2H, Ar–H), 7.28 (m, 2H, Ar–H); EIMS, m/z: 339 (Mþ þ 1); Anal. Calcd.
for C19H15FN2OS: C, 67.44; H, 4.47; N, 8.28%. Found: C, 67.27; H,
4.65; N, 8.12%.
–C]CH); EIMS, m/z: 748 (Mþ
C24H18F17N5OS: C, 38.56; H, 2.43; N, 9.37%. Found: C, 38.86; H,
2.26; N, 9.63%.
þ
1); Anal. Calcd. for
4.2.25. 2,5,6-Trimethyl-3-{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
10- heptadecafluorodecyl)-1H-1,2,3-triazol-4-yl) methyl}thieno
[2,3-d] pyrimidin-4(3H)one (7b)
White solid, yield: 40.22% (289 mg), mp: 176 ꢃC, IR (KBr): 1672
(C]O), 1545 (C]N), 1430 (C]) cmꢀ1; 1H NMR (CDCl3): 2.38 (s, 2H,
CH2), 2.46 (s, 3H, CH3), 2.85 (s, 6H, 2CH3), 4.55–4.68 (m, 2H, NCH2),
5.30 (s, 2H, NCH2), 7.8(s, 1H, –C]CH); EIMS, m/z: 722 (Mþ þ 1);
Anal. Calcd. for C22H16F17N5OS: C, 36.63; H, 2.24; N, 9.71%. Found: C,
36.81; H, 1.99; N, 9.49%.
Preparation of 10-azido-1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadeca
fluorodecane (5) [22]
Preparation of 1H–1,2,3-triazol-4-yl thieno[2,3-d]pyrimidines (6)
and 1H-1,2,3-triazol-4-yl thieno[2,3-d]pyrimidin-4(3H) ones (7).
4.2.20. General procedure
The propargylated thieno [2,3-d] pyrimidines 3 or thieno [2,3-
d] pyrimidin-4-ones
4
(0.001 mol) and copper (I) iodide
4.2.26. 2-(4-Fluorobenzyl)-5,6-dimethyl-3-{1-(3,3,4,4,5,5,6,6,7,7,8,8,
9,9,10,10,10- heptadeca fluorodecyl)-1H-1,2,3-triazol-4-yl)methyl}-
thieno [2,3-d] pyrimidin-4(3H)-one (7c)
White solid, yield: 45% (366 mg), mp: 139 ꢃC; IR (KBr): 1660
(C]O),1530 (C]N),1470 (C]C) cmꢀ1; 1H NMR (CDCl3): 2.40 (s, 3H,
CH3), 2.45 (s, 3H, CH3), 2.72–2.9 (m, 2H, CH2), 4.52 (s, 2H, CH2),
4.65–4.7(t, 2H, NCH2), 5.15 (s, 2H, NCH2), 7.0–7.18 (m, 2H, Ar–H),
7.35–7.42 (m, 2H, Ar–H), 7.8 (s, 1H, –C]CH); EIMS, m/z: 816
(Mþ þ 1); Anal. Calcd for C28H19F18N5OS: C, 41.24; H, 2.35; N, 8.59%.
Found: C, 41.49; H, 2.19; N, 8.76%.
(0.001 mol) was taken in dry THF (5 mL) and cooled to 0 ꢃC.
Perfluoroalkylazide 5 (0.0015 mol) in THF (3 mL) was added
dropwise to the reaction mixture over period of 20 min under
stirring and continued for 12 h at room temperature. The reaction
mixture was concentrated in vacuo and poured into the crushed
ice. The crude triazolyl thienopyrimidine product was collected by
extraction with ethyl acetate. The product was purified by passing
through a column packed with silica gel using n-hexane and
ethylacetate as eluents.
4.2.21. 2-Phenyl-4{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
4.2.27. 2-Methyl-3-{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
10-heptadecafluorodecyl)-1H-1,2,3-tria zol-4-yl)methyl}oxy)-
5,6,7,8-tetrahydro[1] benzothieno[2,3-d]pyrimidine (6a)
White solid, yield: 59.33% (479 mg); mp: 160 ꢃC; IR (KBr): 1569
10-heptadecafluorodecyl)-1H-1,2,3-tria zol-4-yl)methyl}-3,5,6,
7-tetrahydro-4H-cyclopenta [4,5] thieno [2,3-d] pyrimidin-4-one (7d)
White solid, yield: 41.66% (305 mg), mp: 186 ꢃC, IR (KBr):1670
(C]N), 1508 (C]C), 1250 (C–O–C) cmꢀ1
;
1H NMR (CDCl3): 1.85–
(C]O), 1543 (C]N), 1460 (C]C) cmꢀ1 1H NMR (CDCl3): 2.50 (s,
;
1.98 (m, 4H, 2CH2), 2.80–2.86 (m, 4H, 2CH2), 2.92–3.02 (m, 2H,
CH2), 4.55–4.68 (t, 2H, NCH2), 5.85 (s, 2H, OCH2), 7.4–7.52 (m, 3H,
Ar–H), 7.7(s, 1H, –C]CH), 8.48–8.55(m, 2H, Ar–H); EIMS, m/z: 810
(Mþ þ 1); Anal. Calcd. for C29H20F17N5OS: C, 43.03; H, 2.49; N,
8.65%. Found: C, 43.19; H, 2.07; N, 8.88%.
3H, CH3), 2.85–2.90 (m, 4H, 2CH2), 2.95 (s, 2H, CH2), 3.0–3.12 (m,
2H, CH2), 4.58–4.65 (t, 2H, NCH2), 5.3 (s, 2H, NCH2), 7.85 (s, 1H,
–C]CH); EIMS, m/z: 734 (Mþ
þ
1); Anal. Calcd. for
C23H16F17N2OS: C, 37.66; H, 2.20; N, 9.55%. Found: C, 37.45; H,
2.02; N, 9.73%.
4.2.22. 2-Phenyl-4-{1-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
10- heptadeca fluorodecyl)-1H-1,2,3-triazol-4-yl)methyl}oxy)-
5,6-dimethyl thieno[2,3-d] pyrimidine (6b)
4.3. Pharmacological studies
4.3.1. Radioligand binding studies
White solid, yield: 42.45% (332 mg), mp: 202 ꢃC; IR (KBr): 1560
4.3.1.1. Materials. [3H] CCPA was obtained from NEN Life Science
(48.6 Ci/mmol) and [3H] MSX-2 from Amersham (85 Ci/mmol).
;
(C]N), 1490(C]C), 1260 (C–O–C) cmꢀ1 1H NMR (CDCl3):

B. Sirisha et al. / European Journal of Medicinal Chemistry 45 (2010) 1739–1745
1745
4.3.1.2. Membrane preparations. Frozen rat brains were obtained
References
from Pel Freez, Rogers, AR, USA. Rat brains were dissected to obtain
cortical membrane preparations for A1 and striatal membrane
preparations for A2A assay as described [11].
[1] A. Michiel van Rhee, S.M. Siddiqi, N. Melman, D. Shi, W.L. Padgett, J.W. Daly,
K.A. Jacobson, J. Med. Chem. 39 (1996) 398–406.
[2] P.G. Baraldi, US005939432A, 1999.
[3] R. Romagnoli, P.G. Baraldi, A.R. Moorman, M.A. Iaconinoto, M.D. Carrion,
C.L. Cara, M.A. Tabrizi, D. Preti, F. Fruttarolo, S.P. Baker, K. Varani, P.A. Borea,
Bioorg. Med. Chem. Lett. 16 (2006) 5530–5533.
[4] I.O. Donkor, H. Li, S.F. Queener, Eur. J. Med. Chem. 38 (2003) 605–611.
[5] T. Horiuchi, J. Chiba, K. Uoto, T. Soga, Bioorg. Med. Chem. Lett. 19 (2009)
305–308.
[6] B.A. Abdel-rahman, A.R. El-Gazzarhoda, N. Hussein Hend, Hafez, Acta Pharm.
57 (2007) 395–411.
[7] B. Shireesha, K. Uma Shankar, A. Raghuram Rao, K.S. Rajan, M. Raghuprasad,
Int. J. Pharma. Sci. Nanotechnol. 1 (2008) 136–143.
[8] M.R. Prasad, D.P. Kishore, Chem. Pharm. Bull. 55 (2007) 776–779.
[9] K.S. Rajan, M.R. Prasad, D. Harikrishna, M.C. Prabhakar, D. Rama Krishna,
A.R. Rao, Open Med. Chem. J. 2 (2008) 101–111.
[10] B. Shireesha, B. Narsaiah, A. Raghuram Rao, S. Ramakrishna, Madhusudhana,
P.V. Diwan, T. Yakaiah, Thienopyridines as pharmacologicaly active agents,
Indian Patent application No. 10/DEL/2009 dated 5/01/2009.
[11] M. Raghu Prasad, A. Raghuram Rao, P. Shantan Rao, K.S. Rajan, S. Meena,
K. Madhavi, Eur. J. Med. Chem. 43 (2007) 614–620.
4.3.1.3. Radioligand binding assays. Binding assays for A1 and A2A
were performed essentially as described in the literature. Stock
solutions of the compounds were prepared in dimethyl sulphoxide
(DMSO) and the final concentration of DMSO in the assay was 2.5%.
The radioligand concentrations were [3H] CCPA: 0.5 nM (rat A1) and
[3H] MSX-2: 1.0 mM (rat A2A).
Drug solution (50
mL) was added into a 96-well plate followed by
50
mL of radioligand solution and 100
m
L (30 g/vial) of protein
m
suspension of rat brain cortex (A1) or rat brain striatal membranes
(A2A) and incubated at room temperature for 90/30 min. CADO
(2-chloroadenosine)/NECA [50-(N-ethylcarbamoyl)adenosine] was
used for nonspecific binding for A1 and A2A AR binding studies,
respectively. Incubation was terminated by rapid filtration over
Whatman GF/B filters using a Brandell cell harvester (Brandell,
Gaithersburg, MD). To the filter plate, scintillation liquid was added
[12] J.W. Lee, B. Ku Kim, S.H. Jin, Bull. Korean Chem. Soc. 26 (2005) 833–836.
[13] H.C. Kolb, M.G. Finn, K.B. Sharpless, Angew. Chem. Int. Ed. 40 (2001)
2004–2021.
(40
m
l/vail – Microscent-20) and incubated at room temperature for
[14] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, Angew. Chem. Int. Ed. 41
(2002) 2596–2599.
[15] H.C. Kolb, K.B. Sharpless, Drug Discov. Today 8 (2003) 1128–1137.
[16] J.K. Mishra, G. Panda, J.S. Rao, G.N. Sastry, Tetrahedron Lett. 47 (2006)
3357–3360.
10 h and radioactivity was measured bya scintillation counter. Those
compounds whose percentage inhibitionwas found to be more than
20% at a test concentration of 1 mM were taken and a full concen-
[17] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
et al., Gaussian 03, Revision C.02. Gaussian, Inc., Wallingford CT,, 2004.
[18] F. Biegler-Ko¨nig, J. Scho¨nbohm, AIM2000, version 2.0.
[19] K. Gewald, E. Schinke, H. Bo¨ttcher, Chem. Ber. 99 (1966) 94–100.
[20] M.R. Prasad, K.S. Rajan, K. Mogilaiah, P.S. Rao, A.R.R. Rao, Synthesis 14 (2001)
2119–2123.
[21] P. Mani Chandrika, T. Yakaiah, G. Gayatri, K. Pranay Kumar, B. Narsaiah,
U.S.N. Murthy, A. Raghu Ram Rao, Eur. J. Med. Chem. 45 (2010) 78–84.
[22] A. Gheorghe, E. Cuevas-Yan˜ez, J. Horn, W. Bannwarth, B. Narsaiah, O. Reiser,
Syn. Lett. 17 (2006) 2767–2770.
tration – inhibition curve was determined.
4.3.2. Data analysis
Data were analyzed using Graph Pad PRISM version 3.0 (San
Diego, CA). For nonlinear regression analysis, the Cheng–Prushoff
equation and a KD value of 0.5 nM for [3H] CCPA at rat A1ARs was
used to calculate Ki values from EC50 values.
[23] A. Gheorghe, A. Matsuno, O. Reiser, Adv. Synth. Catal. 348 (2006)
1016–1020.
[24] C.W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 67 (2002) 3057–3064.
[25] F. Himo, T. Lovell, R. Hilgraf, V.V. Rostovtsev, L. Noodleman, K.B. Sharpless,
V.V. Fokin, J. Am. Chem. Soc. 127 (2005) 210–216.
[26] G. Gayatri, G.N. Sastry, J. Chem. Sci. 117 (2005) 573–582.
[27] G. Gayatri, G.N. Sastry, J. Phys. Chem. A. 113 (2009) 12013–12021.
[28] K. Fukui, Science 218 (1982) 747–754.
[29] R.G. Parr, W. Yang, J. Am. Chem. Soc. 106 (1984) 4049–4050.
[30] W. Yangs, W.J. Mortier, J. Am. Chem. Soc. 108 (1986) 5708–5711.
[31] P. Geerlings, F.D. Proft, W. Langenaeker, Chem. Rev. 103 (2003) 1793–1874.
Acknowledgments
Authors wish to thank Dr. J.S. Yadav, Director, IICT, Hyderabad,
India for his constant encouragement and CSIR, India for the
research fellowships to two of them (GG and TY). We thankfully
acknowledge the cooperation extended by Prof. Dr. Christa E.
Mueller, University of Bonn, Germany for providing facilities and
guidance for radioligand binding studies.