Assessment of antiplatelet activity of 2-aminopyrimidines

Article abstract of DOI:10.1016/j.ejmech.2012.01.035

A series of 4,6-diaryl-2-aminopyrimidines was developed as antiplatelet agents and their potency was evaluated by in vitro assay. Compound 14k was found to be two times more potent than aspirin. These encouraging results could be helpful for the development of new antiplatelet compounds.

Full text of DOI:10.1016/j.ejmech.2012.01.035

European Journal of Medicinal Chemistry 50 (2012) 428e432
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Assessment of antiplatelet activity of 2-aminopyrimidines
*
Rajani Giridhar , Riyaj S. Tamboli, R. Ramajayam, Dhaval G. Prajapati, M.R. Yadav
Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M. S. University of Baroda, Vadodara 390 001, 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 4,6-diaryl-2-aminopyrimidines was developed as antiplatelet agents and their potency was
evaluated by in vitro assay. Compound 14k was found to be two times more potent than aspirin. These
encouraging results could be helpful for the development of new antiplatelet compounds.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 11 August 2010
Received in revised form
16 January 2012
Accepted 17 January 2012
Available online 24 January 2012
Keywords:
2-Aminopyrimidines
Aspirin
Thrombosis
Antiplatelet agents
1. Introduction
JNK inhibitor [6], while the cyano-substituted pyrimidine structure
4 shows remarkable inhibition for the vascular endothelial growth
Approximately 40% of morbidity and mortality in western
countries is due to atherothrombotic vascular disease [1]. Platelets
play a pivotal role in thrombosis and haemeostasis. The major
physiological role of platelets is to maintain the integrity of the
circulatory system and responds rapidly and robustly at sites of
vascular injury. Platelets are activated when brought into contact
with agents such as collagen, thrombin and ADP. Such activation
leads to a range of responses that play a critical role in arterial
factor receptor 2 (VEGF-R2) [7] and a piperidine-substituted ami-
nopyrrolopyrimidine 5 serves as a Y5 receptor antagonist [8].
Piperazine-substituted 2-aminopyrimidine structure 6 displays
histamine H4 receptor properties [9], while compound 7 acts as
a
reverse-transcriptase inhibitor [10] and benzopyrano-2-
aminopyrimidines [11] and 4,5-diaryl-2-aminopyrimidine
have been shown to possess antiplatelet property [12].
8
9
As a part of our research program, aiming at the discovery of
novel diaryl heterocycles based antiplatelet agents, we recently re-
ported the potent antiaggregatory effect of the lead compound
which contains a diaryl fragment as a key structural element and 1,4-
diazepine as a heterocyclic core [13] 10 (Fig. 3). Encouraged by this
finding and taking into account the well-documented antiplatelet
activity associated with the 2-aminopyrimidine pharmacophoric
unit [11,12], we report the synthesis and preliminary results of
antiplatelet activity of the 4,6-diaryl-2-aminopyrimidines 14.
thrombosis, including platelet aggregation, dense and
a granule
secretion and procoagulant activity [2]. Currently, aspirin 1 and
thienopyridine drugs (clopidrogel 2a and ticlopidine 2b) are widely
used antiplatelet agents (Fig.1). Nearly 50% of the global population
are resistant to these drugs resulting in increased risk of recurrent
myocardial infarction and stroke, or cardiac related mortality [3,4].
Although recent advances for the treatment of coronary syndromes
have been made by implementing dual antiplatelet therapy using
a combination of aspirin with a thienopyridine drug during the
acute phase and for secondary prevention, the atherothrombotic
disease remains a leading cause of morbidity and mortality [5]. The
limitations of the currently available antiplatelet agents have trig-
gered the development of newer drugs.
2. Results and discussion
2.1. Chemistry
Compounds derived from 2-aminopyrimidine have provided
the basis for different medicinal applications (Fig. 2). Cyclohexyl-
substituted aminopyrimidine 3 was found to act as a potential
The diarylpyrimidine derivatives presented in this paper were
prepared according to the synthetic route described in Scheme 1.
The 1,3-diketones 12aeq required for the work were obtained from
the esters 11aeq, by base-catalyzed Baker-Venkataraman trans-
formation [14]. Condensation of 2-hydroxyacetophenones with
various substituted benzoic acids in dry pyridine and POCl3
* Corresponding author. Tel.: þ91 265 2434187; fax: þ91 265 2423898.
E-mail address: rajanimsu@rediffmail.com (R. Giridhar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.035

R. Giridhar et al. / European Journal of Medicinal Chemistry 50 (2012) 428e432
429
O
N
O
H
CH3
O
OH
N
O
CH3
S
S
Cl
Cl
O
2a
2b
1
Fig. 1. Clinically used antiplatelet agents.
furnished the esters 11aeq. In the IR spectra, the 1,3-diketones
showed absorption bands for C]O in the range 1615e1625 cmꢀ1
induced aggregation for aspirin is 6e24
U
. From the comparative
.
study of test compounds (14aeq) at same concentration of aspirin,
eleven compounds were found active as shown in Table 1.
From eleven compounds 14k is the most potent compound
being two times more potent than aspirin. Also 14 (a, d, j, n, o)
produced more than 60% inhibition and 14 (c, g, m and q) found
more active than aspirin (Fig. 4).
Treatment of the diketone with concd H2SO4 leads to formation
of flavone (13aeq) [15]. The formation of the flavones 13aeq was
confirmed by the appearance of C]O absorption bands at
1640e1660 cmꢀ1. Reaction of the flavones 13aeq with a slight
excess of guanidine hydrochloride in alkaline medium afforded 4,6-
diaryl-2-aminopyrimidines 14aeq [10].
Compounds 14aeq showed a characteristic peak at 3200 cmꢀ1
for OeH stretching, asymmetric and symmetric stretching NeH
bands at 3500 and 3350 cmꢀ1 respectively in the IR spectrum. In
2.3. Structureeactivity relationships
The effect of electron withdrawing and donating groups on the
basic scaffold, 4,6-diaryl 2-aminopyrimidine was studied with
respect to antiplatelet activity of the synthesized compounds
(Table 1). Among all the compounds in the series, 14k was found to
be the most potent inhibitor. This suggests that in the 40eOCH3
series; 200,400-dichloro substitution favors antiplatelet activity.
Dichloro substitution at these positions increases the lipophilic
nature of the molecule. This could be one of the reasons for highest
% inhibition of platelet aggregation for compound 14k. Compounds
with mono substituted chlorine at 400 position and methyl at 300
position (14h & 14i respectively) showed drastic decrease in
activity whereas 300-chloro substituted compound (14j) showed
higher % inhibition than that of 14h & 14i. In case of 50eOCH3 series,
substitution of chlorine at 300 position retained similar activity (14d)
as that of 40eOCH3 series. Surprisingly fluorine at 400 position has
shown diminished activity (14e). Replacement of phenyl ring with
a heterocyclic furan ring has shown complete loss of activity (14f).
the 1H NMR spectra, NH2 protons
d
5.1e6.0 and the pyrimidinyl
proton (C5eH) appeared as a sharp singlet at 7.3e7.5. The
aromatic protons appeared as a multiplet at 6.5e8.0. The evidence
of partial ring formation due to internal hydrogen bonding of the
hydroxyl protons is clearly observed at higher
13e14. In the 13C
NMR spectra the values of the carbons are in conformation with
d
d
d
d
the structures of the compounds synthesized.
2.2. Antiplatelet activity
The newly synthesized compounds were studied for their in vitro
platelet aggregation inhibitory activity on whole human blood [16].
The study was performed using Whole Blood Aggregometer, Chro-
nolog Corporation, Haverton, PA, USA. The inhibitory activity of
seventeen compounds was measured to compare with inhibition
induced by standard drug Aspirin (ASA) (10 mg/ml). Each assay was
performed three times, taking control and aspirin for comparative
assay each time. Whole blood aggregometer measures inhibition of
3. Conclusions
platelet aggregation induced by ADP (10 mM) in ohms which is the
resistance produced by accumulation of aggregates on the electrode.
The control or normal platelet aggregation was found to be
To summarize, design and synthesis of a series of 4,6-diaryl-2-
aminopyrimidines was carried out, in which several electron
withdrawing, donating as well as bulky functions were introduced
to replace the phenyl ring substitutions. Biological evaluation
10.92 ꢁ1.92
U
and for aspirin (10
m
g/ml) the inhibition of aggregation
6.82 ꢁ 1.21
U
(37%). The standard range for inhibition of ADP (10
mM)
NH2
N
NH
N
O
O
N
N
OH
H
N
NC
N
N
N
N
N
H
N
N
NH2
H
5
3
4
H3CO
NH2
OH
N
NH2
N
N
O
N
N
N
N
N
N
NH2
N
OPh
NH2
H3CO
6
8
9
7
Fig. 2. Biologically interesting 2-aminopyrimidine compounds.

430
R. Giridhar et al. / European Journal of Medicinal Chemistry 50 (2012) 428e432
Table 1
Structure and activity of compounds 14aeq.
N
N
3"
6"
3'
OH
4''
5"
2"
4'
2'
X
5
Y
4
5'
6
6'
3 N
N 1
10
2
NH2
14a-q
Fig. 3. General structure of the diaryl-1,4-diazepine.
indicated that some compounds showed moderate to potent anti-
platelet activities which will be further helpful for the development
of new antiplatelet compounds.
.
Comp. no.
X
Y
% Inhibition of platelet aggregation
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
14q
Aspirin
50-OCH3
50-OCH3
50-OCH3
50-OCH3
50-OCH3
50-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
40-OCH3
50-OC2H5
e
400-OCH3
300-OCH3
200-Cl
300-Cl
400-F
64.28
NDa
55.13
66.85
8.79
1.46
42.67
8.42
23.08
65.84
77.47
NDa
51.19
64.01
62.82
58.52
39.92
37.55
4. Experimental
Melting points were determined in capillaries using Toshniwal
melting point apparatus and are uncorrected. IR (in cmꢀ1) spectra
2-furyl
H
in KBr pellets on a Shimadzu 8300 instrument; and 1H NMR and 13
C
400-Cl
300-CH3
300-Cl
NMR spectra were recorded in CDCl3 on a Bruker Avance II spec-
trometer (400 MHz), using tetramethylsilane as an internal stan-
dard. Chemical shift data are reported in parts per million (d in
200,400-Cl2
400-OCH3
300-OCH3
400-F
ppm) where s, br, t and m designate singlet, broad, triplet and
multiplet, respectively. Elemental analyses were recorded on
a Perkin Elmer PE 2400 CHNS analyzer. Mass spectra were recorded
on APISciEX mass spectrometer equipped with an electrospray
ionization (ESI) interface. Column chromatography was carried out
using silica gel (100e200 mesh). Thin-layer chromatography (TLC)
was performed on precoated Silica gel Merck plates. Compounds
were visualized by illuminating with UV light (254 nm) or exposure
to iodine vapors. All chemicals and solvents were of reagent grade
and were purified and dried using standard methods. Flavones
13aeq was prepared according to literature method [15].
400-CH3
400-NO2
H
e
NDa:Not determined.
washed with water and recrystallized from methanol to give
14aeq.
4.1.1. 4-(20-hydroxy-50-methoxyphenyl)-6-(400-methoxyphenyl)-2-
aminopyrimidine (14a)
4.1. General method for the preparation of 4,6-diaryl-2-
aminopyrimidine derivatives (14aeq)
Yield 29%, Mp.184e185 ꢂC (Yellow crystals from MeOH), IR (KBr)
nmax: 3520, 3368, 3200,1600 cmꢀ1; 1H NMR (CDCl3, 400 MHz): 3.86
(s, 6H, OCH3), 5.48 (s, 2H, NH2), 7.24e7.26 (d, J ¼ 7.2 Hz, 2H, ArH),
7.30e7.32 (d, J ¼ 7.6 Hz, 2H, ArH), 7.36e7.38 (d, J ¼ 8.4 Hz, 2H, ArH),
7.49 (s, 1H, ArH), 8.06 (s, 1H, pyri). MS m/z; 324.05 (Mþ1). Anal.
Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.0. Found: C, 67.04; H,
5.28; N, 13.10.
A mixture of the respective flavone 13aeq (0.0019 mol),
guanidine hydrochloride (0.01 mol) and potassium hydroxide
(1.0 g) was refluxed in methanol (30 mL) for 4e6 h. After the
completion of the reaction, the mixture was poured on to crushed
ice containing acetic acid. The yellow solid obtained was filtered,
Y
O
COOH
Y
OH
OH
CH3
O
O
b
a
Y
X
X
+
X
CH3
O
O
O
(12a-q)
(11a-q)
Y
OH
N
O
c
X
Y
d
X
N
O
(13a-q)
NH2
(14a-q)
Scheme 1. Synthesis of 4,6-diaryl-2-aminopyrimidines. Reagents and conditions: (a) POCl3, dry pyridine, rt, 2 h; (b) fused KOH, dry pyridine, rt, 2 h; (c) AcOH, catalytic H2SO4, 100 ꢂC,
1 h; (d) Guanidine hydrochloride, KOH, MeOH, reflux, 4e6 h.

R. Giridhar et al. / European Journal of Medicinal Chemistry 50 (2012) 428e432
431
(m, 1H, furan), 13.18 (s, 1H, OH); 13C NMR (CDCl3, 400 MHz):
56.09, 100.00, 111.32, 112.39, 112.48, 117.34, 119.33, 119.97, 145.02,
d
151.89, 152.20, 154.99, 157.27, 160.59, 165.80; MS m/z; 284.1
(M þ 1); Anal. Calcd for C15H13N3O3: C, 63.60; H, 4.63; N, 14.83.
Found: C, 63.64; H, 4.52; N, 14.98.
4.1.7. 4-(20-hydroxy-40-methoxyphenyl)-6-phenyl-2-
aminopyrimidine (14g)
Yield 24%, Mp. 186e189 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax: 3508, 3354, 1625 cmꢀ1 1H NMR (CDCl3, 400 MHz):
;
d
3.88 (s, 3H, OCH3), 5.17 (s, 2H, NH2), 6.57 (m, 2H, ArH), 7.29e7.32
Fig. 4. Graphical illustration of percentage inhibition of platelet aggregation of the test
compounds 14aeq and aspirin.
(m, 2H, ArH), 7.37 (s,1H, pyri.), 7.54e7.57 (m, 4H, ArH), 13.84 (s, 1H,
OH); MS m/z; 294.75 (M þ 1); Anal. Calcd for C17H15N3O2: C, 69.61;
H, 5.15; N, 14.33. Found: C, 69.64; H, 5.02; N, 14.28.
4.1.2. 4-(20-hydroxy-50-methoxyphenyl)-6-(300-methoxyphenyl)-2-
aminopyrimidine (14b)
4.1.8. 4-(20-hydroxy-40-methoxyphenyl)-6-(400-chlorophenyl)-2-
aminopyrimidine (14h)
Yield 54%, Mp. 158e160 ꢂC (Yellow crystals from MeOH), IR
Yield 33%, Mp. 215e217 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax
:
3430, 3320, 3200, 1590 cmꢀ1
;
1H NMR (CDCl3,
(KBr) nmax: ;
3494, 3365, 2923, 1618 cmꢀ1 1H NMR (CDCl3,
400 MHz): 3.84 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.18 (s, 2H, NH2),
6.95e6.97 (m, 1H, ArH), 7.0e7.02 (m, 1H, ArH), 7.05e7.07 (m, 1H,
ArH), 7.34e7.35 (d, 1H, ArH), 7.40e7.44 (m, 1H, ArH), 7.48 (s, 1H,
pyri), 7.59e7.61 (m, 2H, ArH), 13.19 (br, 1H, OH). MS m/z; 324.1
(M þ 1). Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.0.
Found: C, 66.78; H, 5.42; N, 13.04.
400 MHz): d 3.89 (s, 3H, OCH3), 5.19 (s, 2H, NH2), 6.59 (s, 1H, ArH),
7.29e7.30 (m, 2H, ArH), 7.32e7.34 (d, J ¼ 7.6, 2H, ArH), 7.40 (1H,
Pyri), 7.42e7.44 (d, J ¼ 7.4, 2H, ArH); MS m/z; 328.76 (M þ 1); Anal.
Calcd for C17H14ClN3O2: C, 62.30; H, 4.31; N, 12.82. Found: C, 62.35;
H, 4.22; N, 13.02.
4.1.9. 4-(20-hydroxy-40-methoxyphenyl)-6-(300-methylphenyl)-2-
aminopyrimidine (14i)
4.1.3. 4-(20-hydroxy-50-methoxyphenyl)-6-(200-chlorophenyl)-2-
aminopyrimidine (14c)
Yield 26%, Mp. 230e232 ꢂC, (Yellow crystals from MeOH), IR
Yield 52%, Mp.163e165 ꢂC (Yellow crystals from MeOH), IR (KBr)
(KBr) nmax: 3498, 3281, 3173,1618 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
nmax: 3480, 3310 1590 cmꢀ1
;
1H NMR (CDCl3, 400 MHz):
d
3.81 (s,
d 2.45 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 5.94 (s, 2H, NH2), 6.47e6.51
3H, OCH3), 5.24 (s, 2H, NH2), 6.97 (s,1H, ArH), 7.0e7.02 (m,1H, ArH),
7.39e7.42 (m, 3H, ArH), 7.50e7.52 (m, 1H, ArH), 7.60e7.62 (m, 1H,
ArH), 7.83 (s, 1H, pyri), 13.09 (br, 1H, OH); MS m/z; 328.1 (M þ 1);
Anal. Calcd for C17H14ClN3O2: C, 62.30; H, 4.31; N, 12.82. Found: C,
62.18; 4.42; N, 12.76.
(m, 3H, ArH), 7.36e7.39 (m, 3H, ArH), 7.46 (1H, pyri), 7.80e7.86 (m,
1H, ArH), 14.22 (br, 1H, OH); MS m/z; 308.16 (M þ 1); Anal. Calcd for
C18H17N3O2: C, 70.34; H, 5.58; N, 13.67. Found: C, 70.36, H, 5.52; N,
13.49.
4.1.10. 4-(20-hydroxy-40-methoxyphenyl)-6-(300-chlorophenyl)-2-
aminopyrimidine (14j)
4.1.4. 4-(20-hydroxy-50-methoxyphenyl)-6-(300-chlorophenyl)-2-
aminopyrimidine (14d)
Yield 35%, Mp. 189e190 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax: 3425, 3310, 3173, 1618 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
3.86 (s, 3H, OCH3), 5.37 (s, 2H, NH2), 6.51e6.54 (m, 2H, ArH), 7.40 (s,
1H, pyri), 7.44e7.48 (m, 2H, ArH), 7.77e7.79 (d, J ¼ 8.6, 1H, ArH),
7.92e7.94 (d, J ¼ 8.92, 1H, ArH), 8.04 (s, 1H, ArH), 13.74 (br, 1H, OH);
MS m/z; 328.76 (M þ 1); Anal. Calcd for C17H14ClN3O2: C, 62.30; H,
4.31; N, 12.82. Found: C, 62.24; H, 4.44; N, 12.72.
Yield 70%, Mp.178e179 ꢂC (Yellow crystals from MeOH), IR (KBr)
nmax: 3456, 3352, 1639, 1571 cmꢀ1 1H NMR (CDCl3, 400 MHz):
;
d
3.85 (s, 3H, OCH3), 5.26 (s, 2H, NH2), 7.04e7.26 (m, 3H, ArH),
7.32e7.37 (m, 2H, ArH), 7.53 (s, 1H, pyri), 7.98e8.12 (m, 2H, ArH),
13.12 (br, 1H, OH); 13C NMR (CDCl3, 400 MHz):
56.13, 102.10,
d
111.48, 117.36, 119.37, 119.84, 125.27, 127.38, 130.08, 130.78, 135.01,
139.14, 152.24, 154.94, 160.79, 165.16, 166.00; MS m/z; 328.1
(M þ 1); Anal. Calcd for C17H14ClN3O2: C, 62.30; H, 4.31; N, 12.82.
Found: C, 62.24; 4.25; N, 12.92.
4.1.11. 4-(20-hydroxy-40-methoxyphenyl)-6-(200,400-dichlorophenyl)-
2-aminopyrimidine (14k)
Yield 54%, Mp. 204e206 ꢂC, (Yellow crystals from MeOH), IR
4.1.5. 4-(20-hydroxy-50-methoxyphenyl)-6-(400-flurophenyl)-2-
aminopyrimidine (14e)
(KBr) nmax
:
3649, 3575, 3173, 1635 cmꢀ1 1H NMR (CDCl3,
;
400 MHz):
d
3.84 (s, 3H, OCH3), 5.15 (s, 2H, NH2), 6.48e6.51 (m, 2H,
Yield 56%; Mp. 202e204 ꢂC, (Yellow crystals from MeOH), IR
ArH), 7.33 (s,1H, pyri), 7.36e7.38 (dd, J ¼ 8.36, 1H, ArH), 7.52e7.53
(m, 1H, ArH), 7.56e7.58 (d, J ¼ 8.32, 1H, ArH), 7.67e7.69 (dd,
J ¼ 9.52, 1H, ArH), 13.19 (s, 1H, OH); 13C NMR (CDCl3, 400 MHz):
(KBr) nmax: 3410, 3220, 1652 cmꢀ1 1H NMR (CDCl3, 400 MHz):
;
d
3.85 (s, 3H, OCH3), 5.16 (s, 2H, NH2), 6.95 (s, 1H, ArH), 7.0e7.03 (m,
1H, ArH), 7.17e7.19 (d, J ¼ 6.68, 2H), 7.33e7.44 (m, 1H, ArH), 7.45 (s,
1H, pyri) 8.06e8.08 (d, J ¼ 6.8, 2H), 13.16 (s, 1H, OH); MS m/z; 312.1
(M þ 1); Anal. Calcd for C17H14FN3O2: C, 65.59; H, 4.53; N, 13.50.
Found: C, 65.78; H, 4.64; N, 13.46.
d 55.48, 101.89, 105.84, 107.69, 110.34, 127.60, 128.66, 130.28, 131.79,
132.94, 135.56, 136.12, 160.05, 163.19, 163.90, 164.05, 165.55; MS m/
z; 362.1 (M þ 1); Anal. Calcd for C17H13Cl2N3O2: C, 56.37; H, 3.62; N,
11.60. Found: C, 56.48; H, 3.46; N, 11.72.
4.1.6. 4-(20-hydroxy-50-methoxyphenyl)-6-(200-furyl)-2-
aminopyrimidine (14f)
4.1.12. 4-(20-hydroxy-40-methoxyphenyl)-6-(400-methoxyphenyl)-2-
aminopyrimidine (14l)
Yield 48%, Mp. 216e217 ꢂC, (Yellow crystals from MeOH), IR
Yield 35%, Mp. 223e225 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax
:
3460, 3310, 3220, 1652 cmꢀ1
;
1H NMR (CDCl3,
(KBr) nmax: 3410, 3300 (NH2), 3173.9 (OH), 1635 cmꢀ1 1H NMR
;
400 MHz):
d
3.85 (s, 3H, OCH3), 5.12 (s, 2H, NH2), 6.58e6.60 (m, 1H,
(CDCl3, 400 MHz): d 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.15 (s,
furan) 6.94e6.96 (m, 1H, furan), 7.0e7.02 (m, 1H, ArH) 7.22e7.23
(m, 1H, ArH) 7.33e7.34 (m, 1H, ArH), 7.46 (s, 1H, pyri), 7.62e7.63
2H, NH2), 6.54e6.57 (m, 2H, ArH), 6.60 (s,1H, ArH), 7.35 (s,1H, pyri),
7.36e7.37 (d, J ¼ 7.8, 2H, ArH), 7.42e7.43 (d, J ¼ 7.84, 2H, ArH); MS

432
R. Giridhar et al. / European Journal of Medicinal Chemistry 50 (2012) 428e432
m/z; 324.13 (M þ 1); Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30;
and maintained at 37 ꢂC throughout the experiment. The diluted
blood sample was equilibrated for at least 2 min until a stable
N, 13.00. Found: C, 66.78; H, 5.21, N, 13.0.
baseline reading was achieved and 10
was added. After 2 min incubation, 10
m
l of test sample (or control)
4.1.13. 4-(20-hydroxy-40-methoxyphenyl)-6-(300-methoxyphenyl)-2-
aminopyrimidine (14m)
m
l of ADP (10 M of final
m
concentration) was added to initiate the aggregation. The aggrega-
tion was monitored for 6 min and the results were analyzed using
Aggro/link version 4.75 software. The impedance value (in ohms)
was determined from the impedance graph, starting from the lowest
point after the start of reaction to the end of the 6 min time interval.
The blood samples were used within 4 h after collection. To ensure
consistent platelet function during the experimental period, controls
were analyzed at regular intervals between the test samples. The
results of antiplatelet activity are given in Table 1. Percentage inhi-
bition was calculated using the following formula:
Yield 22%, Mp. 186e188 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax
:
3390, 3260, 3173, 1635 cmꢀ1 1H NMR (CDCl3,
;
400 MHz):
d
3.84 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.09 (s, 2H, NH2),
6.46e6.48 (m, 1H, ArH), 6.49e6.51 (m, 1H, ArH), 7.01e7.04 (m, 1H,
ArH), 7.38e7.42 (m, 2H, ArH), 7.56 (s, 1H, pyri), 7.61e7.63 (m, 1H,
ArH), 7.81 (d, 1H, ArH), 14.25 (s, 1H, OH); MS m/z; 324.1 (M þ 1);
Anal. Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
66.90; H, 5.24, N, 13.02.
4.1.14. 4-(20-hydroxy-40-methoxyphenyl)-6-(400-fluorophenyl)-2-
aminopyrimidine (14n)
ðA ꢀ BÞ
Percentage inhibition ¼
ꢃ 100
Yield 25%, Mp. 205e207 ꢂC, (Yellow crystals from MeOH), IR
A
(KBr) nmax: 3500, 3380, 3173,1635 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
where A is maximal impedance value (in ohms) obtained in the
presence of control and B is maximal impedance value (in ohms)
obtained in the presence of sample.
d
3.88 (s, 3H, OCH3), 5.12 (s, 2H, NH2), 6.50e6.53 (m, 2H, ArH),
7.16e7.18 (d, J ¼ 8.4 Hz, 2H, ArH), 7.39 (s,1H, pyri), 7.75e7.78 (m,1H,
ArH), 8.04e8.06 (d, J ¼ 8.84 Hz, 2H, ArH),14.08 (br,1H, OH); 13C NMR
(CDCl3, 400 MHz):
d 55.44, 100.91, 101.90, 107.47, 110.56, 115.78,
Acknowledgments
116.00,128.35,129.20,129.28,133.25,160.22,163.08,163.28,163.80,
164.47, 165.87; MS m/z; 312.1 (M þ 1); Anal. Calcd for C17H14FN3O2:
C, 65.59; H, 4.53; N, 13.50. Found: C, 65.67; H, 4.56; N, 13.52.
We thank All India Council for Technical Education (AICTE),
New Delhi, India, for financial support in the form of Research
Promotion Scheme [File No. 8023/RID/RPS/46/2004-05] to RG,
National Doctoral Fellowship to RST [File No. 1-10/RID/NDF/PG/
36/2009-2010] and Indian Council of Medical Research (ICMR),
New Delhi, India, for the award of Senior Research Fellowship
to RR.
4.1.15. 4-(20-hydroxy-40-methoxyphenyl)-6-(400-methylphenyl)-2-
aminopyrimidine (14o)
Yield 16%, Mp. 195e196 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax
: ;
3400, 3390, 3173, 1635 cmꢀ1 1H NMR (CDCl3,
400 MHz):
d 2.44 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 5.94 (s, 2H, NH2),
6.47e6.51 (m, 2H, ArH), 7.36e7.39 (m, 2H, ArH), 7.46 (s, 1H, pyri),
7.80e7.86 (m, 3H, ArH), 13.96 (s, 1H, OH); MS m/z; 308.1 (M þ 1);
Anal. Calcd for C18H17N3O2: C, 70.34; H, 5.58; N, 13.67. Found: C,
70.36, H, 5.50; N, 13.48.
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.01.035.
4.1.16. 4-(20-hydroxy-40-methoxyphenyl)-6-(400-nitrophenyl)-2-
aminopyrimidine (14p)
References
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4.1.17. 4-(20-hydroxy-50-ethoxyphenyl)-6-phenyl-2-
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Yield 42%, Mp. 148e150 ꢂC, (Yellow crystals from MeOH), IR
(KBr) nmax: 3410, 3390, 3173,1635 cmꢀ1; 1H NMR (CDCl3, 400 MHz):
d
1.42e1.45 (t, J ¼ 13.96 Hz, 3H, CH3), 4.12e4.16 (q, J ¼ 13.96 Hz, 2H,
CH2), 5.86 (s, 2H, NH2), 6.82 (s, 1H, pyri), 6.96e7.01 (m, 3H, ArH),
7.91e7.94 (m, 3H, ArH), 8.03e8.06 (m, 2H, ArH); MS m/z; 308.2
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Platelet aggregation experiments were carried out using Chrono-
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from Chrono-Log Corporation (Havertown, PA, USA). The whole
blood aggregation assay was conducted using the electrical imped-
ance method. 450 m
l of pre-warmed (37 ꢂC) whole blood sample was
diluted with an equal volume of pre-warmed PBS (pH 7.4). The
diluted blood sample was stirred at a constant speed of 1000 rpm
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