Novel synthesis approach and antiplatelet activity evaluation of 6-alkylamino-2,4-dialkyl(aryl)thiopyrimidines

Article abstract of DOI:10.1016/j.tet.2011.05.056

A new and efficient procedure has been designed for the preparation of 6-alkylamino-2,4-dialkyl(aryl)thiopyrimidines. The first alkylthio group was introduced into the pyrimidine ring by S-alkylation. The introduction of the second one was successfully ac

Full text of DOI:10.1016/j.tet.2011.05.056

Tetrahedron 67 (2011) 5156e5161
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel synthesis approach and antiplatelet activity evaluation
of 6-alkylamino-2,4-dialkyl(aryl)thiopyrimidines
Guocheng Liu a, Jiaxi Xu a, Ki Chul Park b, Ning Chen a, Si Zhang c, Zhongren Ding c, Feng Wang d,
,
Hongguang Du a
*
a State Key Laboratory of Chemical Resource Engineering, Department of Organic Chemistry, College of Science, Beijing University of Chemical Technology, Beijing 100029, China
b Institute of Carbon Science and Technology (ICST), Shinshu University, Nagano 380-8553, Japan
c Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College,
Shanghai 200032, China
d State Key Laboratory of Chemical Resource Engineering, Institute of Carbon Fibers and Composites, Beijing University of Chemical Technology, Beijing 100029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new and efficient procedure has been designed for the preparation of 6-alkylamino-2,4-dialkyl(aryl)
thiopyrimidines. The first alkylthio group was introduced into the pyrimidine ring by S-alkylation. The
introduction of the second one was successfully achieved using the diazotizationealkylthionation method
to afford 2,4-dialkyl(aryl)thio-6-chloropyrimidines. Subsequent nucleophilic displacement by the corre-
sponding amines conveniently gave a series of the target compounds. Thus, the two same or different
alkylthio groups were easily introduced into the pyrimidine ring through the two different approaches.
The human anti-platelet aggregation activity of the newly synthesized compounds is also described.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 7 March 2011
Received in revised form 4 May 2011
Accepted 13 May 2011
Available online 19 May 2011
Keywords:
Pyrimidine
Diazotization
Disulfide
Synthesis
1. Introduction
a free amino group for the activity. As for adenine nucleotide ana-
logues against P2T receptor, the effective enhancement of the activity
Pyrimidines play an essential role in several biological processes
and have considerable chemical and pharmacological importance in
terms that the pyrimidine ring can be found in nucleoside antibiotics,
antibacterial and cardiovascular.1e6 Pyrimidine derivatives, espe-
cially, alkylthio-substituted pyrimidines, such as 2-propylthio-tri-
azolopyrimidines7 and thienopyrimidines8 have attracted much
attention because of their quite high anti-platelet aggregation
activity as inhibitors for P2-receptor family. As related works, there
have been active attempts to develop the antagonist of P2Y receptors
(which mediate platelet aggregation induced by adenosine
diphosphate, ADP) by employing adenine nucleotide derivatives
containing two phosphate groups, i.e., adenosine-30,50-bisphosphate
analogues, as P2Y1 receptor antagonists9,10 and 4-alkoxyl-
2-alkylthio-6-aminopyrimidine derivatives as P2Y12 receptor an-
tagonists.11 The evaluated anti-platelet aggregation ability of a series
of the synthesized pyrimidine derivatives proves their potential as
lead compounds to develop a new series of P2Y12 antagonists.11
Furthermore, the results appear to suggest the importance of the
chemical structure of alkylthio substituents and of the presence of
by N-monoalkylation at the 6-position of the adenine moiety has
been found out by Ingall et al.12 Considering such findings on the
structure of the antagonist candidates, we achieved one hypothesis
that N-monoalkylation of pyrimidine compounds might also in-
crease anti-platelet aggregation activity. Furthermore, to date, there
are few report on the synthesis and evaluation of dialkyl(aryl)thio-
substituted pyrimidines as platelet aggregation inhibitors. Hence,
we designed to introduce another alkylthio group into the pyrimi-
dine ring (Fig. 1). As it is well known, the introduction of alkyl/
arylthio groups into the pyrimidine ring is commonly achieved
through either the alkylation of thiol groups or the nucleophilic
substitution of halogens by alkylmercaptides. However, these
R2
NH2
HN
N
N
R1
R
R1
R
O
N
S
S
N
S
Fig. 1. Structures of 4-alkoxyl-2-alkylthio-6-aminopyrimidines and 6-alkylamino-2,4-
* Corresponding author. E-mail address: dhg@mail.buct.edu.cn (H. Du).
dialkyl(aryl)thiopyrimidines.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.056

G. Liu et al. / Tetrahedron 67 (2011) 5156e5161
5157
processes must be conducted under harsh conditions, especially in
OH
Cl
N
OH
the absence of activating substituents. In the present study, we suc-
cessfully introduced the two same or different alkyl(aryl)thio groups
into the pyrimidine ring with two different approaches, S-alkylation
and diazotizationealkylthionation, giving the key intermediate 2,4-
dialkyl(aryl)thio-6-chloropyrimidines. Thus far, there has been no
report on the application of the diazotizationealkylthionation re-
action to aminopyrimidine derivatives. The subsequent nucleophilic
displacement of the chloro groups by the corresponding amines af-
POCl3, PhNMe2
reflux, 1 h
RX, NaOH
r.t., 1-3 d
N
N
N
H2N
N
H
S
H2N
SR
H2N
N
SR
3a-b
2a-b
1
R = Me, n-Pr.
Scheme 1. Synthesis of compounds 3 from 1 via S-alkylation and chlorination.
The introduction of dialkyl(aryl)thio groups into the pyrimidine
rings was a key step. Initially, we attempted to prepare the designed
compounds 5 from 3 via displacement of sodium alkylmercaptides
and N-alkylation. However, the N-alkylation of 4-amino-2,6-
dialkyl(aryl)thiopyrimidines would give a mixture of two prod-
ucts, monoalkyl and dialkyl products.19 Thus, we took into account
the need to employ an alternative method.
fords
a series of 6-alkylamino-2,4-dialkyl(aryl)thiopyrimidines.
Herein, we describe the details of the convenient synthesis and the
evaluation results of all the synthesized compounds as human
platelet aggregation inhibitors.
It is already known that primary aromatic amines,20 such as ani-
line,21 pyridine,20 and guanosine derivatives,12 can be directly con-
verted into sulfides through diazotization under nonaqueous and
neutral conditions. However, the diazotizationealkylthionation re-
action of aminopyrimidine derivatives has not been reported in
earlier studies. We have found the addition of isoamyl nitrite
(i-AmONO) into the mixture of 2-alkylthio-4-amino-6-
chloropyrimidine derivatives 3 and excess dialkyl(aryl) disulfide at
60 ꢁC yields the corresponding alkyl(aryl)thio derivatives. In the
reaction process, the mixed solution became deep brown with
the immediate evolution of gas, which lasted until the completion of
the reaction. As a result, we have succeeded in transforming the free
amino groups of the compounds 3a,b to alkyl(aryl)thio groups,
whereby 2,4-dialkyl(aryl)thio-6-chloropyrimidines (4aee) were
obtained (Scheme 2). However, the mechanism of this reaction
presently has not been identified.21ꢀ24
2. Results and discussion
2.1. Chemistry
6-Alkylamino-2,4-dialkyl(aryl)thiopyrimidines have been pre-
viously prepared by two methods. One was the treatment of
3-phenyl-l,2,3-triaxolo[4,5-d]pyrimidine-5,7-dithione with butyl
lithium and an alkylating agent.13 Though the route was short, the
reaction was conducted at a low temperature (ꢀ70 ꢁC), resulting in
a
low yield (30%). Another method was the conversion of
4,6-dihydroxyl-2-mercaptopyrimidine to 6-alkylamino-2,4-
dialkylthiopyrimidines via S-alkylation, chlorination, and nucleo-
philic substitution.14 However, it was likely to afford the byproduct
of 2,4,6-trialkylthiopyrimidines.
During the last few years, our group has been working on the
development of new platelet aggregation antagonists with the
sulfur-substituted pyrimidine derivatives. In our method described
below, 6-alkylamino-2,4-dialkyl(aryl)thiopyrimidines can be syn-
thesized in good yields from a commercially available starting
material, 4-amino-6-hydroxyl-2-mercaptopyrimidine 1, under
simple and mild reaction conditions.
Cl
Cl
NHR2
H2NR2
N
N
N
N
i-AmONO
R1SSR1
R1S
N
SR
R1S
SR
EtOH, reflux
H2N
N
SR
We first synthesized 2-alkylthio-4-amino-6-chloropyrimidines
(3a,b) from the starting compound 1 by a well-established pro-
cedure (Scheme 1).15e17 The tautomerized thiol group was alky-
lated under basic conditions in the presence of the appropriate
alkyl halide to obtain 2a,b. Then, the hydroxyl group was chlori-
nated with phosphoryl chloride (POCl3)18 in combination with
dimethylaniline to increase the yield. After the excess POCl3 was
removed in vacuo, the residue was poured into the mixture of the
cooled concentrated ammonium hydroxide and chloroform. When
the residue was dissolved completely, it was extracted with chlo-
roform, concentrated, and then purified by column chromatogra-
phy to afford 2-alkylthio-4-amino-6-chloropyrimidines (3a,b).
3a-b
4a-e
5a-i
R = Me, n-Pr; R1 = n-Pr, i-Pr, Ph, Bn; R2 = Bn, CH2CH2Ph.
Scheme 2. Preparation of compounds 5 from 3.
To optimize the synthesis conditions, we selected 4-amino-6-
chloro-2-propylthiopyrimidines 3b and dipropyl disulfide as model
substrates, and examined the effects of different amount of the dia-
lkyl(aryl) disulfide in the presence of i-AmONO (6.2 equiv of 3b) in
anhydrous acetonitrile at 60 ꢁC. The examination was initially com-
menced with 10 M equiv of dipropyl disulfide according to the pre-
vious literature. However, the yield at the molar ratio of 7:1 (Table 1,
Table 1
Optimizing reaction conditions for synthesis of 4b
Cl
Cl
N
N
CH3CN
60 oC
(n-PrS)2
+
i-AmONO
+
N
H2N
S
S
N
S
3b
4b
Entry
Compound
(n-PrS)2
i-AmONO
Catalyst
Time (h)
Product
3b (equiv)
(equiv)
(equiv)
4b (Yield, %)
1
2
3
4
5
6
7
1
1
1
1
1
1
1
10
7
5
1
7
6.2
6.2
6.2
6.2
6.2
6.2
6.2
No
No
No
No
CuCl
CuCl
No
2
2
2
2.5
1
76
75
65
46
77
78
55
7
7
2
1

5158
G. Liu et al. / Tetrahedron 67 (2011) 5156e5161
entry 2) was comparable to that at 10:1, although it became lower
with furtherdecrease of themolar ratio (Table 1, entries3 and 4). That
is, the examination results with other molar ratios indicate that the
reaction can be sufficiently carried out with 7:1 of dipropyl disulfide
to 3. In the series of examinations, more importantly, we have found
that the reaction is definitelyaccelerated by the addition of CuCl even
under the same condition (Table 1, entries 2, 5, 6, and 7), which in-
dicates that the reaction is probably based on a radical mechanism.
Isoamyl nitrite would react with 3 under nonaqueous and neutral
conditions to immediately release N2 gas and concomitantly produce
the pyrimidinyl radical, which would efficiently be trapped by the
surrounding abundant dialkyl(aryl) disulfide.
Under our optimized conditions, we examined the reaction ef-
ficiency of dipropyl disulfide, diisopropyl disulfide, diphenyl
disulfide, and dibenzyl disulfide with (3a,b) in the presence of
i-AmONO, respectively. As can be seen from Table 2, almost all the
reactions were efficiently carried out to give 4 in good yields
(72e82%). However, the reaction yield on diphenyl disulfide be-
entry 2) was used as the nucleophilic reagent, the reaction proceeds
more slowly to keep the yield lower even for the reaction time
extended to 38 h (the yield: 73%). As for aniline, unfortunately, no
reaction occurred due to the low nucleophilicity of aniline, even
though the reaction time was prolonged.
2.2. Antiplatelet activity evaluation
The antiplatelet activity of all the synthetic compounds was
assayed on human platelet-rich plasma by using the Born’s
reported turbidimetric method,25 where ADP (adenosine 50-di-
phosphate) was employed as the agonist, and Cangrelor
(AR-C69931MX) was used as the positive control (PIC50 was 9.4,26
30
mM ADP, human washed platelets), in the experiment, the
percentage of platelet aggregation was 0% at the final concentra-
tions of 100 nM. The results indicate that compounds 5a and 5h
show some inhibitive effect on antiplatelet activities both at their
final concentrations of 10
compounds 5ceg and 5i show partial inhibition effect (ꢂ10%) on
platelet aggregation at the high concentration of 100 M. The
mM and 100 mM (Table 4). In contrast,
came lower (52%). It is assumed that a p-p conjugative effect in the
phenylthio radical would lower its reactivity compared with other
alkylthio radicals.
m
anitplatelet activities are comparable to those of 4-alkoxyl-2-
alkylthio-6-aminopyrimidines reported by Cattaneo et al.11 in
Table 2
the case that the same concentration of ADP (10 mM) was used. For
The diazotizationealkylthionation reaction of 3
all the observed partial antiplatelet activity, however, the struc-
tural difference of the alkyl(aryl)thio groups and N-mono-
substituents resulted in no remarkable variation on the
antiplatelet-activity.
Cl
Cl
N
N
CuCl, CH3CN
60 oC
N
R1SSR1
+
i-AmONO
+
R1S
SR
H2N
N
SR
3a-b
4a-e
Table 4
Entry
Compound 3
R
R1
Time (h)
Product 4
Yield (%)
The antiplatelet activity of compounds 5
1
2
3
4
5
3a
3b
3b
3b
3b
Me
n-Pr
n-Pr
i-Pr
C6H5
CH2C6H5
1
2
2.5
2.5
1.5
4a
4b
4c
4d
4e
81
75
72
52
82
Compounds 5
10
M (%)a
M (%)a
5a
5b
5c
5d
5e
5f
5g
5h
5i
n-Pr
n-Pr
n-Pr
n-Pr
m
107
104
101
99
98
91
101
94
103
97
102
96
97
90
102
100
99
94
100 m
a
The antiplatelet activity was expressed as percentage of the platelet aggregation
measured in the presence of each compound to that in presence of the vehicle
(DMSO) alone as blank. ADP was used as 10 mM.
Subsequent nucleophilic displacement of the chlorine atom of 4
by the various amines conveniently afforded the target compounds
5, as is shown in Table 3. The reaction is compatible with different
primary amines in the presence of triethylamine. However, the
results indicate that the effect of steric hindrance has a negative
influence on the nucleophilic substitution. When a sterically hin-
dered structure of amine, such as 1-phenylethylamine (Table 3,
3. Conclusions
We have developed a newand efficient synthesis of 6-alkylamino-
2,4-dialkyl(aryl)thiopyrimidines, where two same or different kinds
of alkylthio groups were introduced with two different methods into
the pyrimidine ring skeleton. Commercially available starting mate-
rial, 4-amino-6-hydroxyl-2-mercaptopyrimidine 1 was converted to
2-alkylthio-4-amino-6-chloropyrimidines (3a,b) via S-alkylation and
chlorination. Then, the second alkylthio group was introduced by
diazotization of 3 with i-AmONO and then reacted with dialkyl(aryl)
disulfide to afford 2,4-dialkyl(aryl)thio-6-chloropyrimidines (4aee).
The conversion of aminopyrimidine derivatives via the diazo-
tizationealkylthionation has been achieved in this study for the first
time. Subsequently, nucleophilic substitution with various amines
successfully gives the designed compounds (5aei). All the synthe-
sized compounds were evaluated for their inhibition activities on the
human platelet aggregation caused by ADP as agonist (ADP concen-
Table 3
Nucleophilic substitution of 2,4-dialkyl(aryl)thio-6-chloropyrimidines (4aee)
R2
Cl
HN
Et3N, EtOH
N
N
H2N-R2
+
reflux
R1S
N
SR
R1S
N
SR
5a-i
4a-e
Entry Compound 4
R
R1
R2
Time Product Yield
(h)
5
(%)
tration: 10
(ꢂ10%) against platelet aggregation at their final concentration of
100 M. Although the examined series of N-monoalkylated and
dialkyl(aryl)thio-substituted pyrimidines currently have no remark-
able activity, the observed partial effect seems to suggest the im-
portance of further screening forother substituents for the purpose of
increasing the activity. In this view, our developed synthesis is ef-
fective tool for the access of various 6-alkylamino-2,4-dialkyl(aryl)
thiopyrimidines not to be investigated yet.
mM). Some compounds (5ceg and 5i) show partial effect
1
2
3
4
5
6
7
8
9
4a
4a
4b
4b
4b
4c
4d
4d
4e
4c
Me n-Pr
Me n-Pr
n-Pr n-Pr
n-Pr n-Pr
n-Pr n-Pr
n-Pr i-Pr
n-Pr C6H5
n-Pr C6H5
C6H5CH2CH2
C6H5CH(CH3)
C6H5CH2
p-MeC6H4CH2
m-MeOC6H4CH2CH2 16
p-MeOC6H4CH2
cyclohexyl
C6H5CH2CH2
12
38
13
14
5a
5b
5c
5d
5e
5f
5g
5h
5i
84
73
85
84
84
86
83
88
83
m
15
10
16
15
40
n-Pr CH2C6H5 p-MeOC6H4CH2
n-Pr i-Pr C6H5
10
No reaction

G. Liu et al. / Tetrahedron 67 (2011) 5156e5161
5159
4. Experimental section
4.1. General
NMR (300 MHz, CDCl3)
d
¼6.16 (s,1H, CH), 5.18 (br s, 2H, NH2), 2.50 (s,
3H, SCH3). 13C NMR (75 MHz, CDCl3)
d
¼172.4, 163.2, 159.3, 99.1, 13.9.
4.3.2. 4-Amino-6-chloro-2-propylthiopyrimidine (3b). Colorless crys-
tals, yield: 3.08 g (56%), mp 98e100 ꢁC (lit.16a 101e102 ꢁC). 1H NMR
The chemicals were obtained from commercial sources, and the
solvents used in reactions were dried by standard procedures prior
to use. Melting points were measured on a Yanaco MP-500 melting
point apparatus without being corrected. 1H and 13C NMR spectra
were recorded on a Varian Mercury 200 spectrometer, Plus 300
spectrometer or Bruker 400 plus. Chemical shifts are reported
(300 MHz, CDCl3)
d
¼6.11 (s, 1H, CH), 5.03 (br s, 2H, NH2), 3.04 (t,
J¼7.3 Hz, 2H, SCH2), 1.70 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.00 (t,
J¼7.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3)
¼172.2, 163.2, 159.4,
d
99.0, 32.7, 22.4, 13.4.
relative to TMS (1H) or DMSO-d6
(
13C). IR spectra were recorded
4.4. General procedure for the synthesis of 2,4-dialkyl(aryl)
on a PerkineElmer Fourier transform infrared spectrometer Spec-
trum 100. High Resolution Mass spectra (HRMS-ESI) were obtained
on an Agilent LC/TOF mass spectrometer. P.E. denotes petroleum
ether (bp 60e90 ꢁC). Cangrelor (AR-C69931MX) was a gift from
AstraZeneca (Loughborough, UK). ADP was purchased from
Chrono-Log (Havertown, PA, USA); sodium citrate was purchased
from Sigma (St Louis, MO).
thio-6-chloropyrimidines (4aee)
Compound 3a or 3b (5.85 mmol) was added into the dry ace-
tonitrile (35 mL) containing appropriate dialkyl(aryl) disulfide
(40.95 mmol) and CuCl (0.29 m mol). The mixture was purged
thoroughly with N2 and stirred at room temperature for 30 min.
Isoamyl nitrite (4.25 g, 36.3 mmol) was added to the solution im-
mediately, the reaction was stirred for 30 min at room temperature,
and then the mixture was heated at 60 ꢁC. The reaction time was
listed in Table 2. The disappearance of the starting material was
monitored by TLC (EtOAc/P.E., 1:10, v/v). After removing CuCl, the
solvent and other volatiles were removed under reduced pressure.
The residue was purified by column chromatography (Et3N-neu-
tralized silica gel, gradient elution separation with EtOAc/P.E.,
1:40e1:20, v/v) to afford the product as colorless or yellow oil.
4.2. General procedure for the synthesis of 2-alkylthio-4-
amino-6-hydroxylpyrimidines (2a,b)
Haloalkane (12.4 mmol) was added dropwise into a solution of
4-amino-6-hydroxyl-2-mercaptopyrimidine (1) (1.78 g, 12.4 mmol)
in 1 mol/L NaOH (12.4 mL). The reaction mixture was kept under
stirring at room temperature for 1e3 days, until TLC (EtOAc/MeOH,
15:1, v/v) monitoring indicated no unreacted materials existed. The
mixture was neutralized with acetic acid. The resulting solid was
collected by filtration, washed with P.E. (10 mL) and water (10 mL),
and dried.
4.4.1. 4-Chloro-2-methylthio-6-propylthiopyrimidine (4a). Colorless
oil, yield: 1.11 g (81%), IR (film): 2961, 2875,1732,1358, 810 cmꢀ1. 1H
NMR (400 MHz, CDCl3)
d
¼6.80 (s, 1H, CH), 3.12 (t, J¼7.3 Hz, 2H,
SCH2), 2.52 (s, 3H, SCH3), 1.72 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.01
(t, J¼7.3 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3)
¼172.6, 171.9,
d
158.7, 113.0, 31.7, 22.6, 14.3, 13.4. ESI-HRMS: m/z [MþH]þ calcd for
4.2.1. 4-Amino-6-hydroxyl-2-methylthiopyrimidine (2a). Colorless
crystals, yield: 1.85 g (95%), mp 264e266 ꢁC (lit.15 268e269 ꢁC). 1H
C8H12ClN2S2: 235.0125; found: 235.0138.
NMR (300 MHz, DMSO-d6)
NH2), 4.89 (s, 1H, CH), 2.41 (s, 3H, SCH3). 13C NMR (50 MHz, DMSO-
d6)
¼164.6, 163.6, 163.0, 81.3, 12.7.
d
¼11.84 (br s, 1H, OH), 6.44 (br s, 2H,
4.4.2. 4-Chloro-2,6-dipropylthiopyrimidine (4b). Yellow oil, yield:
1.15 g (75%), IR (film): 2965, 2872, 1665, 1361, 811 cmꢀ1 1H NMR
.
d
(300 MHz, CDCl3)
d
¼6.72 (s, 1H, CH), 3.04 (t, J¼7.3 Hz, 2H, SCH2),
3.01 (t, J¼7.3 Hz, 2H, SCH2),1.67 (sextet, J¼7.3 Hz, 2H, SCH2CH2),1.65
4.2.2. 4-Amino-6-hydroxyl-2-propylthiopyrimidine (2b). Colorless
crystals, yield: 2.09 g (91%), mp 206e208 ꢁC (lit.15 209e210 ꢁC).
(sextet, J¼7.3 Hz, 2H, SCH2CH2), 0.94 (t, J¼7.3 Hz, 3H, CH3), 0.95 (t,
J¼7.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3)
¼172.3, 171.7, 158.5,
d
1H NMR (300 MHz, DMSO-d6)
d
¼11.50 (br s, 1H, OH), 6.46 (br s,
112.9, 33.0, 31.5, 22.5 (2 carbons),13.4,13.3. ESI-HRMS: m/z [MþH]þ
2H, NH2), 4.87 (s, 1H, CH), 3.03 (t, J¼7.2 Hz, 2H, SCH2), 1.62
calcd for C10H16ClN2S2: 263.0438; found: 263.0447.
(sextet, J¼7.2 Hz, 2H, SCH2CH2), 0.95 (t, J¼7.2 Hz, 3H, CH3). 13C
NMR (50 MHz, DMSO-d6)
13.2.
d
¼164.4, 163.5, 162.2, 81.3, 31.2, 22.4,
4.4.3. 4-Chloro-6-isopropylthio-2-propylthiopyrimidine (4c). Yellow
oil, yield: 1.11 g (72%), IR (film): 2962, 2872, 1700, 1361, 808 cmꢀ1
.
1H NMR (300 MHz, CDCl3)
d
¼6.78 (s,1H, CH), 4.05 (heptet, J¼6.8 Hz,
1H, SCH), 3.10 (t, J¼7.3 Hz, 2H, SCH2), 1.77 (sextet, J¼7.3 Hz, 2H,
SCH2CH2), 1.42 (d, J¼6.8 Hz, 6H, SCH(CH3)2), 1.05 (t, J¼7.3 Hz, 3H,
4.3. General procedure for the synthesis of 2-alkylthio-4-
amino-6-chloropyrimidines (3a,b)
CH3). 13C NMR (50 MHz, CDCl3)
d
¼172.4, 171.8, 158.6, 112.9, 35.1,
32.9, 22.8 (2 carbons), 22.6, 13.4. ESI-HRMS: m/z [MþH]þ calcd for
2-Alkylthio-4-amino-6-hydroxylpyrimidine (2a,b) (27 mmol),
POCl3 (15 mL, 162 mmol), and PhNMe2 (6.8 mL, 54 m mol) were
refluxed together for 1 h. The excess POCl3 was removed in vacuo,
and then the residue was poured into 60 mL (1:1, v/v) of cooled
concentrated ammonium hydroxide and chloroform. The solution
was kept stirring at room temperature. After POCl3 was dissolved
completely, the aqueous phase was extracted with CHCl3
(3ꢃ20 mL), the combined organic phases were dried with MgSO4,
concentrated, and purified by flash column chromatography (Et3N-
neutralized silica gel, gradient elution separation with EtOAc/P.E.,
1:50e1:2, v/v) and then by recrystallization from cyclohexane to
afford the product as colorless crystals.
C10H16ClN2S2: 263.0438; found: 263.0447.
4.4.4. 4-Chloro-6-phenylthio-2-propylthiopyrimidine (4d). Yellow oil,
yield: 0.90 g (52%), IR (film): 2962, 2872, 1732, 1359, 809, 748,
690 cmꢀ1. 1H NMR (300 MHz, CDCl3)
d
¼7.61e7.47 (m, 5H, ArH), 6.43
(s, 1H, CH), 2.94 (t, J¼7.3 Hz, 2H, SCH2), 1.62 (sextet, J¼7.3 Hz, 2H,
SCH2CH2), 0.95 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (50 MHz, CDCl3)
d
¼173.7, 172.6, 159.8, 135.8, 130.3, 129.8, 126.9, 111.2, 32.9, 22.3, 13.3.
ESI-HRMS: m/z [MþH]þ calcd for C13H14ClN2S2: 297.0281; found:
297.0263.
4.4.5. 4-Benzylthio-6-chloro-2-propylthiopyrimidine (4e). Yellow oil,
yield: 1.49 g (82%), IR: 2962, 2867, 1718, 1361, 808, 774, 698 cmꢀ1. 1H
4.3.1. 4-Amino-6-chloro-2-methylthiopyrimidine (3a). Colorless
crystals, yield: 3.94 g (83%), mp 122e124 ꢁC (lit.18 126e127 ꢁC). 1H
NMR (300 MHz, CDCl3)
4.44 (s, 2H, PhCH2), 3.11 (t, J¼7.3 Hz, 2H, SCH2), 1.76 (sextet, J¼7.3 Hz,
d
¼7.40e7.23 (m, 5H, ArH), 6.83 (s, 1H, CH),

5160
G. Liu et al. / Tetrahedron 67 (2011) 5156e5161
2H, SCH2CH2), 1.03 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (50 MHz, CDCl3)
(neat): 3253, 2964, 829, 772, 693 cmꢀ1. 1H NMR (200 MHz, CDCl3)
d¼172.6, 170.9, 158.8, 136.3, 128.8, 128.7, 127.5, 112.6, 33.8, 33.0, 22.4,
d
¼7.26e7.18 (m, 1H, ArH), 6.80e6.74 (m, 3H, ArH), 5.85 (s, 1H, CH),
13.4. ESI-HRMS: m/z [MþH]þ calcd for C14H16ClN2S2: 311.0438;
4.89 (br s, 1H, NH), 3.79 (s, 3H, OCH3), 3.51 (dt, J¼6.5 Hz, 2H,
NHCH2), 3.08 (t, J¼7.3 Hz, 2H, SCH2), 3.07 (t, J¼7.3 Hz, 2H, SCH2),
2.84 (t, J¼6.9 Hz, 2H, NHCH2CH2), 1.75 (sextet, J¼7.3 Hz, 2H,
SCH2CH2), 1.71 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.03 (t, J¼7.3 Hz, 3H,
found: 311.0409.
4.5. General procedure for the synthesis of 6-alkylamino-2,4-
dialky(aryl)thiopyrimidines (5aei)
CH3), 1.02 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3)
¼170.2,
d
167.5, 160.8, 159.8, 140.1, 129.6, 121.0, 114.5, 111.8, 94.6, 55.1, 42.3,
35.4, 32.6, 31.1, 23.1 (2 carbons), 13.5, 13.4. ESI-HRMS: m/z [MþH]þ
calcd for C19H28N3OS2: 378.1668; found: 378.1675.
A solution of 2,4-dialkyl(aryl)thio-6-chloropyrimidine (4aee)
(0.7 mmol), the appropriate amine (4.2 mmol) and triethylamine
(1.4 mmol) in anhydrous ethanol (20 mL) was refluxed for the given
time as reported in Table 3. Then the reaction mixture was evap-
orated in vacuo, and the residue was purified by flash column
chromatography (Et3N-neutralized silica gel, gradient elution sep-
aration with EtOAc/P.E., 1:40e1:20, v/v) and then by re-
crystallization from cyclohexane to afford the product as colorless
crystals.
4. 5. 6. 4-(4-Methoxybenzyl)amino-6-isopropylthio-2-
propylthiopyrimidine (5f). Colorless crystals, yield: 244.3 mg (86%),
mp 112e113 ꢁC. IR (neat): 3248, 2964, 1169, 803, 743, 679 cmꢀ1. 1H
NMR (200 MHz, CDCl3)
d
¼7.20 (d, J¼8.7 Hz, 2H, ArH), 6.85 (d,
J¼8.7 Hz, 2H, ArH), 5.85 (s, 1H, CH), 5.35 (br s, 1H, NH), 4.37 (d,
J¼5.7 Hz, 2H, NHCH2), 3.98 (heptet, J¼6.8 Hz, 1H, SCH), 3.78 (s, 3H,
OCH3), 3.04 (t, J¼7.3 Hz, 2H, SCH2), 1.73 (sextet, J¼7.3 Hz, 2H,
SCH2CH2), 1.36 (d, J¼6.8 Hz, 6H, SCH(CH3)2), 1.00 (t, J¼7.3 Hz, 3H,
4.5.1. 2-Methylthio-4-phenethylamino-6-propylthiopyrimidine
(5a). Colorless crystals, yield: 187.9 mg (84%), mp 96e98 ꢁC. IR
(neat): 3260, 2959, 698, 796, 833 cmꢀ1. 1H NMR (300 MHz, CDCl3)
CH3). 13C NMR (50 MHz, CDCl3)
d
¼170.2, 167.6, 161.0, 158.9, 129.8,
128.6, 114.0, 95.1, 55.2, 44.8, 34.3, 32.6, 23.2, 23.1, 13.5. ESI-HRMS:
d
¼7.34e7.29 (m, 3H, ArH), 7.24e7.18 (m, 2H, ArH), 5.87 (s, 1H, CH),
m/z [MþH]þ calcd for C18H26N3OS2: 364.1512; found: 364.1518.
4.76 (br s, 1H, NH), 3.52 (m, 2H, NHCH2), 3.09 (t, J¼7.3 Hz, 2H, SCH2),
2.86 (t, J¼6.9 Hz, 2H, NHCH2CH2), 2.50 (s, 3H, SCH3), 1.72 (sextet,
J¼7.3 Hz, 2H, SCH2CH2), 1.01 (t, J¼7.3 Hz, 3H, CH3). 13C NMR
4.5.7. 4-Cyclohexylamino-6-phenylthio-2-propylthiopyrimidine
(5g). Colorless crystals, yield: 208.9 mg (83%), mp 80e81 ꢁC. IR
(neat): 3252, 2927, 827, 749, 690 cmꢀ1. 1H NMR (200 MHz, CDCl3)
(50 MHz, CDCl3)
d
¼170.4, 167.6, 160.8, 138.5, 128.7, 128.6, 126.5,
94.6, 42.5, 35.4, 31.1, 23.0, 13.9, 13.4. ESI-HRMS: m/z [MþH]þ calcd
d
¼7.61e7.40 (m, 5H, ArH), 5.40 (s, 1H, CH), 4.68 (br s, 1H, NH), 2.92
for C16H22N3S2: 320.1250; found: 320.1256.
(t, J¼7.3 Hz, 2H, SCH2), 1.90e1.06 (m, 13H, SCH2CH2þcyclohexyl),
0.95 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (50 MHz, CDCl3)
¼170.6, 169.5,
d
4.5.2. 2-Methylthio-4-(1-phenylethyl)amino-6-propylthiopyrimidine
(5b). Colorless crystals, yield: 163.3 mg (73%), mp 80e82 ꢁC. IR
(neat): 3352, 2967, 814, 758, 695 cmꢀ1. 1H NMR (200 MHz, CDCl3)
160.5,135.8,129.4,129.3,129.1, 94.2, 49.7, 32.7, 32.5, 25.4, 24.6, 22.9,
13.4. ESI-HRMS: m/z [MþH]þ calcd for C19H26N3S2: 360.1563;
found: 360.1570.
d
¼7.39e7.20 (m, 5H, ArH), 5.70 (s, 1H, CH), 5.16 (m, 1H, NHCH), 4.67
(br s, 1H, NH), 2.98 (t, J¼7.3 Hz, 2H, SCH2), 2.45 (s, 3H, SCH3), 1.62
(sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.50 (d, J¼6.8 Hz, 3H, NHCH(CH3)),
4.5.8. 4-Phenethylamino-6-phenylthio-2-propylthiopyrimidine
(5h). Colorless crystals, yield: 235.0 mg (88%), mp 98e100 ꢁC. IR
(neat): 3251, 2964, 802, 751, 700 cmꢀ1. 1H NMR (200 MHz, CDCl3)
0.95 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (50 MHz, CDCl3)
¼170.2, 168.0,
d
160.2, 143.3, 128.7, 127.3, 125.6, 94.8, 51.3, 31.1, 23.7, 22.9, 13.8, 13.4.
ESI-HRMS: m/z [MþH]þ calcd for C16H22N3S2: 320.1250; found:
320.1256.
d
¼7.63e7.09 (m, 10H, ArH), 5.49 (s, 1H, CH), 5.19 (br s, 1H, NH), 3.42
(m, 2H, NHCH2), 2.98 (t, J¼7.3 Hz, 2H, SCH2), 2.80 (t, J¼7.2 Hz, 2H,
NHCH2CH2), 1.67 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 0.98 (t, J¼7.3 Hz,
3H, CH3). 13C NMR (50 MHz, CDCl3)
d¼170.2, 161.1, 138.4, 135.9,
4.5.3. 4-Benzylamino-2,6-dipropylthiopyrimidine (5c). Colorless crys-
tals, yield: 181.8 mg (85%), mp 66e67 ꢁC. IR (neat): 3250, 2965, 849,
129.6, 129.5, 128.9, 128.7, 128.6, 126.6, 94.0, 42.5, 35.4, 32.7, 22.9,
13.5. ESI-HRMS: m/z [MþH]þ calcd for C21H24N3S2: 382.1406;
found: 382.1399.
799, 695 cmꢀ1. 1H NMR (300 MHz, CDCl3)
d
¼7.35e7.27 (m, 5H, ArH),
5.86 (s,1H, CH), 5.47 (br s,1H, NH), 4.45 (d, J¼5.6 Hz, 2H, NHCH2), 3.05
(t, J¼7.3 Hz, 2H, SCH2), 3.04 (t, J¼7.3 Hz, 2H, SCH2), 1.72 (sextet,
J¼7.3 Hz, 2H, SCH2CH2), 1.68 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.00 (t,
J¼7.3 Hz, 3H, CH3), 0.99 (t, J¼7.3 Hz, 3H, CH3). 13C NMR (75 MHz,
4.5.9. 4-Benzylthio-6-(4-methoxybenzyl)amino-2-propylthiopyrim-
idine (5i). Colorless crystals, yield: 239.1 mg (83%), mp 110e112 ꢁC.
IR (neat): 3253, 2965, 1175, 833, 712, 696 cmꢀ1. 1H NMR (300 MHz,
CDCl3)
d
¼170.1, 167.7, 161.0, 137.9, 128.6, 127.4, 127.2, 94.8, 45.3, 32.6,
CDCl3)
d
¼7.23e7.19 (m, 5H, ArH), 7.20 (d, J¼8.7 Hz, 2H, ArH), 6.86
31.1, 23.0 (2 carbons), 13.5, 13.4. ESI-HRMS: m/z [MþH]þ calcd for
(d, J¼8.7 Hz, 2H, NHCH2), 5.88 (s, 1H, CH), 5.01 (br s,1H, NH), 4.39 (s,
2H, SCH2), 4.36 (m, 2H, NCH2), 3.80 (s, 3H, OCH3), 3.06 (t, J¼7.3 Hz,
2H, SCH2), 1.73 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 0.99 (t, J¼7.3 Hz, 3H,
C17H24N3S2: 334.1406; found: 334.1412.
4.5.4. 4-(4-Methylbenzyl)amino-2,6-dipropylthiopyrimidine
(5d). Colorless crystals, yield: 204.4 mg (84%), mp 92e94 ꢁC. IR
(neat): 3249, 2966, 828, 799, 685 cmꢀ1. 1H NMR (300 MHz, CDCl3)
CH3). 13C NMR (50 MHz, CDCl3)
d¼170.4, 167.0, 161.0, 159.0, 137.7,
129.8, 128.8, 128.6, 128.5, 127.1, 114.1, 94.8, 55.2, 44.9, 33.3, 32.6,
23.0, 13.5. ESI-HRMS: m/z [MþH]þ calcd for C22H26N3OS2: 412.1512;
found: 412.1526.
d
¼7.19 (d, J¼8.1 Hz, 2H, ArH), 7.14 (d, J¼8.1 Hz, 2H, ArH), 5.88 (s, 1H,
CH), 5.32 (br s, 1H, NH), 4.42 (d, J¼5.2 Hz, 2H, NHCH2), 3.06 (t,
J¼7.3 Hz, 2H, SCH2), 3.05 (t, J¼7.3 Hz, 2H, SCH2), 2.35 (s, 3H,
C6H4CH3), 1.75 (sextet, J¼7.3 Hz, 2H, SCH2CH2), 1.70 (sextet,
J¼7.3 Hz, 2H, SCH2CH2), 1.02 (t, J¼7.3 Hz, 3H, CH3), 1.03 (t, J¼7.3 Hz,
4.6. In vitro examination for inhibition of platelet
aggregation
3H, CH3). 13C NMR (75 MHz, CDCl3)
d¼170.2, 167.7, 161.0, 137.1,
Blood was sampled from the cubital vein of healthy volunteers
without taking aspirin or other nonsteroidal anti-inflammatory
drugs for at least 14 days on the basis of informed consent before
blood collection. The blood sample was collected in 50 mL sample
tubes containing 3.8% sodium citrate (1:9, v/v), and centrifuged at
300 rpm for 20 min to generate platelet-rich plasma (PRP). The
residual blood was centrifuged at 900 rpm for 10 min. The
134.8, 129.3, 127.2, 94.8, 45.1, 32.6, 31.1, 23.1 (2 carbons), 21.0, 13.5,
13.4. ESI-HRMS: m/z [MþH]þ calcd for C18H26N3S2: 348.1563;
found: 348.1569.
4.5.5. 4-(3-Methoxyphenethyl)amino-2,6-dipropylthiopyrimidine
(5e). Colorless crystals, yield: 222.0 mg (84%), mp 46e48 ꢁC. IR

G. Liu et al. / Tetrahedron 67 (2011) 5156e5161
5161
6. Nargund, L. V. G.; Badiger, V. V.; Yarna, S. U. Eur. J. Med. Chem. 1994, 29,
245e247.
7. Zhang, H. C.; Maryanoff, B. E.; Ye, H.; Chen, C. PCT Int. Appl. WO2008054795,
supernatant fraction was called platelet-poor plasma (PPP). Aliquots
of 500 L of PRP were distributed in test cuvettes and inserted into
m
the incubation chamber of an aggregometer (Model 400VS, Chrono-
Log, Haverston, PA) at 37 ꢁC. Platelet aggregation was measured on
the aggregometer using the PRP fractions after activation by ADP
2008 Chem. Abstr. 2008, 148, 517743.
8. Kortum, S. W.; Lachance, R. M.; Schweitzer, B. M.; Yalamanchili, G.; Rahman, H.;
Ennis, M. D.; Huff, R. M.; Tenbrinkt, R. E. Bioorg. Med. Chem. Lett. 2009, 19,
5919e5923.
9. Kim, H. S.; Barak, D.; Harden, T. K.; Boyer, J. L.; Jacobson, K. A. J. Med. Chem. 2001,
44, 3092e3108.
10. Xu, B.; Stephens, A.; Kirschenheuter, G.; Greslin, A. F.; Cheng, X.; Sennelo, J.;
Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S. A.; Kim, H. S.; Bischofberger, N.;
Cook, G.; Jacobson, K. A. J. Med. Chem. 2002, 45, 5694e5709.
11. Crepaldi, P.; Crepaldi, B.; Cacciari, B.; Bonache, M. C.; Spalluto, G.; Varani, K.;
Borea, P. A.; von Kuegelgen, I.; Hoffmann, K.; Pugliano, M.; Razzari, C.; Cattaneo,
M. Bioorg. Med. Chem. 2009, 17, 4612e4621.
12. Ingall, A. H.; Dixon, J.; Bailey, A.; Coombs, M. E.; Cox, D.; McInally, J. I.; Hunt, S.
F.; Kindon, N. D.; Teobald, B. J.; Willis, P. A.; Humphries, R. G.; Leff, P.; Clegg, J. A.;
Smith, J. A.; Tomlinson, W. J. Med. Chem. 1999, 42, 213e220.
13. Dooley, M. J.; Quinn, R. J.; Patalinghug, W. C.; White, A. H. Tetrahedron Lett. 1990,
31, 6103e6104.
(final concentration 10 m
M) according to Born,25 DMSO was used as
a vehicle control and Cangrelor was used as the positive control. The
test compounds were dissolved in DMSO (below at 0.5% final con-
centration) and added to the PRP for 1 min before activation with
agonist. The extent of aggregation was quantified by determining
the maximum height of the analysis curve. Each sample was allowed
to aggregate for at least 3 min. The chart recorder (Model 707,
Chrono-Log, Haverston, PA, USA) was set for 1 cm/min. The baseline
was set using the PPP fraction as blank. The anti-platelet aggregation
activity was expressed as percent inhibition by comparison with
that measured in presence of vehicle (DMSO) alone.
14. Cheshire, D. R.; Cox, R. J.; Meghani, P.; Preston, C. F.; Smith, N. M.; Stonehouse, J.
P. PCT Int. Appl. WO2006024823, 2006 Chem. Abstr. 2006, 144, 292773.
15. Cosimelli, B.; Greco, G.; Ehlardo, M.; Novellino, E.; Settimo, F. D.; Taliani, S.;
Motta, C. L.; Bellandi, M.; Tuccinardi, T.; Martinelli, A.; Ciampi, O.; Trincavelli,
M. L.; Martini, C. J. Med. Chem. 2008, 51, 1764e1770.
Supplementary data
16. (a) Tsuji, T.; Watanabe, S.; Nakadai, Y.; Toyoshima, S. Chem. Pharm. Bull. 1962, 10,
9e13; (b) Slivka1, N. Y.; Gevaza, Y. I.; Staninets, V. I. Chem. Heterocycl. Compd.
2004, 40, 660e666; (c) Kang, Y.; Kim, S.; Myoung, Y.; Baek, D. Heterocycles
2000, 53, 1551e1557.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.05.056.
17. (a) Baddiley, J.; Topham, A. J. Chem. Soc. 1944, 678e679; (b) Pfleiderer, W.; Fink,
H. Justus Liebigs Ann. Chem. 1962, 657, 149e155.
References and notes
18. Baker, B. R.; Joseph, J. P.; Schaub, R. E. J. Org. Chem. 1954, 19, 631e637.
19. Sechi, M.; Rizzi, G.; Bacchi, A.; Carcelli, M.; Rogolino, D.; Pala, N.; Sanchez, T. W.;
Taheri, L.; Dayam, R.; Neamati, N. Bioorg. Med. Chem. 2009, 17, 2925e2935.
20. Giam, C. S.; Kikukawa, K. J. Chem. Soc., Chem. Commun. 1980, 756e757.
21. Allaire, F. S.; Lyga, J. W. Synth. Commun. 2001, 31, 1857e1861.
22. Nair, V.; Richardson, S. G. Synthesis 1982, 670e672.
23. Reszka, K.; Naghipur, A.; Lownz, J. W. Free Radical Res. Commun. 1990, 10, 47e56.
24. Naghipur, A.; Reszka, K.; Lown, J. W. Can. J. Chem. 1990, 68, 1950e1960.
25. Born, G. V. R. Nature (London) 1962, 194, 927e929.
1. Clark, J.; Shohhet, M. S.; Korakas, D.; Varvounis, G. J. Heterocycl. Chem. 1993, 30,
1065e1072.
2. Tozcoparan, B.; Ertan, M.; Kelicen, P.; Demirdamar, R. Farmaco 1999, 54,
588e593.
3. Quiroga, J.; Insuasty, B.; Craz, S.; Herrandez, P.; Bolafios, A.; Moreno, R.;
Harmoza, A.; DeAlmeidas, R. H. J. Heterocycl. Chem. 1998, 35, 1333e1338.
4. Santagati, M.; Modica, M.; Santagati, A.; Russo, F.; Spampinato, S. Pharmazie
1996, 51, 7e15.
26. Cattaneo, M. Circulation 2010, 121, 171e179.
5. Ahluwalia, V. K.; Chopra, M.; Chandra, R. J. Chem. Res. 2000, 162e163.