New approach to synthesize symmetrical and unsymmetrical 6-(N-Alkyl(Aryl)amino)-and 6-(N, N-dialkyl(Aryl)amino)-2,4-bis(Alkyl(Aryl)Thio) pyrimidines as anti-platelet agents

Article abstract of DOI:10.1080/10426507.2011.636782

A new and straightforward procedure has been developed for the preparation of symmetrical and unsymmetrical 6-(N-alkyl(aryl)amino)-and 6-(N,N- bisalkyl(aryl)amino)-2,4-bis(alkyl(aryl)thio)pyrimidines. The two identical or different alkylthio groups were s

Full text of DOI:10.1080/10426507.2011.636782

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Phosphorus, Sulfur, and Silicon and the
Related Elements
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New Approach to Synthesize
Symmetrical and Unsymmetrical
6-(N-Alkyl(Aryl)Amino)- and 6-
(N,N-Dialkyl(Aryl)Amino)-2,4-
Bis(Alkyl(Aryl)Thio)Pyrimidines as anti-
Platelet Agents
Guocheng Liu a , Jiaxi Xu a , Mingwu Yu a , Ning Chen a , Si Zhang b ,
Zhongren Ding b & Hongguang Du a
a State Key Laboratory of Chemical Resource Engingeering,
Department of Organic Chemistry, College of Science , Beijing
University of Chemical Technology , Beijing , China
b Key Laboratory of Molecular Medicine, Ministry of Education,
and Department of Biochemistry and Molecular Biology , Fudan
University Shanghai Medical College , Shanghai , China
Accepted author version posted online: 04 Jan 2012.Published
online: 27 Mar 2012.
To cite this article: Guocheng Liu , Jiaxi Xu , Mingwu Yu , Ning Chen , Si Zhang , Zhongren Ding
& Hongguang Du (2012) New Approach to Synthesize Symmetrical and Unsymmetrical 6-(N-
Alkyl(Aryl)Amino)- and 6-(N,N-Dialkyl(Aryl)Amino)-2,4-Bis(Alkyl(Aryl)Thio)Pyrimidines as anti-
Platelet Agents, Phosphorus, Sulfur, and Silicon and the Related Elements, 187:5, 650-659, DOI:
10.1080/10426507.2011.636782
To link to this article: http://dx.doi.org/10.1080/10426507.2011.636782
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Phosphorus, Sulfur, and Silicon, 187:650–659, 2012
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2011.636782
NEW APPROACH TO SYNTHESIZE SYMMETRICAL
AND UNSYMMETRICAL 6-(N-ALKYL(ARYL)AMINO)-
AND 6-(N,N-DIALKYL(ARYL)AMINO)-2,4-
BIS(ALKYL(ARYL)THIO)PYRIMIDINES
AS ANTI-PLATELET AGENTS
Guocheng Liu,1 Jiaxi Xu,1 Mingwu Yu,1 Ning Chen,1 Si Zhang,2
Zhongren Ding,2 and Hongguang Du1
1State Key Laboratory of Chemical Resource Engingeering, Department of Organic
Chemistry, College of Science, Beijing University of Chemical Technology,
Beijing China
2Key Laboratory of Molecular Medicine, Ministry of Education, and Department of
Biochemistry and Molecular Biology, Fudan University Shanghai Medical College,
Shanghai, China
GRAPHICAL ABSTRACT
Abstract A new and straightforward procedure has been developed for the preparation of
symmetrical and unsymmetrical 6-(N-alkyl(aryl)amino)- and 6-(N,N-bisalkyl(aryl)amino)-
2,4-bis(alkyl(aryl)thio)pyrimidines. The two identical or different alkylthio groups were
successfully introduced into the pyrimidine ring of 4-amino-6-hydroxy-2- mercaptopy-
rimidine via S-alkylation with alkyl halides, and via a nucleophilic displacement with
sodium alkylmercaptides, affording the key intermediate symmetrical and unsymmet-
rical 2,4-bis(alkyl(aryl)thio)-6-aminopyrimidines. Subsequently, N-alkylation of the
2,4-bis(alkyl(aryl)thio)-6-aminopyrimidines with alkyl halides conveniently gave the desired
products. The human anti-platelet activities of all the synthesized new compounds were also
evaluated.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.
Keywords Pyrimidines; nucleophilic displacement; N-alkylation; anti-platelet activity
Received 20 September 2011; accepted 26 October 2011.
Address correspondence to Hongguang Du, State Key Laboratory of Chemical Resource Engingeering,
Department of Organic Chemistry, College of Science, Beijing University of Chemical Technology, Beijing
100029, China. E-mail: dhg@mail.buct.edu.cn
650

SYNTHESIZING OF POTENTIAL HUMAN PLATELET AGGREGATION INHIBITORS 651
INTRODUCTION
Many heterocyclic compounds have been found to be effective platelet aggregation
inhibitors.1–6 Especially, alkylthio-substituted pyrimidines have attracted much attention
for their dramatic anti-platelet aggregation activities.7–11 Some of them have been widely
attempted to develop the antagonist of adenosine diphosphate (ADP) receptor.8,9 Thus,
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 ADP
receptor antagonists.
In 2009, Cattaneo’s group reported 4-alkoxy-2-alkylthio-6-aminopyrimidines as ADP
receptor antagonists, and suggested that the presence of alkylthio substituents and a free
amino group were important for the activity.10 However, according to Ingall’s results that
N-monoalkylation at the 6-position of adenosine derivatives can effectively enhance the ac-
tivity,12 we presume that the introduction of substituents at the free amino group, such as N-
monoalkylation and N,N-dialkylation, would increase the activity. Thus, a new series of 6-
(N-alkyl(aryl)amino)- and 6-(N,N-dialkyl(aryl)amino)-2,4-bis(alkyl(aryl)thio)pyrimidines
were designed to evaluate their human anti-platelet activities.
There are a few reports on the synthesis and anti-platelet activity evaluation of 6-
alkyl(aryl)amino-2,4-bis(alkyl(aryl)thio)pyrimidines. They have been previously prepared
via the treatment of 3-phenyl-1,2,3-triaxolo[4,5-d]pyrimidine-5,7-dithione with an alkylat-
ing agent in the presence of butyllithium in low yield (30%),13 via the conversion of 4,6-
dihydroxy-2-mercaptopyrimidine to 6-alkylamino-2,4-bis(alkylthio)pyrimidines through
S-alkylation, chlorination, and nucleophilic substitution with 2,4,6-trialkylthiopyrimidines
as byproducts,14 and via diazotation-alkylthionation and ammonolysis.11 However, the last
method needs large excess of raw materials, such as dialkyl/aryl disulfides (5.0 equiv.) and
amines (6.0 equiv.).
In this paper, a novel strategy for the preparation of symmetrical and unsym-
metrical 6-(N-alkyl(aryl)amino)- and 6-(N,N-bisalkyl(aryl)amino)-2,4-bis(alkyl(aryl)thio)
pyrimidines from the starting material 4-amino-6-hydroxy-2-mercaptopyrimidine has been
developed. Herein, we present the details of the convenient synthesis and evaluation of 6-
(N-alkyl(aryl)amino)- and 6-(N,N-dialkyl(aryl)amino)-2,4-bis(alkyl(aryl)thio)pyrimidines
as human anti-platelet agents.
RESULTS AND DISCUSSION
Chemistry
The synthesis of the title compounds required access to a series of symmetrical and
unsymmetrical 2,6-dialkyl/arylthio-4-aminopyrimidine derivatives. Commercially avail-
able starting material, 4-amino-6-hydroxy-2-mercaptopyrimidine (1) was converted to 2-
alkylthio-4-amino-6-chloropyrimidines (3a,b) by S-alkylation and chlorination according
to our previously reported procedure.11 The first alkylthio group was conveniently intro-
duced into the pyrimidine ring by S-alkylation, while the introduction of the second alkylthio
group into the pyrimidine skeleton was a key step. In our previously reported procedure, the
second alkylthio group was introduced by diazotation-alkylthionation of aminopyrimidine
derivatives 3.11
In the present study, the second alkylthio group was introduced into the pyrimi-
dine skeleton of 3 via nucleophilic displacement with various sodium alkylmercaptides
affording the desired dialkyl/arylthioated-pyrimidine derivatives 4. Subsequently, the title

652
GUOCHENG LIU ET AL.
compounds 5 and 6 were generated via N-alkylation with the corresponding alkyl halides
(Scheme 1).
Scheme 1 Synthesis of compounds 5 and 6 from 1.
Initially, we selected the reaction of 4-amino-6-chloro-2-propylthiopyrimidine (3b)
with sodium propanethiolate as the model reaction to optimize the reaction conditions
of nucleophilic displacement. Sodium propanethiolate and 3b were stirred at room tem-
perature for 12 h. Unfortunately, we did not obtain the desired product, i.e., 4-amino-2,6-
bis(propylthio)pyrimidine (4b) even though we increased the amount of sodium propanethi-
olate to 3.0 equiv. and extended the reaction time to 48 h (Table 1, entries 1–4). Subsequently,
3b and sodium propanethiolate in the ratio 1:1.1 were refluxed for 12 h. However, the 1H
NMR spectrum of the major product revealed that it was a mixture of two compounds (3b
and 4b in a ratio 1:0.8), which could not be separated by column chromatography due to
similar polarity (Table 1, entry 5). To our delight, we obtained the desired product 4b in
80% yield after increasing the ratio to 1:2, and a similar result was obtained at ratio 1:3
(Table 1, entries 6 and 7).
Table 1 Optimizing reaction conditions of nucleophilic displacement
Entry
NaSPr (equiv.)
Conditions
Time (h)
Product 4b yield (%)
1
2
3
4
5
6
7
1.1
2.0
3.0
3.0
1.1
2.0
3.0
r.t.
r.t.
r.t.
12
12
12
48
12
12
12
No reaction
No reaction
No reaction
No reaction
36
r.t.
Reflux
Reflux
Reflux
80
81

SYNTHESIZING OF POTENTIAL HUMAN PLATELET AGGREGATION INHIBITORS 653
Table 2 Nucleophilic displacement of compounds (3a,b) and sodium alkylmercaptides
Entry
R
R1
Time (h)
Product 4
Yield (%)
1
2
3
4
5
Me
n-Pr
n-Pr
i-Pr
n-Bu
CH2Ph
12
12
12
12
12
4a
4b
4c
4d
4e
85
83
82
84
80
n-Pr
n-Pr
n-Pr
n-Pr
Under the optimized conditions, we examined the reaction efficiency of sodium
propanethiolate, sodium propane-2-thiolate, sodium butanethiolate, and sodium benzyl
mercaptide with (3a,b). As can be seen from Table 2, all the reactions were efficiently
carried out to give 4a–e in good yields (80%–85%).
Subsequently, the N-alkylation reaction of 2,6-bis(alkyl(aryl)thio)-4-amino
pyrimidines (4) and various alkyl halides conveniently afforded the desired compounds 5
and 6.
The examination of N-alkylation was initially commenced with 4a and iodoethane
(1.0 equiv.) in the presence of triethylamine (1.0 equiv.) in CH3CN under reflux for 24 h.15
Unfortunately, no desired product 5a was observed even though the amount of iodoethane
was increased to 2.0 equiv. (Table 3, entries 1 and 2). Afterwards, we selected sodium
hydride (NaH) as the base to carry out the reaction.16 We were surprised to find that the
desired product 5a was generated with only 6% yield, while 90% of the substrate 4a was
recovered (Table 3, entry 3). When the reaction was repeated in the presence of 2.0 equiv.
of NaH at ratio 1:2, the yield was improved to 20%, and it was noteworthy that a small
Table 3 Optimizing reaction conditions of N-alkylation reaction
EtI
(equiv.)
Catalyst
(equiv.)
Time
(h)
Product 5a
yield (%)
Product 6a
yield (%)
Unreacted 4a
yield (%)
Entry
1
2
3
4
5
6
7
1.0
2.0
2.0
2.0
2.0
3.0
2.0
Et3N (1.0)
Et3N (1.0)
NaH (1.0)
NaH (2.0)
NaH (3.0)
NaH (3.0)
NaH (3.0)
24
24
24
24
24
24
48
No reaction
No reaction
6.0
0
0
0
100
100
90
80
20
20
52.3
52.5
52.7
Trace
21.6
21.7
21.5
19
20

654
GUOCHENG LIU ET AL.
Table 4 N-Alkylation reaction of 4-amino-2,6-bis(alkyl(aryl)thio)pyrimidines (4a–e).
Substrate
Product 5,
yield (%)
Product 6,
yield (%)
Unreacted 4,
yield (%)
Entry
4
R
R1
R2X
EtI
PhCH2Br
MeI
n-PrI
EtI
n-PrI
1
2
3
4
5
6
4a
4b
4c
4d
4e
4e
Me
n-Pr
n-Pr
i-Pr
n-Bu
PhCH2
PhCH2
5a, 52.3
5b, 31.9
5c, 47.3
5d, 51.4
5e, 51.3
5f, 51.3
6a, 21.6
6b, 6.6
6c, 14.9
6d, -
6e, 8.9
6f, -
4a, 20.0
4b, 29.6
4c, 25.0
4d, 20.5
4e, 17.6
4e, 21.6
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
amount of the N,N-dialkylated product 6a was detected by LC-MS (Table 3, entry 4). The
N,N-dialkylated product 6a was readily separated by column chromatography when the
reaction was conducted with 3.0 equiv. of NaH at the same ratio (1:2), and the yield of
the monoalkylated product 5a was increased to 52.3% (Table 3, entry 5). As shown in Table
3, the N-alkylation gave a mixture of monoalkylated and dialkylated products. Furthermore,
the reaction conversion could not be achieved completely even when the reaction time was
extended to 48 h and the amount of iodoethane was increased to 3.0 equiv. (Table 3, entries 6
and 7). Similarly, as for 2,4-dinitroaniline, the two nitrogen atoms in the pyrimidine ring
decreases the electron density of the free amino group of dialkylthiopyrimidine derivative
4, which results in a weaker nucleophilicity of the amino group, and thus alkylation hardly
occurs. Hence, the reaction conversion cannot be achieved completely.
The N-alkylation of 2,4-bisalkyl(aryl)thio-6-aminopyrimidines (4) was readily ex-
tended to other alkyl halides, such as iodomethane, 1-iodopropane, and benzyl bromide,
under the optimized reaction conditions (Table 4). Even though an excess of alkyl halides
was used, the corresponding desired N-monoallylated products were always produced as
major products. However, we did not obtain the desired dialkylated products 6d and 6f due
to their low yields.
Anti-Platelet Activity Evaluation
The anti-platelet activity of all the synthesized compounds was assayed on human
platelet-rich plasma by using the Born’s method,17 where ADP and Cangrelor (pIC50 was
9.4,18 30 µM ADP, human washed platelets) were used as agonist and positive control,
respectively. In the experiment, the percentage of platelet aggregation of Cangrelor was
0% at the final concentrations of 100 nM. The results are presented in the Supplemental
Materials.
CONCLUSIONS
We have developed a novel protocol to prepare symmetrical and unsymmetrical 6-
(N-alkyl(aryl)amino)- and 6-(N,N-bisalkyl(aryl)amino)-2,4-bisalkyl(aryl)thiopyrimidines
as potential human platelet aggregation inhibitors, in which two identical or different

SYNTHESIZING OF POTENTIAL HUMAN PLATELET AGGREGATION INHIBITORS 655
alkyl/arylthio groups wereintroducedintothepyrimidineskeletonbytwodifferent methods.
All the synthesized compounds were evaluated for their inhibition activities on the human
platelet aggregation induced by ADP. The results demonstrate that the introduction of
substituents at the free amino group of pyrimidine derivatives is beneficial for the activity.
EXPERIMENTAL
General
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 (400 MHz) and
13C NMR (100 MHz) spectra (CDCl3) were recorded on a Bruker 400 plus spectrometer.
Chemical shifts are reported relative to TMS (1H) or CDCl3 (13C). IR spectra (KBr, cm−1
)
were recorded on a Perkin Elmer Fourier transform infrared spectrometer, Spectrum 100.
High Resolution Mass Spectra (HRMS-ESI) were obtained on an Agilent LC/TOF mass
◦
◦
spectrometer. P.E. denotes petroleum ether (b.p.: 60 C–90 C). Cangrelor was obtained
from AstraZeneca. ADP was purchased from Chrono-Log.
Synthesis of 2-Alkylthio-4-amino-6-chloropyrimidines (3a,b)
The known 2-alkylthio-4-amino-6-chloropyrimidines (3a,b) were synthesized from
4-amino-6-hydroxy-2-mercaptopyrimidine (1) according to our reported procedure.11
General Procedure for the Synthesis of Symmetrical and
Unsymmetrical 2,4-Bis(alkyl(aryl)thio)-6-aminopyrimidines (4a–e)
A solution of compound 3 (1.0 mmol) and sodium alkylmercaptide (2.0 mmol) in
CH3CN (15 mL) was refluxed for 12 h. Next, the reaction mixture was evaporated in
vacuo, and the residue was dissolved in 20-mL CHCl3 and 20 mL water. The aqueous
phase was extracted with CHCl3 (3 × 20 mL), and the combined organic phases were
dried with anhydrous Na2SO4. After evaporation of the organic solvent, the residue was
recrystallized from cyclohexane to afford the desired products, 4a–e, as colorless crystals.
1H and 13C NMR spectra for 4a–e, 5a–f, and 6a–c,e are presented in the Supplemental
Materials (Figures S1–S29).
4-Amino-2-methylthio-6-propylthiopyrimidine (4a). Colorless crystals,
yield: 85%, m.p.: 60 ◦C–62 ◦C; IR: 3309, 3169, 2961, 2928, 1556, 1360, 1274; 1H NMR:
δ = 6.00 (s, 1H, CH), 4.72 (br s, 2H, NH2), 3.10 (t, J = 7.3 Hz, 2H, SCH2), 2.51 (s, 3H,
SCH3), 1.73 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.03 (t, J = 7.3 Hz, 3H, CH3); 13C NMR:
δ = 170.9, 168.4, 161.3, 96.4, 31.2, 23.1, 13.9, 13.5; HRMS (ESI): m/z [M + H]+ calcd.
for C8H14N3S2: 216.0624, found: 216.0629.
4-Amino-2,6-bis(propylthio)pyrimidine (4b). Colorless crystals, yield: 83%,
m.p.: 62 ◦C–64 ◦C; IR: 3312, 3165, 2961, 2928, 1556, 1355, 1273; 1H NMR: δ = 5.98 (s,
1H, CH), 4.72 (br s, 2H, NH2), 3.09 (t, J = 7.3 Hz, 2H, SCH2), 3.07 (t, J = 7.3 Hz, 2H,
SCH2), 1.74 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.73 (sextet, J = 7.3 Hz, 2H, SCH2CH2),
1.03 (t, J = 7.3 Hz, 3H, CH3), 1.02 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.6,
168.1, 161.3, 96.5, 32.6, 31.1, 23.1, 23.0, 13.5, 13.4; HRMS (ESI): m/z [M + H]+ calcd.
for C10H18N3S2: 244.0937, found:244.0936.

656
GUOCHENG LIU ET AL.
4-Amino-6-isopropylthio-2-propylthiopyrimidine (4c). Colorless crystals,
yield: 82%, m.p.: 74 ◦C–76 ◦C; IR: 3308, 3166, 2962, 2925, 15576, 1362, 1270; 1H NMR:
δ = 5.96 (s, 1H, CH), 4.78 (br s, 2H, NH2), 4.02 (septet, J = 6.8 Hz, 1H, SCH), 3.07 (t,
J = 7.3 Hz, 2H, SCH2), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.39 (d, J = 6.8 Hz,
6H, 2CH3), 1.02 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.6, 168.1, 161.4, 96.7, 34.3,
32.6, 23.2 (2C), 23.1, 13.5; HRMS (ESI): m/z [M + H]+ calcd. for C10H18N3S2: 244.0937,
found: 244.0937.
4-Amino-6-butylthio-2-propylthiopyrimidine (4d). Colorless crystals, yield:
◦
◦
1
84%, m.p.: 92 C–94 C; IR: 3303, 3161, 2962, 2929, 1560, 1360, 1272; H NMR: δ =
5.98 (s, 1H, CH), 4.67 (br s, 2H, NH2), 3.10 (t, J = 7.3 Hz, 2H, SCH2), 3.09 (t, J =
7.3 Hz, 2H, SCH2), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.67 (quint, J = 7.3 Hz, 2H,
SCH2CH2CH2), 1.45 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.03 (t, J = 7.3 Hz, 3H, CH3),
0.94 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 172.0, 163.3, 159.1, 99.0, 38.7, 32.6, 31.1,
22.3, 21.4, 13.5, 13.2; HRMS (ESI): m/z [M + H]+ calcd. for C11H20ClN3S2: 258.1093,
found: 258.1109.
4-Amino-6-benzylthio-2-propylthiopyrimidine (4e). Colorless crystals,
yield: 80%, m.p.: 73 ◦C–75 ◦C; IR: 3309, 3160, 2965, 2924, 1557, 1362, 1275, 890, 722,
1
698; H NMR: δ = 7.37–7.28 (m, 4H, ArH), 7.25–7.22 (m, 1H, ArH), 5.99 (s, 1H, CH),
4.69 (br s, 2H, NH2), 4.42 (s, 2H, SCH2), 3.07 (t, J = 7.3 Hz, 2H, SCH2), 1.73 (sextet,
J = 7.3 Hz, 2H, SCH2CH2), 0.99 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.67, 167.3,
161.4, 137.5, 128.7, 128.5, 127.2, 96.3, 33.3, 32.6, 22.9, 13.4; HRMS (ESI): m/z [M +
H]+ calcd. for C14H18N3S2: 292.0937, found: 292.0937.
General Procedure for the Synthesis of
6-(N-Alkyl(aryl)amino)-2,4-bis(alky(aryl)thio)pyrimidines (5a–f) and
6-(N-Alkyl(aryl)amino)-2,4-bis(alky(aryl)thio)pyrimidines (5a–f) (6a–c, 6e)
Under the nitrogen atmosphere, a solution of compound 4 (1.0 mmol) and alkyl
halide (2.0 mmol) in dry CH3CN (15 mL) was stirred at room temperature for 30 min. NaH
(3.0 mmol) was then added to the solution, and the mixture was refluxed for 24 h. After
quenching by water, the solvent was removed under reduced pressure. The residue was
dissolved in water (20 mL) and extracted with ethyl acetate (3 × 20 mL), and the combined
organic phases were dried with anhydrous Na2SO4. After evaporation of organic solvent,
the residue was purified by flash chromatography (Et3N-neutralized silica gel, gradient
elution separation with EtOAc/P.E., 1:10–1:5 v/v) to afford the monoalkylated products
5a–f, the dialkylated products 6a–c, 6e, and unreacted 4.
4-Ethylamino-2-methylthio-6-propylthiopyrimidine (5a). Colorless oil,
1
yield: 52.3%; IR (film): 3393, 3265, 2965, 2920, 1572, 1271; H NMR: δ = 5.88 (s, 1H,
CH), 4.73 (br s, 1H, NH), 3.26 (t, J = 6.3 Hz, 2H, NCH2), 3.10 (t, J = 7.2 Hz, 2H, SCH2),
2.50 (s, 3H, SCH3), 1.72 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.22 (t, J = 7.2 Hz, 3H,
CH3), 1.02 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.4, 167.7, 161.0, 94.4, 36.3, 31.2,
23.2, 14.6, 13.9, 13.5; HRMS (ESI): m/z [M + H]+ calcd. for C10H18N3S2: 244.0937,
found: 244.0938.
4-Benzylamino-2,6-bis(propylthio)pyrimidine (5b)11. Colorless crystals,
◦
◦
1
yield: 31.9%, m.p.: 66 C–67 C; H NMR: δ = 7.35–7.27 (m, 5H, ArH), 5.88 (s, 1H,
CH), 5.14 (br s, 1H, NH), 4.46 (d, J = 5.5 Hz, 2H, NCH2), 3.06 (t, J = 7.3 Hz, 2H, SCH2),
3.05 (t, J = 7.3 Hz, 2H, SCH2), 1.73 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.69 (sextet, J
= 7.3 Hz, 2H, SCH2CH2), 1.01 (t, J = 7.3 Hz, 3H, CH3), 1.00 (t, J = 7.3 Hz, 3H, CH3).

SYNTHESIZING OF POTENTIAL HUMAN PLATELET AGGREGATION INHIBITORS 657
4-Methylamino-6-isopropylthio-2-propylthiopyrimidine (5c). Colorless
crystals, yield: 47.3%, m.p.: 58 ◦C–60 ◦C; IR (film): 3409, 3259, 2961, 2930, 1567, 1275;
1H NMR: δ = 5.86 (s, 1H, CH), 5.16 (br s, 1H, NH), 4.03 (septet, J = 6.8 Hz, 1H, SCH),
3.05 (t, J = 7.3 Hz, 2H, SCH2), 2.86 (d, J = 5.0 Hz, 3H, NCH3), 1.74 (sextet, J =
7.3 Hz, 2H, SCH2CH2), 1.38 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.01 (t, J = 7.3 Hz, 3H,
SCH2CH2CH3); 13C NMR: δ = 170.0, 167.7, 161.9, 94.4, 34.3, 32.6, 28.2, 23.3 (2C), 23.2,
13.5; HRMS (ESI): m/z [M + H]+ calcd. for C11H20N3S2: 258.1093, found: 258.1096.
4-Butylthio-6-proplyamino-2-propylthiopyrimidine (5d). Colorless crys-
tals, yield: 51.4%, m.p.: 86 ◦C–88 ◦C; IR (film): 3432, 2969, 2926, 1578, 1268; 1H NMR:
δ = 5.85 (s, 1H, CH), 5.03 (br s, 1H, NH), 3.15 (t, J = 6.1 Hz, 2H, NCH2), 3.08 (t, J =
7.3 Hz, 2H, SCH2), 3.03 (t, J = 7.3 Hz, 2H, SCH2), 1.72 (sextet, J = 7.3 Hz, 2H,
SCH2CH2), 1.64 (quint, J = 7.3 Hz, 2H, SCH2CH2), 1.56 (sextet, J = 7.4 Hz, 2H,
NCH2CH2), 1.41 (sextet, J = 7.3 Hz, 2H, SCH2CH2CH2), 1.00 (t, J = 7.3 Hz, 3H, CH3),
0.91 (t, J = 7.3 Hz, 6H, 2CH3); 13C NMR: δ = 170.0, 167.3, 161.0, 94.2, 43.1, 32.3,
31.7, 28.8, 23.0, 22.4, 21.9, 13.5, 13.4, 11.2; HRMS (ESI): m/z [M + H]+ calcd. for
C14H26ClN3S2: 300.1563, found: 300.1562.
4-Benzylthio-6-ethylamino-2-propylthiopyrimidine (5e). Colorless crys-
tals, yield: 51.3%, m.p.: 50 ◦C–52 ◦C, IR (film): 3409, 3262, 2965, 2929, 1567, 1274; 1H
NMR: δ = 7.37 (d, J = 7.4 Hz, 2H, ArH), 7.29 (dd, J = 7.2, 7.6 Hz, 2H, ArH), 7.23 (dd,
J = 7.1, 7.3 Hz, 1H, ArH), 5.86 (s, 1H, CH), 4.77 (br s, 1H, NH), 4.41 (s, 2H, SCH2Ph),
3.24 (t, J = 6.0 Hz, 2H, NCH2), 3.06 (t, J = 7.3 Hz, 2H, SCH2), 1.73 (sextet, J = 7.3 Hz,
2H, SCH2CH2), 1.19 (t, J = 7.1 Hz, 3H, CH3), 0.99 (t, J = 7.3 Hz, 3H, CH3); 13C NMR:
δ = 170.4, 166.8, 161.1, 137.9, 128.9, 128.6, 127.2, 94.2, 36.2, 33.3, 32.6, 23.0, 14.5,
13.5. HRMS (ESI): m/z [M + H]+ calcd. for C16H22N3S2: 320.1250, found: 320.1250.
4-Benzylthio-6-propylamino-2-propylthiopyrimidine (5f). Colorless crys-
tals, yield: 51.3%, m.p.: 40 ◦C–42 ◦C, IR (film): 3393, 2964, 2922, 1574, 1277; 1H NMR:
δ = 7.40 (d, J = 7.5 Hz, 2H, ArH), 7.33 (dd, J = 7.3, 7.5 Hz, 2H, ArH), 7.26 (dd, J = 7.0,
7.5 Hz, 1H, ArH), 5.90 (s, 1H, CH), 4.89 (br s, 1H, NH), 4.44 (s, 2H, SCH2Ph), 3.18 (br s,
2H, NCH2), 3.09 (t, J = 7.3 Hz, 2H, SCH2), 1.77 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.61
(sextet, J = 7.2 Hz, 2H, NCH2CH2), 1.03 (t, J = 7.3 Hz, 3H, CH3), 0.98 (t, J = 7.3 Hz,
3H, CH3); 13C NMR: δ = 170.3, 166.8, 161.2, 137.9, 128.9, 128.6, 127.2, 94.3, 43.3, 33.4,
32.7, 23.1, 22.6, 13.6, 11.4; HRMS (ESI): m/z [M + H]+ calcd. for C17H24N3S2: 334.1406,
found: 334.1409.
4-Diethylamino-6-propylthio-2-methylthiopyrimidine (6a). Colorless oil,
yield: 21.6%; IR (film): 2962, 2926, 1560, 1281; 1H NMR: δ = 5.94 (s, 1H, CH), 3.45 (br
s, 4H, 2NCH2), 3.10 (t, J = 7.3 Hz, 2H, SCH2), 2.48 (s, 3H, SCH3), 1.71 (sextet, J = 7.3
Hz, 2H, SCH2CH2), 1.15 (t, J = 7.3 Hz, 6H, 2CH3), 1.01 (t, J = 7.3 Hz, 3H, CH3); 13C
NMR: δ = 170.1, 166.8, 159.4, 94.0, 42.2 (2C), 31.3, 23.2, 14.0, 13.5, 12.8 (2C); HRMS
(ESI): m/z [M + H]+ calcd. for C12H22N3S2: 272.1250, found: 272.1254.
4-Dibenzylamino-2,6-bis(propylthio)pyrimidine (6b). Colorless oil, yield:
1
6.6%, IR (film): 2962, 2929, 1554, 1294, 828, 798, 698; H NMR: δ = 7.39–7.30 (m,
6H, ArH), 7.25 (d, J = 7.2 Hz, 4H, ArH), 6.08 (s, 1H, CH), 4.76 (br s, 4H, NCH2Ph), 3.11
(t, J = 7.3 Hz, 2H, SCH2), 3.09 (t, J = 7.3 Hz, 2H, SCH2), 1.77 (sextet, J = 7.3 Hz, 2H,
SCH2CH2), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.05 (t, J = 7.3 Hz, 3H, CH3), 1.02
(t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.0, 168.0, 160.8, 136.9, 128.6, 127.2, 127.0,
94.1, 50.0 (2C), 32.6, 31.1, 23.0, 22.9, 13.4 (2C); HRMS (ESI): m/z [M + H]+ calcd. for
C24H30N3S2: 424.1876, found: 424.1886.

658
GUOCHENG LIU ET AL.
4-Dimethylamino-6-isopropylthio-2-propylthiopyrimidine (6c). Colorless
oil, yield: 14.9%, IR (film): 2962, 2928, 1566, 1295; 1H NMR: δ = 5.96 (s, 1H, CH), 4.04
(septet, J = 6.8 Hz, 1H, SCH), 3.06 (t, J = 7.3 Hz, 2H, SCH2), 3.03 (s, 6H, N(CH3)2),
1.76 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.37 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.02 (t,
J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 169.8, 167.0, 160.9, 94.6, 37.0 (2C), 34.4, 32.7, 23.3
(2C), 23.2, 13.6; HRMS (ESI): m/z [M + H]+ calcd. for C12H22N3S2: 272.1250, found:
272.1250.
4-Benzylthio-6-diethylamino-2-propylthiopyrimidine (6e). Colorless oil,
yield: 8.9%, IR (film): 2967, 2930, 1557, 1278, 830, 804, 699; 1H NMR: δ = 7.38 (d, J =
7.3 Hz, 2H, ArH), 7.29 (dd, J = 7.1, 7.7 Hz, 2H, ArH), 7.22 (dd, J = 7.2, 7.3 Hz, 1H,
ArH), 5.90 (s, 1H, CH), 4.40 (s, 2H, SCH2Ph), 3.41 (br s, 4H, 2NCH2), 3.04 (t, J = 7.3
Hz, 2H, SCH2), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2CH2), 1.12 (t, J = 7.0 Hz, 6H, 2CH3),
1.01 (t, J = 7.3 Hz, 3H, CH3); 13C NMR: δ = 170.0, 166.2, 159.5, 138.2, 128.9, 128.5,
127.1, 93.8, 42.2 (2C), 33.5 (2C), 32.8, 23.3, 13.6, 12.9; HRMS (ESI): m/z [M + H]+
calcd. for C18H26N3S2: 348.1563, found: 348.1552.
In Vitro Human Anti-Platelet Aggregation Activity
All experiments using human subjects were performed in accordance with the Dec-
laration of Helsinki and approved by the Institutional Review Board, Fudan University.
Blood was sampled from the cubital vein of healthy volunteers without taking aspirin or
other nonsteroidal anti-inflammatory drugs for at least 14 days and the informed consent
was obtained before blood collection. The blood sample was collected in 50-mL plastic
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 supernatant fraction was called platelet-poor plasma (PPP). Aliquots of 500
µL of PRP were distributed in test cuvettes and inserted into the incubation ◦chamber of
an aggregometer (Model 400VS, Chrono-Log, Haverston, PA, USA) at 37 C. Platelet
aggregation was measured on the aggregometer using the PRP fractions after activation by
ADP (final concentration 10 µM) according to the Born’s method.11,17 The test compounds
were dissolved in DMSO (below at 0.5% final concentration) and added to the PRP for
1 min before platelet activation with agonists, the extent of aggregation was quantified by
determining the maximum height of the aggregation tracing. 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−1. The baseline was set using PPP as blank. The platelet
aggregation inhibitory activity was expressed as percentage inhibition by comparison with
that measured in the presence of an equivalent amount of vehicle (DMSO) alone.
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