(N-Alkyl(Aryl)Amino)

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New Approach to Synthesize Symmetrical and Unsymmetrical 6-(N-Alkyl(Aryl)Amino)- and 6(N,N-Dialkyl(Aryl)Amino)-2,4Bis(Alkyl(Aryl)Thio)Pyrimidines as antiPlatelet Agents a

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Guocheng Liu , Jiaxi Xu , Mingwu Yu , Ning Chen , Si Zhang , b

Zhongren Ding & Hongguang Du

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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 Available online: 04 Jan 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-(NAlkyl(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 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 C Taylor & Francis Group, LLC Copyright 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)-2,4BIS(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 1

State Key Laboratory of Chemical Resource Engingeering, Department of Organic Chemistry, College of Science, Beijing University of Chemical Technology, Beijing China 2 Key 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- mercaptopyrimidine via S-alkylation with alkyl halides, and via a nucleophilic displacement with sodium alkylmercaptides, affording the key intermediate symmetrical and unsymmetrical 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: [email protected] 650

SYNTHESIZING OF POTENTIAL HUMAN PLATELET AGGREGATION INHIBITORS

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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 activity,12 we presume that the introduction of substituents at the free amino group, such as Nmonoalkylation 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 6alkyl(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 alkylating agent in the presence of butyllithium in low yield (30%),13 via the conversion of 4,6dihydroxy-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 unsymmetrical 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 available starting material, 4-amino-6-hydroxy-2-mercaptopyrimidine (1) was converted to 2alkylthio-4-amino-6-chloropyrimidines (3a,b) by S-alkylation and chlorination according to our previously reported procedure.11 The first alkylthio group was conveniently introduced 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 pyrimidine skeleton of 3 via nucleophilic displacement with various sodium alkylmercaptides affording the desired dialkyl/arylthioated-pyrimidine derivatives 4. Subsequently, the title

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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 temperature for 12 h. Unfortunately, we did not obtain the desired product, i.e., 4-amino-2,6bis(propylthio)pyrimidine (4b) even though we increased the amount of sodium propanethiolate 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 1 2 3 4 5 6 7

NaSPr (equiv.)

Conditions

Time (h)

Product 4b yield (%)

1.1 2.0 3.0 3.0 1.1 2.0 3.0

r.t. r.t. r.t. r.t. Reflux Reflux Reflux

12 12 12 48 12 12 12

No reaction No reaction No reaction No reaction 36 80 81


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Table 2 Nucleophilic displacement of compounds (3a,b) and sodium alkylmercaptides


Entry 1 2 3 4 5

R

R1

Time (h)

Product 4

Yield (%)

Me n-Pr n-Pr n-Pr n-Pr

n-Pr n-Pr i-Pr n-Bu CH2 Ph

12 12 12 12 12

4a 4b 4c 4d 4e

85 83 82 84 80

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 CH3 CN 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

Entry 1 2 3 4 5 6 7

EtI (equiv.)

Catalyst (equiv.)

Time (h)

Product 5a yield (%)

Product 6a yield (%)

Unreacted 4a yield (%)

1.0 2.0 2.0 2.0 2.0 3.0 2.0

Et3 N (1.0) Et3 N (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 20 52.3 52.5 52.7

0 0 0 Trace 21.6 21.7 21.5

100 100 90 80 20 19 20

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Table 4 N-Alkylation reaction of 4-amino-2,6-bis(alkyl(aryl)thio)pyrimidines (4a–e).


Entry 1 2 3 4 5 6

Substrate 4

R

R1

R2X

4a 4b 4c 4d 4e 4e

Me n-Pr n-Pr n-Pr n-Pr n-Pr

n-Pr n-Pr i-Pr n-Bu PhCH2 PhCH2

EtI PhCH2 Br MeI n-PrI EtI n-PrI

Product 5, yield (%)

Product 6, yield (%)

Unreacted 4, yield (%)

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

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 extended 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


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alkyl/arylthio groups were introduced into the pyrimidine skeleton by two different 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 13 C 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 CH3 CN (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 Na2 SO4. After evaporation of the organic solvent, the residue was recrystallized from cyclohexane to afford the desired products, 4a–e, as colorless crystals. 1 H 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, SCH2 CH2 ), 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 C8 H14 N3 S2 : 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, SCH2 CH2 ), 1.73 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 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 C10 H18 N3 S2 : 244.0937, found:244.0936.


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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, SCH2 CH2 ), 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 C10 H18 N3 S2 : 244.0937, found: 244.0937. 4-Amino-6-butylthio-2-propylthiopyrimidine (4d). Colorless crystals, yield: 84%, m.p.: 92 ◦ C–94 ◦ C; IR: 3303, 3161, 2962, 2929, 1560, 1360, 1272; 1H 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, SCH2 CH2 ), 1.67 (quint, J = 7.3 Hz, 2H, SCH2 CH2 CH2 ), 1.45 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 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 C11 H20 ClN3 S2 : 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, 698; 1H 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, SCH2 CH2 ), 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 C14 H18 N3 S2 : 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 CH3 CN (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 Na2 SO4 . After evaporation of organic solvent, the residue was purified by flash chromatography (Et3 N-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, yield: 52.3%; IR (film): 3393, 3265, 2965, 2920, 1572, 1271; 1H 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, SCH2 CH2 ), 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 C10 H18 N3 S2 : 244.0937, found: 244.0938. 4-Benzylamino-2,6-bis(propylthio)pyrimidine (5b)11. Colorless crystals, yield: 31.9%, m.p.: 66 ◦ C–67 ◦ C; 1H 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, SCH2 CH2 ), 1.69 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 1.01 (t, J = 7.3 Hz, 3H, CH3 ), 1.00 (t, J = 7.3 Hz, 3H, CH3 ).



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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; 1 H 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, SCH2 CH2 ), 1.38 (d, J = 6.8 Hz, 6H, CH(CH3 )2 ), 1.01 (t, J = 7.3 Hz, 3H, SCH2 CH2 CH3 ); 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 C11 H20 N3 S2 : 258.1093, found: 258.1096. 4-Butylthio-6-proplyamino-2-propylthiopyrimidine (5d). Colorless crystals, 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, SCH2 CH2 ), 1.64 (quint, J = 7.3 Hz, 2H, SCH2 CH2 ), 1.56 (sextet, J = 7.4 Hz, 2H, NCH2 CH2 ), 1.41 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 CH2 ), 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 C14 H26 ClN3 S2 : 300.1563, found: 300.1562. 4-Benzylthio-6-ethylamino-2-propylthiopyrimidine (5e). Colorless crystals, 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, SCH 2 Ph), 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, SCH2 CH2 ), 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 C16 H22 N3 S2 : 320.1250, found: 320.1250. 4-Benzylthio-6-propylamino-2-propylthiopyrimidine (5f). Colorless crystals, 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, SCH 2 Ph), 3.18 (br s, 2H, NCH2 ), 3.09 (t, J = 7.3 Hz, 2H, SCH2 ), 1.77 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 1.61 (sextet, J = 7.2 Hz, 2H, NCH2 CH2 ), 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 C17 H24 N3 S2 : 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, SCH2 CH2 ), 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 C12 H22 N3 S2 : 272.1250, found: 272.1254. 4-Dibenzylamino-2,6-bis(propylthio)pyrimidine (6b). Colorless oil, yield: 6.6%, IR (film): 2962, 2929, 1554, 1294, 828, 798, 698; 1H 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, NCH2 Ph), 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, SCH2 CH2 ), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 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 C24 H30 N3 S2 : 424.1876, found: 424.1886.


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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, SCH2 CH2 ), 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 C12 H22 N3 S2 : 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, SCH 2 Ph), 3.41 (br s, 4H, 2NCH2 ), 3.04 (t, J = 7.3 Hz, 2H, SCH2 ), 1.75 (sextet, J = 7.3 Hz, 2H, SCH2 CH2 ), 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 C18 H26 N3 S2 : 348.1563, found: 348.1552.

In Vitro Human Anti-Platelet Aggregation Activity All experiments using human subjects were performed in accordance with the Declaration 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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