Design, synthesis and antiproliferative activity of novel 5-nitropyrimidine-2,4-diamine derivatives bearing alkyl acetate moiety

European Journal of Medicinal Chemistry 118 (2016) 161e169

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and antiproliferative activity of novel 5-nitropyrimidine-2,4-diamine derivatives bearing alkyl acetate moiety Pei-Liang Zhao*, 1, Yan-Hong Li 1, Hai-Kui Yang, Peng Chen, Bei Zhang, Qi Sun, Qiu Li, Wen-Wei You** Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern Medical University, Guangzhou 510515, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 December 2015 Received in revised form 16 March 2016 Accepted 14 April 2016 Available online 16 April 2016

In order to discover new anticancer drug leads, a series of novel alkylamino pyrimidine derivatives were designed and synthesized based on our previous work via a ring-opening strategy. Biological evaluation with four human cancer cell lines (MDA-MB-231, A549, HepG2, and MCF-7) showed that most of these compounds possessed moderate to potent antiproliferative activities. The most promising compound 7w displayed a three-fold improvement compared with commercial anticancer drug fluorouracil in inhibiting HepG2 cell proliferation with IC50 value of 10.37 mM. Moreover, flow-activated cell sorting analysis suggested that compound 7w mainly arrested HepG2 cells in G2/M stage. Hence, it could serve as a promising lead for the design of novel anticancer small-molecule drugs. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: 2,4-Diaminopyrimidines Alkylamino pyrimidines Antiproliferative activity Synthesis

1. Introduction Cancer today still remains one of the leading causes of death worldwide, which makes the identification of novel drugs crucial to address this disease [1]. In this context, 2,4-diaminopyrimidine scaffold, as one of the most widely naturally-occurring products and also in clinically useful molecules, has attracted great attention due to its potential anticancer effect for decades [2e8]. For instance, 5-nitrosopyrimidine analog NU6027 (1, Fig. 1), designed as an alternative to purine, has been developed as potential antitumor agent and 5-nitroso has been confirmed as a crucial pharmacophore group due to an intramolecular hydrogen bond between the adjacent 5-nitroso and 4-amino group [9,10]. Meanwhile, recent literature survey has revealed that N-alkylation of diaminopyrimidine by introduction of amino side chain in pyrimidine core has been reported to further enhance the antitumor activity of candidate compounds. For example, ZK-304709 (2), an oral multi-target tumor growth inhibitor has been reported to block tumor cell

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (P.-L. Zhao), (W.-W. You). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2016.04.038 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

[email protected]

proliferation and induce apoptosis by inhibiting a combination of disease progression driving pathways [11,12]. While methyl acetate containing 2,4-diaminopyrimidine 3 has been found to inhibit antiIgE, which can be further explored as a potential therapeutic against HER2/neu overexpressing tumors, such as breast and ovarian cancers [13,14]. More recently, through optimization of the linker-length of alkylamine in pyrimidine ring, Chen et al. discovered compound 4 had most potent activities both for inhibiting breast cancer cells growth and migration, which indicated that the flexibility of the alkylamine moiety might be a vital factor for antiproliferative activity [15]. Recently, we combined a 2,4-diaminopyrimidine scaffold by introducing N-methylpiperazine moiety into pyrimidine core with phenylalkylamine as a flexible linkage, resulting in identification of a series of pyrimidine-piperazine compounds 5 (Fig. 2) with favorable antiproliferative activities [16]. Thus, in view of the previous rationale and as a continuation of our interest in developing nitrogen-containing heterocycles as novel bioactive substances [17e21], we undertook a lead-optimization program with the aim of discovering a new lead structure with antiproliferative activity. As shown in Fig. 2, keeping in mind the similarity of nitro and nitroso in hydrogen bond acceptor, our optimization efforts were firstly directed toward the ring-opening of piperazine to give the scaffold 6. As an important kind of oxygen-bearing functional

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Fig. 1. Reported 2,4-diaminopyrimidines with antitumor activity.

Fig. 2. Design strategy of the title compounds 7a ~ w.

group, the acetic acid ester appears in many antitumor agents [22e25]. Hence, we replaced the terminal amine group at the 4position of the pyrimidine 6 with an acetic acid ester to design the novel flexible scaffold 7. Herein, we described the detailed synthetic routes, screening results, and structure-activity relationships of these compounds 7a ~ w. 2. Chemistry As shown in Scheme 1, the target 2,4-disubstituted aminopyrimidine derivatives 7a ~ w were prepared by a two or three-step synthetic route. According to existing methods [26], the key intermediates 10 were firstly synthesized from the 2,4dichloropyrimidine 8 as starting material by a nucleophilic substitution reaction at C-4 using a base such as potassium carbonate (K2CO3), and resulted in good to excellent yields. In the second step, the C-2 chlorine was displaced by various alkyl or aromatic amines. This reaction was run under rigorous conditions (90
C) for 3e6 h

using absolute 2-methoxyethanol as a solvent to afford the target 2,4-disubstituted pyrimidine derivatives 7a ~ g in moderate to excellent yield (68e95%). Meanwhile, the N-alkylated another intermediates 11 were obtained by reaction of intermediates 10 with the methyl iodide and benzyl bromide in DMF with potassium carbonate as base (Scheme 1). The intermediates were subsequently reacted with various amines by heating at 90
C in 2methoxyethanol to successfully afford the desirable target compounds 7h ~ w in moderate to excellent isolated yields ranging from 69% to 92%. The structures of all synthesized compounds 7a ~ w were characterized by 1H NMR, 13C NMR, ESI-MS, melting point, and elemental analysis. Detailed synthetic procedures were described in the Experimental Section. In addition, the representative compound 7q was further confirmed by single-crystal X-ray diffraction. As shown in Fig. 3, the crystal structure showed that 7q had a Cshape, and the dihedral angles made by the pyrimidine ring with benzene and chloro-substituted benzene ring are 71.17 and 53.56
,

Scheme 1. Synthesis of the target compounds 7a ~ w.

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163

Fig. 3. Molecular structure of compound 7q.

respectively. 3. Pharmacology results and discussion The in vitro antiproliferative activities of the synthesized compounds 7a ~ w were tested in four cancer cell lines, including MDAMB-231 (human breast cancer cells), A549 (human alveolar epithelial cells), HepG2 (human hepatoma cells), and MCF-7 (human mammary adenocarcinoma cells). Fluorouracil which is one of the most popular and effective anticancer drugs and also possesses a 2,4,5-trisubstituted pyrimidine scaffold was used as the reference drug. Cell proliferative activities were assayed with the MTT method [21] and the results, expressed as IC50, were summarized in Table 1. As can be seen in Table 1, unfortunately, the results indicated that most of the compounds were ineffective (IC50 > 100 mM) on 549 cell lines. In contrast, it is clear that nearly all synthesized compounds exhibit moderate to strong antiproliferative activities against MDA-MB-231 cell lines. Among them, three representative compounds (7f, 7g, 7j) demonstrated much higher antiproliferative activities than positive control fluorouracil. And another three compounds (7b, 7d, 7i) displayed comparable potency with that of fluorouracil. Furthermore, most of the tested compounds showed significant antiproliferative to HepG2 cell lines and nine compounds (7i, 7o ~ r, 7t ~ w) displayed much stronger cytotoxicity than fluorouracil. Especially, compound 7w showed three-fold improvement compared to fluorouracil in inhibiting HepG2 cell proliferation with the IC50 value of 10.37 mM. More interestingly, compound 7w also exhibited remarkable cytotoxic activities against MCF-7 cell lines with the IC50 value of 20.96 mM, which was also found to be much higher than fluorouracil. It is worth noting that water solubility of compound 7w exhibited remarkable improvement with its values 3.0 mg/mL and 80.5 mg/mL at pH 6.4 and pH 1.0, respectively. Further investigations on the structureeactivity relationship were carried out in detail to study the effects of several substituents (n, R1, R2, R3) of the pyrimidine ring. In general, linker-length of

alkylamine in C-2 position of pyrimidine ring has profound effects on inhibitory activity and the activities decrease in the following order: n ¼ 2 > 1 > 0. For example, compound 7w (n ¼ 2) showed most promising antiproliferative activities against four cancer cell lines. After changing the linkage, the antitumor activities of compounds 7v (n ¼ 1) and 7o (n ¼ 0) were significantly reduced. Whereas, the presence of methyl or ethyl of the R1 in acetic acid ester substituent has practically no obvious effect on antitumor activities. Besides, the compounds bearing a benzyl (R2) group showed much higher antiproliferative activities against HepG2 cell lines than the corresponding hydrogen substituted pyrimidine analogues (7a vs 7o, 7b vs 7p, 7c vs 7r, 7e vs 7s, 7f vs 7v, 7g vs 7w). In most cases, compounds bearing phenyl group (R3) at the C-2 amine chain of pyrimidine ring (7a ~ d, 7i ~ k and 7p ~ r) displayed obviously higher cytotoxic activity than those of the corresponding compounds with piperidine group at the same position (7b, 7l, 7s). It indicated that the substituents of R3 on the amine chain group are crucial to the antiproliferative activities. To study the effect of the target compounds on cell cycle progression, we performed flow-activated cell sorting analysis for the representative compound 7w which exhibited most promising antiproliferative activities. HepG2 cells were treated at four different concentrations (1, 5, 10, 20 mM). As shown in Fig. 4 and Table 2, compound 7w arrested the HepG2 cell cycle in G2/M phase with a concentration-dependent effect. The G2/M peak significantly increased from 21.54% to 25.46% (1 mM), 30.83% (5 mM), 31.94% (10 mM), and 36.85% (20 mM) after 48 h of treatment. 4. Conclusion In conclusion, based on the structures of our previously discovered antiproliferative compounds, a new series of alkylamino pyrimidine derivatives were designed and synthesized via a strategy of ring-opening. Bioassay indicated that most of these compounds possessed moderate to potent antiproliferative activities. Particularly, compound 7w displayed a three-fold improvement compared to fluorouracil in inhibiting HepG2 cell proliferation with

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Table 1 Cytotoxic activities of compounds 7a ~ w against human tumor cells.

Comp.

n

R1

R2

R3

7a 7b 7c 7d 7e

0 0 0 0 0

CH3 CH3 CH3 CH3 CH3

H H H H H

Ph 3-MeOPh 4-ClPh 4-MeOPh

7f 7g 7h 7i 7j 7k 7l

1 2 0 0 0 0 0

CH3 CH3 H H H H H

H H CH3 CH3 CH3 CH3 CH3

7m 7n 7o 7p 7q 7r 7s

1 2 0 0 0 0 0

H H CH3 CH3 CH3 CH3 CH3

7t 7u 7v 7w fluorouracil

1 1 1 2

CH3 CH3 CH3 CH3

In vitro cytotoxicity IC50(mM)a MDA-MB-231

a

A549

HepG2

MCF-7

54.34 31.27 55.54 32.57 80.68

>100 36.11 >100 >100 >100

>100 >100 >100 >100 >100

>100 35.8 >100 >100 >100

Ph Ph Ph 3,4,5-trMeOPh 4-ClPh 3-MeOPh

22.29 21.94 >100 25.94 21.20 58.00 59.58

74.41 48.25 >100 >100 23.46 31.88 >100

>100 71.08 >100 26.16 49.80 73.42 >100

52.38 >100 >100 41.97 60.68 93.68 >100

CH3 CH3 CH2Ph CH2Ph CH2Ph CH2Ph CH2Ph

Ph Ph Ph 3-MeOPh 3,4,5-trMeOPh 4-ClPh

61.14 44.81 >100 47.21 40.37 34.35 75.84

79.42 36.98 >100 >100 >100 >100 >100

>100 86.70 44.38 27.63 37.83 30.86 57.11

85.85 32.54 >100 >100 >100 >100 >100

CH2Ph CH2Ph CH2Ph CH2Ph

COOCH2CH3 3-MeOPh Ph Ph

92.46 80.68 82.43 22.29 24.60

>100 >100 >100 87.29 35.41

44.51 33.56 20.95 10.37 46.83

>100 57.44 >100 20.96 30.41

IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients of variation were <10%.

IC50 value of 10.37 mM. Furthermore, flow-activated cell sorting analysis revealed that compound 7w arrested the cell cycle of HepG2 cells in the G2/M phase with a concentration-dependent effect. The initial structureeactivity relationship studies indicated that linker-length of alkylamine in C-4 position of pyrimidine ring play a crucial role in modulating the antitumor activity, which could be of help in the rational design of 2,4-diaminopyrimidines as novel anticancer drugs. 5. Experimental protocols 5.1. Chemistry 1

H NMR and 13C NMR spectra were performed using a MercuryPlus 400 spectrometer in CDCl3 or DMSO-d6 solution and chemical shifts were recorded in parts per million (ppm) with TMS as the internal standard. MS spectra were determined using a Micromass ZQ 4000 mass spectrometer, and signals were given in m/z. Elemental analyses were performed on a Vario EL III elemental analysis instrument. All melting points (mp) were obtained on a Buchi B-545 melting point apparatus and were uncorrected. Unless otherwise noted, reagents were purchased from commercial suppliers and used without further purification while all solvents were redistilled before use. Yields were not optimized. 5.1.1. General procedure for the 4-alkylaminopyrimidine 10 A mixture of 2,4-dichloro-5-nitropyrimidine 8 (1.92 g 10 mmol), ethyl 2-aminoacetate hydrochloride or methyl 2-aminoacetate

hydrochloride 9 (10 mmol), and anhydrous K2CO3 (3.04 g, 22 mmol) in 20 mL of anhydrous acetone were stirred at room temperature for 4e6 h. After the reaction was complete according to the TLC detection, the precipitate was filtered off and solvent was removed under reduced pressure and the residue was purified by column chromatography to give the key intermediates 10 in excellent yields. 5.1.1.1. Methyl 2-(2-chloro-5-nitropyrimidin-4-ylamino)acetate (10a). Yield, 87%; 1H NMR (400 MHz, CDCl3) d: 3.87 (s, 3H, OCH3), 4.45 (s, 2H, CH2), 8.78 (br, 1H, NH), 9.11 (s, 1H, ArH). ESI-MS: m/ z ¼ 247.1 [Mþ1]þ. 5.1.1.2. Ethyl 2-(2-chloro-5-nitropyrimidin-4-ylamino)acetate (10b). Yield, 84%; 1H NMR (400 MHz, CDCl3) d: 1.36 (t, J ¼ 7.2 Hz, 3H, CH3), 4.32 (dd, J1 ¼ 7.0 Hz, J2 ¼ 14.2 Hz, 2H, CH2), 4.42 (d, J ¼ 5.2 Hz, 2H, CH2), 8.78 (br, 1H, NH), 9.11 (s, 1H, ArH). ESI-MS: m/z ¼ 261.9 [Mþ1]þ. 5.1.2. General procedure for the N-alkylated aminopyrimidine 11 To a solution of 4-alkylaminopyrimidine 10 (20 mmol) in DMF (10 mL) was added anhydrous K2CO3 (1.66 g, 12 mmol). After 30 min of stirring at room temperature, halogen derivative (ICH3 or benzyl bromide, 10 mmol) in DMF solution was added dropwise to the mixture. The resulting solution reacted for about 2e4 h and then was filtered and concentrated. The residue was purified via flash chromatography to give the pure product 11 in moderate yields.

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Fig. 4. Effect of compound 7w on cell cycle and apoptosis in HepG2 cells. Flow cytometry analysis of HepG2 cells treated with 7w for 48 h. (A) Control; (B) 7w, 1 mM; (C) 7w, 5 mM; (D) 7w, 10 mM; (E) 7w, 20 mM.

Table 2 Effect of compound 7w on cell cycle distribution in HepG2 cells. Concentration

Sub-G1(%)

G0/G1(%)

S(%)

G2/M(%)

0 mM 1 mM 5 mM 10 mM 20 mM

2.09 5.08 3.85 4.80 4.91

44.28 34.03 31.26 30.91 29.86

28.95 28.08 28.28 27.02 25.75

21.54 25.46 30.83 31.94 36.85

5.1.2.1. Methyl 2-((2-chloro-5-nitropyrimidin-4-yl) (methyl)amino) acetate (11a). Yield, 63%; 1H NMR (400 MHz, CDCl3) d: 3.05 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 4.39 (s, 2H, CH2), 8.76 (s, 1H, ArH). ESI-MS: m/z ¼ 261.7 [Mþ1]þ. 5.1.2.2. Ethyl 2-(benzyl(2-chloro-5-nitropyrimidin-4-yl)amino)acetate (11b). Yield, 67%; 1H NMR (400 MHz, CDCl3) d: 1.31 (t, J ¼ 7.0 Hz, 3H, CH3), 4.18 (s, 2H, CH2), 4.25 (dd, J1 ¼ 7.2 Hz, J2 ¼ 14.4 Hz, 2H, CH2), 4.78 (s, 2H, CH2), 7.39e7.29 (m, 5H, ArH), 8.73 (s, 1H, ArH). ESI-MS: m/z ¼ 351.5 [Mþ1]. 5.1.3. General procedure for the target compounds 7a ~ w A mixture of intermediates 10 or 11 (1.0 mmol) and various amines (1.0 mmol) in 10.0 mL of 2-methoxyethanol was stirred and heated at 90
C for 3e6 h. After the reaction was complete

according to the TLC detection, the solvent was evaporated to give the crude product followed by recrystallisation from ethanol to give the target compounds 7a ~ w in yields of 68e95%. 5.1.3.1. Ethyl 2-(5-nitro-2-(phenylamino)pyrimidin-4-ylamino)acetate (7a). Yield, 87%; mp 154.4e156.1
C; 1H NMR (400 MHz, CDCl3) d: 1.32 (t, J ¼ 7.2 Hz, 3H, CH3), 4.27 (dd, J1 ¼ 7.2 Hz, J2 ¼ 14 Hz, 2H, CH2), 4.36 (d, J ¼ 5.6 Hz, 2H, CH2), 7.18 (t, J ¼ 7.2 Hz, 1H, ArH), 7.40 (t, J ¼ 7.8 Hz, 2H, ArH), 7.58 (d, J ¼ 8.0 Hz, 2H, ArH), 7.74 (br, 1H, NH), 8.86 (br, 1H, NH), 9.08 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 14.5, 43.2, 61.2, 120.7, 123.9, 128.9, 139.2, 156.1, 157.8, 159.7, 169.6. ESI-MS: m/z ¼ 340.6 [MþNa]þ, 318.7 [Mþ1]þ. Anal. Calcd. for C14H15N5O4: C, 52.99; H, 4.76; N, 22.07; Found C 52.70, H 4.90, N 22.36. 5.1.3.2. Ethyl 2-((2-((3-methoxyphenyl)amino)-5-nitropyrimidin-4yl)amino)acetate (7b). Yield, 68%; mp 150.6e152.6
C; 1H NMR (400 MHz, DMSO-d6) d: 1.16 (t, J ¼ 7.0 Hz, 3H, CH3), 3.76 (s, 3H, OCH3), 4.08 (dd, J1 ¼ 6.8 Hz, J2 ¼ 14.0 Hz, 2H, CH2), 4.33 (t, J ¼ 6.0 Hz, 2H, CH2), 6.67 (dd, J1 ¼ 1.6 Hz, J2 ¼ 8.0 Hz, 1H, ArH), 7.21 (t, J ¼ 8.0 Hz, 1H, ArH), 7.27 (d, J ¼ 8.4 Hz, 1H, ArH), 7.33 (t, J ¼ 2.0 Hz, 1H, ArH), 9.03 (s, 1H, ArH), 9.14 (br, 1H, NH), 10.38 (br, 1H, NH). 13C NMR (100 MHz, CDCl3) d: 14.1, 42.9, 55.3, 61.8, 106.5, 109.8, 112.8, 129.7, 138.9, 156.0, 157.4, 160.1, 168.8. ESI-MS: m/z ¼ 348.6 [Mþ1]þ. Anal. Calcd. for C15H17N5O5: C 51.87, H 4.93, N 20.16; Found C 51.75,

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H 5.03, N 20.41.

22.35.

5.1.3.3. Ethyl 2-((2-((4-chlorophenyl)amino)-5-nitropyrimidin-4-yl) amino)acetate (7c). Yield, 71%; mp 178.8e180.6
C; 1H NMR (400 MHz, DMSO-d6) d: 1.15 (t, J ¼ 7.0 Hz, 3H, CH3), 4.08 (dd, J1 ¼ 7.0 Hz, J2 ¼ 13.8 Hz, 2H, CH2), 4.30 (d, J ¼ 6.0 Hz, 2H, CH2), 7.34 (d, J ¼ 8.8 Hz, 2H, ArH), 7.68 (d, J ¼ 8.8 Hz, 2H, ArH), 9.02 (s, 1H, ArH), 9.15 (br, 1H, NH), 10.51 (br, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d: 14.4, 43.2, 61.2, 122.3, 127.5, 128.7, 138.1, 155.9, 157.8, 159.7, 169.6. ESI-MS: m/z ¼ 374.6 [MþNa]þ, 352.6[Mþ1]þ. Anal. Calcd. for C14H14N5O4: C 47.80, H 4.01, N 19.91; Found C 48.07, H 4.23, N 20.06.

5.1.3.9. Methyl2-(methyl(5-nitro-2-((3,4,5-trimethoxyphenyl)amino) pyrimidin-4-yl)amino)acetate (7i). Yield, 91%; mp 142.6e143.6
C; 1 H NMR (400 MHz, CDCl3) d: 3.10 (s, 3H, CH3), 3.65 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.90 (s, 6H, 2  OCH3), 4.26 (s, 2H, CH2), 6.75 (s, 2H, ArH), 7.43 (br, 1H, NH), 8.88 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 40.6, 52.1, 52.7, 56.1, 60.9, 99.1, 124.8, 133.8, 135.0, 153.2, 157.2, 157.9, 158.2, 168.9. ESI-MS: m/z ¼ 430.8 [MþNa]þ, 408.9 [Mþ1]þ. Anal. Calcd. for C17H21N5O7: C, 50.12; H, 5.20; N, 17.19; Found C 49.98, H 4.95, N 17.05.

5.1.3.4. Ethyl 2-((2-((4-methoxyphenyl)amino)-5-nitropyrimidin-4yl)amino)acetate (7d). Yield, 95%; mp 184.8e185.7
C; 1H NMR (400 MHz, CDCl3) d: 1.32 (t, J ¼ 7.0 Hz, 3H, CH3), 3.85 (s, 3H, OCH3), 4.26 (dd, J1 ¼ 6.8 Hz, J2 ¼ 14.0 Hz, 2H, CH2), 4.32 (d, J ¼ 5.6 Hz, 2H, CH2), 6.93 (d, J ¼ 8.8 Hz, 2H, ArH), 7.46 (s, J ¼ 8.8 Hz, 2H, ArH), 7.69 (br, 1H, NH), 8.56 (br, 1H, NH), 9.05 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 14.4, 43.1, 55.6, 61.1, 114.0, 120.8, 122.2, 132.1, 156.0, 157.6, 159.5, 169.6. ESI-MS: m/z ¼ 370.8 [MþNa]þ, 348.8 [Mþ1]þ. Anal. Calcd. for C15H17N5O5: C C 51.87, H 4.93, N 20.16; Found C 51.72, H 4.67, N 19.87.

5.1.3.10. Methyl 2-((2-((4-chlorophenyl)amino)-5-nitropyrimidin-4yl) (methyl)amino)acetate (7j). Yield, 86%; mp 158.7e160.5
C; 1H NMR (400 MHz, DMSO-d6) d: 2.97 (s, 3H, CH3), 3.58 (s, 3H, OCH3), 4.39 (s, 2H, CH2), 7.35e7.31 (m, 2H, ArH), 7.63 (d, J ¼ 8.8 Hz, 2H, ArH), 8.86 (s, 1H, ArH), 10.33 (br, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d; 40.7, 52.2, 52.8, 122.1, 124.8, 127.2, 128.7, 138.2, 156.7, 158.0, 169.5. ESI-MS: m/z ¼ 374.8 [MþNa]þ, 352.9 [Mþ1]þ. Anal. Calcd. for C14H14ClN5O4: C, 47.80; H, 4.01; N, 19.91; Found C 47.66, H 3.98, N 19.73.

5.1.3.5. Ethyl2-((2-((1-(methylsulfonyl)piperidin-4-yl)amino)-5nitropyrimidin-4-yl)amino)acetate (7e). Yield, 70%; mp 179.8e181.6
C; 1H NMR (400 MHz, CDCl3)d: 1.35 (t,J ¼ 7.2 Hz, 3H, CH3), 1.85 (s, 2H, CH2), 2.10 (s, 2H, CH2), 2.89 (s,3H, CH3), 3.30 (s, 2H, CH2), 3.56 (s, 2H, CH2), 4.30 (t, J ¼ 6.8 Hz, 2H, CH2), 4.35 (d, J ¼ 4.4 Hz, 2H, CH2), 8.63 (s, 1H, ArH), 8.98 (s, 1H, NH), 9.78 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d: 14.6, 30.6, 34.6, 34.8, 43.0, 44.7, 45.0, 48.1, 61.1, 120.2, 155.9, 158.0, 161.0, 169.9. ESI-MS: m/ z ¼ 425.5 [MþNa]þ, 403.5[Mþ1]þ. Anal. Calcd. for C14H22N6O6S: C, 41.78; H, 5.51; N, 20.88; S, 7.97; Found C 41.95, H 5.30, N 20.61, S, 8.192. 5.1.3.6. Ethyl 2-((2-(benzylamino)-5-nitropyrimidin-4-yl)amino)acetate (7f). Yield, 76%; mp 159.4e161.3
C; 1H NMR (400 MHz, CDCl3) d: 1.29 (t, J ¼ 4.0 Hz, 3H, CH3), 4.23 (dd, J1 ¼ 7.2 Hz, J2 ¼ 14.4 Hz, 2H, CH2), 4.29 (d, J ¼ 5.2 Hz, 2H, CH2), 7.40e7.31 (m, 5H, ArH), 8.81 (s, 1H, ArH), 8.90 (br, 1H, NH). 13C NMR (100 MHz, DMSOd6) d: 14.4, 42.9, 44.9, 60.9, 120.3, 127.3, 127.7, 128.7, 139.4, 155.9, 158.0, 161.8, 169.7. ESI-MS: m/z ¼ 354.6[MþNa]þ. Anal. Calcd. for C15H17N5O4: C 54.38, H 5.17, N 21.14; Found C 54.01, H 4.97, N 20.87. 5.1.3.7. Ethyl 2-((5-nitro-2-(phenethylamino)pyrimidin-4-yl)amino) acetate (7g). Yield, 73%; mp168.8e170.5
C; 1H NMR (400 MHz, DMSO-d6) d: 1.09 (t, J ¼ 7.2 Hz, 3H, CH3), 2.82 (t, J ¼ 7.4 Hz, 2H, CH2), 3.45 (dd, J1 ¼ 6.4 Hz, J2 ¼ 14.4 Hz, 2H, CH2), 4.05 (dd, J1 ¼ 7.0 Hz, J2 ¼ 14.2 Hz, 2H, CH2), 4.26 (d, J ¼ 5.6 Hz, 2H, CH2), 7.23 (d, J ¼ 7.2 Hz, 3H, ArH), 7.30 (t, J ¼ 7.2 Hz, 2, ArH), 8.45 (t, J ¼ 5.4 Hz, 1H, ArH), 8.87 (br, 1H, NH), 9.02 (t, J ¼ 5.4 Hz, 1H, NH). 13C NMR (100 MHz, DMSOd6) d: 14.4, 34.9, 42.9, 43.2, 61.0, 120.1, 126.6, 127.7, 128.7, 129.0, 139.5, 155.9, 157.9, 161.7, 169.8. ESI-MS: m/z ¼ 346.6[MþNa]þ. Anal. Calcd. For C16H19N5O4: C 55.64, H 5.55, N 20.28; Found C, 55.36 H, 5.74; N, 20.41. 5.1.3.8. Methyl 2-(methyl(5-nitro-2-(phenylamino)pyrimidin-4-yl) amino)acetate (7h). Yield, 92%; mp 173.7e174.6
C; 1H NMR (400 MHz, CDCl3) d: 3.10 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 4.29 (s, 2H, CH2), 7.17 (t, J ¼ 7.4 Hz, 1H, ArH), 7.37 (t, J ¼ 8.0 Hz, 2H, ArH), 7.49 (d, J ¼ 7.6 Hz, 2H, ArH), 7.93 (br, 1H, NH), 8.85 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 40.7, 52.2, 52.7, 120.5, 124.1, 125.1, 128.8, 137.9, 157.0, 157.8, 158.0, 169.1. ESI-MS: m/z ¼ 340.9 [MþNa]þ. Anal. Calcd. for C14H15N5O4: C, 52.99; H, 4.76; N, 22.07; Found C 53.06, H 4.53, N

5.1.3.11. Methyl 2-((2-((3-methoxyphenyl)amino)-5-nitropyrimidin4-yl) (methyl)amino)acetate (7k). Yield, 74%; mp 123.8e125.6
C;1H NMR (400 MHz, CDCl3) d: 3.09 (s, 3H, CH3), 3.70 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.29 (s, 2H, CH2), 6.69 (dd, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, 1H, ArH), 7.07e7.05 (m, 1H, ArH), 7.10 (s, 1H, ArH), 7.25 (t, J ¼ 8.0 Hz, 1H, ArH), 7.92 (br, 1H, NH), 8.84 (s, 1H, ArH). 13C NMR (100 MHz, DMSOd6) d: 40.6, 52.1, 52.7, 55.4, 106.5, 108.9, 112.8, 124.6, 129.6, 140.4, 156.9, 158.1, 158.2, 159.9, 169.5. ESI-MS: m/z ¼ 370.9 [MþNa]þ, 348.9 [Mþ1]þ. Anal. Calcd. for C15H17N5O5: C, 51.87; H, 4.93; N, 20.16; Found C,51.76, H, 4.771, N, 20.35. 5.1.3.12. Ethyl2-(methyl(2-((1-(methylsulfonyl)piperidin-4-yl) amino)-5-nitropyrimidin-4-yl)amino) acetate (7l). Yield, 71%; mp 144.1e146.9
C; 1H NMR (400 MHz, CDCl3) d: 2.10 (s, 2H, CH2), 2.19 (s, 1H, CH), 2.84 (s, 3H, CH3), 2.93 (d, J ¼ 10.4 Hz, 3H, CH3), 3.08 (s, 3H, OCH3), 3.76 (s, 6H, 3  CH2), 4.20 (s, 2H, CH2), 5.57 (br, 1H, NH), 8.78 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 30.6, 34.7, 40.7, 44.8, 48.1, 52.3, 52.7, 123.4, 124.5, 129.0, 131.9, 132.1, 157.0, 158.5, 159.7, 167.4, 169.6, 170.2. ESI-MS: m/z ¼ 425.9 [MþNa]þ, 403.9 [Mþ1]þ. Anal. Calcd. for C14H22N6O6S: C, 41.78; H, 5.51; N, 20.88; S, 7.97; Found C 41.62, H 5.34, N 20.76, S 7.783. 5.1.3.13. Methyl 2-((2-(benzylamino)-5-nitropyrimidin-4-yl) (methyl)amino)acetate (7m). Yield, 69%; mp 131.3e133.5
C; 1H NMR (400 MHz, CDCl3) d: 3.05 (s, 3H, CH3), 3.65 (s, 3H, OCH3), 4.16 (s, 2H, CH2), 4.51 (d, J ¼ 5.6 Hz, 2H, CH2), 7.39e7.29 (m, 5H, ArH), 8.33 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 44.8, 52.0, 52.2, 52.7, 123.5, 127.2, 127.5, 128.7, 139.7, 156.9, 158.6, 160.6, 169.9. ESIMS: m/z ¼ 354.9 [MþNa]þ. Anal. Calcd. for C15H17N5O4: C, 54.38; H, 5.17; N, 21.14; Found C 54.57, H 5.02, N 21.25. 5.1.3.14. Methyl 2-(methyl(5-nitro-2-(phenethylamino)pyrimidin-4yl)amino)acetate(7n). Yield, 73%; mp 100.8e103.5
C; 1H NMR (400 MHz, CDCl3) d: 2.88 (t, J ¼ 6.8 Hz, 2H, CH2), 3.09 (s, 3H, CH3), 3.60 (d, J ¼ 6.4 Hz, 2H, CH2), 3.73 (s, 3H, OCH3), 4.25 (s, 2H, CH2), 5.86 (br, 1H, NH), 7.23 (t, J ¼ 9.6 Hz, 3H, ArH), 7.34 (t, J ¼ 7.2 Hz, 2H, ArH), 8.72 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 26.7, 35.0, 40.7, 43.1, 52.0, 52.7, 123.3, 126.5, 128.7, 129.0, 139.6, 157.1, 158.4, 160.4, 170.0. ESI-MS: m/z ¼ 369.9 [MþNa]þ, 347.0[Mþ1]þ. Anal. Calcd. for C16H19N5O4: C, 55.64; H, 5.55; N, 20.28; Found C 55.36, H 5.42, N 20.46.

P.-L. Zhao et al. / European Journal of Medicinal Chemistry 118 (2016) 161e169

5.1.3.15. Ethyl 2-(benzyl(5-nitro-2-(phenylamino)pyrimidin-4-yl) amino)acetate (7o). Yield, 80%; mp 99.7e102.1
C; 1H NMR (400 MHz, CDCl3) d: 1.23 (t, J ¼ 7.0 Hz, 3H, CH3), 4.15 (d, J ¼ 8.8 Hz, 4H, CH2), 4.80 (s, 2H, CH2), 7.12 (t, J ¼ 7.2 Hz, 1H, ArH), 7.34 (dd, J1 ¼ 6.8 Hz, J2 ¼ 15.6 Hz, 7H, ArH), 7.45 (d, J ¼ 7.6 Hz, 2H, ArH), 7.54 (br, 1H, NH), 8.89 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 14.0, 50.7, 55.1, 61.3, 120.6, 124.2, 127.8, 128.7, 128.9, 134.9, 137.8, 157.3, 158.2, 158.3, 168.6. ESI-MS: m/z ¼ 430.6 [MþNa]þ, 408.6[Mþ1]þ. Anal. Calcd. for C21H21N5O4: C, 61.91; H, 5.20; N, 17.19; Found C 61.69, H 5.48, N 17.07. 5 .1. 3 .16 . Et hyl 2 - ( b e n z yl ( 2 - ( ( 3- met h ox y ph e nyl ) am i n o) - 5 nitropyrimidin-4-yl)amino)acetate (7p). Yield, 75%; mp 193.2e194.6
C; 1H NMR (400 MHz, DMSO-d6) d: 1.11 (t, J ¼ 6.8 Hz, 3H, CH3), 3.70 (s, 3H, OCH3), 4.02 (dd, J1 ¼ 7.2 Hz, J2 ¼ 14.4 Hz, 2H, CH2), 4.24 (s, 2H, CH2), 4.79 (s, 2H, CH2), 6.64e6.61 (m, 1H, ArH), 7.15 (d, J ¼ 6.0 Hz, 2H, ArH), 7.20 (s, 1H, ArH), 7.32e7.25 (m, 5H, ArH), 8.83 (s, 1H, ArH), 10.20 (br, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d: 14.2, 52.0, 55.1, 55.3, 61.1, 106.4, 109.2, 113.0, 125.1, 127.7, 127.9, 128.7, 129.6, 136.1, 140.3, 157.0, 158.2, 158.4, 159.9, 168.8. ESI-MS: m/z ¼ 460.5 [MþNa]þ, 438.6 [Mþ1]þ. Anal. Calcd. for C22H23N5O5: C, 60.40; H, 5.30; N, 16.01; Found C 60.12, H 5.58, N 15.97. 5.1.3.17. Ethyl2-(benzyl(5-nitro-2-((3,4,5-trimethoxyphenyl)amino) pyrimidin-4-yl)amino)acetate (7q). Yield, 81%; mp 140.3e142.1
C; 1 H NMR (400 MHz, CDCl3) d: 1.21 (t, J ¼ 7.0 Hz, 3H, CH3), 3.79 (s, 6H, 2  OCH3), 3.83 (s, 3H, OCH3), 4.07 (s, 2H, CH2), 4.11 (t, J ¼ 7.0 Hz, 2H, CH2), 4.87 (s, 2H, CH2), 6.77 (s, 2H, ArH), 7.38e7.28 (m, 5H, ArH), 7.74 (br, 1H, NH), 8.89 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 13.9, 50.3, 56.0, 60.9, 61.4, 98.6, 128.0, 128.8, 133.7, 134.8, 134.9, 153.3, 158.2, 158.4, 168.4. ESI-MS: m/z ¼ 498.7 [Mþ1]þ. Anal. Calcd. for C24H27N5O7: C, 57.94; H, 5.47; N, 14.08; Found C 57.83, H 5.63, N 14.32. 5 .1. 3 .18 . E t h yl 2 - ( b e n z y l ( 2 - ( ( 4 - c h l o r o b e n z yl ) a m i n o ) - 5 nitropyrimidin-4-yl)amino)acetate (7r). Yield, 77%; mp 107.2e108.1
C; 1H NMR (400 MHz, CDCl3) d: 1.24 (t, J ¼ 7.0 Hz, 3H, CH3), 4.15 (dd, J1 ¼ 6.8 Hz, J2 ¼ 14.0 Hz, 4H, CH2), 4.79 (s, 2H, CH2), 7.24 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.39e7.30 (m, 7H, ArH), 7.83 (br, 1H, NH), 8.85 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 14.0, 50.8, 55.1, 61.5, 99.9, 121.8, 125.6, 127.7, 127.9, 128.7, 128.9, 129.2, 134.7, 136.4, 157.2, 158.0, 158.1, 168.5. ESI-MS: m/z ¼ 465.1 [MþNa]þ, 443.1 [Mþ1]þ. Anal. Calcd. for C21H20ClN5O4: C, 57.08; H, 4.56; Cl, 8.02; N, 15.85; Found C 56.78, H 4.72, N 15.93. 5.1.3.19. Ethyl2-(benzyl(2-((1-(methylsulfonyl)piperidin-4-yl) amino)-5-nitropyrimidin-4-yl)amino)acetate (7s). Yield, 71%; mp 149.5e151.7
C; 1H NMR (400 MHz, CDCl3) d: 1.27 (t, J ¼ 7.0 Hz, 3H, CH3), 1.59 (d, J ¼ 9.6 Hz, 2H, CH2), 1.97 (D, J ¼ 11.6 Hz, 2H, CH2), 2.81 (s, 5H, CH3CH2), 3.68 (d, J ¼ 10.8 Hz, 2H, CH2), 3.76 (s, 1H, CH), 4.04 (s, 2H, CH2), 4.21 (dd, J1 ¼ 7.0 Hz, J2 ¼ 14.2 Hz, 2H, CH2), 4.79 (s, 2H, CH2), 5.85 (d, J ¼ 6.0 Hz, 1H, NH), 7.37e7.30 (m, 5H, ArH), 8.79 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 14.1, 31.2, 35.1, 44.5, 48.1, 51.3, 55.0, 61.3, 124.5, 127.4, 127.7, 128.7, 135.2, 157.3, 158.6, 159.7, 169.0. ESI-MS: m/z ¼ 515.5 [MþNa]þ, 493.6 [Mþ1]þ. Anal. Calcd. for C21H28N6O6S: C, 51.21; H, 5.73; N, 17.06; S, 6.51; Found C 51.49, H 5.94, N 16.78. 5.1.3.20. Ethyl 2-(benzyl(2-((2-ethoxy-2-oxoethyl)amino)-5nitropyrimidin-4-yl)amino)acetate (7t). Yield, 70%; mp 128.1e129.9
C; 1H NMR (400 MHz, DMSO-d6) d: 1.21e1.15 (m, 6H, 2  CH3), 3.92 (d, J ¼ 4.4 Hz, 2H, CH2), 4.08 (dd, J1 ¼ 11.6 Hz, J2 ¼ 23.8 Hz, 6H, 3  CH2), 4.72 (d, J ¼ 10.0 Hz, 2H, CH2), 7.32e7.28 (m, 5H, ArH), 8.43 (br, 1H, NH), 8.72 (s, 1H, ArH). 13C NMR (100 MHz,

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CDCl3) d: 14.06, 14.08, 43.6, 50.7, 55.0, 61.2, 61.5, 124.9, 127.8, 128.6, 135.1, 157.2, 158.6, 160.1, 168.9, 169.4. ESI-MS: m/z ¼ 440.5 [MþNa]þ, 418.6 [Mþ1]þ. Anal. Calcd. for C19H23N5O6: C, 54.67; H, 5.55; N, 16.78; Found C 54.43, H 5.63, N 16.86. 5 .1. 3 . 21. E t hyl 2 - ( b e n z yl ( 2 - ( (3 - m e t h o xy b e n z yl ) a m i n o ) - 5 nitropyrimidin-4-yl)amino)acetate (7u). Yield, 72%; mp 78.1e80.2
C; 1H NMR (400 MHz, CDCl3) d: 1.23 (t, J ¼ 7.0 Hz, 3H, CH3), 3.81 (s, 3H, OCH3), 4.02 (s, 2H, CH2), 4.15 (dd, J1 ¼ 7.2 Hz, J2 ¼ 14.0 Hz, 1H, CH2), 4.51 (d, J ¼ 5.2 Hz, 2H, CH2), 4.77 (s, 2H, CH2), 6.72 (br, 1H, NH), 6.85 (d, J ¼ 8.8 Hz, 3H, ArH), 7.26 (d, J ¼ 7.6 Hz, 1H, ArH), 7.33 (d, J ¼ 8.4 Hz, 5H, ArH), 8.59 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 14.1, 45.9, 50.5, 55.2, 61.2, 112.8, 113.4, 119.7, 124.3, 127.7, 127.9, 128.6, 129.8, 135.2, 139.2, 157.4, 158.4, 159.9, 160.3, 169.0. ESI-MS: m/z ¼ 452.6 [Mþ1]þ. Anal. Calcd. for C23H25N5O5: C, 61.19; H, 5.58; N, 15.51; Found C 61.59, H 5.86, N 15.80. 5.1.3.22. Ethyl 2-(benzyl(2-(benzylamino)-5-nitropyrimidin-4-yl) amino)acetate (7v). Yield, 91%; mp 91.5e93.1
C; 1H NMR (400 MHz, CDCl3) d: 1.22 (t, J ¼ 7.0 Hz 3H, CH3), 4.02 (s, 2H, CH2), 4.14 (dd, J1 ¼ 7.0 Hz, J2 ¼ 14.2 Hz, 2H, CH2), 4.53 (d, J ¼ 5.6 Hz, 2H, CH2), 4.76 (s, 2H, CH2), 6.96 (br, 1H,NH), 7.36e7.30 (m, 10H, ArH), 8.46 (d, J ¼ 2.8 Hz, 2H, ArH), 7.77 (d, J ¼ 8.0 Hz, 1H, ArH), 7.87e7.83 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3) d: 14.1, 45.9, 50.6, 55.0, 61.2, 124.3, 127.5, 127.7, 127.9, 128.6, 128.7, 135.2, 137.6, 157.4, 158.4, 160.3, 169.0. ESI-MS: m/z ¼ 444.8 [MþNa]þ, 422.9 [Mþ1]þ. Anal. Calcd. for C22H23N5O4: C, 62.70; H, 5.50; N, 16.62; Found C 62.50, H 5.70, N 16.68. 5.1.3.23. Ethyl 2-(benzyl(5-nitro-2-(phenethylamino)pyrimidin-4-yl) amino)acetate (7w). Yield, 71%; mp 104.3e105.2
C; 1H NMR (400 MHz, CDCl3) d: 1.23 (d, J ¼ 7.2 Hz, 3H, CH3), 2.85 (t, J ¼ 6.8 Hz, 2H, CH2), 3.61 (dd, J1 ¼ 6.4 Hz, J2 ¼ 13.2 Hz, 2H, CH2), 4.08 (s, 2H, CH2), 4.19 (dd, J1 ¼ 7.0 Hz, J2 ¼ 14.0 Hz, CH2), 4.81 (s, 2H, CH2), 6.17 (br, 1H, NH),7.16 (d, J ¼ 7.2 Hz, 2H, ArH), 7.25 (d, J ¼ 7.2 Hz, 1H, ArH), 7.37e7.31 (m, 7H, ArH), 8.73 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d: 14.3, 35.1, 43.1, 52.0, 55.2, 60.9, 123.8, 126.5, 127.5, 127.7, 128.2, 128.7, 129.0, 136.5, 139.5, 157.2, 158.5, 160.6, 169.3. ESIMS: m/z ¼ 459.2 [MþNa]þ, 437.2 [Mþ1]þ. Anal. Calcd. for C23H25N5O4: C, 63.44; H, 5.79; N, 16.08; Found C 63.16, H 5.95, N 16.03. 5.2. Crystallographic analysis Colorless blocks of 7q were mounted on a quartz fiber. Cell dimensions and intensities were measured at 298 K on a Bruker SMART CCD area detector diffractometer with graphite monochromated Mo Ka radiation (l ¼ 0.71073 Å); the unit cell dimensions were determined to be: a ¼ 30.382, b ¼ 8.3532, c ¼ 18.065 Å and a ¼ 90.00, b ¼ 115.01, g ¼ 90.00
in the space group C2/c; qmax ¼ 27.48; 18,672 measured reflections; 4732 independent reflections (Rint ¼ 0.0997) of which 3378 had jFoj > 2jFoj. Data were corrected for Lorentz and polarization effects and for absorption (Tmin ¼ 0.9567; Tmax ¼ 0.9780). The structure was solved by direct methods using SHELXS-97 [27]; all other calculations were performed with Bruker SAINT System and Bruker SMART programs [28]. Full-matrix least-squares refinement based on F2 using the weight of 1/[s2(Fo 2) þ (0.0570P)2 þ 0.5025P] gave final values of R ¼ 0.0539, uR ¼ 0.1106, and GOF(F) ¼ 0.993 for 281 variables and 4731 contributing reflections. Maximum shift/ error ¼ 0.001(3), max/min residual electron density ¼ 0.257/ 0.239 e Å3. Hydrogen atoms were observed and refined with a fixed value of their isotropic displacement parameter.

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5.3. Pharmacology evaluation 5.3.1. Antitumor activity The antitumor activities of compounds 7a ~ w were evaluated with MDA-MB-231, A549, HepG2, and MCF-7 cell lines by the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay in vitro. The cancer cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were splitted at 70e80% confluence, about twice a week by trypsinization. Exponentially growing cells were plated in 96-well plates (5  103 cells/well) and incubated at 37
C for 24 h for attachment. Test compounds were prepared by dissolving in dimethyl sulfoxide (DMSO) at 20 mM and diluted with the medium into a series of concentrations. The culture medium was then changed, and cells grew in medium with the test compounds. DMSO (0.1%) was used as negative control. Cells were incubated at 37
C with 5% CO2 for 48 h. Then the medium was replaced with MTT solution (5 mg/mL, 200 mL) followed by incubation for another 4 h. The medium was then aspirated and formazan crystals were dissolved in DMSO (150 mL) for about 10 min. The absorbance at 570 nm (Abs) of the suspension was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The inhibition percentage was calculated using the following formula: % inhibition ¼ (Abscontrol ¼ Abscompound)/ Abscontrol  100%. The IC50 values of the test compounds and fluorouracil were measured by treating cells with drugs of various concentrations, and analyzed by use of the prism statistical package (GraphPad Software, San Diego, CA, U.S.A.). 5.3.2. Flow-activating cell sorting analysis (FACS) The effect of compound 7w on cell cycle phase distribution of human breast cancer HepG2 was assessed using flow cytometry. When the cells grew to about 70% confluence in 6-well microplates, they were treated with compound 7w at given concentrations (1, 5, 10, 20 mM). After 48 h, cells were harvested by trypsinization, washed with PBS, and fixed in 70% ice cold (4
C) ethanol overnight. They were then washed with PBS, incubated with RNase (50 mg/mL final concentration) at 37
C for 30 min, stained with propidium iodide (50 mg/mL final concentration), and analyzed by flow cytometry (Beckman Coulter). Acknowledgments This work was supported by National Natural Science Foundation of China (21102069 and 21372113), and the project of the Outstanding Young Teachers in Guangdong Province, and Science and Technology New Star in Zhujiang Guangzhou City (No. 2012J2200051). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.04.038. References [1] P. Vineis, C.P. Wild, Global cancer patterns: causes and prevention, Lancet 383 (2014) 549e557. [2] W.M. Seganish, W.T. McElroy, R.J. Herr, S. Brumfield, W.J. Greenlee, J. Harding, V. Komanduri, J. Matasi, K.C. Prakash, D. Tulshian, J. Yang, L. Yet, K. Devito, J. Fossetta, C.G. Garlisi, D. Lundell, X. Niu, C. Sondey, Initial optimization and series evolution of diaminopyrimidine inhibitors of interleukin-1 receptor associated kinase 4, Bioorg. Med. Chem. Lett. 25 (2015) 3203e3207. [3] S.J. Robinson, J.P. Petzer, G. Terre'Blanche, A. Petzer, M.M. van der Walt, J.J. Bergh, A.C. Lourens, 2-Aminopyrimidines as dual adenosine A1/A2A antagonists, Eur. J. Med. Chem. 104 (2015) 177e188.

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