Accepted Manuscript One-pot synthesis and antiproliferative activity of novel 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties Wei-Feng Ma, Hai-Kui Yang, Meng-Jin Hu, Qian Li, Tian-Zhu Ma, Zhong-Zhen Zhou, Rui-Yuan Liu, Wen-Wei You, Pei-Liang Zhao PII:

S0223-5234(14)00623-0

DOI:

10.1016/j.ejmech.2014.07.017

Reference:

EJMECH 7137

To appear in:

European Journal of Medicinal Chemistry

Received Date: 13 May 2014 Revised Date:

10 June 2014

Accepted Date: 6 July 2014

Please cite this article as: W.-F. Ma, H.-K. Yang, M.-J. Hu, Q. Li, T.-Z. Ma, Z.-Z. Zhou, R.-Y. Liu, W.W. You, P.-L. Zhao, One-pot synthesis and antiproliferative activity of novel 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.07.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

One-pot synthesis and antiproliferative activity of novel 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties

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Wei-Feng Mab,#, Hai-Kui Yanga,#, Meng-Jin Hua, Qian Lia, Tian-Zhu Maa, Zhong-Zhen Zhoua, Wen-Wei Youa, *, Pei-Liang Zhaoa, *

N H

N

N H

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27

28

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Anticancer activities against HepG2, A549, MDA-MB-231 and MCF-7, IC50 27 = 19.98, 21.41, 15.83, 32.76 µM 28 = 20.19, 12.78, 7.46, 16.24 µM fluorouracil = 46.83, 35.41, 24.60, 39.88 µM

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ACCEPTED MANUSCRIPT

One-pot synthesis and antiproliferative activity of novel 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties

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Wei-Feng Mab,#, Hai-Kui Yanga,#, Meng-Jin Hua, Qian Lia, Tian-Zhu Maa, Zhong-Zhen Zhoua,

5

Rui-Yuan Liua, Wen-Wei Youa, *, Pei-Liang Zhaoa, * a

Department of Chemistry, School of Pharmaceutical Science, Southern Medical University,

b

SC

Guangzhou 510515, P.R.China

Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R.China

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ABSTRACT: A series of novel 2,4-diaminopyrimidines containing piperidine and piperazine moieties were synthesized via an efficient one-pot methodology. The bioassay tests demonstrated that compounds 27 and 28 displayed much stronger antitumor activities against four human cancer 15

cell lines (HepG2, A549, MDA-MB-231 and MCF-7) than positive control fluorouracil. Particularly,

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compound 28 showed a two-fold improvement compared to fluorouracil in inhibiting MDA-MB-231 and A549 cell proliferation with IC50 values of 7.46 and 12.78 µM, respectively. Further flow-activated cell sorting analysis revealed that the most promising compound 28 displayed a significant effect on G2/M cell-cycle arrest in a dose-dependent manner in MDA-MB-231 cells.

25

Keywords: 2,4-Diaminopyrimidines; Piperidine; Piperazine; Antiproliferative activity; One-pot synthesis

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—————————— ∗ Corresponding author. Tel./fax: +86(0)20 61648196. ∗

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Corresponding author. E-mail: [email protected] (P.-L. Zhao), [email protected] (W.-W. You)

#

These authors contributed equally to this work.

1

ACCEPTED MANUSCRIPT 1. Introduction Pyrimidines are an important class of heterocyclic structures found in many synthetic and 35

natural occurring products with a remarkable spectrum of biological activities [1-6]. Among the existing large numbers of structurally diverse pyrimidine derivatives, 2,4-diaminopyrimidines have

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attracted considerable attention due to their important chemopreventive and chemotherapeutic effects on cancer [7-15]. For example, as shown in Fig. 1, NU6027 (1), originally identified as a cyclin-dependent kinase 2 inhibitor [16, 17], more recently has emerged as a potential antitumor 40

agent inhibiting ataxia telangiectasia mutated and Rad3-related kinase (ATR) [18]. On the other

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hand, literature survey has revealed that the structural modification of 2,4-diaminopyrimidines by introduction of nitrogen-containing piperidine and piperazine heterocycles, can further enhance the

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antitumor activity of candidate compounds. For example, the piperidine-pyrimidine analog R547 (2), shown in Fig. 1, was identified as a selective adenosine triphosphate-competitive 45

cyclin-dependent kinase inhibitor and is currently being tested in phase I clinical trials [19,20]. While the piperazine-pyrimidine compound XL228 (3), a multitargeted protein kinase inhibitor, has been proved effective in patients with solid tumors or hematologic malignancies in phase I clinical

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trials [21-23]. More interestingly, Font et al. documented that the length of amino side chain in pyrimidine 2- and 4-position of compound 4 had an important effect on its antiproliferative activity 50

[24], which indicated that the flexibility of the amino group might be a vital factor for the activity of

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compound 4.



Based on the above considerations, and in a continuation of our interest in the synthesis of

55

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nitrogen-containing heterocycles with anticancer activity [25-27], we developed an idea that introducing 4-amino piperidine and N-methylpiperazine moiety into the C2 or C4 -position of the pyrimidine scaffold with phenylalkylamine as a flexible linkage might result in new 2,4-diaminopyrimidine derivatives with high cytotoxic activity (Fig. 2). In this study, we described the one-pot synthesis and screening results, and structure-activity relationships of these compounds 8-31. 60



2

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2. Chemistry Owing to their important biological roles in chemopreventive action and the broad range of

65

pharmacological

properties,

many

methods

have

been

developed

for

synthesis

of

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2,4-diaminopyrimidine derivatives [14, 28-31]. All of the methodology involved two steps from the substituted 2,4-dichloropyrimidine as starting material. The halopyrimidines were firstly converted to 4-aminopyrimidine by a nucleophilic substitution reaction at C-4, and following purification, the 70

C-2 chlorine was displaced by various amines in the second step. Although, many efforts were

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made to synthesize new 2,4-diaminopyrimidine derivatives, no publications described the one-pot reaction between the substituted 2,4-dichloropyrimidine and amine. Herein, Scheme 1 shows the

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newly developed an efficient one-pot methodology for synthesis of 2,4-diaminopyrimidine derivatives bearing piperidine and piperazine moieties 8-31. The reliable model procedure involved 75

the treatment of 5-substituted 2,4-diaminopyrimidine (5) with 1.0 equiv of amine (6) and 2.0 equiv of N,N-diisopropylethylamine (DIPEA) as base in absolute 2-methoxyethanol at room temperature for about 12 h under N2 flow. The resulting mixture reacted with 1.1 equiv of various another amine

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(7) at 90°C for 12-18 h to successfully afford the desirable target compounds 8-31 in moderate to good isolated yields (58-84%).

The structures of the prepared 2,4-diaminopyrimidine derivatives 8-31 were characterized by

80

H NMR, 13C NMR, ESI-MS and elemental analysis and the results are shown in the Experimental

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1

section. In addition, the representative compound 25 was further confirmed by single-crystal X-ray

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diffraction. As shown in Fig. 3, the crystal structure showed that 25 had a C-shape, while the six-membered piperidine ring adopts a chair conformation. 85



3. Pharmacology Results and Discussion 90

The in vitro antitumor activities of the synthesized compounds 8-31 against four human cancer cell lines, including HepG2 (human hepatoma cells), A549 (human alveolar epithelial cells), MDA-MB-231 (human breast cancer cells) and MCF-7 (human mammary adenocarcinoma cells), 3

ACCEPTED MANUSCRIPT were assayed by MTT method [32]. Fluorouracil which is one of the most effective anticancer agents was used as the reference drug and the results expressed as IC50 (µM) were summarized in 95

Table 1. Here, the IC50 value represents the concentration of one compound resulting in a 50% inhibition in cell growth after a 48 h incubation, and is the average of three independent



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experiments.

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For the convenience of structure-activity relationship analysis, compounds 8-20, 21-23, 24-28 and 29-31 were defined as C2-piperidine, C2-piperazine, C4-piperidine and C4-piperazine

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pyrimidine derivatives, respectively. As indicated in Table 1 and Fig. 4, unfortunately, the results indicated that all C2- and C4-piperazine pyrimidine derivatives (21-23, 29-31) were ineffective (IC50 > 200 µM). Remarkably, most of the C2- and C4-piperidine pyrimidines (8-20, 24-28) displayed substantial antiproliferative activities. For example, the IC50 values of compounds 27 and 28 ranged from 7.46 to 32.76 µM against all of the tested cell lines. The IC50 values of compound

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16 were 11.73, 32.41 and 46.80 µM against MDA-MB-231, MCF-7 and A549 cells, respectively. In most cases, C4-piperidine pyrimidine derivatives displayed much higher antitumor activities against HepG2, A549 and MCF-7 than C2-piperidine pyrimidine derivatives, for example, compounds 9

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and 25 (n = 0, R1 = H, R2 = 3-MeOC6H4), 10 and 28 (n = 1, R1 = H, R2 = 4-MeOC6H4CH2). Further analysis on the structure–activity relationship investigated the effects of several

115

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substituents on the pyrimidine ring. Among the series of C2-piperidine pyrimidine derivatives, as showed in Table 1, the antitumor activities were influenced by the C5-substituents on the pyrimidine ring, and overall, hydrogen substitution (8-11) resulted in higher activities against all of the tested cell lines than those of the corresponding compounds with chloro substitution (12-20). However, compound 16 is an exception, which exhibited broad-spectrum antiproliferative activities against most of the tested cell lines. Nevertheless, within the series of C4-piperidine pyrimidine 120

derivatives, most compounds displayed potent antitumor activities. Particularly, two potent compounds 27 and 28 exhibited significantly higher activity than fluorouracil against all of the tested cell lines.

4

ACCEPTED MANUSCRIPT More interestingly, both of the most promising compounds 27 and 28 have a methoxy group in the 4-position of their aromatic rings, which illustrates that 4-methoxy group may be helpful to 125

increase their cytotoxicity. Furthermore, these two compounds have a CH2 or CH2CH2 bridge between the aromatic moiety and the pyrimidine ring. After removing the linkage, the

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antiproliferative activities (compound 24) were significantly reduced, which indicated that the flexibility of the bridge might play a crucial role in modulating the antitumor activity.

To study the effect of the synthesized compounds on cell cycle progression, flow-activated cell 130

sorting analysis was performed. The most promising compound 28 was tested against

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MDA-MB-231 cell lines at three different concentrations. As can be seen in Fig. 5 and Table 2, compound 28 did not induce apoptosis after 48 h treatment. However, The G2/M peak significantly

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increased from 18.12% to 33.79% (20 µM), 36.51% (40 µM), and 40.95% (80 µM) after 48 h of treatment. These data suggest that compound 28 induced significant cell cycle arrest in the G2/M 135

phase in a dose-dependent manner, compared to untreated cells



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4. Conclusion

In conclusion, the incorporation of piperidine or piperazine group at C2 or C4 of the

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2,4-diaminopyrimidine scaffold with a flexible linkage was designed and efficiently synthesized via a one-pot, three-component reaction. More importantly, the preliminary biological evaluation indicated that two compounds 27 and 28 exhibited more potent in vitro cytotoxic activities against

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HepG2, A549, MDA-MB-231 and MCF-7 cell lines than positive control fluorouracil. Particularly, compound 28 showed a two-fold improvement compared to fluorouracil in inhibiting MDA-MB-231 and A549 cell proliferation with IC50 values of 7.46 and 12.78 µM, respectively. These results indicated that this novel class of 2,4-diaminopyrimidine analogs may find their 150

medicinal applications after further structural modulation and biological studies.

5. Experimental protocols 5.1 Chemistry 5

ACCEPTED MANUSCRIPT 1

155

H NMR and 13C NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3

or DMSO-d6 solution and chemical shifts were recorded in parts per million (ppm) with TMS as the internal reference. 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

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analysis instrument. Melting points (mp) were taken on a Buchi B-545 melting point apparatus and are uncorrected. Unless otherwise noted, reagents were purchased from commercial suppliers and 160

used without further purification while all solvents were redistilled before use.

5.1.1 General procedure for the one-pot reactions of 5-substituted-2,4-dichloropyrimidine,

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1-(methylsulfonyl)piperidin-4-amine or 1-methylpiperazine, and various amino compounds. A mixture of a 5-substituted-2,4-dichloropyrimidine 5 (1.0 mmol), amino compound 6 (1.0

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mmol) and N,N-diisopropylethylamine (2.0 mmol, 0.258 g) in 8.0 mL of dry 2-methoxyethanol was stirred at room temperature for about 12 h under N2 flow. Then another amino compound 7 (1.1 mmol) was added. The reaction mixture was heated at 90°C for another 12-18 h. After the reaction was complete according to the TLC detection, solvent was removed under reduced pressure and the residue was purified by column chromatography using a mixture of trichloromethane and methanol,

170

5.1.1.1.

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(60:1) as an eluent to give the target compounds in yields of 58-84%.

N4-(4-Chlorophenyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine

(8).

Yield, 61%; mp 169.7-171.1°C; H NMR (400 MHz , CDCl3) δ: 1.72 (dd, J1 = 10.4 Hz, J2 = 19.8 Hz, 1

2H CH2), 2.16 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 2H, CH2), 2.85 (s, 3H, SO2-CH3), 3.06 (t, J = 11.8 Hz,

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2H, CH2), 3.70 (d, J = 12.0 Hz, 2H, CH2), 4.02 (s, 1H, CH), 6.13 (d, J = 5.9 Hz, 1H, pyrimidine-H), 7.35 (d, J = 8.3 Hz, 2H, ArH), 7.42 (d, J = 8.4 Hz, 2H, ArH), 7.83 (d, J = 4.3 Hz, 1H, 175

pyrimidine-H).

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C NMR (100 MHz, DMSO-d6) δ: 31.4, 34.6, 45.1, 47.5, 120.9, 125.3, 128.8,

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140.0, 156.5, 160.8, 161.4. ESI-MS: m/z = 404.5 [M+Na]+, 382.6 [M+1]+. Anal. Calcd. for C16H20ClN5O2S: C 50.32, H 5.28, N 18.34, S 8.40; Found C 50.58, H 5.05, N 18.53, S 8.03. 5.1.1.2. N4-(3-Methoxyphenyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (9). Yield, 72%; mp 175.4-176.5°C; 1H NMR (400 MHz , DMSO-d6) δ: 1.56 (dd, J1 = 10.6 Hz , J2= 180

20.4 Hz, 2H, CH2), 1.99 (d, J = 11.6 Hz, 2H, CH2), 2.83 (d, J = 11.2 Hz, 2H, CH2), 2.89 (s, 3H, SO2-CH3), 3.57 (d, J = 12.0 Hz, 2H, CH2), 3.75 (s, 3H, O-CH3), 3.84 (m, 1H, CH), 6.01 (d, J = 5.6 Hz, 1H, pyrimidine-H), 6.54 (d, J = 8.4 Hz, 1H, ArH), 6.77 (s, 1H, NH), 7.19 (t, J = 8.0 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.40 (s, 1H, ArH), 7.85 (d, J = 5.6 Hz, 1H, pyrimidine-H), 9.14 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 31.3, 34.6, 45.1, 47.4, 55.4, 105.8, 106.9, 112.1, 129.7,

185

142.7, 156.5, 159.9, 161.0, 161.5. ESI-MS: m/z = 400.4 [M+Na]+, 378.5 [M+1]+. Anal. Calcd. for 6

ACCEPTED MANUSCRIPT C17H23N5O3S: C 54.09, H 6.14, N 18.55, S, 8.49; Found C 54.34, H 6.33, N 18.29, S 8.72. 5.1.1.3. N4-(4-Methoxybenzyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (10). Yield, 66%; mp 94.9-96.0°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.48 (dd, J1 = 9.8 Hz, J2 = 20.4 Hz, 2H, CH2), 1.87 (d, J = 11.0 Hz, 2H, CH2), 2.80 (t, J = 10.8 Hz, 2H, CH2), 2.86 (s, 3H, SO2-CH3), 190

3.49 (d, J = 11.6 Hz, 2H, CH2), 3.73 (s, 3H, O-CH3), 3.76 (m, 1H, CH), 4.38(s, 2H, CH2), 5.75(d, J

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= 5.6 Hz, 1H, pyrimidine-H), 6.40 (s, 1H, NH), 6.88 (d, J = 8.4 Hz, 2H, ArH), 7.22(t, J = 8.4 Hz, 2H, ArH), 7.34 (s, 1H, NH), 7.64 (d, J = 5.6 Hz, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.4, 34.5, 43.0, 45.0, 46.9, 55.5, 114.0, 114.2, 128.9, 129.3, 132.4, 155.1, 158.5, 161.7, 162.9. ESI-MS: m/z = 414.6 [M+Na]+, 392.6 [M+1]+. Anal. Calcd. for C18H25N5O3S: C 55.22, H 6.44, N 17.89, S 8.19; Found C 55.33, H 6.73, N 17.26, S 8.22.

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195

5.1.1.4. N4-Benzyl-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (11). Yield, 63%; mp 127.0-128.3°C; 1H NMR (400 MHz, CDCl3) δ: 1.60 (dd, J1 = 10.0 Hz, J2 = 20.8 Hz, 2H, CH2),

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2.11 (d, J = 12.0 Hz, 2H, CH2), 2.80 (s, 3H, SO2-CH3), 2.91 (t, J = 10.8 Hz, 2H, CH2), 3.70 (d, J = 9.6 Hz, 2H, CH2), 3.92 (m, 1H, CH), 4.52 (s, 2H, CH2), 4.97 (s, 1H, NH), 5.10 (s, 1H, NH), 5.77 (s, 200

1H, pyrimidine-H), 7.28-7.34 (m , 5H, ArH), 7.83 (s, 1H, pyrimidine-H).

13

C NMR (100 MHz,

DMSO-d6) δ: 31.3, 34.5, 43.6, 45.0, 46.9, 126.9, 127.6, 128.6, 140.6, 155.4, 161.7, 162.9. ESI-MS: m/z = 384.6 [M+Na]+, 362.6 [M+1]+. Anal. Calcd. for C17H23N5O2S: C 56.49, H 6.41, N 19.38, S 8.87; Found C 56.78, H 6.64, N 19.12, S 8.85.

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5-Chloro-N4-(4-methoxybenzyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-

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5.1.1.5.

diamine (12). Yield, 61%; mp 127.0-128.3°C; 1H NMR (400 MHz, CDCl3) δ: 1.60 (dd, J1 = 10.0 Hz, J2 = 20.4 Hz, 2H, CH2), 2.12 (d, J = 11.6 Hz, 2H, CH2), 2.81 (s, 3H, SO2-CH3), 2.91 (t, J = 11.2 Hz, 2H, CH2), 3.72 (d, J = 12.0 Hz, 2H, CH2), 3.83 (s, 3H, O-CH3), 3.87-3.89 (m, 1H, CH), 4.58 (d,

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J = 5.2 Hz, 2H, CH2), 4.90 (s, 1H, NH), 5.48 (s, 1H, NH), 6.91 (d, J = 8.0 Hz, 2H, ArH), 7.28 (d, J = 4.4 Hz, 2H, ArH), 7.82 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.6, 43.2, 45.0, 47.4, 55.4, 114.0, 128.8, 132.4, 153.5, 157.6, 158.5, 160.1. ESI-MS: m/z = 448.5 [M+Na]+,

AC C

210

426.6 [M+1]+. Anal. Calcd. for C18H24ClN5O3S: C 50.76, H 5.68, N 16.44, S 7.53; Found C 51.03, H 5.50, N 16.23, S 7.35. 5.1.1.6.

5-Chloro-N4-(3-methoxyphenyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-

diamine (13). Yield, 67%; mp 205.0-205.3°C; 1H NMR (400 MHz, CDCl3) δ: 1.65 (dd, J1 = 10.2 215

Hz, J2 = 20.2 Hz, 2H, CH2), 2.19 (d, J = 11.4 Hz, 2H, CH2), 2.83 (s, 3H, SO2-CH3), 2.97 (t, J = 11.4 Hz, 2H, CH2), 3.76 (d, J = 12.0 Hz, 2H, CH2), 3.85 (s, 3H, O-CH3), 3.92-3.93 (m, 1H, CH), 5.21 (s, 1H, NH), 6.70 (d, J = 8.4 Hz, 1H, ArH), 7.07 (d, J = 7.2 Hz, 2H , ArH), 7.28 (d, J = 16.0 Hz, 1H, ArH), 7.42 (s, 1H, NH), 7.96 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.5, 45.1, 47.8, 55.5, 108.1, 108.8, 114.4, 129.4, 140.6, 155.9, 159.7, 159.9. ESI-MS: m/z = 434.4

220

[M+Na]+, 412.4 [M+1]+. Anal. Calcd. for C18H24ClN5O3S: C 50.76, H 5.68, N 16.44, S 7.53; Found 7

ACCEPTED MANUSCRIPT C 51.05, H 6.03, N 15.95, S 7.43. 5.1.1.7. 5-Chloro-N4-(4-chlorophenyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (14). Yield, 75%; mp 201.2-201.5°C; 1H NMR (400 MHz, CDCl3) δ: 1.65 (dd, J1 = 10.4 Hz, J2 = 20.4 Hz, 2H, CH2), 2.17 (d, J = 16.8 Hz, 2H, CH2), 2.84 (s, 3H, SO2-CH3), 2.93 (t, J = 11.2 Hz, 2H, 225

CH2), 3.77 (d, J = 12.0 Hz, 2H, CH2), 3.87-3.89 (m, 1H, CH), 5.14 (s, 1H, NH), 7.04 (s, 1H, NH),

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7.34 (d, J = 8.0 Hz, 2H, ArH), 7.56 (d, J = 8.0 Hz, 2H, ArH), 7.97 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.7, 45.0, 47.8, 123.7, 127.2, 128.5, 138.6, 155.8, 159.8. ESI-MS: m/z = 438.3 [M+Na]+, 416.4 [M+1]+. Anal. Calcd. for C16H19Cl2N5O2S: C 46.16, H 4.60, N 16.82, S 7.70; Found C 45.96, H 4.24, N 16.37, S 7.28. 5.1.1.8.

5-Chloro-N4-(4-methoxyphenyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-

SC

230

diamine (15). Yield, 76%; mp 183.2-185.0°C; 1H NMR (400 MHz, CDCl3) δ: 1.60 (dd, J1 = 10.4 Hz, J2 = 20.0 Hz, 2H, CH2), 2.13 (d, J = 10.0 Hz, 2H, CH2), 2.81 (s, 3H, SO2-CH3), 2.89 (t, J = 10.4

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Hz, 2H, CH2), 3.73 (d, J = 12.0 Hz, 2H, CH2), 3.84 (s, 4H, O-CH3, CH), 5.03 (s, 1H, NH), 6.91 (d, J = 9.0 Hz, 3H, ArH, NH), 7.46 (d, J = 9.0 Hz, 2H, ArH), 7.92 (s, 1H, pyrimidine-H). 235

13

C NMR

(100 MHz, DMSO-d6) δ: 31.2, 34.6, 45.1, 47.8, 55.6, 113.9, 124.4, 132.3, 155.0, 155.9, 156.2, 159.9. ESI-MS: m/z = 434.4 [M+Na]+, 412.5 [M+1]+. Anal. Calcd. for C17H22ClN5O3S : C 49.57, H 5.38, N 17.00, S 7.78; Found C 49.18, H 5.69, N 16.72, S 7.95.

5.1.1.9. 5-Chloro-N4-benzyl-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (16). 240

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Yield, 59%; mp 149.8-151.0°C; 1H NMR (400 MHz, CDCl3) δ: 1.46-1.53 (m, 2H, CH2), 1.98 (d, J = 12.0 Hz, 2H, CH2), 2.71 (s, 3H, SO2-CH3), 2.74-2.81 (m, 2H, CH2), 3.60 (d, J = 12.2 Hz, 2H, CH2), 3.74 (dd, J1 = 4.0 Hz, J2 = 8.0 Hz, 1H, CH), 4.57 (d, J = 8.0 Hz, 2H, CH2), 4.87 (s, 1H, NH), 5.50 (s, 1H, NH), 7.19-7.28 (m, 5H, ArH), 7.74 (s, 1H, pyrimidine-H).

13

C NMR (100 MHz,

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DMSO-d6) δ: 31.1, 34.6, 43.9, 45.0, 47.4, 126.9, 127.4, 128.6, 140.5, 153.6, 157.7, 160.1. ESI-MS: m/z = 418.4 [M+Na]+, 396.5 [M+1]+. Anal. Calcd. for C17H22ClN5O2S : C 51.57, H 5.60, N 17.69, S 8.10; Found C 52.08, H 5.91, N 17.14, S 8.92.

AC C

245

5.1.1.10. 5-Chloro-N2-(1-(methylsulfonyl)piperidin-4-yl)-N4-phenylpyrimidine-2,4-diamine (17). Yield, 70%; mp 188.6-189.5°C; 1H NMR (400 MHz, CDCl3) δ: 1.62-1.70 (m, 2H, CH2), 2.13-2.20 (m, 2H, CH2), 2.83 (s, 3H, SO2-CH3), 2.96 (t, J = 10.4 Hz, 2H, CH2), 3.73 (d, J = 12.0 Hz, 2H, CH2), 3.85-3.94 (m, 1H, CH), 5.50 (s, 1H, NH), 7.12 (s, 1H, ArH), 7.17 (t, J = 7.2 Hz, 1H, ArH), 250

7.38 (t, J = 8.0 Hz, 2H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 7.95 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.6, 45.0, 47.8, 122.3, 123.6, 128.7, 139.5, 155.2, 156.0, 159.9. ESI-MS: m/z = 404.4 [M+Na]+, 382.5 [M+1]+. Anal. Calcd. for C16H20ClN5O2S: C 50.32, H 5.28, N 18.34, S 8.40; Found C 50.59, H 5.17, N 18.53, S 8.11. 5.1.1.11.

5-Chloro-N4-(4-chlorophenethyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,48

ACCEPTED MANUSCRIPT 255

diamine (18). Yield, 68%; mp 171.9-172.5°C; 1H NMR (400 MHz, CDCl3) δ: 1.62-1.70 (m, 2H, CH2), 2.14-2.19 (m, 2H, CH2), 2.83 (s, 3H, SO2-CH3), 2.91 (t, J = 7.0 Hz, 2H, CH2), 2.96 (t, J = 10.0 Hz, 2H, CH2), 3.66-3.75 (m, 4H, 2×CH2), 3.88-3.95 (m, 1H, CH), 5.10 (s, 1H, NH), 5.29 (s, 1H, NH), 7.31 (s, 1H, ArH), 7.17 (s, 1H, ArH), 7.15 (s, 1H, ArH), 7.33 (s, 1H, ArH), 7.79 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.5, 34.8, 41.7, 45.0, 47.4, 128.6, 130.9,

260

131.1, 139.0, 153.4, 157.7, 160.2. ESI-MS: m/z = 468.4 [M+Na]+, 446.4 [M+1]+. Anal. Calcd. for

RI PT

C18H23Cl2N5O2S: C 48.65, H 5.22, N 15.76, S 7.22; Found C 48.79, H 5.03, N 15.87, S 7.29. 5.1.1.12. 5-Chloro-N4-(4-methoxyphenethyl)-N2-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4diamine (19). Yield, 77%; mp 157.5-159.8°C; 1H NMR (400 MHz, CDCl3) δ: 1.67 (dd, J1 = 3.6 Hz, J2 = 10.2 Hz, 2H, CH2), 2.16 (dd, J1 = 4.0 Hz, J2 = 12.0 Hz, 2H, CH2), 2.83 (s, 3H, SO2-CH3), 2.87 (t, J = 8.0 Hz, 2H, CH2), 2.99 (t, J = 10.2 Hz, 2H, CH2), 3.53-3.77 (m, 4H, 2×CH2), 3.82 (s, 3H,

SC

265

O-CH3), 3.91-4.01 (m, 1H, CH), 5.36 (s, 1H, NH), 6.89 (d, J = 8.4 Hz, 2H, ArH), 7.14 (d, J = 8.4

M AN U

Hz, 2H, ArH), 7.77 (s, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.5, 42.3, 45.0, 47.5, 55.4, 114.2, 129.9, 131.8, 153.2, 157.8, 158.1, 160.1. ESI-MS: m/z = 462.4 [M+Na]+, 440.4 [M+1]+. Anal. Calcd. for C19H26ClN5O3S: C 51.87, H 5.96, N 15.92, S 7.29; Found C 52.55, H 6.19, 270

N 15.53, S 7.82.

5.1.1.13. 5-Chloro-N2-(1-(methylsulfonyl)piperidin-4-yl)-N4-phenethylpyrimidine-2,4-diamine (20). Yield, 81% ; mp 162.4-163.9°C; 1H NMR (400 MHz, CDCl3) δ: 1.63-1.71 (m, 2H, CH2), 2.14-2.18

TE D

(m, 2H, CH2), 2.82 (s, 3H, SO2-CH3), 2.87-3.01 (m, 4H, 2×CH2), 3.72 (dd, J1 = 6.8 Hz, J2 = 13.2 Hz, 4H, 2×CH2), 3.90-4.00 (m, 1H, CH), 5.37 (s, 1H, NH), 7.23 (d, J = 7.0 Hz, 2H, ArH), 7.27-7.30 275

(m, 2H, ArH), 7.35 (t, J = 7.2 Hz, 2H, ArH), 7.78 (s, 1H, pyrimidine-H).

13

C NMR (100 MHz,

DMSO-d6) δ: 31.2, 34.5, 35.4, 42.1, 45.0, 47.5, 126.5, 128.8, 129.0, 139.9, 153.1, 157.8, 160.0.

EP

ESI-MS: m/z = 432.3 [M+Na]+, 410.4 [M+1]+. Anal. Calcd. for C18H24ClN5O2S:C 52.74, H 5.90, N 17.08, S 7.82; Found C 52.84, H 4.56, N 17.27, S 7.69.

280

N-Benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine

(21).

Yield,

83%;

mp

AC C

5.1.1.14.

149.0-150.1°C; H NMR (400 MHz, DMSO-d6) δ: 2.18 (s, 3H, N-CH3), 2.23-2.32 (m, 4H, 2×CH2), 1

3.56-3.67 (m, 4H, 2×CH2), 4.45 (d, J = 4.2 Hz, 2H, CH2), 5.79 (d, J = 5.6 Hz, 1H, pyrimidine-H), 7.23 (dd, J1 = 4.2 Hz, J2 = 8.0 Hz, 1H, ArH), 7.31 (d, J = 4.4 Hz, 4H, ArH), 7.50 (s, 1H, NH), 7.72 (d, J = 4.0 Hz, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 43.6, 43.7, 46.3, 54.9, 96.1, 127.0, 127.8, 128.6, 140.6, 155.3, 161.6, 162.7, 172.4. ESI-MS: m/z = 307.2 [M+Na]+, 285.2 285

[M+1]+. Anal. Calcd. for C16H21N5: C 67.82, H 7.47, N, 24.71; Found C 67.40, H 7.87, N 24.81. 5.1.1.15. N-(4-Methoxybenzyl)-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (22). Yield, 82%; mp 108.1-110.2°C; 1H NMR (400 MHz , DMSO-d6) δ: 2.19 (s, 3H, O-CH3), 2.24-2.33 (m, 4H, 2×CH2), 3.57-3.67 (m, 4H, 2×CH2), 3.72 (s, 3H, O-CH3), 4.38 (s, 2H, CH2), 5.77 (d, J = 5.7 Hz, 1H, pyrimidine-H), 6.88 (d, J = 8.6 Hz, 2H, ArH), 7.24 (d, J = 8.6 Hz, 2H, ArH), 7.41 (s, 1H, NH), 7.71 9

ACCEPTED MANUSCRIPT 290

(d, J = 5.6 Hz, 1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 43.2, 43.6, 46.3, 54.9, 55.4, 96.1, 114.0, 129.1, 132.4, 155.3, 158.6, 161.6, 162.7. ESI-MS: m/z = 336.5 [M+Na]+, 314.5 [M+1]+. Anal. Calcd. for C17H23N5O: C 65.15, H 7.40; N 22.35; Found C 64.97, H 7.60, N 22.19. 5.1.1.16. N-(3-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (23). Yield, 58%; mp 139.5-141.4°C; 1H NMR (400 MHz, CDCl3) δ: 2.36 (s, 3H, N-CH3), 2.50 (t, J = 4.8 Hz, 4H, 2×CH2), 3.69 (t, J = 4.8 Hz, 4H, 2×CH2), 3.83 (s, 3H, O-CH3), 6.05 (d, J = 6.0 Hz, 1H,

RI PT

295

pyrimidine-H), 6.57 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H, ArH), 7.04 (d, J = 9.6 Hz, 1H, ArH), 7.09 (s, 1H, NH), 7.21 (t, J = 8.0 Hz, 1H, ArH), 7.41 (t, J = 2.0 Hz, 1H, ArH), 8.01 (d, J = 6.0 Hz, 1H, pyrimidine-H). 13C NMR (100 MHz, CDCl3) δ: 43.9, 46.1, 54.6, 55.1, 95.0, 104.8, 107.5, 111.5, 129.3, 141.4, 156.6, 159.5, 160.0, 162.3. ESI-MS: m/z = 322.7 [M+Na]+, 300.8 [M+1]+. Anal. Calcd. for C16H21N5O: C 64.19, H 7.07, N 23.39; Found C 64.03, H 7.36, N 23.47.

SC

300

5.1.1.17. N2-(4-Methoxyphenyl)-N4-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (24).

M AN U

Yield, 77%; mp 187.4-189.0°C; 1H NMR (400 MHz, CDCl3) δ: 2.36 (s, 3H, SO2-CH3), 2.49 (t, J = 4.8 Hz, 4H, 2×CH2), 3.66 (t, J = 4.8 Hz, 4H, 2×CH2), 3.82 (s, 3H, O-CH3), 6.01 (d, J = 6.4 Hz, 1H, pyrimidine-H), 7.45 (d, J = 9.2 Hz, 2H, ArH), 7.47 (d, J = 9.2 Hz, 2H, ArH), 7.97 (d, J = 6.0 Hz, 305

1H, pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.3, 44.9, 46.6, 55.6, 113.9, 114.3, 120.5, 134.9, 153.9, 155.3, 160.2, 162.2. ESI-MS: m/z = 400.6 [M+Na]+, 378.6 [M+1]+. Anal. Calcd. for C17H23N5O3S: C 54.09, H 6.14, N 18.55, S 8.49; Found C 54.39, H 6.44, N 18.27, S 8.20.

TE D

5.1.1.18. N2-(3-Methoxyphenyl)-N4-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (25). Yield, 60%; mp 203.5-204.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.52 (dd, J1 = 10.4 Hz, J2 = 20.0 310

Hz, 2H, CH2), 2.04 (d, J = 10.4 Hz, 2H, CH2), 2.89 (d, J = 2.0 Hz, 1H, CH), 2.91 (s, 3H, SO2-CH3), 3.57 (d, J = 12.4 Hz, 2H, CH2), 3.73 (s, 3H, O-CH3), 3.96 (s, 1H, NH), 5.95 (d, J = 5.8 Hz, 1H,

EP

pyrimidine-H), 6.46 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H, ArH), 7.13 (t, J = 8.2 Hz, 1H, ArH), 7.30 (d, J = 8.0 Hz, 1H, ArH), 7.51 (s, 1H, ArH), 7.82 (d , J = 5.6 Hz, 1H, pyrimidine-H), 8.93 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.6, 44.9, 46.7, 55.3, 105.1, 105.7, 111.4, 129.4, 142.9,

315

AC C

13

155.3, 159.9, 159.9, 160.1, 162.2. ESI-MS: m/z = 400.3 [M+Na]+, 378.4 [M+1]+. Anal. Calcd. for C17H23N5O3S: C 54.09, H 6.14, N 18.55, S 8.49; Found C 54.28, H 6.42, N 18.26, S 8.55. 5.1.1.19. N2-(4-Chlorophenyl)-N4-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (26). Yield, 79%; mp 208.8-209.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.51 (dd, J1 = 8.0 Hz, J2 = 20.0 Hz, 2H, CH2), 2.02 (d, J = 12.0 Hz, 2H, CH2), 2.90 (d, J = 12.0 Hz, 1H, CH), 2.92 (s, 3H,

320

SO2-CH3), 3.58 (d, J = 12.0 Hz, 2H, CH2), 3.96 (s, 1H, NH), 5.97 (d, J = 4.0 Hz, 1H, pyrimidine-H), 7.29 (d, J = 8.0 Hz, 2H, ArH), 7.80 (d, J = 2.4 Hz, 2H, ArH), 8.31 (s, 1H, pyrimidine-H), 9.12 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 31.3, 34.7, 44.9, 46.7, 120.0, 124.2, 128.5, 140.8, 155.2, 159.9, 162.2. ESI-MS: m/z = 404.5 [M+Na]+, 382.5 [M+1]+. Anal. Calcd. for C16H20ClN5O2S: C 50.32, H 5.28, N 18.34, S 8.40; Found C 50.13, H 5.39, N 18.12, S 10

ACCEPTED MANUSCRIPT 325

8.13. 5.1.1.20.

N2-(2-Methoxyphenethyl)-N4-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine

(27). Yield, 84 %; mp 94.1-96.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.49 (d, J = 10.0 Hz, 2H, CH2), 1.98 (d, J = 11.4 Hz, 2H, CH2), 2.81 (dd, J1 = 6.0 Hz, J2 = 14.0 Hz, 4H, 2×CH2), 2.87 (s, 3H, SO2-CH3), 3.41 (dd, J1 = 6.0 Hz, J2 = 14.0 Hz, 2H, CH2), 3.52 (d, J = 12.0 Hz, 2H, CH2), 3.78 (s, 3H, O-CH3), 5.74 (d, J = 8.0 Hz, 1H, pyrimidine-H), 6.51 (s, 1H, NH), 6.87 (t, J = 8.0 Hz, 1H,

RI PT

330

ArH), 6.95 (d, J = 8.0 Hz, 1H, ArH), 7.18 (dd, J1 = 8.0 Hz, J2 = 17.0 Hz, 2H ArH), 7.65 (d, J = 4.0 Hz, 1H pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 31.2, 34.6, 35.0, 42.9, 44.8, 46.2, 55.4, 114.1, 129.9, 131.7, 132.2, 158.0, 162.3. ESI-MS: m/z = 444.5 [M+K]+, 406.3 [M+1]+. Anal. Calcd. for C19H27N5O3S: C 56.28, H 6.71, N 17.27, S 7.91; Found C 55.92, H 6.53, N 17.01, S 8.19.

.

SC

335

5.1.1.21. N2-(4-Methoxybenzyl)-N4-(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (28).

M AN U

Yield, 62%; mp 42.7-43.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.45 (dd, J1 = 10.8 Hz, J2 = 20.8 Hz, 2H, CH2), 1.85-1.93 (m, 2H, CH2), 2.81-2.88 (m, 2H, CH2), 2.90 (s, 3H, SO2-CH3), 3.51 (d, J = 12.4 Hz, 2H, CH2), 3.72 (s, 3H, O-CH3), 3.90 (s, 1H, CH), 4.40 (d, J = 5.6 Hz, 2H, CH2), 5.90 (d, J 340

= 6.4 Hz, 1H, pyrimidine-H), 6.89 (d, J = 8.6 Hz, 2H, ArH), 7.25 (d, J = 8.6 Hz, 2H, ArH), 7.66 (d, J = 6.2 Hz, 1H, pyrimidine-H), 7.98 (s, 1H, NH).

13

C NMR (100 MHz, DMSO-d6) δ: 30.6, 34.8,

43.8, 44.6, 47.0, 55.5, 97.5, 114.1, 129.0, 131.3, 158.7, 162.0. ESI-MS: m/z = 422.4 [M+Na]+, 392.4 N 18.10, S 7.81. 345

TE D

[M+1]+. Anal. Calcd. for C18H25N5O3S: C 55.22, H 6.44, N 17.89, S 8.19; Found C 55.32, H 6.60,

5.1.1.22. N-(4-Methoxyphenyl)-4-(4-methylpiperazin-1-yl)pyrimidin-2-amine (29). Yield, 73%; mp 132.5-133.8°C; 1H NMR (400 MHz, CDCl3) δ: 2.36 (s, 3H, N-CH3), 2.49 (t, J = 5.2 Hz, 4H,

EP

2×CH2), 3.65 (t, J = 5.0 Hz, 4H, 2×CH2), 3.82 (s, 3H, O-CH3), 6.01 (d, J = 6.4 Hz, 1H, pyrimidine-H), 6.89 (d, J = 9.2 Hz, 2H, ArH), 7.46 (d, J = 9.2 Hz, 2H, ArH), 7.97 (d, J = 6.0 Hz, 1H, 350

AC C

pyrimidine-H). 13C NMR (100 MHz, DMSO-d6) δ: 43.8, 46.1, 54.6, 55.5, 95.0, 114.0, 120.6, 134.8, 154.1, 157.0, 160.0, 162.4. ESI-MS: m/z = 322.7 [M+Na]+, 300.8 [M+1]+. Anal. Calcd. for C16H21N5O: C 64.19, H 7.07, N 23.39; Found C 64.50, H 7.39, N 22.89. 5.1.1.23. N-(4-Chlorophenyl)-4-(4-methylpiperazin-1-yl)pyrimidin-2-amine (30). Yield, 75%; mp 148.6-150.1°C; 1H NMR (400 MHz, CDCl3) δ: 2.37 (s, 3H, N-CH3), 2.50 (t, J = 5.0 Hz, 4H, 2×CH2), 3.67 (t, J = 4.6 Hz, 4H, 2×CH2), 6.06 (d, J = 6.4 Hz, 1H, pyrimidine-H), 7.19 (s, 1H, NH), 355

7.27 (d, J = 8.8 Hz, 2H, ArH), 7.53 (d, J = 8.8 Hz, 2H, ArH), 7.99 (d, J = 6.4 Hz, 1H, pyrimidine-H). C NMR (100 MHz, CDCl3) δ: 29.6, 43.8, 46.1, 54.6, 95.2, 120.4, 126.5, 128.6, 138.7, 156.4,

13

159.3, 162.3. ESI-MS: m/z = 326.6 [M+Na]+, 304.6 [M+1]+. Anal. Calcd. for C15H18ClN5: C 59.30, H 5.97, N 23.05; Found C 59.65, H 6.30, N 22.67.

11

ACCEPTED MANUSCRIPT 5.1.1.24. N-(2-Methoxyphenethyl)-4-(4-methylpiperidin-1-yl)pyrimidin-2-amine (31). Yield, 60%; 360

mp 41.0-41.3°C; 1H NMR (400 MHz, CDCl3) δ: 2.35 (s, 3H, N-CH3), 2.47 (t, J = 5.0 Hz, 2H, CH2), 2.51 (d, J = 3.6 Hz, 2H, CH2), 2.93 (t, J = 7.0 Hz, 2H, CH2), 3.58-3.64 (m, 6H, 3×CH2), 3.85 (s, 3H, O-CH3), 5.32 (s, 1H, NH), 5.87 (d, J = 6.4 Hz, 1H, pyrimidine-H), 6.90 (dd, J = 7.8 Hz, J = 15.4 Hz, 2H, ArH), 7.16-7.24 (m, 2H, ArH), 7.85 (d, J = 8.0 Hz, 1H, pyrimidine-H). 13C NMR (100 MHz, CDCl3) δ: 30.5, 41.5, 43.6, 46.1, 54.6, 55.2, 92.9, 110.3, 120.5, 127.5, 127.9, 130.5, 156.1, 157.6, 161.7, 162.4. ESI-MS: m/z = 350.7 [M+Na]+, 328.7 [M+1]+. Anal. Calcd. for C18H25N5O: C 66.03, H 7.70, N 21.39; Found C 65.61, H 7.42, N 20.92.

370

SC

5.2 Crystallographic analysis

RI PT

365

Colorless blocks of 25 were mounted on a quartz fiber. Cell dimensions and intensities were

mono-chromated Mo Kα radiation

M AN U

measured at 293 K on a Bruker SMART CCD area detector diffractometer with graphite (λ = 0.71073 Å); the unit cell dimensions were determined to

be: a = 7.997, b = 9.5775, c = 12.1841 Å and α = 90.847, β = 95.958, γ = 92.656° in the space group P-1; θmax = 27.47; 9095 measured reflections; 4207 independent reflections (Rint = 0.1156) of which 375

1181 had |Fo| > 2|Fo|. Data were corrected for Lorentz and polarization effects and for absorption

TE D

(Tmin = 0.9801; Tmax = 0.9801). The structure was solved by direct methods using SHELXS-97 [33]; all other calculations were performed with Bruker SAINT System and Bruker SMART programs [34]. Full-matrix least-squares refinement based on F2 using the weight of 1/[σ2(Fo 2) + (0.1605P)2 + 0.0000P] gave final values of R = 0.0728, ωR = 0.3241, and GOF(F) = 0.906 for 237 variables and 4207 contributing reflections. Maximum shift/error

EP

380

0.000(3), max/min residual electron

density = 0.443/-0.644 e Å-3. Hydrogen atoms were observed and refined with a fixed value of their

AC C

isotropic displacement parameter.

Crystallographic data for the structures 25 has been deposited with the Cambridge Crystallographic Data Center (CCDC) under the number 997012. Copies of the data can be 385

obtained, free of charge, on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK [Fax: t44-1223-336033; e-mail: [email protected] or at www.ccdc.cam.ac.uk].

5.3 Pharmacology evaluation 5.3.1 Antitumor activity 390

The antitumor activities of compounds 8-31 were evaluated with HepG2, A549, 12

ACCEPTED MANUSCRIPT MDA-MB-231,

and

MCF-7

cell

lines

by

the

standard

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [32] 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 70-80% confluence, about twice a week by trypsinization.

RI PT

395

Exponentially growing cells were plated in 96-well plates (1×104 cells/well) and incubated at 37 °C for 24 h for attachment. Test compounds were prepared by dissolving in dimethyl sulfoxide (DMSO) at 10 mM and diluted with the medium into a series of concentrations. The culture

400

SC

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 for 48 h. Then the medium was replaced

M AN U

with MTT solution (5 mg/mL, 100 µL) followed by incubation for another 4 h. The medium was then aspirated and formazan crystals were dissolved in DMSO (100 µL) 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: % 405

inhibition = (Abscontrol Abscompound)/Abscontrol×100%. The IC50 values of the test compounds and

TE D

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.).

410

EP

5.3.2 Flow-activating cell sorting analysis (FACS) The effect of compound 28 on cell cycle phase distribution of human breast cancer

AC C

MDA-MB-231 was assessed using flow cytometry. When the cells grew to about 70% confluence in 6-well microplates, they were treated with compound 28 at given concentrations (20, 40, 80 µM). 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 µg/mL final 415

concentration) at 37 °C for 30 min, stained with propidium iodide (50 µg/mL final concentration), and analyzed by flow cytometry (Beckman Coulter).

Acknowledgments This work was supported by National Natural Science Foundation of China (21102069, 420

21372113 and 81101732), and the project of science and technology new star in Zhujiang 13

ACCEPTED MANUSCRIPT Guangzhou city (No. 2012J2200051 and 2013J2200047).

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RI PT

425

albicans: a remarkable reaction between histidine and pyridoxal phosphate, J. Am. Chem. Soc. 134 (2012) 9157-9159

[2] Y.L. Kang, T. Taldone, H.J. Patel, P.D. Patel, A. Rodina, A. Gozman, R. Maharaj, C.C. Clement,

430

SC

M.R. Patel, J.L. Brodsky, J.C. Young, G. Chiosis. Heat shock protein 70 inhibitors. 1. 2,5′-thiodipyrimidine and 5-(Phenylthio)pyrimidine acrylamides as irreversible binders to an

M AN U

allosteric site on Heat shock protein 70, J. Med. Chem. 66 (2014) 1188-1207. [3] B. Kuppast, K. Spyridaki, G. Liapakis, H. Fahmy. Synthesis of substituted pyrimidines as corticotropin releasing factor (CRF) receptor ligands, Eur. J. Med. Chem. 78 (2014) 1-9. [4] J.B. He, L.L. Feng, J. Li, R.J. Tao, Y.L. Ren, J. Wan, H.W. He. Design, synthesis and molecular 435

modeling of novel N-acylhydrazone derivatives as pyruvate dehydrogenase complex E1

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inhibitors, Bioorg. Med. Chem. 22 (2014) 89-94.

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EP

440

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2,4,8,22-tetraazatetracyclo[14.3.1.1³, ⁷ .1 ⁹ ,¹³]docosa-1(20),3(22),4,6,9(21),10,12,16,18-nonaene macrocycles, J. Med. Chem. 55 (2012) 449-464.

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[33] Sheldrick, G. M. (2001). SHELXTL (Version 5.0). University of Gőttingen: Germany, 2001. [34] Bruker SMART V5.628, SAINT V6.45. & SADABS Bruker AXS Inc., Madison, Wisconsin, USA, 2001.

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Figure captions

545

Fig. 2. Design strategy of the title compounds 8-31. 550

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Fig. 3. Molecular structure of compound 25.

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Fig. 4. SAR of compounds 8-31.

555

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

Fig. 5. Effect of compound 28 on cell cycle and apoptosis in MDA-MB-231 cells. Flow cytometry analysis of MDA-MB-231 cells treated with 28 for 48 h. (A) Control; (B) 28, 20 µM; (C) 28, 40 µM;

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560

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(D) 28, 80 µM

570

18

ACCEPTED MANUSCRIPT 575

Table 1 Cytotoxic Activities of Compounds 8-31 against human tumor cells.

N H

N

N H

n R2

N N

8-20

R1

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

0 0 1

H H H H Cl Cl Cl Cl Cl Cl Cl Cl Cl H H H H H H H H H H H

0 0 0 0 2 1 0 0 2

H

24-28

R2 4-ClC6H4 3-MeOC6H4 4-MeOC6H4 C 6H 5 4-MeOC6H4 3 - MeOC6H4 4-ClC6H4 4-MeOC6H4 C 6H 5 C 6H 5 4-ClC6H4 4-MeOC6H4 C 6H 5 C 6H 5 4-MeOC6H4 3 - MeOC6H4 4-MeOC6H4 3-MeOC6H4 4-ClC6H4 4-MeOC6H4 4-MeOC6H4 4-MeOC6H4 4-ClC6H4 2-MeOC6H4 fluorouracil

N H

N

HepG2 78.61 86.63 51.74 148.59 >200 >200 >200 >200 >200 >200 >200 >200 123.13 >200 >200 >200 86.19 67.94 92.62 19.98 20.19 >200 >200 >200 46.83

R1

N R2

nN

H

N

N N

29-31

In vitro cytotoxicity IC50(µM) a A549 MDA-MB-231 >200 >200 >200 29.44 51.6 33.69 42.52 60.76 60.93 34.08 >200 >200 >200 >200 >200 >200 46.80 11.73 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 91.65 57.74 >200 108.74 >200 102.48 21.41 15.83 12.78 7.46 >200 >200 >200 >200 >200 >200 35.41 24.60

MCF-7 >200 77.81 57.16 98.55 64.83 >200 >200 >200 32.41 >200 >200 >200 >200 >200 >200 >200 >200 72.13 >200 32.76 16.24 >200 >200 >200 39.88

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



N H

O S N O

R1

N N H

N

N H

N n R2

n R2

21-23 R1

N nN

H

N

N

RI PT

R2

n=1> 2>0

N H

>>

8-20

24-28

N

N

29-31

n=1> 2>0 R 1 = H > Cl (except 16)

Fig. 4. SAR of the compounds 8-31

B

AC C

EP

A

TE D

M AN U

SC

595

600 C

D

Fig. 5. Effect of compound 28 on cell cycle and apoptosis in MDA-MB-231 cells. Flow cytometry analysis of MDA-MB-231 cells treated with 28 for 48 h. (A) Control; (B) 28, 20 µM; (C) 28, 40 µM; (D) 28, 80 µM.

21

ACCEPTED MANUSCRIPT 605

R1

N

+ N

N

N H

R2

90oC 7

6

R1

N R3

N H

N

N H

R2

8-31

RI PT

5

2-methoxyethanol Cl r.t.

Cl

R 3 NH2

EP

TE D

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Scheme 1 One-pot synthesis of compounds 8-31

AC C

Cl

R 2 NH2

R1

N

DIPEA

22

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Highlights

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► 24 novel 2,4-diaminopyrimidines were synthesized via a one-pot reaction. ► Antiproliferative activity of these compounds was evaluated. ► Two compounds displayed much stronger antitumor activity than Fluorouracil. ►Compound 28

AC C

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displayed a significant effect on G2/M cell-cycle arrest in MDA-MB-231.

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One-pot synthesis and antiproliferative activity of novel 2,4-diaminopyrimidine derivatives bearing piperidine and

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piperazine moieties Wei-Feng Mab,#, Hai-Kui Yanga,#, Meng-Jin Hua, Qian Lia, Tian-Zhu Maa, Zhong-Zhen Zhoua, Rui-Yuan Liua, Wen-Wei Youa, *, Pei-Liang Zhaoa, * a

Department of Chemistry, School of Pharmaceutical Science, Southern Medical

Department of Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R.China

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b

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University, Guangzhou 510515, P.R.China

Supporting Information No.

List of Figures

Page

No.

List of Figures

Page

S3

25

1

S4

26

13

C NMR Spectrum of Compound 20

S28

S5

27

1

H NMR Spectrum of Compound 21

S29

C NMR Spectrum of Compound 9

S6

28

13

C NMR Spectrum of Compound 21

S30

H NMR Spectrum of Compound 10

S7

29

1

H NMR Spectrum of Compound 22

S31

S8

30

13

C NMR Spectrum of Compound 22

S32

S9

31

1

H NMR Spectrum of Compound 23

S33

1

1

2

13

3

1

4

13

5

1

6

13

7

1

8

13

C NMR Spectrum of Compound 11

S10

32

13

C NMR Spectrum of Compound 23

S34

1

H NMR Spectrum of Compound 12

S11

33

1

H NMR Spectrum of Compound 24

S35

13

C NMR Spectrum of Compound 12

S12

34

13

C NMR Spectrum of Compound 24

S36

1

H NMR Spectrum of Compound 13

S13

35

1

H NMR Spectrum of Compound 25

S37

10 11

TE D

C NMR Spectrum of Compound 8

EP

H NMR Spectrum of Compound 9

C NMR Spectrum of Compound 10

H NMR Spectrum of Compound 11

AC C

9

H NMR Spectrum of Compound 8

H NMR Spectrum of Compound 20

S27

12

13

C NMR Spectrum of Compound 13

S14

36

13

C NMR Spectrum of Compound 25

S38

13

1

H NMR Spectrum of Compound 14

S15

37

1

H NMR Spectrum of Compound 26

S39

14

13

C NMR Spectrum of Compound 14

S16

38

13

C NMR Spectrum of Compound 26

S40

15

1

H NMR Spectrum of Compound 15

S17

39

1

H NMR Spectrum of Compound 27

S41

S1

ACCEPTED MANUSCRIPT 16

13

C NMR Spectrum of Compound 15

S18

40

13

C NMR Spectrum of Compound 27

S42

17

1

H NMR Spectrum of Compound 16

S19

41

1

H NMR Spectrum of Compound 28

S43

18

13

C NMR Spectrum of Compound 6

S20

42

13

C NMR Spectrum of Compound 28

S44

19

1

H NMR Spectrum of Compound 17

S21

43

1

H NMR Spectrum of Compound 29

S45

20

13

C NMR Spectrum of Compound 17

S22

44

13

21

1

H NMR Spectrum of Compound 18

S23

45

1

22

13

C NMR Spectrum of Compound 18

S24

46

13

23

1

H NMR Spectrum of Compound 19

S25

47

1

24

13

S26

48

13

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H NMR Spectrum of Compound 30

S47 S48

H NMR Spectrum of Compound 31

S49

C NMR Spectrum of Compound 31

M AN U TE D EP AC C S2

S46

C NMR Spectrum of Compound 30

SC

C NMR Spectrum of Compound 19

C NMR Spectrum of Compound 29

S50

N N H

N

N H

M AN U

S N O

SC

Cl

O

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AC C

EP

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8

Figure S1. 1H NMR spectrum (400 MHz, CDCl3) of compound 8.

S3

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Cl

S N O

N N H

N

N H

AC C

EP

TE D

M AN U

8

SC

O

Figure S2. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 8.

S4

O N N H

N

AC C

EP

TE D

9

O

N H

M AN U

S N O

SC

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Figure S3. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 9.

S5

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S N O

N N H

N H

N

O

AC C

EP

TE D

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9

SC

O

Figure S4. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 9.

S6

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N N H

N H

N

O

AC C

EP

TE D

10

M AN U

S N O

SC

O

Figure S5. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 10.

S7

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ACCEPTED MANUSCRIPT

O N H

N

N H

O

AC C

EP

TE D

M AN U

10

SC

N

S N O

Figure S6. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 10.

S8

O N

S N O

N H

N

M AN U

N H

SC

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ACCEPTED MANUSCRIPT

AC C

EP

TE D

11

Figure S7. 1H NMR spectrum (400 MHz, CDCl3) of compound 11.

S9

O S N O

N N

N H

AC C

EP

TE D

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11

N H

SC

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Figure S8. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 11.

S10

N

N H

SC

Cl

N

O

AC C

EP

TE D

12

N H

M AN U

O S N O

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Figure S9. 1H NMR spectrum (400 MHz, CDCl3) of compound 12.

S11

O S N O

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Cl

N H

SC

N N

N H

O

AC C

EP

TE D

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12

Figure S10. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 12.

S12

N H

N

AC C

EP

TE D

13

O

N H

SC

Cl

N

M AN U

O S N O

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Figure S11. 1H NMR spectrum (400 MHz, CDCl3) of compound 13.

S13

N H

N

SC

Cl

N

N H

O

M AN U

O S N O

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AC C

EP

TE D

13

Figure S12. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 13.

S14

N H

Cl

Cl

N N

SC

O S N O

RI PT

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N H

AC C

EP

TE D

M AN U

14

Figure S13. 1H NMR spectrum (400 MHz, CDCl3) of compound 14.

S15

Cl

N N

N H

AC C

EP

TE D

M AN U

14

N H

Cl

SC

O S N O

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Figure S14. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 14.

S16

Cl

N N H

N H

N

AC C

EP

TE D

15

O

M AN U

O S N O

SC

RI PT

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Figure S15. 1H NMR spectrum (400 MHz, CDCl3) of compound 15.

S17

N N H

Cl

O

SC

O S N O

RI PT

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N

N H

AC C

EP

TE D

M AN U

15

Figure S16. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 15.

S18

N H

N

N H

SC

Cl

N

M AN U

O S N O

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ACCEPTED MANUSCRIPT

AC C

EP

TE D

16

Figure S17. 1H NMR spectrum (400 MHz, CDCl3) of compound 16.

S19

Cl

N N H

N

N H

AC C

EP

TE D

M AN U

16

SC

O S N O

RI PT

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Figure S18. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 16.

S20

Cl

N

N H

N

M AN U

N H

SC

O S N O

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AC C

EP

TE D

17

Figure S19. 1H NMR spectrum (400 MHz, CDCl3) of compound 17.

S21

Cl

N

SC

O S N O

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N H

N

N H

AC C

EP

TE D

M AN U

17

Figure S20. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 17.

S22

Cl N

AC C

EP

TE D

18

N H

M AN U

N N H

SC

Cl

O S N O

RI PT

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Figure S21. 1H NMR spectrum (400 MHz, CDCl3) of compound 18.

S23

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Cl

Cl

N N

N H

M AN U

N H

SC

O S N O

AC C

EP

TE D

18

Figure S22. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 18.

S24

O

N H

N

N H

AC C

EP

TE D

19

SC

Cl

N

M AN U

O S N O

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Figure S23. 1H NMR spectrum (400 MHz, CDCl3) of compound 19.

S25

O

SC

Cl

N

N H

N

M AN U

O S N O

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N H

AC C

EP

TE D

19

Figure S24. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 19.

S26

O S N O

Cl

N N

N H

M AN U

N H

SC

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ACCEPTED MANUSCRIPT

AC C

EP

TE D

20

Figure S25. 1H NMR spectrum (400 MHz, CDCl3) of compound 20.

S27

Cl

N N H

N

N H

AC C

EP

TE D

M AN U

20

SC

O S N O

RI PT

ACCEPTED MANUSCRIPT

Figure S26. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 20.

S28

N

N

N

N H

M AN U

N

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

21

Figure S27. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 21.

S29

N

N

N

N H

M AN U

N

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

21

Figure S28. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 21.

S30

SC

RI PT

ACCEPTED MANUSCRIPT

N N

N

O

AC C

EP

TE D

22

N H

M AN U

N

Figure S29. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 22.

S31

N N

N

O

AC C

EP

TE D

M AN U

22

N H

SC

N

RI PT

ACCEPTED MANUSCRIPT

Figure S30. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 22.

S32

N

N

N

N H

O

M AN U

N

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

23

Figure S31. 1H NMR spectrum (400 MHz, CDCl3) of compound 23.

S33

N N

N H

N

O

M AN U

N

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

23

Figure S32. 13C NMR spectrum (100 MHz, CDCl3) of compound 23.

S34

N N H

N

M AN U

N H

SC

O S N O

O

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

24

Figure S33. 1H NMR spectrum (400 MHz, CDCl3) of compound 24.

S35

RI PT

ACCEPTED MANUSCRIPT

SC

O S N O

O N

N

N H

M AN U

N H

AC C

EP

TE D

24

Figure S34. 13C NMR spectrum (100 MHz, CDCl3) of compound 24.

S36

SC

O S N O

N N H

N

AC C

EP

TE D

25

N H

M AN U

O

RI PT

ACCEPTED MANUSCRIPT

Figure S35. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 25.

S37

SC

RI PT

ACCEPTED MANUSCRIPT

O

M AN U

N N H

N

O S N O

N H

AC C

EP

TE D

25

Figure S36. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 25.

S38

N N H

N

M AN U

N H

SC

O S N O

Cl

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

26

Figure S37. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 26.

S39

RI PT

ACCEPTED MANUSCRIPT

N N

N H

M AN U

N H

SC

O S N O

Cl

AC C

EP

TE D

26

Figure S38. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 26.

S40

N N H

N

N H

AC C

EP

TE D

27

M AN U

O S N O

O

SC

RI PT

ACCEPTED MANUSCRIPT

Figure S39. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 27.

S41

RI PT

ACCEPTED MANUSCRIPT

O S N O

N

N

N H

M AN U

N H

SC

O

AC C

EP

TE D

27

Figure S40. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 27.

S42

N N

AC C

EP

TE D

28

N H

M AN U

N H

O

SC

O S N O

RI PT

ACCEPTED MANUSCRIPT

Figure S41. 1H NMR spectrum (400 MHz, DMSO-d6) of compound 28.

S43

RI PT

ACCEPTED MANUSCRIPT

O S N O

N H

O

SC

N N

N H

AC C

EP

TE D

M AN U

28

Figure S42. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 28.

S44

O N N

N

M AN U

N H

SC

RI PT

ACCEPTED MANUSCRIPT

N

AC C

EP

TE D

29

Figure S43. 1H NMR spectrum (400 MHz, CDCl3) of compound 29.

S45

SC

RI PT

ACCEPTED MANUSCRIPT

O

M AN U

N N

N H

N

N

AC C

EP

TE D

29

Figure S44. 13C NMR spectrum (100 MHz, DMSO-d6) of compound 29.

S46

SC

RI PT

ACCEPTED MANUSCRIPT

Cl

N H

M AN U

N N

N

N

AC C

EP

TE D

30

Figure S45. 1H NMR spectrum (400 MHz, CDCl3) of compound 30.

S47

Cl N N

N N

AC C

EP

TE D

30

M AN U

N H

SC

RI PT

ACCEPTED MANUSCRIPT

Figure S46. 13C NMR spectrum (100 MHz, CDCl3) of compound 30.

S48

O N N H

N

M AN U

N

SC

RI PT

ACCEPTED MANUSCRIPT

N

AC C

EP

TE D

31

Figure S47. 1H NMR spectrum (400 MHz, CDCl3) of compound 31.

S49

SC

RI PT

ACCEPTED MANUSCRIPT

O N H

N

M AN U

N N

N

AC C

EP

TE D

31

Figure S48. 13C NMR spectrum (100 MHz, CDCl3) of compound 31.

S50