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

Article abstract of DOI:10.1016/j.ejmech.2014.07.017

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 cell lines (HepG2, A549, MDA-MB-231 and MCF-7) 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. Further flow-activated cell sorting analysis revealed that the most promising compound 28 displayed a significant effect on G ₂/M cell-cycle arrest in a dose-dependent manner in MDA-MB-231 cells.

Full text of DOI:10.1016/j.ejmech.2014.07.017

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
10.1016/j.ejmech.2014.07.017
EJMECH 7137
DOI:
Reference:
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.
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ACCEPTED MANUSCRIPT
One-pot synthesis and antiproliferative activity of novel
,4-diaminopyrimidine derivatives bearing piperidine and piperazine
moieties
2
b,#
a,#
a
a
a
a
Wei-Feng Ma , Hai-Kui Yang , Meng-Jin Hu , Qian Li , Tian-Zhu Ma , Zhong-Zhen Zhou ,
a,
a,
Wen-Wei You *, Pei-Liang Zhao *
O
S
O
S
O
N
N
N
N
O
O
N
N
N
N
N
N
H
H
H
H
O
2
7
28
Anticancer activities against HepG2, A549, MDA-MB-231 and MCF-7, IC50
2
2
7 = 19.98, 21.41, 15.83, 32.76 µM
8 = 20.19, 12.78, 7.46, 16.24 µM
fluorouracil = 46.83, 35.41, 24.60, 39.88 µM

ACCEPTED MANUSCRIPT
One-pot synthesis and antiproliferative activity of novel
,4-diaminopyrimidine derivatives bearing piperidine and piperazine
moieties
2
b,#
a,#
a
a
a
a
5
Wei-Feng Ma , Hai-Kui Yang , Meng-Jin Hu , Qian Li , Tian-Zhu Ma , Zhong-Zhen Zhou ,
a
a,
a,
Rui-Yuan Liu , Wen-Wei You *, Pei-Liang Zhao *
Department of Chemistry, School of Pharmaceutical Science, Southern Medical University,
Guangzhou 510515, P.R.China
a
b
Department of Microbiology, School of Public Health and Tropical Medicine, Southern
1
0
Medical University, Guangzhou 510515, P.R.China
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
cell lines (HepG2, A549, MDA-MB-231 and MCF-7) 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 IC values of 7.46 and 12.78 ꢀM, respectively.
1
2
5
0
5
0
Further flow-activated cell sorting analysis revealed that the most promising compound 28
displayed a significant effect on G /M cell-cycle arrest in a dose-dependent manner in
2
MDA-MB-231 cells.
2
3
5
0
Keywords: 2,4-Diaminopyrimidines; Piperidine; Piperazine; Antiproliferative activity; One-pot
synthesis
∗—
—————————
Corresponding author. Tel./fax: +86(0)20 61648196.
∗
Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao), youww@smu.edu.cn (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
3
4
4
5
5
6
5
0
5
0
5
0
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
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
agent inhibiting ataxia telangiectasia mutated and Rad3-related kinase (ATR) [18]. On the other
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
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
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
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
[24], which indicated that the flexibility of the amino group might be a vital factor for the activity of
compound 4.
<Insert figure 1 here>
Based on the above considerations, and in a continuation of our interest in the synthesis of
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
-31.
8
<Insert figure 2 here>
2

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2. Chemistry
6
7
7
8
5
0
5
0
Owing to their important biological roles in chemopreventive action and the broad range of
pharmacological properties, many methods have been developed for synthesis of
,4-diaminopyrimidine derivatives [14, 28-31]. All of the methodology involved two steps from the
2
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
C-2 chlorine was displaced by various amines in the second step. Although, many efforts were
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
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
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 N flow. The resulting mixture reacted with 1.1 equiv of various another amine
2
(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
1
13
H NMR, C NMR, ESI-MS and elemental analysis and the results are shown in the Experimental
section. In addition, the representative compound 25 was further confirmed by single-crystal X-ray
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.
8
5
<Insert scheme 1 here>
<Insert figure 3 here>
3. Pharmacology Results and Discussion
9
0
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 IC (ꢀM) were summarized in
5
0
9
5
Table 1. Here, the IC value represents the concentration of one compound resulting in a 50%
50
inhibition in cell growth after a 48 h incubation, and is the average of three independent
experiments.
<Insert table 1 here>
100
<Insert figure 4 here>
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
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
105
(
IC50 > 200 ꢀM). Remarkably, most of the C2- and C4-piperidine pyrimidines (8-20, 24-28)
displayed substantial antiproliferative activities. For example, the IC values of compounds 27 and
5
0
2
1
8 ranged from 7.46 to 32.76 ꢀM against all of the tested cell lines. The IC values of compound
50
6 were 11.73, 32.41 and 46.80 ꢀM against MDA-MB-231, MCF-7 and A549 cells, respectively. In
110
115
120
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
1
2
1
2
and 25 (n = 0, R = H, R = 3-MeOC H ), 10 and 28 (n = 1, R = H, R = 4-MeOC H CH ).
6
4
6
4
2
Further analysis on the structure–activity relationship investigated the effects of several
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
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
increase their cytotoxicity. Furthermore, these two compounds have a CH or CH CH bridge
125
2
2
2
between the aromatic moiety and the pyrimidine ring. After removing the linkage, the
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
sorting analysis was performed. The most promising compound 28 was tested against
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
increased from 18.12% to 33.79% (20 ꢀM), 36.51% (40 ꢀM), and 40.95% (80 ꢀM) after 48 h of
130
treatment. These data suggest that compound 28 induced significant cell cycle arrest in the G /M
2
1
35
40
phase in a dose-dependent manner, compared to untreated cells
<Insert figure 5 here>
<Insert table 2 here>
1
4. Conclusion
In conclusion, the incorporation of piperidine or piperazine group at C2 or C4 of the
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
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 IC values of 7.46 and 12.78 ꢀM, respectively.
145
5
0
These results indicated that this novel class of 2,4-diaminopyrimidine analogs may find their
medicinal applications after further structural modulation and biological studies.
150
5. Experimental protocols
5.1 Chemistry
5

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1
13
H NMR and C NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3
155
160
165
or DMSO-d solution and chemical shifts were recorded in parts per million (ppm) with TMS as the
6
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
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
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,
-(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
1
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 N flow. Then another amino compound 7 (1.1
2
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,
(60:1) as an eluent to give the target compounds in yields of 58-84%.
4
2
1
70
75
5.1.1.1. N -(4-Chlorophenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (8).
1
Yield, 61%; mp 169.7-171.1°C; H NMR (400 MHz , CDCl ) δ: 1.72 (dd, J = 10.4 Hz, J = 19.8 Hz,
3
1
2
2
2
7
H CH ), 2.16 (dd, J = 4.0 Hz, J = 8.0 Hz, 2H, CH ), 2.85 (s, 3H, SO -CH ), 3.06 (t, J = 11.8 Hz,
2 1 2 2 2 3
H, CH ), 3.70 (d, J = 12.0 Hz, 2H, CH ), 4.02 (s, 1H, CH), 6.13 (d, J = 5.9 Hz, 1H, pyrimidine-H),
2
2
.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,
1
3
1
pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.4, 34.6, 45.1, 47.5, 120.9, 125.3, 128.8,
6
+
+
1
40.0, 156.5, 160.8, 161.4. ESI-MS: m/z = 404.5 [M+Na] , 382.6 [M+1] . Anal. Calcd. for
C H ClN O S: 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.
16
20
5
2
4
2
5
.1.1.2. N -(3-Methoxyphenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (9).
1
Yield, 72%; mp 175.4-176.5°C; H NMR (400 MHz , DMSO-d ) δ: 1.56 (dd, J = 10.6 Hz , J =
6
1
2
1
80
85
20.4 Hz, 2H, CH ), 1.99 (d, J = 11.6 Hz, 2H, CH ), 2.83 (d, J = 11.2 Hz, 2H, CH ), 2.89 (s, 3H,
2 2 2
SO -CH ), 3.57 (d, J = 12.0 Hz, 2H, CH ), 3.75 (s, 3H, O-CH ), 3.84 (m, 1H, CH), 6.01 (d, J = 5.6
2
3
2
3
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,
1
3
NH). C NMR (100 MHz, DMSO-d ) δ: 31.3, 34.6, 45.1, 47.4, 55.4, 105.8, 106.9, 112.1, 129.7,
6
+
+
1
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
C H N O S: 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.
17
23
5
3
4
2
5
.1.1.3. N -(4-Methoxybenzyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (10).
1
Yield, 66%; mp 94.9-96.0°C; H NMR (400 MHz, DMSO-d ) δ: 1.48 (dd, J = 9.8 Hz, J = 20.4 Hz,
6
1
2
2
H, CH ), 1.87 (d, J = 11.0 Hz, 2H, CH ), 2.80 (t, J = 10.8 Hz, 2H, CH ), 2.86 (s, 3H, SO -CH ),
2 2 2 2 3
1
90
95
3.49 (d, J = 11.6 Hz, 2H, CH ), 3.73 (s, 3H, O-CH ), 3.76 (m, 1H, CH), 4.38(s, 2H, CH ), 5.75(d, J
2 3 2
=
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,
1
3
2
H, ArH), 7.34 (s, 1H, NH), 7.64 (d, J = 5.6 Hz, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ:
6
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 C H N O S: C 55.22, H 6.44, N
1
8
25
5
3
1
17.89, S 8.19; Found C 55.33, H 6.73, N 17.26, S 8.22.
4
2
5
.1.1.4. N -Benzyl-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (11). Yield, 63%;
1
mp 127.0-128.3°C; H NMR (400 MHz, CDCl ) δ: 1.60 (dd, J = 10.0 Hz, J = 20.8 Hz, 2H, CH ),
3
1
2
2
2
9
.11 (d, J = 12.0 Hz, 2H, CH ), 2.80 (s, 3H, SO -CH ), 2.91 (t, J = 10.8 Hz, 2H, CH ), 3.70 (d, J =
2 2 3 2
.6 Hz, 2H, CH ), 3.92 (m, 1H, CH), 4.52 (s, 2H, CH ), 4.97 (s, 1H, NH), 5.10 (s, 1H, NH), 5.77 (s,
2
2
1
3
200
1H, pyrimidine-H), 7.28-7.34 (m , 5H, ArH), 7.83 (s, 1H, pyrimidine-H). C NMR (100 MHz,
DMSO-d ) 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:
6
+
+
m/z = 384.6 [M+Na] , 362.6 [M+1] . Anal. Calcd. for C H N O S: C 56.49, H 6.41, N 19.38, S
1
7
23
5
2
8.87; Found C 56.78, H 6.64, N 19.12, S 8.85.
4
2
5.1.1.5.
5-Chloro-N -(4-methoxybenzyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-
1
2
05
10
diamine (12). Yield, 61%; mp 127.0-128.3°C; H NMR (400 MHz, CDCl ) δ: 1.60 (dd, J = 10.0
3
1
Hz, J = 20.4 Hz, 2H, CH ), 2.12 (d, J = 11.6 Hz, 2H, CH ), 2.81 (s, 3H, SO -CH ), 2.91 (t, J = 11.2
2
2
2
2
3
Hz, 2H, CH ), 3.72 (d, J = 12.0 Hz, 2H, CH ), 3.83 (s, 3H, O-CH ), 3.87-3.89 (m, 1H, CH), 4.58 (d,
2
2
3
J = 5.2 Hz, 2H, CH ), 4.90 (s, 1H, NH), 5.48 (s, 1H, NH), 6.91 (d, J = 8.0 Hz, 2H, ArH), 7.28 (d, J
2
1
3
=
4.4 Hz, 2H, ArH), 7.82 (s, 1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.6, 43.2,
6
+
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] ,
+
4
26.6 [M+1] . Anal. Calcd. for C H ClN O S: C 50.76, H 5.68, N 16.44, S 7.53; Found C 51.03,
1
8
24
5
3
H 5.50, N 16.23, S 7.35.
4
2
5.1.1.6.
5-Chloro-N -(3-methoxyphenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-
1
diamine (13). Yield, 67%; mp 205.0-205.3°C; H NMR (400 MHz, CDCl ) δ: 1.65 (dd, J = 10.2
3
1
2
15
20
Hz, J = 20.2 Hz, 2H, CH ), 2.19 (d, J = 11.4 Hz, 2H, CH ), 2.83 (s, 3H, SO -CH ), 2.97 (t, J = 11.4
2 2 2 2 3
Hz, 2H, CH ), 3.76 (d, J = 12.0 Hz, 2H, CH ), 3.85 (s, 3H, O-CH ), 3.92-3.93 (m, 1H, CH), 5.21 (s,
2
2
3
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,
1
3
ArH), 7.42 (s, 1H, NH), 7.96 (s, 1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.5,
6
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
+
+
2
[M+Na] , 412.4 [M+1] . Anal. Calcd. for C H ClN O S: C 50.76, H 5.68, N 16.44, S 7.53; Found
1
8
24
5
3
7

ACCEPTED MANUSCRIPT
C 51.05, H 6.03, N 15.95, S 7.43.
4
2
5
.1.1.7. 5-Chloro-N -(4-chlorophenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine
1
(
14). Yield, 75%; mp 201.2-201.5°C; H NMR (400 MHz, CDCl ) δ: 1.65 (dd, J = 10.4 Hz, J =
3
1
2
2
0.4 Hz, 2H, CH ), 2.17 (d, J = 16.8 Hz, 2H, CH ), 2.84 (s, 3H, SO -CH ), 2.93 (t, J = 11.2 Hz, 2H,
2 2 2 3
225
CH ), 3.77 (d, J = 12.0 Hz, 2H, CH ), 3.87-3.89 (m, 1H, CH), 5.14 (s, 1H, NH), 7.04 (s, 1H, NH),
2 2
13
7
.34 (d, J = 8.0 Hz, 2H, ArH), 7.56 (d, J = 8.0 Hz, 2H, ArH), 7.97 (s, 1H, pyrimidine-H). C NMR
(
100 MHz, DMSO-d ) δ: 31.2, 34.7, 45.0, 47.8, 123.7, 127.2, 128.5, 138.6, 155.8, 159.8. ESI-MS:
6
+
+
m/z = 438.3 [M+Na] , 416.4 [M+1] . Anal. Calcd. for C H Cl N O S: C 46.16, H 4.60, N 16.82, S
1
6
19
2
5
2
7.70; Found C 45.96, H 4.24, N 16.37, S 7.28.
4
2
2
30
35
5.1.1.8.
5-Chloro-N -(4-methoxyphenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-
1
diamine (15). Yield, 76%; mp 183.2-185.0°C; H NMR (400 MHz, CDCl ) δ: 1.60 (dd, J = 10.4
3
1
Hz, J = 20.0 Hz, 2H, CH ), 2.13 (d, J = 10.0 Hz, 2H, CH ), 2.81 (s, 3H, SO -CH ), 2.89 (t, J = 10.4
2
2
2
2
3
Hz, 2H, CH ), 3.73 (d, J = 12.0 Hz, 2H, CH ), 3.84 (s, 4H, O-CH , CH), 5.03 (s, 1H, NH), 6.91 (d,
2
2
3
1
3
J = 9.0 Hz, 3H, ArH, NH), 7.46 (d, J = 9.0 Hz, 2H, ArH), 7.92 (s, 1H, pyrimidine-H). C NMR
2
(100 MHz, DMSO-d ) δ: 31.2, 34.6, 45.1, 47.8, 55.6, 113.9, 124.4, 132.3, 155.0, 155.9, 156.2,
6
+
+
1
59.9. ESI-MS: m/z = 434.4 [M+Na] , 412.5 [M+1] . Anal. Calcd. for C H ClN O S : C 49.57, H
17 22 5 3
5.38, N 17.00, S 7.78; Found C 49.18, H 5.69, N 16.72, S 7.95.
4
2
5
.1.1.9. 5-Chloro-N -benzyl-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (16).
1
Yield, 59%; mp 149.8-151.0°C; H NMR (400 MHz, CDCl ) δ: 1.46-1.53 (m, 2H, CH ), 1.98 (d, J
3
2
2
40
45
= 12.0 Hz, 2H, CH ), 2.71 (s, 3H, SO -CH ), 2.74-2.81 (m, 2H, CH ), 3.60 (d, J = 12.2 Hz, 2H,
2 2 3 2
CH ), 3.74 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H, CH), 4.57 (d, J = 8.0 Hz, 2H, CH ), 4.87 (s, 1H, NH),
2
1
2
2
1
3
5
.50 (s, 1H, NH), 7.19-7.28 (m, 5H, ArH), 7.74 (s, 1H, pyrimidine-H). C NMR (100 MHz,
DMSO-d ) 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:
6
+
+
m/z = 418.4 [M+Na] , 396.5 [M+1] . Anal. Calcd. for C H ClN O S : C 51.57, H 5.60, N 17.69, S
1
7
22
5
2
2
8.10; Found C 52.08, H 5.91, N 17.14, S 8.92.
2
4
5
.1.1.10. 5-Chloro-N -(1-(methylsulfonyl)piperidin-4-yl)-N -phenylpyrimidine-2,4-diamine (17).
1
Yield, 70%; mp 188.6-189.5°C; H NMR (400 MHz, CDCl ) δ: 1.62-1.70 (m, 2H, CH ), 2.13-2.20
3
2
(
m, 2H, CH ), 2.83 (s, 3H, SO -CH ), 2.96 (t, J = 10.4 Hz, 2H, CH ), 3.73 (d, J = 12.0 Hz, 2H,
2 2 3 2
CH ), 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),
2
1
3
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). C NMR
(
100 MHz, DMSO-d ) δ: 31.2, 34.6, 45.0, 47.8, 122.3, 123.6, 128.7, 139.5, 155.2, 156.0, 159.9.
6
+
+
ESI-MS: m/z = 404.4 [M+Na] , 382.5 [M+1] . Anal. Calcd. for C H ClN O S: C 50.32, H 5.28, N
16
20
5
2
18.34, S 8.40; Found C 50.59, H 5.17, N 18.53, S 8.11.
4
2
5.1.1.11.
5-Chloro-N -(4-chlorophenethyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-
8

ACCEPTED MANUSCRIPT
1
2
55
60
diamine (18). Yield, 68%; mp 171.9-172.5°C; H NMR (400 MHz, CDCl ) δ: 1.62-1.70 (m, 2H,
3
CH ), 2.14-2.19 (m, 2H, CH ), 2.83 (s, 3H, SO -CH ), 2.91 (t, J = 7.0 Hz, 2H, CH ), 2.96 (t, J =
2
2
2
3
2
1
1
0.0 Hz, 2H, CH ), 3.66-3.75 (m, 4H, 2×CH ), 3.88-3.95 (m, 1H, CH), 5.10 (s, 1H, NH), 5.29 (s,
2 2
H, 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,
1
3
pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.5, 34.8, 41.7, 45.0, 47.4, 128.6, 130.9,
6
+
+
2
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
C H Cl N O S: 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.
18
23
2
5
2
4
2
5
.1.1.12. 5-Chloro-N -(4-methoxyphenethyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-
1
diamine (19). Yield, 77%; mp 157.5-159.8°C; H NMR (400 MHz, CDCl ) δ: 1.67 (dd, J = 3.6 Hz,
3
1
J = 10.2 Hz, 2H, CH ), 2.16 (dd, J = 4.0 Hz, J = 12.0 Hz, 2H, CH ), 2.83 (s, 3H, SO -CH ), 2.87
2
2
1
2
2
2
3
2
65
70
(t, J = 8.0 Hz, 2H, CH ), 2.99 (t, J = 10.2 Hz, 2H, CH ), 3.53-3.77 (m, 4H, 2×CH ), 3.82 (s, 3H,
2 2 2
O-CH ), 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
3
1
3
Hz, 2H, ArH), 7.77 (s, 1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.5, 42.3, 45.0,
6
+
4
7.5, 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 C H ClN O S: C 51.87, H 5.96, N 15.92, S 7.29; Found C 52.55, H 6.19,
19
26
5
3
2
N 15.53, S 7.82.
2
4
5
.1.1.13. 5-Chloro-N -(1-(methylsulfonyl)piperidin-4-yl)-N -phenethylpyrimidine-2,4-diamine (20).
1
Yield, 81% ; mp 162.4-163.9°C; H NMR (400 MHz, CDCl ) δ: 1.63-1.71 (m, 2H, CH ), 2.14-2.18
3
2
(
m, 2H, CH ), 2.82 (s, 3H, SO -CH ), 2.87-3.01 (m, 4H, 2×CH ), 3.72 (dd, J = 6.8 Hz, J = 13.2
2 2 3 2 1 2
Hz, 4H, 2×CH ), 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
2
1
3
275
(m, 2H, ArH), 7.35 (t, J = 7.2 Hz, 2H, ArH), 7.78 (s, 1H, pyrimidine-H). C NMR (100 MHz,
DMSO-d ) 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.
6
+
+
ESI-MS: m/z = 432.3 [M+Na] , 410.4 [M+1] . Anal. Calcd. for C H ClN O S:C 52.74, H 5.90, N
1
8
24
5
2
17.08, S 7.82; Found C 52.84, H 4.56, N 17.27, S 7.69.
5.1.1.14. N-Benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (21). Yield, 83%; mp
1
2
80
85
149.0-150.1°C; H NMR (400 MHz, DMSO-d ) δ: 2.18 (s, 3H, N-CH ), 2.23-2.32 (m, 4H, 2×CH ),
6
3
2
3
7
.56-3.67 (m, 4H, 2×CH ), 4.45 (d, J = 4.2 Hz, 2H, CH ), 5.79 (d, J = 5.6 Hz, 1H, pyrimidine-H),
2 2
.23 (dd, J = 4.2 Hz, J = 8.0 Hz, 1H, ArH), 7.31 (d, J = 4.4 Hz, 4H, ArH), 7.50 (s, 1H, NH), 7.72
1
2
1
3
(
d, J = 4.0 Hz, 1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 43.6, 43.7, 46.3, 54.9, 96.1,
6
+
1
27.0, 127.8, 128.6, 140.6, 155.3, 161.6, 162.7, 172.4. ESI-MS: m/z = 307.2 [M+Na] , 285.2
+
2
[M+1] . Anal. Calcd. for C H N : C 67.82, H 7.47, N, 24.71; Found C 67.40, H 7.87, N 24.81.
16
21
5
5
1
3
.1.1.15. N-(4-Methoxybenzyl)-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (22). Yield, 82%; mp
1
08.1-110.2°C; H NMR (400 MHz , DMSO-d ) δ: 2.19 (s, 3H, O-CH ), 2.24-2.33 (m, 4H, 2×CH ),
6
3
2
.57-3.67 (m, 4H, 2×CH ), 3.72 (s, 3H, O-CH ), 4.38 (s, 2H, CH ), 5.77 (d, J = 5.7 Hz, 1H,
2
3
2
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
1
3
290
(d, J = 5.6 Hz, 1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 43.2, 43.6, 46.3, 54.9, 55.4,
6
+
+
9
6.1, 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 C H N O: C 65.15, H 7.40; N 22.35; Found C 64.97, H 7.60, N 22.19.
1
7
23
5
5.1.1.16. N-(3-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (23). Yield, 58%; mp
1
1
39.5-141.4°C; H NMR (400 MHz, CDCl ) δ: 2.36 (s, 3H, N-CH ), 2.50 (t, J = 4.8 Hz, 4H,
3
3
2
95
00
2×CH ), 3.69 (t, J = 4.8 Hz, 4H, 2×CH ), 3.83 (s, 3H, O-CH ), 6.05 (d, J = 6.0 Hz, 1H,
2 2 3
pyrimidine-H), 6.57 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H, ArH), 7.04 (d, J = 9.6 Hz, 1H, ArH), 7.09 (s,
1
2
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,
1
3
pyrimidine-H). C NMR (100 MHz, CDCl ) δ: 43.9, 46.1, 54.6, 55.1, 95.0, 104.8, 107.5, 111.5,
3
+
+
1
29.3, 141.4, 156.6, 159.5, 160.0, 162.3. ESI-MS: m/z = 322.7 [M+Na] , 300.8 [M+1] . Anal. Calcd.
3
for C H N O: C 64.19, H 7.07, N 23.39; Found C 64.03, H 7.36, N 23.47.
1
6
21
5
2
4
5
.1.1.17. N -(4-Methoxyphenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (24).
1
Yield, 77%; mp 187.4-189.0°C; H NMR (400 MHz, CDCl ) δ: 2.36 (s, 3H, SO -CH ), 2.49 (t, J =
3
2
3
4
.8 Hz, 4H, 2×CH ), 3.66 (t, J = 4.8 Hz, 4H, 2×CH ), 3.82 (s, 3H, O-CH ), 6.01 (d, J = 6.4 Hz, 1H,
2 2 3
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,
1
3
305
1H, pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.3, 44.9, 46.6, 55.6, 113.9, 114.3, 120.5,
6
+
+
1
34.9, 153.9, 155.3, 160.2, 162.2. ESI-MS: m/z = 400.6 [M+Na] , 378.6 [M+1] . Anal. Calcd. for
C H N O S: 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.
17
23
5
3
2
4
5
.1.1.18. N -(3-Methoxyphenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (25).
1
Yield, 60%; mp 203.5-204.6°C; H NMR (400 MHz, DMSO-d ) δ: 1.52 (dd, J = 10.4 Hz, J = 20.0
6
1
2
3
10
15
Hz, 2H, CH ), 2.04 (d, J = 10.4 Hz, 2H, CH ), 2.89 (d, J = 2.0 Hz, 1H, CH), 2.91 (s, 3H, SO -CH ),
2 2 2 3
3
.57 (d, J = 12.4 Hz, 2H, CH ), 3.73 (s, 3H, O-CH ), 3.96 (s, 1H, NH), 5.95 (d, J = 5.8 Hz, 1H,
2 3
pyrimidine-H), 6.46 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H, ArH), 7.13 (t, J = 8.2 Hz, 1H, ArH), 7.30 (d, J
1
2
=
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).
1
3
C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.6, 44.9, 46.7, 55.3, 105.1, 105.7, 111.4, 129.4, 142.9,
6
+
+
3
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
C H N O S: 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.
17
23
5
3
2
4
5
.1.1.19. N -(4-Chlorophenyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (26).
1
Yield, 79%; mp 208.8-209.6°C; H NMR (400 MHz, DMSO-d ) δ: 1.51 (dd, J = 8.0 Hz, J = 20.0
6
1
2
Hz, 2H, CH ), 2.02 (d, J = 12.0 Hz, 2H, CH ), 2.90 (d, J = 12.0 Hz, 1H, CH), 2.92 (s, 3H,
2
2
320
SO -CH ), 3.58 (d, J = 12.0 Hz, 2H, CH ), 3.96 (s, 1H, NH), 5.97 (d, J = 4.0 Hz, 1H,
2 3 2
pyrimidine-H), 7.29 (d, J = 8.0 Hz, 2H, ArH), 7.80 (d, J = 2.4 Hz, 2H, ArH), 8.31 (s, 1H,
1
3
pyrimidine-H), 9.12 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 31.3, 34.7, 44.9, 46.7, 120.0,
6
+
+
1
24.2, 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 C H ClN O S: C 50.32, H 5.28, N 18.34, S 8.40; Found C 50.13, H 5.39, N 18.12, S
1
6
20
5
2
1
0

ACCEPTED MANUSCRIPT
325
8.13.
.1.1.20.
2
4
5
N -(2-Methoxyphenethyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine
1
(
27). Yield, 84 %; mp 94.1-96.6°C; H NMR (400 MHz, DMSO-d ) δ: 1.49 (d, J = 10.0 Hz, 2H,
6
CH ), 1.98 (d, J = 11.4 Hz, 2H, CH ), 2.81 (dd, J = 6.0 Hz, J = 14.0 Hz, 4H, 2×CH ), 2.87 (s, 3H,
2
2
1
2
2
SO -CH ), 3.41 (dd, J = 6.0 Hz, J = 14.0 Hz, 2H, CH ), 3.52 (d, J = 12.0 Hz, 2H, CH ), 3.78 (s,
2
3
1
2
2
2
3
30
35
3H, O-CH ), 5.74 (d, J = 8.0 Hz, 1H, pyrimidine-H), 6.51 (s, 1H, NH), 6.87 (t, J = 8.0 Hz, 1H,
3
ArH), 6.95 (d, J = 8.0 Hz, 1H, ArH), 7.18 (dd, J = 8.0 Hz, J = 17.0 Hz, 2H ArH), 7.65 (d, J = 4.0
1
2
1
3
Hz, 1H pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 31.2, 34.6, 35.0, 42.9, 44.8, 46.2, 55.4,
6
+
+
1
14.1, 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 C H N O S: C 56.28, H 6.71, N 17.27, S 7.91; Found C 55.92, H 6.53, N 17.01, S
1
9
27
5
3
3
8.19.
.
2
4
5
.1.1.21. N -(4-Methoxybenzyl)-N -(1-(methylsulfonyl)piperidin-4-yl)pyrimidine-2,4-diamine (28).
1
Yield, 62%; mp 42.7-43.8°C; H NMR (400 MHz, DMSO-d ) δ: 1.45 (dd, J = 10.8 Hz, J = 20.8
6
1
2
Hz, 2H, CH ), 1.85-1.93 (m, 2H, CH ), 2.81-2.88 (m, 2H, CH ), 2.90 (s, 3H, SO -CH ), 3.51 (d, J =
2
2
2
2
3
1
2.4 Hz, 2H, CH ), 3.72 (s, 3H, O-CH ), 3.90 (s, 1H, CH), 4.40 (d, J = 5.6 Hz, 2H, CH ), 5.90 (d, J
2
3
2
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,
1
3
J = 6.2 Hz, 1H, pyrimidine-H), 7.98 (s, 1H, NH). C NMR (100 MHz, DMSO-d ) δ: 30.6, 34.8,
6
+
4
3.8, 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
+
[
M+1] . Anal. Calcd. for C H N O S: C 55.22, H 6.44, N 17.89, S 8.19; Found C 55.32, H 6.60,
1
8
25
5
3
N 18.10, S 7.81.
3
45
50
5.1.1.22. N-(4-Methoxyphenyl)-4-(4-methylpiperazin-1-yl)pyrimidin-2-amine (29). Yield, 73%; mp
1
1
2
32.5-133.8°C; H NMR (400 MHz, CDCl ) δ: 2.36 (s, 3H, N-CH ), 2.49 (t, J = 5.2 Hz, 4H,
3
3
×CH ), 3.65 (t, J = 5.0 Hz, 4H, 2×CH ), 3.82 (s, 3H, O-CH ), 6.01 (d, J = 6.4 Hz, 1H,
2
2
3
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,
1
3
pyrimidine-H). C NMR (100 MHz, DMSO-d ) δ: 43.8, 46.1, 54.6, 55.5, 95.0, 114.0, 120.6, 134.8,
6
+
+
3
154.1, 157.0, 160.0, 162.4. ESI-MS: m/z = 322.7 [M+Na] , 300.8 [M+1] . Anal. Calcd. for
C H N O: C 64.19, H 7.07, N 23.39; Found C 64.50, H 7.39, N 22.89.
16
21
5
5
1
2
.1.1.23. N-(4-Chlorophenyl)-4-(4-methylpiperazin-1-yl)pyrimidin-2-amine (30). Yield, 75%; mp
1
48.6-150.1°C; H NMR (400 MHz, CDCl ) δ: 2.37 (s, 3H, N-CH ), 2.50 (t, J = 5.0 Hz, 4H,
3
3
×CH ), 3.67 (t, J = 4.6 Hz, 4H, 2×CH ), 6.06 (d, J = 6.4 Hz, 1H, pyrimidine-H), 7.19 (s, 1H, NH),
2
2
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).
1
3
C NMR (100 MHz, CDCl ) δ: 29.6, 43.8, 46.1, 54.6, 95.2, 120.4, 126.5, 128.6, 138.7, 156.4,
3
+
+
1
59.3, 162.3. ESI-MS: m/z = 326.6 [M+Na] , 304.6 [M+1] . Anal. Calcd. for C H ClN : C 59.30,
15 18 5
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%;
1
360
mp 41.0-41.3°C; H NMR (400 MHz, CDCl ) δ: 2.35 (s, 3H, N-CH ), 2.47 (t, J = 5.0 Hz, 2H, CH ),
3
3
2
2
.51 (d, J = 3.6 Hz, 2H, CH ), 2.93 (t, J = 7.0 Hz, 2H, CH ), 3.58-3.64 (m, 6H, 3×CH ), 3.85 (s, 3H,
2 2 2
O-CH ), 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,
3
1
3
2
H, ArH), 7.16-7.24 (m, 2H, ArH), 7.85 (d, J = 8.0 Hz, 1H, pyrimidine-H). C NMR (100 MHz,
CDCl ) 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,
3
+
+
3
65
70
161.7, 162.4. ESI-MS: m/z = 350.7 [M+Na] , 328.7 [M+1] . Anal. Calcd. for C H N O: C 66.03,
18 25 5
H 7.70, N 21.39; Found C 65.61, H 7.42, N 20.92.
5.2 Crystallographic analysis
3
Colorless blocks of 25 were mounted on a quartz fiber. Cell dimensions and intensities were
measured at 293 K on a Bruker SMART CCD area detector diffractometer with graphite
mono-chromated Mo Kα radiation (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 (R = 0.1156) of which
int
375
380
385
390
1181 had |F | > 2|F |. Data were corrected for Lorentz and polarization effects and for absorption
o
o
(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
2
2
2
2
[
34]. Full-matrix least-squares refinement based on F using the weight of 1/[σ (F ) + (0.1605P)
o
+
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/errorꢀ0.000(3), max/min residual electron
-3
density = 0.443/-0.644 e Å . Hydrogen atoms were observed and refined with a fixed value of their
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
obtained, free of charge, on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK [Fax:
t44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or at www.ccdc.cam.ac.uk].
5
5
.3 Pharmacology evaluation
.3.1 Antitumor activity
The antitumor activities of compounds 8-31 were evaluated with HepG2, A549,
1
2

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.
395
400
405
4
Exponentially growing cells were plated in 96-well plates (1×10 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
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
with MTT solution (5 mg/mL, 100 ꢀL) followed by incubation for another 4 h. The medium was
then aspirated and formazan crystals were dissolved in DMSO (100 ꢀL) 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 = (AbscontrolꢁAbscompound)/Abscontrol×100%. The IC values of the test compounds and
50
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)
410
The effect of compound 28 on cell cycle phase distribution of human breast cancer
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 ꢀM).
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 ꢀg/mL final
415
concentration) at 37 °C for 30 min, stained with propidium iodide (50 ꢀg/mL final concentration),
and analyzed by flow cytometry (Beckman Coulter).
Acknowledgments
This work was supported by National Natural Science Foundation of China (21102069,
21372113 and 81101732), and the project of science and technology new star in Zhujiang
420
1
3

ACCEPTED MANUSCRIPT
Guangzhou city (No. 2012J2200051 and 2013J2200047).
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545
Figure captions
Fig. 1. Reported 2,4-diaminopyrimidines with antitumor activity.
Fig. 2. Design strategy of the title compounds 8-31.
Fig. 3. Molecular structure of compound 25.
Fig. 4. SAR of compounds 8-31.
5
50
55
5
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 ꢀM; (C) 28, 40 ꢀM;
(D) 28, 80 ꢀM
560
565
570
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8

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575
Table 1 Cytotoxic Activities of Compounds 8-31 against human tumor cells.
R1
O
R1
N
O
S
R1
R1
N
N
S
N
N
R2
N
O
N
n
R2
N
N
n
N
N
n
N
R2
O
n
N
N
N
H
H
R2
N
N
N
H
H
H
N
H
N
8
-20
21-23
24-28
29-31
a
In vitro cytotoxicity IC (ꢀM)
1
2
50
Comp.
n
R
R
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
A549
>200
>200
51.6
MDA-MB-231
>200
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
8
9
0
0
1
H
H
H
4-ClC H
6
3-MeOC H
4
6
4
4
29.44
33.69
60.76
34.08
>200
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4-MeOC H
6
1
1
0
H
Cl
Cl
C
6
H
5
42.52
60.93
>200
>200
>200
46.80
>200
>200
>200
>200
>200
>200
>200
91.65
>200
>200
21.41
12.78
>200
>200
>200
35.41
4-MeOC H
6
4
3 - MeOC H
6 4
0
0
1
0
2
2
2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
4-ClC
4-MeOC
6
H
4
>200
>200
11.73
>200
>200
H
6
4
C
C
H
H
6
6
5
5
4-ClC
4-MeOC
H
6
4
H
>200
>200
>200
>200
6
4
4
C
C
H
H
6
6
5
5
1
1
H
4-MeOC H
3 - MeOC
4-MeOC
6
0
0
0
H
H
H
H
6
4
>200
H
57.74
108.74
102.48
15.83
7.46
6
4
4
3-MeOC H
6
0
2
1
H
H
H
4-ClC H
6
4
4-MeOC
4-MeOC
4-MeOC
H
H
H
6
6
6
4
4
4
0
0
2
H
H
H
>200
>200
>200
24.60
4-ClC
2-MeOC
fluorouracil
H
6
4
H
6
4
4
6.83
a
IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients of variation were
<10%.
580
Table 2 Effect of compound 28 on cell cycle distribution in MDA-MB-231 cells.
Concentration Sub-G (%) G /G (%)
S(%)
42.22
32.03
29.91
26.78
G /M(%)
1
1.69
1.09
1.03
1.26
0
1
2
0
ꢀM
37.97
33.09
32.55
31.01
18.12
33.79
36.51
40.95
20 ꢀM
40 ꢀM
80 ꢀM
1
9

ACCEPTED MANUSCRIPT
F
F
HN
N
O
O
O
N
O
S
O
N
N
O
H
N
N
N
O
m
H
H
n
N
N
4
N
N
N
2
N
N
N
N
N
N
1
N
H
H
H
H
H
H
N
H
O
N
3
NU6027
R547
XL228
Fig. 1. Reported 2,4-diaminopyrimidines with antitumor activity.
585
Heterocycle-amine
Side Chain
Flexible
Linker
O
X
Y
S
N
N
NH
Ar
n
O
N
N
H
Pyrimidine
Scaffold
N
1
X = N, Y = C-R (8-23)
1
X = C-R , Y = N (24-31)
Fig. 2. Design strategy of the title compounds 8-31
590
Fig. 3. Molecular structure of compound 25.
2
0

ACCEPTED MANUSCRIPT
R1
N
R2
n
N
N
N
H
O
S
O
R1
O
N
R1
N
S
N
N
N
21-23
R2
n
O
>
N
H
N
N
>>
R2
n
N
N
N
H
H
R1
H
N
8
-20
24-28
R2
n
N
H
N
N
N
29-31
n = 1 > 2 > 0
n = 1 > 2 > 0
1
R = H > Cl (except 16)
Fig. 4. SAR of the compounds 8-31
595
A
B
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 ꢀM; (C) 28, 40 ꢀM; (D) 28, 80 ꢀM.
2
1

ACCEPTED MANUSCRIPT
605
R1
R1
R1
Cl
R3 NH2
N
N
N
DIPEA
+
R2 NH2
R2
R3
o
R2
2
-methoxyethanol Cl
r.t.
N
N
90 C
Cl
N
5
N
N
N
H
H
H
6
7
8-31
Scheme 1 One-pot synthesis of compounds 8-31
2
2

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

ACCEPTED MANUSCRIPT
One-pot synthesis and antiproliferative activity of novel
,4-diaminopyrimidine derivatives bearing piperidine and
piperazine moieties
2
b,#
a,#
a
a
a
a
Wei-Feng Ma , Hai-Kui Yang , Meng-Jin Hu , Qian Li , Tian-Zhu Ma , Zhong-Zhen Zhou ,
a
a,
a,
Rui-Yuan Liu , Wen-Wei You *, Pei-Liang Zhao *
Department of Chemistry, School of Pharmaceutical Science, Southern Medical
University, Guangzhou 510515, P.R.China
a
b
Department of Microbiology, School of Public Health and Tropical Medicine, Southern
Medical University, Guangzhou 510515, P.R.China
Supporting Information
No.
1
List of Figures
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List of Figures
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1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
H NMR Spectrum of Compound 8
S3
H NMR Spectrum of Compound 20
3
13
2
C NMR Spectrum of Compound 8
S4
C NMR Spectrum of Compound 20
1
3
H NMR Spectrum of Compound 9
S5
H NMR Spectrum of Compound 21
3
13
4
C NMR Spectrum of Compound 9
S6
C NMR Spectrum of Compound 21
H NMR Spectrum of Compound 22
1
5
H NMR Spectrum of Compound 10
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3
13
6
C NMR Spectrum of Compound 10
S8
C NMR Spectrum of Compound 22
1
7
H NMR Spectrum of Compound 11
S9
H NMR Spectrum of Compound 23
3
13
8
C NMR Spectrum of Compound 11
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S16
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C NMR Spectrum of Compound 23
H NMR Spectrum of Compound 24
1
9
H NMR Spectrum of Compound 12
3
13
1
1
0
1
C NMR Spectrum of Compound 12
C NMR Spectrum of Compound 24
1
H NMR Spectrum of Compound 13
H NMR Spectrum of Compound 25
3
13
12
13
14
15
C NMR Spectrum of Compound 13
C NMR Spectrum of Compound 25
H NMR Spectrum of Compound 26
1
H NMR Spectrum of Compound 14
3
13
C NMR Spectrum of Compound 14
C NMR Spectrum of Compound 26
1
H NMR Spectrum of Compound 15
H NMR Spectrum of Compound 27
S1

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
3
13
16
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20
21
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C NMR Spectrum of Compound 15
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C NMR Spectrum of Compound 27
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1
H NMR Spectrum of Compound 16
H NMR Spectrum of Compound 28
3
13
1
C NMR Spectrum of Compound 6
C NMR Spectrum of Compound 28
H NMR Spectrum of Compound 17
H NMR Spectrum of Compound 29
3
13
C NMR Spectrum of Compound 17
C NMR Spectrum of Compound 29
H NMR Spectrum of Compound 30
1
H NMR Spectrum of Compound 18
3
13
1
C NMR Spectrum of Compound 18
C NMR Spectrum of Compound 30
H NMR Spectrum of Compound 19
H NMR Spectrum of Compound 31
3
13
C NMR Spectrum of Compound 19
C NMR Spectrum of Compound 31
S2

ACCEPTED MANUSCRIPT
Cl
O
S
N
N
O
N
N
N
H
H
8
1
3
Figure S1. H NMR spectrum (400 MHz, CDCl ) of compound 8.
S3

ACCEPTED MANUSCRIPT
Cl
O
N
S
N
O
N
N
N
H
H
8
1
3
6
Figure S2. C NMR spectrum (100 MHz, DMSO-d ) of compound 8.
S4

ACCEPTED MANUSCRIPT
O
S
N
N
O
O
N
N
N
H
H
9
1
6
Figure S3. H NMR spectrum (400 MHz, DMSO-d ) of compound 9.
S5