Design, synthesis and biological evaluation of novel 2,4-diaminopyrimidine derivatives as potent antitumor agents

Article abstract of DOI:10.1039/c9nj02154j

For developing novel therapeutic agents with anticancer activities, two series of novel 2,4-diaminopyrimidine derivatives possessing triazolopiperazine or 1,4,8-triazaspiro[4.5]decan-3-one scaffolds were designed and synthesized. Preliminary investigations showed that some compounds exhibited moderate to excellent potency against four tested cancer cell lines as compared with palbociclib and momelotinib. In particular, the most promising compounds 9k and 13f showed the most potent antitumor activities with IC₅₀ values of 2.14/1.98 μM, 3.59/2.78 μM, 5.52/4.27 μM, and 3.69/4.01 μM against A549, HCT-116, PC-3 and MCF-7 cell lines, respectively. Structure-activity relationship (SAR) studies were conducted based on the variation of the moiety of the aromatic ring and the terminal aniline on the pyrimidine core. Furthermore, the mechanism of their anticancer activity was clarified by further exploring the bioactivity. The results showed that compound 9k obviously inhibited the proliferation of A549 cell lines, induced a great decrease in the mitochondrial membrane potential leading to apoptosis of cancer cells, suppressed the migration of tumor cells and prolonged the A549 cell cycle distribution, representing blockage at the G2-M phase and accumulation at the S phase.

Full text of DOI:10.1039/c9nj02154j

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Design, synthesis and biological evaluation of novel 2,4-diaminopyrimidine derivaDtOiIv: e10s.10a3s9/C9NJ02154J
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potent antitumor agents
Gang Hu, Chu Wang，Xin Xin，Shuaikang Li，Zefei Li，Yanfang Zhao, Ping Gong
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China.
Abstract
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For developing novel therapeutic agents with anticancer activities, two series of novel
2,4-diaminopyrimidine
derivatives
possessing
triazolopiperazine
or
1,4,8-triazaspiro[4.5]decan-3-one scaffolds were designed and synthesized. The preliminary
investigation showed that some compounds displayed moderate to excellent potency against four
tested cancer cell lines as compared with palbociclib and momelotinib. In particular, the most
promising compounds 9k and 13f showed the most potent antitumor activities with IC50 values of
2.14/1.98 μM, 3.59/2.78 μM, 5.52/4.27 μM, and 3.69/4.01 μM against A549, HCT-116, PC-3 and
MCF-7 cell lines, respectively. The structure-activity relationships (SARs) studies were conducted
through the variation in the moiety of aromatic ring and the terminal aniline on pyrimidine core.
Furthermore, the mechanism of their anticancer activity was clarified by further explorations in
the bioactivity. The results showed that compound 9k obviously inhibited proliferation of A549
cell lines, induced a great decrease in mitochondrial membrane potential leading to apoptosis of
cancer cells, suppressed the migration of tumor cells and prolonged the A549 cells cycle
distribution, represented the blockage at G2-M phase and accumulation at S phase.
Key words: Antiproliferative activity; Synthesis; aminopyrimidine derivatives; Structure-activity
relationships
Corresponding author. E-mail address: gongpinggp@126.com (P. Gong). yanfangzhao@126.com(Y.F. Zhao).

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1. Introduction
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Cancer is one of the most serious diseases and the leading cause of morbidity and mortality[1].
There were an estimated 18.1 million new cases of cancer and 9.6 million deaths from cancer
worldwide in 2018[2]. This casts great socioeconomic burdens. Cancer treatment and prevention
has long been a crucial problem and a challenge to the medical profession as well. Thus, for
several decades, researchers have been devoting themselves to finding effective clinical
approaches for the treatment of cancer and searching for novel anticancer agents[3]. For these
purposes, combination principle strategy has been widely used to develop new antineoplastic
drugs that act synergistically on multiple targets by covalently fusing two or more active
pharmacophores in a single-hybrid molecule with dual or multiple anticancer activitives[4].
More and more evidence indicated that the heterocyclic scaffold was a significant tool for
finding new active substances with many potential applications[5-6]. Especially, aminopyrimidine
and its bioisosteric structures have been recommended as a privileged structure, and an important
class of chemotherapeutic drugs have emerged for the treatment of cancers, such as CDK4/6
inhibitor(palbociclib), JAK inbibitor(momelotinib), VEGFR inhibitor(pazopanib) and Aurora A
inhibitor(alisertib), et al[7-9]. These characteristics demonstrated that the extent of ongoing interests
toward new 2-aminopyrimidine derivatives and prompted us to develop this pharmacophore as
novel potential bioactive molecules(Fig.1).
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HN
O
O
O
N
N
N
N
N
N
N
N
N
N
H
H
H
N
CN
9
O
Palbociclib
CDK4/6 inhibitor
Momelotinib
JAK1/2 inhibitor
N
N
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N
H
O
O
privileged
structure
O
N
N
N
HO
N
O
S
N
H2N
N
N
N
N
N
H
H
F
O
Alisertib
AuroraA inhibitor
Pazopanib
VEGFR inhibitor
Cl
Fig. 1 Structures of representative kinase inhibitors containing aminopyrimidine nuclei
Triazolopiperazine derivatives had a variety of biological activities, such as hypoglycemic[10]
,
anti-inflammatory[11], anti-virus[12],antitumor activity [13], and so on. In recent years, a series of
antitumor compounds with triazolopiperazines moiety have been designed and synthesized.
Among these derivatives, AZ0108 has been reported to demonstrate significant inhibitory activity
against PARP. The co-crystal structure of AZ0108 in complex with PARP domains revealed that
the 1,2,4-triazole framework played key roles in the interaction with PARP kinase due to their
binding modes and the presence of hydrogen bonds. Thus, the development of such small
molecules with triazolopiperazines framework, which can readily bind with various enzymes and
receptors through hydrogen bonds, is a viable approach to develop new activity compounds.
Spiropiperidines belongs to a relevant class of molecules for various GPCR target such as
anti-inflammatory angents or δ-orpioid agonists, and could offer a useful structure for the
discovery of new active compounds. To our knowledge, the spiropiperidines structure was rarely
reported in the antitumor filed. For enriching the structural diversity, we also introduced the
1,4,8-triazaspiro[4.5]decan-3-one fragment to replace triazolopiperazines moiety as its good

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DOI: 10.1039/C9NJ02154J
bioactivity as well as had the hydrogen bond donor and acceptor, which could have the interaction
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with the receptors[14]. (Fig.2).
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F
F
F
O
NH2
N
N
N
N
Sitagliptin
DPPIV inhibitors
F3C
scaffold
hopping
N
H
N
O
N
O
N
N
N
NH
N
N
O
F
F
N
N
N
N
OCH3
F
Bn
N
HO
Cl
O
O
H
F
AZD0108 H3C
PARP inhibitor
H
O
N
N
N S
O
N
N
N
N
H
O
O
H
Cl
H
anti-inflammatory angents
-orpioid agonists
Fig.
2 Structures of representative triazolopiperazines derivatives and scaffold hopping to
1,4,8-triazaspiro[4.5]decan-3-ones framework
Judging from these observations, it was thought of interest to study the combination of
2-aminopyrimidine moiety and triazolopiperazine/1,4,8-triazaspiro[4.5]decan-3-one framework
combination, which were expected to exhibit synergistic antitumor effect. Accordingly, two series
of 2-aminopyrimidine derivatives have been designed and synthesized(Fig.3). Various substituents
were introduced to the terminal phenyl ring with the purpose of exploring the influence of
substituents on anticancer activity by regulating the electronic and steric effects. Further
modifications were performed by introducing various aliphatic amino groups into the hydrophilic
tail which exposing to the solvent [15-16]
.

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various aliphatic amines
3R2RN
N
N
N
N
N
N
N
N
N
H
N
N
R1
9a-9l
R1
terminal phenyl ring
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N
+
fused
HN
N
various aliphatic amines
3R2RN
N
N
H
N
N
N
N
H
O
H
N
N
R1
O
13a-13l
N
R1
terminal phenyl ring
Fig.
3
Design strategy for the aminopyrimidine derivatives containing triazolopiperazine or
1,4,8-triazaspiro[4.5]decan-3-one scaffolds.
2. Chemistry
The general synthetic routes of the target compounds are shown in Scheme 1-3. The
intermediate 2-hydrazinylpyrazine (2) was prepared from the commercially available
2-chloropyrazine (1) and hydrazine hydrate in good yield according to the known procedure[17]
.
Intermediate 2 was condensed with appropriate benzaldehydes in ethanol at room temperature to
give the corresponding hydrazones which was not isolated in the next step and was treated by
chloramine T trihydrate though the oxidative cyclisation cascade to afford the fused
triazolopyrazine derivatives (4). The key intermediates (5) were obtained via the substitution
reaction of intermediates 4 with 2,4-dichloropyrimidine. These above were mentioned in Scheme
1. Side chain anilines (8) were prepared in two steps as shown in Scheme 2. Aromatic
nucleophilic substitution of fluorine in 4-fluoronitrobenzene by aliphatic amines were readily
achieved in DMF at 120 °C. The resulting nitrobenzenes were reduced to anilines using Pd/C in
EtOH and generally used without further purification. The synthetic route to compounds 9a-j and

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13a-j was outlined in Scheme 3. The intermediate (11) could easily obtain by folloDwOinI: g10.t1h03e9/C9NJ02154J
2,4-dichloropyrimidine in the presence of K2CO3 and the 4-position that was displaced by
4-piperidone[18]. Subsequently, amination of 5 and 11 by corresponding aryl amines (8) in the
presence of p-toluenesulfonic acid furnished target compounds 9a-l and 12. Finally, target
compounds 13a-j were successfully afforded via the condensation of 12 with appropriate
2-(phenylamino)acetamides in 1,2-dichloroethane (DCE) at 90 °C with amounts of
camphorsulfonic acid. Their spectral data were in full agreement with the proposed structures.
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N
N
N
N
N
N
N
c
a
b
N
N
HN
N
R1
R1
NH2
Cl
N
2
N
H
4
1
3
N
d
N
Cl
N
N
N
N
5
R1
Scheme 1. General scheme for the synthesis of key intermediates 5; Reagents and conditions:
o
(a)80% hydrazine hydrate, 65 C, 10 h; (b)i: appropriate aromatic aldehyde, EtOH, r.t. 1 h; ii:
chloramine
T
trihydrate, r.t. ovevnight; (c) H2, 10
%
Pd/C, EtOH, r.t., 18 h;
(d)2,4-dichloropyrimidine, TEA, DMF, r.t., 2 h;
a
b
O2N
NR2R3
O2N
F
H2N
NR2R3
8
7
6
Scheme 2. Preparation of the side chain anilines; Reagents and conditions: (a) Amines R1R2NH,
K2CO3, DMF, 120 °C, 3 h; (b) H2, 10 % Pd/C, EtOH, r.t., 5 h.

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N
N
a
N
Cl
N
10
Cl
Cl
N
N
N
HN
N
N
11
N
O
N
8
9
R1
b
H2N
NR2R3
NR2R3
10
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12
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9a-9l
N
N
HN
N
N
c
HN
N
N
H
N
O
O
N
R1
13a-13l
NR2R3
12
NR2R3
Scheme 3. General scheme for the synthesis of target compounds; Reagents and conditions: (a)
4-piperidone hydrate hydrochloride, K2CO3, MeCN, r.t. (b) p-Toluenesulfonic acid, isopropanol,
90 oC, 24 h; (c) appropriate 2-(phenylamino)acetamide , Camphorsulfonic acid, DCE, 90 oC, 24 h.
3.Results and discussion
3.1 In vitro antitumor activity and SARs study
The in vitro antitumor activities of the target compounds were evaluated in A549 (human
lung cancer), HCT-116 (human colon cancer), PC-3 (human prostate cancer) cell lines and MCF-7
(human breast cancer) by the MTT assay. Using palbociclib and Momelotinib as the positive
controls, the bioactivity data summarized in Tables 1 that are presented as half-maximal
inhibitory concentration (IC50) values.
As illustrated in Table 1, most of the prepared compounds showed moderate-to-excellent
potency against four tested cell lines in the μM range, which suggested that the combination of
2-aminopyrimidine moiety and triazolopiperazines/1,4,8-triazaspiro[4.5]decan-3-one framework
exhibit potent synergistic antitumor effect. Our initial effort was to explore the cytotoxic activity
of compounds with different substituents on the phenyl ring in order to understand the
structure-activity relationship. A small set of compounds (9a-9e) with different aryl groups were
synthesized and evaluated for their cytotoxic activity. Compound 9e (R1=3-Cl-4-F-Ph) exhibited
good activity against A549 and HCT-116 cells with IC50 values of 4.95 and 10.76 μM. According

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to this result, the introduction of electron-withdrawing groups(EWGs) exhibited a positive effect
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6
7
on the cytotoxic activity on the A549 cells, such as compound 9b (R1 = 4-fluorophenyl IC50
=
8
9
7.99 μM), 9e (R1 = 3-chloro-4-fluorophenyl, IC50 = 4.95 μM), which are more potent than 9a (R1
= phenyl IC50 = 19.97 μM) and indicated monosubstitution of phenyl is more preferred. By
contrast, electron-donating groups (EDGs) such as 4-methyl (9c) exhibited a negative effect. After
the optimization of R1 moiety, the modification of amino groups NR2R3 was carried out.
Preliminary SARs indicated that the introduction of different amino groups at the C-2 position of
pyrimidine moiety had a significant influence on activity, which suggested that the hydrophilic
group contributed much to their potency. Replacement of the oxygen atom of morpholine group in
9e by sulfur atom or carbon atom(9h and 9f) led to the increased potency, indicating that the
presence of carbon atom was a critical factor in anti-proliferative activities. Generally,
five-membered pyrrolidine analogs were less active than corresponding six-membered inhibitors
(9f vs. 9g) despite of possessing fairly basic nitrogen atom, highlighting the importance of ring
size. 2,6-Dimethypiperazinyl group, showed the most potent antitumor activities with the IC50
values of 2.14 μM, 3.59 μM, 5.52 μM, and 3.69 μM against A549, HCT-116, PC-3 and MCF-7
cell lines, respectively. In order to decrease the polarity of piperazine ring, N-methyl was embeded
on the piperazine ring, and the results indicated that the hydrophilic N-methylpiperazinyl (9j) had
negative impact on the activities. Besides, a loss in potency was observed once the piperazinyl
group in 9f was “opened” to dimethylamine (9i). Meanwhile, 4-methylpiperidine also showed
negative impact on the activities. Obviously, all target compounds possessed selectivity for A549
and HCT-116 cancer cell lines, and had the makings of good candidate for lung and colorectal
cancer.
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Further investigations were performed to investigate the influence DoOfI: 10.1039/C9NJ02154J
the
5
6
7
8
9
1,4,8-triazaspiro[4.5]decan-3-one moiety at the 4-position of pyrimidine core on the cytotoxic
activity. To begin with, we discussed the cytotoxic activity of different substituents on the phenyl
ring. 4-Fluorophenyl (13c) was selected as a positive group than other EWGs, such as
3-chloro-4-fluorophenyl (13d) and 4-chlorophenyl(13e). Compared with the EWGs,
4-methylphenyl (13b) as an EDG exhibited weaker activity. Replacement of the sulfur atom of
thiomorpholine group (13f, IC50 = 2.78μM and 4.27μM against HCT-116 and PC-3) by oxygen
atom (13c), caused the potency to be lowered by 1.7- and 2-fold, respectively.
Five-membered pyrrolidine analogs were more active than corresponding six-membered inhibitors.
Notably, the most promising compound 13f displayed stronger potency than momelotinib and
palbociclib in A549, HCT-116, PC-3 and cells with IC50 values of 1.98 μM, 2.78 μM and 4.27 μM,
respectively. When the pyrrolidino group in 13g was “opened” to diethylamine (13l), the potency
decreased slightly. These encouraging results provided a valuable lead compounds 9k and 13f and
highlighted the potential for further development.
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Table 1. Structures and cytotoxicity of compounds (9a-9l and 13a-13l).
3R2RN
3R2RN
N
N
N
N
N
N
N
N
N
H
N
H
H
N
N
O
13a-13l
9a-9l
N
R1
R1
IC50(μM)
Compd.
R1
NR2R3
A549
HCT-11
PC-3
>30
MCF-7
>30
6
N
N
O
9a
9b
-Ph
4-F-Ph
19.97
7.99
>30
O
13.72
>30
>30

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D1O5I.:4108.1039/C9NJ02154J
N
N
N
N
O
O
O
9c
4-Me-Ph
4-CF3-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
3-Cl-4-F-Ph
-Ph
18.35
16.27
4.95
16.16
12.95
10.76
3.50
4.51
9.71
6.47
>30
>30
>30
9d
>30
9e
>30
12.08
12.62
15.55
13.90
10.36
>30
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12
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14
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9f
2.99
>30
N
9g
8.91
14.68
14.21
8.83
>30
N
S
9h
5.17
N
9i
6.27
N
N
9j
20.60
2.14
N
NH
9k
3.59
>30
5.52
>30
3.69
N
9l
13a
15.56
21.63
16.18
4.26
21.02
>30
N
N
N
N
N
N
O
16.02
18.34
4.87
14.78
11.79
2.78
3.98
4.03
7.01
15.20
5.90
3.62
3.69
3.25
>30
O
O
O
O
S
13b
4-Me-Ph
4-F-Ph
21.40
8.42
16.78
>30
17.33
6.65
13c
13d
3-Cl-4-F-Ph
4-Cl-Ph
11.20
17.70
1.98
6.64
13e
>30
13f
4-F-Ph
4.27
3.63
4.71
4.56
29.90
10.56
5.93
6.82
4.21
4.01
N
13g
4-F-Ph
3.18
6.20
N
13h
4-F-Ph
4.41
3.28
N
13i
4-F-Ph
3.00
6.78
N
OH
13j
4-F-Ph
>30
19.49
6.78
N
13k
4-F-Ph
10.57
4.34
N
13l
4-F-Ph
5.40
Palbociclib
Momelotinib
2.02
3.52
1.56
3.30
3.2 Wound-healing assay.

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The effect of compound 9k on migration of cancer cells was investigated by thDeOwI: 1o0u.1n0d39/C9NJ02154J
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healing assay for migration, which was an important feature of metastatic cancers. Fig 4 and 5
8
9
indicated that concentrations markedly suppressed the wound healing in
concentration-dependent manner.
a
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Fig 4 In vitro wound healing assay on A549 cells. Phase contrast images were obtained by the
treatment of compound 9k at indicated concentrations for 0, 12, 24 and 36 h.

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100.0
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49.3
Control
9k (0.5μM)
34.5
29.5
30.5
26.5
9k(2μM)
23.5
0
0
0
0h
12h
24h
36h
Time (h)
Fig. 5 In vitro wound healing on A549 cells of 9k. Columnar graph represents the transferred
percentage distribution at different concentrations and at different time
3.3 Cell apoptosis study
To investigate the molecular mechanisms of action preliminarily, cell apoptosis analysis of
A549 cells treated with the optimal compounds 9k and 13g were performed using Annexin
V/propidium iodide (PI) double staining. Annexin V was able to penetrate through intact
membranes of live cells and colors DNA as green fluorescence, while PI was only taken
up by apoptotic cells with damaged membranes coloring DNA as red fluorescence. As
shown in Fig. 6, flow cytometric analysis data showed the percentage of Annexin V-positive cells
was significantly increased after exposure to increasing dose of the optimal compounds, compared
with medium control (4.3% vs 92.7% in 9k and 4.3% vs 20.4% in 13g at 10 μM). These results
suggested that the optimal compounds can induce apoptosis in vitro and this is one of mechanisms
by which the compounds exerted its antiproliferative activity.

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Fig. 6 Effect of compound 9k and 13g on cell apoptosis in A549 cells.
3.4 Cell cycle analysis
The cell cycle analysis was performed to investigate the prevention of proliferation in A549
cells with the potent compound 9k and 13g. After treatment of A549 cells with the test
compounds for 24 h at indicated concentrations (9k: 0.08 μM and 2μM; 13g:0.16 μM and 4 μM),
the cells were fixed and stained with PI, the DNA content was analyzed by flow cytometry. The
obtained results were compared with non-treated A549 cells, as control. As shown in Fig. 7,
treatment of A549 cells with 9k at 0.08, 2 μM concentrations increased the percentage of S-phase
cells from 13.43% (as control group) to 26.78%, 30.24%, and G2-M phase from 2.35% (as control
group) to 10.81%, 8.30% and with 13g at 0.16, 4 μM concentrations increased the percentage of
S-phase cells from 13.43% (as control group) to 25.63%, 28.02%, and G2-M phase from 2.35%
(as control group) to 12.53%, 11.23%, respectively. These results confirmed that the test
compounds significantly prolonged the A549 cells cycle distribution, represented the blockage at
G2-M phase and accumulation at S phase.

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Fig. 7 Effect of compound 9k and 13g on the cell cycle distribution of A549 cells.
3.5 In vitro enzyme assay
Based on the cellular assays, potent compounds were selected for further in vitro CDK4,
JAK2, VEGFR2, PI3Kα and FLT3% inhibition at 1 μM. The results were summarized in Table 2.
As shown in Table 2, all compounds poorly inhibited CDK4, VEGFR2 , PI3Kα and FLT3 kinases
with % inhibition values ranging from 1% to 20%. However, most of compounds displayed more
highly inhibition against JAK2 kinases with % inhibition values ranging from 42.9% to 72.3%.
Even though it showed more selectivity against JAK2, the inhibitory activity of compound 9k was
still less than momelotinib. These results indicated that two series of novel compounds worth
further studying as new potential anticancer agent for the treatment of human cancers.
Table 2 Some kinases activity of selected compounds and positive control
Inhibition(%)
VEGFR2
9.7
Compd.
JAK2
55.5
CDK4
4.3
PI3Kα
FLT3
7.3
9f
8.7

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9k
13f
72.3
69.1
42.9
88.7
-
2.9
3.1
3.4
-
20.0
8.4
9.9
-
5.6
2.5
1.8
-
9.4
3.2
0.5
-
13k
Momelotinib
Palbociclib
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92.5
-
-
-
3.6 molecular docking
Fig. 8 A): The JAK2 active site in complex with compound 9k and its hydrophobic surface (brown:
highly hydrophobic, blue: highly hydrophilic). The protein was displayed by secondary structure.
Compounds were shown in colored sticks (cyan: carbon atom, blue: nitrogen atom, red: oxygen
atom, white: hydrogen atom, brown: sulfur atom). B): 9k overlapping with XL019(pink sticks)
In order to better understand the binding mode, molecular docking models of 9k were
performed based upon the cocrystal structure of JAK2 with XL019 (PDB code: 4BBF),
respectively. As shown in Fig.8, compound 9k occupied the kinase domain in a similar way to
XL019, forming two hydrogen bonds via 1-position nitrogen atom and 2-position NH of the
pyrimidine nuclei with Leu 932 at the hinge area. In addition, 1-position NH of the
2,6-dimethypiperazinyl formed another hydrogen bond with Gln853 and the N atom of
triazolopiperazine formed hydrogen bond with Lys882. In the meantime, the π-akyl interaction
between triazolopiperazine and Val863, Met929 and Gly861 was also present.

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3.7 Physicochemical and ADME parameters
5
6
7
8
9
Furthermore, SwissADME (a free web tool to evaluate pharmacokinetics, drug likeness and
medicinal chemistry friendliness of small molecules) was used to predict the physicochemical and
ADME properties of the synthesized compounds.
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As demonstrated in Table 3, the active compounds (9k 、 13f and 13g) shown variable
permeability based on gastrointestinal absorption (GI), according to the BOILED-Egg predictive
model (Brain Or IntestinaL EstimateD permeation method). All predicted compounds showed
high gastrointestinal absorption. With respect to oral bioavailability, it's expected 0.55 of
probability of oral bioavailability score >10% in the rat for all compounds.
Table 3. Physicochemical properties and ADME properties of most active compounds.
H-bon Log
MW
H-bond
Violation aPSA
Lipinski (Ų)
Rotatable
bonds
d
P
(g/mol) acceptors
bBBB cGI
dBS
donors o/w
Compd.
<500
<10
Rule of 5 <140
<10
<5
<5
9k
534.03
519.64
487.57
6
4
4
2
3.99
3.61
3.74
1
1
0
87.03
5
5
5
No
No
Yes
High 0.55
High 0.55
13f
13g
2
101.93
2
76.63
High 0.55
b
c
d
aPSA - Polar surface area. BBB – blood-brain barrier. GI – Gastrointestinal absorption. BS –
Bioavailability Score.
4. Conclusion
In current investigation, two series of novel 2,4-diaminopyrimidine derivatives with
triazolopiperazine or 1,4,8-triazaspiro[4.5]decan-3-one framework, were firstly designed and
synthesized. The structure-activity relationship (SAR) was discussed in details and the results
demonstrated that the most promising compound 9k and 13f showed the most potent antitumor
activities. Combined with the results of the molecular docking and enzymatic studies, the JAK2
was very likely to be one of the drug targets of the compounds. In summary, the SARs studies

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together with the pharmacological assays identified these series of compounds as promising
anti-cancer agents, which will lead to the promising development of new drugs.
5. Experimental procedures
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5.1 Chemistry
All melting points were acquired on a Mettler Melting Point MP70 apparatus (Mettler,
Toledo, Switzerland) and uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC-MS (Agilent, palo Alto, CA, USA). Reactions were monitored by thin-layer
chromatography (TLC) on silica plates (F-254) and visualized under UV light.1H NMR and
13C NMR spectra were performed using Bruker spectrometers (Bruker Bioscience, respectively,
Billerica, MA, USA) with TMS as an internal standard. Column chromatography was run on silica
gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). Unless
otherwise noted, all materials were obtained from commercially available sources and used
without further purification.
5.1.1 General procedure for preparation of compounds (9a)
5.1.1.1 2-hydrazinylpyrazine (2)
Under nitrogen atmosphere, 2-chloropyrazine (96 mL, 1073 mmol) was added dropwise to
aqueous hydrazine (544 mL, 6009 mmol) at 65 °C over 1 h. After the addition, stirring was
continued at 65 °C for 10 h. The mixture was cooled to room temperature and the yellow flakes
were filtered off. The solid was washed with a small amount of water and dried under vacuum to
afford pale yellow solid (60 g). m.p.: 111.7 – 113.3 °C.MS (ESI) m/z: 111.2 [M+H]+
5.1.1.2 3-phenyl -[1,2,4]triazolo[4,3-a]pyrazines (3a)

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To a stirred suspension of 2 (5 g, 34.5 mmol) in ethanol (80 mL) was added the benzDaOldI:e1h0y.1d03e9/C9NJ02154J
(4.82g ，34.5 mmol) and this resulted mixturewas stirred at room temperature for 60 min. The
completion of the reaction was confirmed by TLC. After disappearance of the starting aldehyde,
chloramine T trihydrate (15.44g, 51.75 mmol) was added to the reaction mixture, which was
stirred overnight, until consumption of the intermediate hydrazine 3 formed in the reaction. The
mixture was diluted with addition of chilled water and the precipitate was filtered off, washed
with water followed by cold ethanol and air-dried to give the corresponding
3-penyl-[1,2,4]triazolo[4,3-a]pyrazine 3a as a yellow solid (6.2g). Yield: 70%; mp: 159.2–
4
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1
162.0 °C. MS (ESI) m/z (%): 197.98 [M+H]+. H NMR (400MHz, DMSO-d6) δ 9.50 (s, 1 H),
8.64 (d, J = 5.0 Hz, 1 H), 7.99 – 7.96 (m, 3 H), 7.66 - 7.73 (m, 3 H).
5.1.1.3 3-phenyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine(4a)
3-Phenyl-[1,2,4]triazolo[4,3-a]pyrazine 3 (2.01 g) was hydrogenated under atmospheric hydrogen
with 0.5g 10% Pd/C as a catalyst in ethanol (20 mL) at room temperature for 18 h. The reaction
mixture was filtered through and concentrated. Purification by flash chromatography (silica gel,
10%
methanol/dichloromethane)
afforded
3-phenyl
-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (4a) as a white solid(1.5g). Yield: 75%; MS
(ESI) m/z: 201.20[M+H]+. 1H NMR (400 MHz, DMSO) δ 7.94 – 7.64 (m, 2H), 7.59 – 7.44 (m, 3H),
4.13 (t, J = 5.5 Hz, 2H), 3.77 (s, 2H), 2.84 (t, J = 5.5 Hz, 2H)
5.1.1.4
-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine(5a)
2,4-dichloropyrimidine (0.82g, 5.5mmol) was dissolved in 10mL N,N-dimethylformamide (DMF),
trimethylamine(0.75g, 7.5mmol) was added, followed by
7-(2-chloropyrimidin-4-yl)-3-
phenyl

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3-phenyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine 4a(1g, 5.0mmol). The mixture was
stirred at room temperature for 2 h and then poured into stirring ice-water. The resulting
precipitate was filtered and dried to obtain compound 5a as a white solid (0.96g). Yield: 62%; MS
(ESI) m/z: 312.22 [M+H]+. 1H NMR (400 MHz, DMSO) δ 8.23 (d, J = 6.1 Hz, 1H), 7.81-7.75 (m, 2H),
7.59-7.53 (m, 2H), 7.10 (d, J = 6.2 Hz, 1H), 5.06 (s, 2H), 4.26 (t, J = 5.2 Hz, 2H), 4.13 (t, J = 5.3 Hz,
2H).
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5.1.1.5
N-(4-morpholinophenyl)-4-(3-phenyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)pyrimidi
n-2-amine(9a)
To a stirred solution of compound 5a (1 mmol) in 8 mL isopropanol was added
4-morpholinoaniline 8 (1.1 mmol) and p-toluenesulfonic acid (1.1 mmol). The mixture was heated
to 90 °C for 24 h. The reaction mixture was quenched by saturated Na2CO3 aqueous solution, and
then was extracted with DCM and the organic phase was washed with water, dried over anhydrous
Na2SO4. The combined organic layer was concentrated under reduced pressure and was further
purified by flash column chromatography using dichloromethane/methanol as eluent to afford
product 9a as a white solid.
Yield: 32%; m.p.: 225.4 – 227.5 °C; HRMS (ESI-Q-TOF, m/z) : 455.2301 [M+H]+; H NMR
1
(400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.04 (d, J = 5.9 Hz, 1H), 7.90 – 7.72 (m, 3H), 7.66 – 7.46
(m, 5H), 6.88 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 6.0 Hz, 1H), 5.04 (s, 2H), 4.24 (t, J = 5.2 Hz, 2H),
4.07 (t, J = 5.3 Hz, 2H), 3.78 – 3.68 (m, 4H), 3.12 – 2.98 (m, 4H). 13C NMR (101 MHz, DMSO) δ
162.2, 160.0, 157.6, 152.8, 149.3, 146.2, 133.8, 130.1, 129.3, 128.3, 127.3, 120.5, 116.0, 95.3,

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66.6, 49.8, 43.5, 41.5, 41.1; Anal. Calcd for C25H26N8O: C, 66.06; H, 5.77; N, 24.65; Found: C,
5
6
7
66.07; H, 5.74; N, 24.62.
8
9
Compounds 9b-9l were synthesized by the general procedure of compound 9a with corresponding
raw materials.
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4-(3-(4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-morpholinopheny
l)pyrimidin-2-amine (9b)
Yield: 37%; m.p.: 283.0 – 285.5 °C; HRMS (ESI-Q-TOF, m/z): 473.2215 [M+H]+;1H NMR
(400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.04 (d, J = 5.9 Hz, 1H), 7.86 – 7.79 (m, 2H), 7.56 (d, J =
8.9 Hz, 2H), 7.40 (t, J = 8.8 Hz, 2H), 6.92 – 6.86 (m, 2H), 6.43 (d, J = 6.0 Hz, 1H), 5.04 (s, 2H),
4.23 (t, J = 5.0 Hz, 2H), 4.7 (t, J = 5.1 Hz, 2H), 3.78 – 3.68 (m, 4H), 3.08 – 2.96 (m, 4H).13C
NMR (101 MHz, DMSO) δ 164.5, 162.2, 160.0, 157.6, 152.1, 149.3, 146.2, 133.8, 130.8, 123.9,
120.5, 116.4, 116.08, 95.3, 66.6, 49.8, 43.4, 41.5, 41.0. Anal. Calcd for C25H25FN8O: C, 63.55; H,
5.33; N, 23.71; Found: C, 63.52; H, 5.35; N, 23.73.
N-(4-morpholinophenyl)-4-(3-(p-tolyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)pyrimi
din-2-amine(9c)
Yield: 34%; m.p.: 270.5 – 273.5 °C; HRMS (ESI-Q-TOF, m/z): 469.2468 [M+H]+;1H NMR
(400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.04 (d, J = 5.9 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.57 (d, J
= 9.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 6.0 Hz, 1H), 5.03 (s,
2H), 4.22 (t, J = 5.1 Hz, 2H), 4.06 (t, J = 5.3 Hz, 2H), 3.78 – 3.68 (m, 4H), 3.08 – 2.96 (m, 4H),
2.38 (s, 3H). 13C NMR (101 MHz, DMSO) δ 162.2, 160.0, 157.6, 152.8, 149.1, 146.2, 139.8,
133.8, 129.9, 128.2, 124.5, 120.5, 116.0, 95.3, 66.6, 49.8, 43.4, 41.5, 41.1, 21.3. Anal. Calcd for
C26H28N8O: C, 66.65; H, 6.02; N, 23.91; Found: C, 66.67; H, 6.04; N, 23.96.

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N-(4-morpholinophenyl)-4-(3-(4-(trifluoromethyl)phenyl)-5,6-dihydro-[1,2,4]triazolo[4,3D-aO]Ip: 1y0r.1a0z39/C9NJ02154J
5
6
7
in-7(8H)-yl)pyrimidin-2-amine (9d)
8
9
Yield: 35%; m.p.: 272.4 – 275.2 °C; HRMS (ESI-Q-TOF, m/z): 523.2187 [M+H]+;1H NMR
(400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.05-8.01 (m, 3H), 7.92 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 9.0
Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 6.0 Hz, 1H), 5.07 (s, 2H), 4.30 (t, J = 5.2 Hz, 2H),
4.09 (t, J = 5.4 Hz, 2H), 3.78 – 3.68 (m, 4H), 3.08 – 2.96 (m, 4H). 13C NMR (101 MHz, DMSO) δ
162.2, 160.0, 157.7, 151.8, 149.9, 146.3, 133.8, 131.3, 130.3, 130.1, 129.1, 126.3, 125.8, 123.1,
120.6, 116.0, 95.3, 66.6, 49.8, 43.6, 41.4, 41.0. Anal. Calcd for C26H25F3N8O: C, 59.76; H, 4.82;
N, 21.44; Found: C, 59.73; H, 4.81; N, 21.46.
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4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-morphol
inophenyl)pyrimidin-2-amine (9e)
Yield: 38%; m.p.: 253.4 – 255.2 °C; HRMS (ESI-Q-TOF, m/z):507.1828[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 8.97 (s, 1H), 8.04 (d, J = 6.0 Hz, 1H), 8.00 (dd, J = 7.1, 2.1 Hz, 1H), 7.82-7.77
(m, 1H), 7.63-7.55 (m, 3H), 6.88 (d, J = 9.0 Hz, 2H), 6.44 (d, J = 6.0 Hz, 1H), 5.06 (s, 2H), 4.26 (t,
J = 5.1 Hz, 2H), 4.06 (t, J = 5.3 Hz, 2H), 3.78 – 3.68 (m, 4H), 3.08 – 2.96 (m, 4H). 13C NMR (101
MHz, DMSO) δ 162.2, 160.0, 157.6, 157.2, 151.1, 149.6, 146.2, 133.8, 130.4, 129.4, 125.2, 120.5,
118.1, 116.0, 95.3, 66.6, 49.8, 43.3, 41.4, 41.0. Anal. Calcd for C25H24ClFN8O: C, 59.23; H, 4.77;
N, 22.10; Found: C, 59.20; H, 4.78; N, 22.13.
4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-(piperidi
n-1-yl)phenyl)pyrimidin-2-amine (9f)
Yield: 30%; m.p.: 293.4 – 295.7°C; HRMS (ESI-Q-TOF, m/z): 505.2028[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 8.94 (s, 1H), 8.08 – 7.97 (m, 2H), 7.82-7.78(m, 1H), 7.62 (t, J = 8.9 Hz, 1H),

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7.52 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 6.0 Hz, 1H), 5.06 (s, 2H), D4O.2I:510(.t1,03J9/C9NJ02154J
= 5.3 Hz, 2H), 4.06 (t, J = 4.9 Hz, 2H), 3.06 – 2.99 (m, 4H), 1.67-1.59 (m, 4H), 1.53-1.46 (m, 2H).
13C NMR (101 MHz, DMSO) δ 162.2, 160.0, 158.4, 157.6, 151.1, 149.6, 147.2, 133.3, 130.4,
129.4, 125.2, 120.7, 120.6, 118.1, 116.9, 95.3, 51.0, 43.3, 41.4, 41.0, 25.9, 24.3. Anal. Calcd for
C26H26ClFN8: C, 61.84; H, 5.19; N, 22.19; Found: C, 61.88; H, 5.16; N, 22.20.
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4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-(pyrrolid
in-1-yl)phenyl)pyrimidin-2-amine (9g)
Yield: 30%; m.p.: 264.4 – 266.7°C; HRMS (ESI-Q-TOF, m/z): 491.1880[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 8.77 (s, 1H), 8.00 (d, J = 5.5 Hz, 2H), 7.82-7.77 (m, 1H), 7.61 (t, J = 8.9 Hz,
1H), 7.45 (d, J = 8.7 Hz, 2H), 6.49 (d, J = 8.7 Hz, 2H), 6.38 (d, J = 5.9 Hz, 1H), 5.04 (s, 2H), 4.24
(t, J = 5.3 Hz, 2H), 4.05 (t, J = 5.1 Hz, 2H), 3.24-3.14(d, J = 6.3 Hz, 4H), 1.99-1.89 (q, J = 4.8, 3.3
Hz, 4H). 13C NMR (101 MHz, DMSO) δ 162.2, 160.2, 158.4, 157.7, 151.1, 149.6, 143.9, 130.4,
130.1, 129.5, 125.2, 121.6, 120.5, 118.1, 111.9, 94.8, 48.0, 43.3, 41.3, 40.9, 25.3. Anal. Calcd for
C25H24ClFN8: C, 61.16; H, 4.93; N, 22.82; Found: C, 61.18; H, 4.97; N, 21.82.
4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-thiomor
pholinophenyl)pyrimidin-2-amine (9h)
Yield: 38%; m.p.: 240.4 – 242.3°C; HRMS (ESI-Q-TOF, m/z): 523.1601[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 8.99 (s, 1H), 8.09 – 7.92 (m, 2H), 7.83-7.77 (m, 1H), 7.60 (m, 3H), 6.88 (d, J
= 9.0 Hz, 2H), 6.45 (d, J = 6.0 Hz,1H), 5.06 (s, 2H), 4.26 (t, J = 5.3 Hz, 2H), 4.07 (d, J = 5.3 Hz,
2H), 3.41 – 3.36 (m, 4H), 2.74 – 2.66 (m, 4H). 13C NMR (101 MHz, DMSO) δ 162.2, 160.0, 158.4,
157.6, 151.1, 149.6, 146.4, 133.8, 130.4, 129.5, 125.2, 120.6, 118.1, 117.9, 117.7, 95.4, 52.6, 43.3, 41.4,

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41.0, 26.6. Anal. Calcd for C25H24ClFN8S: C, 57.41; H, 4.63; N, 21.42; Found: C, 47.42;DHOI,: 140..61063;9/C9NJ02154J
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N, 21.40.
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N1-(4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)pyrimidin-
2-yl)-N4,N4-dimethylbenzene-1,4-diamine (9i)
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Yield: 43%; m.p.: 260.8 – 263.3°C; HRMS (ESI-Q-TOF, m/z): 465.1628[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 8.87 (s, 1H), 8.08 – 7.97 (m, 2H), 7.83-7.77 (m, 1H), 7.62 (t, J = 9.0 Hz, 1H),
7.49 (d, J = 9.1 Hz, 2H), 6.70 (d, J = 9.1 Hz, 2H), 6.41 (d, J = 6.0 Hz, 1H), 5.05 (s, 2H), 4.25 (t, J
= 5.3 Hz, 2H), 4.06 (t, J = 5.3 Hz, 2H), 2.83 (s, 6H). 13C NMR (101 MHz, DMSO) δ 162.2, 160.1,
158.4, 157.7, 151.1, 149.6, 146.4, 131.3, 130.5, 129.4, 125.2, 121.1, 120.5, 117.9, 113.44, 95.06,
43.3, 41.4, 41.2, 41.0. Anal. Calcd for C23H22N8: C, 59.42; H, 4.77; N, 24.10; Found: C, 59.44; H,
4.74; N, 24.08.
4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-(4-meth
ylpiperazin-1-yl)phenyl)pyrimidin-2-amine (9j)
Yield: 39.8%; m.p.: 229.4– 232.2°C; HRMS (ESI-Q-TOF, m/z): 520.2028 [M+H]+ ; 1H NMR
(400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.07 – 7.97 (m, 2H), 7.83-7.77 (m, 1H), 7.61-7.52 (m, 3H),
6.89 (d, J = 8.9 Hz, 2H), 6.44 (d, J = 6.0 Hz, 1H), 5.06 (s, 2H), 4.25 (t, J = 5.1 Hz, 2H), 4.07 (d, J
= 5.3 Hz, 2H), 3.15-3.06 (m, 4H), 2.70 – 2.58 (m, 4H), 2.34 (s, 3H). 13C NMR (151 MHz, DMSO)
δ 162.2, 160.0, 158.4, 157.6, 151.1, 149.6, 146.1, 133.6, 130.5, 129.5, 125.3, 120.7, 120.6, 117.9,
116.4, 95.3, 54.9, 49.1, 45.8, 43.3, 41.4, 41.0, 40.4. Anal. Calcd for C26H27ClFN9: C, 60.05; H,
5.23; N, 24.24; Found: C, 60.01; H, 5.19; N, 24.28.
4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-(3,5-dim
ethylpiperazin-1-yl)phenyl)pyrimidin-2-amine (9k)

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Yield: 30%; m.p.: 174.2– 178.1°C; HRMS (ESI-Q-TOF, m/z): 534.2296[M+H]+ ;1H NDMORI: 1(04.100039/C9NJ02154J
MHz, DMSO-d6) δ 8.96 (s, 1H), 8.06 – 7.97 (m, 2H), 7.80 (ddd, J = 8.7, 4.7, 2.2 Hz, 1H), 7.62 (t,
J = 8.9 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.43 (d, J = 6.0 Hz, 1H), 5.07
(s, 2H), 4.26 (t, J = 5.3 Hz, 2H), 4.07 (d, J = 5.2 Hz, 2H), 3.45-3.39 (m, 2H), 2.87 (m, 2H), 2.07 (t,
J = 10.6 Hz, 2H), 1.03 (d, J = 6.2 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 162.2, 160.0, 158.4,
157.7, 151.1, 149.6, 146.4, 133.2, 130.5, 129.5, 125.2, 120.7, 120.6, 118.1, 116.3, 95.2, 56.4,
50.68, 43.37, 41.4, 41.0, 19.7. Anal. Calcd for C27H29ClFN9: C, 60.73; H, 5.47; N, 23.61; Found:
C, 60.75; H, 5.44; N, 23.58.
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4-(3-(3-chloro-4-fluorophenyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-N-(4-(4-meth
ylpiperidin-1-yl)phenyl)pyrimidin-2-amine (9l)
Yield: 28%; m.p.: 292.6– 298.1°C; HRMS (ESI-Q-TOF, m/z): 519.2192[M+H]+ ;1H NMR (400
MHz, DMSO-d6) δ 8.92 (s, 1H), 8.08 – 7.96 (m, 2H), 7.83-7.77 (m, 1H), 7.62 (t, J = 9.0 Hz, 2H),
7.52 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.9 Hz, 2H), 6.43 (d, J = 6.0 Hz, 1H), 5.05 (s, 2H), 4.25 (t, J
= 5.3 Hz, 2H), 4.06 (t, J = 5.3 Hz, 2H), 3.53 (d, J = 11.0 Hz, 2H), 2.58 (d, J = 12.3 Hz, 2H), 1.69
(d, J = 13.0 Hz, 2H), 1.51-1.41 (m, 1H), 1.31-1.19 (m, 2H), 0.94 (d, J = 6.4 Hz, 3H). 13C NMR
(151 MHz, DMSO) δ 162.2, 160.0, 158.4, 157.6, 151.1, 149.6, 146.9, 130.7, 130.5, 129.4, 125.3,
120.7, 120.6, 118.1, 116.9, 95.3, 50.3, 43.3, 41.4, 41.0, 34.2, 30.5, 22.2. Anal. Calcd for
C27H28ClFN8: C, 62.48; H, 5.44; N, 21.59; Found: C, 62.50; H, 5.46; N, 22.53.
5.1.2 General Procedure for Preparation of (7)
Substituted amine (42 mmol) was added to a mixture of 4-fluoronitrobenzene (35 mmol) and
K2CO3 (70 mmol) in 40 mL DMF. The reaction mixture was stirred at 120 °C and followed by
TLC. After completion of the reaction, the mixture was poured into stirring ice-water. The

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resulting precipitate was filtered and dried to obtain compounds 7 as a yellow solid; Yield:
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7
79.1-90.5%.
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5.1.3 General Procedure for Preparation of (8) c
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The substituted nitro compound 7 (15 mmol) was treated with 10% Pd-carbon (5% w/w). The
reaction was subjected to hydrogenation under hydrogen gas at room temperature and the reaction
was monitored by TLC. After completion of the reaction, the mixture was filtered through a Celite
bed and concentrated in a vacuum to afford product 8. Yield: 72.4-88.6%.
5.1.4 General Procedure for Preparation of (13a)
4.1.4.1 1-(2-chloropyrimidin-4-yl)piperidin-4-one (11)
A mixture of 4-piperidone hydrate hydrochloride (21.7g, 140mmol), 2,4-dichloropyrimidine (20g,
134.2mmol) and potassium carbonate (46.3g, 335.5mmol) in 200mL acetonitrile was stirred for 8
h at room temperature, after completion of the reaction, The reaction mixture was filtered through
and concentrated. Purification by flash chromatography afforded 11 as a coulurless solid(20.6g).
Yield: 73%. MS (ESI) m/z: 212.15 [M+H]+, 1H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 6.2 Hz,
1H), 6.92 (d, J = 6.2 Hz, 1H), 4.01-3.80 (m, 4H), 2.53-2.48 (m, 4H).
5.1.4.2 1-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)piperidin-4-one (12a)
To a stirred solution of compound 11 (0.5g, 2.4 mmol) in 10 mL isopropanol was added
4-morpholinoaniline 8 (0.5g, 2.8 mmol) and p-toluenesulfonic acid (0.49g, 2.8 mmol). The
mixture was heated to 90 °C for 10 h. The reaction mixture was quenched by saturated Na2CO3
aqueous solution, and then was extracted with DCM and the organic phase was washed with water,
dried over anhydrous Na2SO4. The combined organic layer was concentrated under reduced

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pressure and was further purified by flash column chromatographyDOI: 10.1039/C9NJ02154J
using
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9
dichloromethane/methanol as eluent to afford product 12a as a white solid.
Yield: 59%. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 7.98 (d, J = 6.0 Hz, 1H), 7.58 (d, J =
8.9 Hz, 2H), 6.86 (d, J = 8.9 Hz, 2H), 6.30 (d, J = 6.0 Hz, 1H), 3.91 (t, J = 6.1 Hz, 4H), 3.73 (t, J
= 4.7 Hz, 4H), 3.01 (t, J = 4.7 Hz, 4H), 2.45 (t, J = 6.1 Hz, 4H).
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5.1.4.3
8-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)-4-phenyl-1,4,8-triazaspiro[4.5]decan-2-one
(13a)
A solution of 12a (0.35g, 1.0 mmol), 2-(phenylamino)acetamide (0.14g, 1.2mmol) and
camphorsulfonic acid (0.28g, 1.2mmol) in 5mL DCE was heated to 90 °C with stirring for 24 h.
The solvent was then removed by a rotatory evaporator, the residue was dissolved in chloroform,
washed with sodium hydrogen carbonate solution and water, and dried over anhydrous
magnesium sulfate. The combined organic layer was concentrated under reduced pressure and was
further purified by flash column chromatography using dichloromethane/methanol as eluent to
afford product 13a as a brown solid.
1
Yield: 23%. m.p.: 242.3 – 244.1 °C; HRMS (ESI-Q-TOF, m/z): 486.2614 [M+H]+; H NMR
(400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.86 (s, 1H), 7.95 (d, J = 6.1 Hz, 1H), 7.54 (d, J = 8.9 Hz,
2H), 7.17 (t, J = 7.9 Hz, 2H), 6.88-6.68 (m, 5H), 6.29 (d, J = 6.1 Hz, 1H), 4.59-4.20 (m, 2H), 3.86
(s, 2H), 3.77 – 3.66 (m, 4H), 3.20 (t, J = 13.2 Hz,2H), 3.06 – 2.97 (m, 4H), 2.48-2.42(m, 2H), 1.62
(d, J = 13.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 169.9, 162.0, 160.0, 159.4, 156.1, 146.3,
143.8, 133.6, 129.5, 120.6, 118.6, 116.0, 95.0, 77.3, 66.6, 51.1, 49.7, 34.1. Anal. Calcd for
C27H31N7O2: C, 66.78; H, 6.44; N, 20.19; Found: C, 66.80; H, 6.42; N, 20.15.

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Compounds 13b-13l were synthesized by the general procedure of compound 13a with
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7
corresponding raw materials.
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8-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)-4-(p-tolyl)-1,4,8-triazaspiro[4.5]decan-2-one
(13b)
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Yield: 20%. m.p.: 248.4 – 250.2 °C; HRMS (ESI-Q-TOF, m/z): 500.2771 [M+H]+; 1H NMR
(400 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.81 (s, 1H), 7.94 (d, J = 6.0 Hz, 1H), 7.54 (d, J = 9.0 Hz,
2H), 6.99 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 6.25 (d, J = 6.1
Hz, 1H), 4.63-4.25 (m, 2H) 3.82 (s, 2H), 3.76 – 3.66 (m, 4H), 3.16 (t, J = 12.7 Hz, 2H), 3.03 –
2.95 (m, 4H), 2.28 (td, J = 13.1, 5.0 Hz, 2H), 2.16 (s, 3H), 1.61 (d, J = 13.0 Hz, 2H). 13C NMR
(101 MHz, DMSO) δ 170.4, 161.9, 158.9, 155.2, 146.4, 141.5, 133.3, 129.9, 129.6, 128.5, 120.8,
117.7, 116.0, 114.6, 95.0, 77.3, 66.6, 55.3, 51.4, 49.7, 34.4, 20.4. Anal. Calcd for C28H33N7O2: C,
67.31; H, 6.66; N, 19.62; Found: C, 67.33; H, 6.62; N, 19.59.
4-(4-fluorophenyl)-8-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)-1,4,8-triazaspiro[4.5]decan
-2-one (13c)
1
Yield: 19%. m.p.: 259.7 – 262.7 °C; HRMS (ESI-Q-TOF, m/z): 504.2520 [M+H]+; H NMR
(400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.81 (s, 1H), 7.92 (d, J = 6.0 Hz, 1H), 7.53 (d, J = 8.9 Hz,
2H), 7.04 (t, J = 8.8 Hz, 2H), 6.93 – 6.80 (m, 4H), 6.25 (d, J = 6.0 Hz, 1H), 4.60-4.19 (m, 2H),
3.85 (s, 2H), 3.75-3.68 (m, 4H), 3.16 (t, J = 13.2 Hz, 2H), 3.02-2.97 (m, 4H), 2.22 (td, J = 13.2,
5.0 Hz, 2H), 1.65 (d, J = 13.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 170.3, 161.9, 159.9, 156.9,
156.6, 146.1, 140.5, 134.0, 120.3, 119.3, 116.0, 115.8, 94.9, 77.5, 66.6, 51.7, 49.8, 34.4. Anal.
Calcd for C27H30FN7O2: C, 64.40; H, 6.00; N, 19.47; Found: C, 64.39; H, 6.02; N, 19.50.

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4-(3-chloro-4-fluorophenyl)-8-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)-1,4,8-triaDzaOsI:p1i0r.o10[39/C9NJ02154J
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7
4.5]decan-2-one (13d)
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9
1
Yield: 19%. m.p.: 256.6 – 259.6 °C; HRMS (ESI-Q-TOF, m/z): 538.2131[M+H]+; H NMR
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(400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.83 (s, 1H), 8.05 – 7.84 (m, 1H), 7.66 – 7.39 (m, 2H), 7.23
(dt, J = 10.6, 5.2 Hz, 1H), 6.89-6.74(m, 4H), 6.41 – 6.22 (m, 1H), 4.60-4.22 (m, 2H), 3.88 (s, 2H),
3.81 – 3.62 (m, 4H), 3.18 (t, J = 11.5 Hz, 2H), 3.00 (d, J = 5.2 Hz, 4H), 2.36 (d, J = 13.7 Hz, 2H),
1.64 (d, J = 13.0 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 169.6, 162.1, 159.97, 154.6, 152.3,
149.9, 146.1, 141.2, 133.9, 120.4, 119.9, 117.5, 117.3, 116.5, 116.0, 95.0, 77.5, 66.6, 51.4, 49.8,
33.9. Anal. Calcd for C27H29ClFN7O2: C, 60.28; H, 5.43; N, 18.22; Found: C, 60.25; H, 5.45; N,
18.21.
4-(4-chlorophenyl)-8-(2-((4-morpholinophenyl)amino)pyrimidin-4-yl)-1,4,8-triazaspiro[4.5]decan
-2-one (13e)
1
Yield: 19%. m.p.: 211.0 – 213.6 °C; HRMS (ESI-Q-TOF, m/z):520.2224[M+H]+; H NMR
(400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.83 (s, 1H), 7.95 (d, J = 6.0 Hz, 1H), 7.55 (d, J = 9.0 Hz,
2H), 7.20 (d, J = 9.0 Hz, 2H), 6.85 (d, J = 9.1 Hz, 2H), 6.75 (d, J = 9.1 Hz, 2H), 6.27 (d, J = 6.1
Hz, 1H), 4.69-4.22 (m, 2H), 3.86 (s, 2H), 3.75-3.67 (m , 4H), 3.19 (t, J = 13.1 Hz, 2H), 3.06 –
2.90 (m, 4H), 2.44 (td, J = 13.1, 5.0 Hz, 2H), 1.63 (d, J = 12.9 Hz, 2H). 13C NMR (101 MHz,
DMSO) δ 169.6, 162.1, 160.1, 157.2, 146.0, 142.7, 134.1, 129.1, 122.2, 120.3, 117.1, 116.1, 94.9,
77.5, 66.6, 55.3, 51.1, 49.8, 34.0. Anal. Calcd for C27H30ClN7O2: C, 62.36; H, 5.82; N, 18.85;
Found: C, 62.35; H, 5.85; N, 18.82.
4-(4-fluorophenyl)-8-(2-((4-thiomorpholinophenyl)amino)pyrimidin-4-yl)-1,4,8-triazaspiro[4.5]de
can-2-one (13f)

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New Journal of Chemistry
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Yield: 21%. m.p.: 252.0 – 254.2 °C; HRMS (ESI-Q-TOF, m/z):520.2291[M+H]+; 1H NDMORI: 1(04.100039/C9NJ02154J
MHz, DMSO-d6) δ 9.56 (s, 1H), 8.81 (s, 1H), 7.93 (d, J = 5.9 Hz, 1H), 7.54 (d, J = 8.9 Hz, 2H),
7.04 (t, J = 8.8 Hz, 2H), 6.86 (m, 4H), 6.24 (d, J = 6.1 Hz, 1H), 4.74-4.22 (m, 2H), 3.85 (s, 2H),
3.16 (t, J = 13.3 Hz, 2H), 3.33-3.31 (m, 4H), 2.74 – 2.60 (m, 4H), 2.22 (td, J = 13.1, 5.1 Hz, 2H),
1.65 (d, J = 13.0 Hz, 2H).13C NMR (101 MHz, DMSO) δ 170.3, 161.9, 160.0, 157.1, 156.7, 146.1,
140.5, 134.1, 120.3, 119.5, 117.8, 116.0, 94.9, 77.5, 52.6, 51.7, 34.4, 26.6. Anal. Calcd for
C27H30FN7OS: C, 62.41; H, 5.82; N, 18.87; Found: C, 62.38; H, 5.85; N, 18.89.
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4-(4-fluorophenyl)-8-(2-((4-(pyrrolidin-1-yl)phenyl)amino)pyrimidin-4-yl)-1,4,8-triazaspiro[4.5]d
ecan-2-one (13g)
1
Yield: 22%. m.p.: 232.4 – 235.6 °C; HRMS (ESI-Q-TOF, m/z): 488.2570[M+H]+; H NMR
(400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.59 (s, 1H), 7.90 (d, J = 5.9 Hz, 1H), 7.44 (d, J = 8.6 Hz,
2H), 7.04 (t, J = 8.8 Hz, 2H), 6.86 (dd, J = 9.2, 4.4 Hz, 2H), 6.46 (d, J = 8.8 Hz, 2H), 6.19 (d, J =
6.0 Hz, 1H), 4.60-4.21 (m, 2H), 3.85 (s, 2H), 3.24-3.05 (m, 6H), 2.21 (td, J = 13.1, 5.1 Hz, 2H),
1.99 – 1.82 (m, 4H), 1.64 (d, J = 12.8 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 170.3, 162.0,
160.3, 157.8, 157.2, 155.5, 143.6, 140.5, 130.4, 121.3, 119.4, 115.8, 111.9, 94.3, 77.5, 51.7, 48.0,
34.4, 25.3. Anal. Calcd for C27H30FN7O: C, 66.51; H, 6.20; N, 20.11; Found: C, 66.55; H, 6.18; N,
20.11.
4-(4-fluorophenyl)-8-(2-((4-(4-methylpiperidin-1-yl)phenyl)amino)pyrimidin-4-yl)-1,4,8-triazaspi
ro[4.5]decan-2-one (13h)
Yield: 17%. m.p.: 222.4 – 225.1 °C; HRMS (ESI-Q-TOF, m/z):516.2883[M+H]+; 1H NMR (400
MHz, DMSO-d6) δ 9.55 (s, 1H), 8.75 (s, 1H), 7.92 (d, J = 6.0 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H),
7.04 (t, J = 8.8 Hz, 2H), 6.90 – 6.70 (m, 4H), 6.23 (d, J = 6.1 Hz, 1H), 4.62-4.25 (m, 2H), 3.85 (s,