Design, synthesis and antiproliferative activity of novel 2,4-diamino-5-methyleneaminopyrimidine derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2021.128213

In order to discover new anticancer agents, 25 novel 2,4-diamino-5-methyleneaminopyrimidine derivatives were designed and synthesized based on our previous work via a ring-opening strategy. Among them, compared with 5-FU, compound 7i exhibited 4.9-, 2.9-,

Full text of DOI:10.1016/j.bmcl.2021.128213

Bioorg. Med. Chem. Lett. 47 (2021) 128213
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antiproliferative activity of novel 2,4-diamino-5--
methyleneaminopyrimidine derivatives as potential anticancer agents
Qiu Li1, Lin Chen1, Xie-Er Jian, Dong-Xin Lv, Wen-Wei You, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern Medical University, Guangzhou 510515, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In order to discover new anticancer agents, 25 novel 2,4-diamino-5-methyleneaminopyrimidine derivatives were
designed and synthesized based on our previous work via a ring-opening strategy. Among them, compared with
5-FU, compound 7i exhibited 4.9-, 2.9-, 2.1-, and 3.0-fold improvement in inhibiting HCT116, HT-29, MCF-7,
2,4-Diamino-5-methyleneaminopyrimidines
Synthesis
Antiproliferative activity
and HeLa cells proliferation with IC50 values of 4.93, 5.57, 8.84, and 14.16 μM, respectively. Moreover, further
mechanistic studies indicated that compound 7i could concentration-dependently induce cell cycle arrest and
apoptosis in HCT116 cells. These findings revealed that 2,4-diamino-5-methyleneaminopyrimidine scaffold has
potential for further investigation to explore novel anticancer agents.
Introduction
novel 2,4-diamino-5-methyleneaminopyrimidine derivatives 7a–y.
Notably, the imine fragment as a versatile pharmacophore is frequently
Cancer has been currently described as one of the leading causes of
death worldwide. Although much progress has been achieved for the
treatment of cancer, emergence of drug resistance and toxicities makes
development of new classes of anticancer agents more urgent.1–3 Ami-
nopyrimidines are one of the most widely naturally-occurring hetero-
found in a large quantities of small antitumor molecules19–21. Herein, we
described the detailed synthetic routes, antiproliferative activities, and
structure–activity relationships of these new compounds 7a–y.
Synthesis of the new 2,4-diamino-5-methyleneaminopyrimidine de-
rivatives 7a–y was illustrated in Scheme 1. According to our previously
described methodology22, intermediates 9 were firstly accomplished
using a nucleophilic substitution reaction of commercially available 2,4-
dichloro-5-nitropyrimidine 8 with appropriate alkylamines in the pres-
ence of potassium carbonate (K2CO3) in acetone. Compounds 10 were
then obtained through a condensation reaction between pyrimidines 9
and various substituted arylamines in good yields. Subsequently, 10
were reduced by hydrogenation with Pd/C in ethanol to give 5-amino-
pyrimidines 11 which were further condensated with appropriate ary-
cyclic molecules, and exhibit
a range of important biological
activities4–6, especially antitumor property.7–9 Among them, 5-
substituted-2,4-diaminopyrimidine scaffold has draw growing atten-
tion due to the powerful anticancer effect10–12. Its 5-nitroso derivatives
NU6027 (1, Fig. 1), has been explored as potential antitumor agent with
remarkable antiproliferative activities.13 While 5-substituted-2,4-diami-
nopyrimidines 2, 3, and 4 (UNC2881) were found to possess their potent
anticancer properties via inhibition of pan-FGFR, steady-state Mer ki-
nase phosphorylation, and CDK2, respectively14–16
.
laldehydes or α-keto esters using catalytic amount of acetic acid by
In previous work, we reported a series of 2,8-disubstituted-7,8-
dihydropteridin-6(5H)-one analogues with the general structure 5 listed
in Fig. 2, which exerted significant antiproliferative activities against a
panel of cancer cell lines17–18 These results triggered us to perform a
pharmacophore exploration and optimization effort around the dihy-
dropteridinone nucleus. Thus, as shown in Fig. 2, keeping in mind the
effects of 5-substituted-2,4-diaminopyrimidines, we firstly undertook a
ring-opening of piperazinone to obtain a scaffold 6, which was subse-
quently bioisosterically substituted with an imine moiety to generate
heating at 80 ◦C in ethanol to successfully provide the desired analogues
7a–y in good isolated yields ranging from 80% to 93%. The chemical
structures of these compounds were fully characterized by general
spectra including 1H NMR, 13C NMR, and HRMS (shown in Supporting
Information).
All newly prepared analogues 7a–y were carried out to investigate
their antiproliferative activities toward four human cancer cell lines
(HCT116, HT-29, HeLa, and MCF-7) taken from different tissues,
through MTT assay. The preliminary data expressed as the half-maximal
* Corresponding author.
E-mail address: plzhao@smu.edu.cn (P.-L. Zhao).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128213
Received 13 May 2021; Received in revised form 14 June 2021; Accepted 16 June 2021
Available online 19 June 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

Q. Li et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128213
O
O
N
N
H
N
O
N
N
O
N
N
H
N
O
N
O
N
H
C
O
N
O
S
O
O
O
N
N
NH
N
N
N
H
O
S
N
N
NH
H
N
O
H
H2N
N
NH2
H2N
NH2
H
1 NU6027
H
O
2
3
4 UN 2881
C
Fig. 1. Structures of representative 5-substituted-2,4-diaminopyrimidines with anticancer activity.
Fig. 2. Design strategy of the title compounds 7a–y.
R2
N
R2
NH2
N
C
O2
N
O2
O2
N
N
N
c
N
a
b
R1
R1
R1
l
C
N
l
l
C
N
N
H
N
N
N
H
N
N
N
H
H
H
8
1
0
9
11
-
a
R1
R1
(
7
l
)
=
R2
R3
N
N
-
d
-
=
=
m x
7
(
)
N
N
NH
R1
H
R1
7
y
(
)
a
7
y
Scheme 1. Synthesis of the target compounds 7a–y. Reagents and conditions: (a) R1-NH2, 0–5 ◦C, K2CO3, acetone; (b) R2-NH2, K2CO3, ethanol; (c) 10% Pd/C, H2,
70 ◦C; (d) HCOCO2C2H5 or Ar-CHO, EtOH, AcOH, 80 ◦C.
inhibitory concentration (IC50) were summarized in Table 1. 5-Fluoro-
uracil (5-FU), a chemotherapeutical agent used to treat various cancers,
was employed as the reference drug. As depicted in Table 1, most
compounds 7a–l with a cyclopentyl substituent on R1 displayed mod-
erate to excellent antiproliferative activities toward all the tested cancer
cell lines. Particularly, analogue 7i was identified as the most promising
compound against HCT116, HT-29, MCF-7, and HeLa cells with IC50
significantly diminished potency on HT-29 and MCF-7 cells (7i vs 7a and
7i vs 7e). Whereas, ethoxycarbonyl fragment was replaced by phenyl,
thiophenyl, and trifluoromethylphenyl, the activity had a sharp loss (7i
vs 7j, 7k, and 7l). The influence of R1 substituents on the inhibitory
activity was also investigated and findings implied that a much bulkier
group on R1 would be beneficial to antiproliferative activity, when
comparing the activities of 7e (R1 = cyclopentyl), 7q (R1 = isopropyl),
and 7y (R1 = cyclopentyl).
values of 4.93, 5.57, 8.84, and 14.16
μM, respectively, which are
approximately 4.9-, 2.9-, 2.1-, and 3.0-fold over than that of 5-FU.
Interestingly, replacement of morpholino group of compound 7i with
N-methylpiperazino and sulfamoyl moiety basically retained anti-
proliferative efficacy toward HeLa and HCT116 cells, but resulted in the
To investigate the mode of action of these analogues on cancer cells,
the most potent derivative 7i was chosen to evaluate its effects on the
cell cycle by flow cytometry. As depicated in Fig. 3, after treatment of
HCT116 cells with compound 7i for 24 h at indicated concentrations (0,
2

Q. Li et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128213
Table 1
Antiproliferative activities of compounds 7a–y against four human cancer cell lines.
Comp.
R1
R2
R3
In vitro cytotoxicity IC50
(
μ
M) a
HCT116
HT-29
HeLa
MCF-7
7a
7b
7c
CO2C2H5
C6H5
17.02 ± 1.29
15.10 ± 0.54
18.95 ± 2.67
42.31 ± 1.10
35.24 ± 3.41
18.87 ± 0.81
28.58 ± 1.48
21.73 ± 2.75
>100
>100
25.73 ± 4.73
33.93 ± 4.67
7d
7e
4-CF3C6H4
CO2C2H5
>100
80.68 ± 5.32
23.6 ± 2.29
>100
>100
9.64 ± 0.87
20.67 ± 2.34
42.22 ± 3.53
7f
C6H5
26.10 ± 2.86
13.34 ± 1.81
>100
26.99 ± 2.61
9.95 ± 2.26
>100
15.56 ± 4.38
10.92 ± 3.43
21.85
39.08 ± 4.77
22.97 ± 3.05
>100
7g
7h
4-CF3C6H4
7i
7j
CO2C2H5
C6H5
4.93 0.31
>100
5.57 1.55
67.62 ± 4.65
20.76 ± 2.53
14.16 1.9
>100
8.84 1.28
>100
7k
>100
>100
>100
7l
4-CF3C6H4
CO2C2H5
C6H5
97.16 ± 7.90
13.61 ± 1.04
>100
>100
>100
>100
7m
7n
7o
7.59 ± 2.11
12.14 ± 2.79
>100
11.54 ± 3.17
>100
12.29 ± 2.66
>100
>100
77.06 ± 0.94
91.05 ± 7.84
7p
7q
4-CF3C6H4
CO2C2H5
42.22 ± 3.57
49.13 ± 8.78
>100
>100
>100
>100
83.26
58.82 ± 3.03
7r
7s
7t
C6H5
>100
98.20 ± 3.23
>100
>100
>100
>100
>100
82.11 ± 7.92
>100
50.18 ± 2.25
>100
4-CF3C6H4
>100
7u
7v
7w
CO2C2H5
C6H5
23.66 ± 4.17
>100
19.56 ± 3.42
>100
27.72 ± 2.57
>100
15.23 ± 2.42
>100
14.83 ± 1.07
25.34 ± 1.87
>100
>100
7x
7y
4-CF3C6H4
CO2C2H5
>100
>100
>100
>100
>100
>100
>100
>100
5-FU/
37.22 ± 1.06
16.07 ± 0.92
42.74 ± 3.38
18.13 ± 2.07
a
50% inhibitory concentration and mean ± SD of three independent experiments performed in duplicate.
2.5, 5.0, and 10.0
μ
M), the percentage of cells in G0/G1 phase were
percentage of apoptotic and dead cells was observed. In the presence of
vehicle alone for 24 h, only 7.0% of the cells were in apoptosis. Whereas,
the percentages of apoptosis obviously rose to 19.5%, 35.9%, and
30.60%, 34.63%, 36.46 and 41.55%, respectively. These findings clearly
demonstrated that 7i concentration-dependently induced an obvious
G0/G1 phase cell cycle arrest.
52.4%, after treatment with 2.5, 5.0, and 10.0 μM of analogue 7i for 24
Next, the cell apoptosis of HCT116 cells was also analysed after
treatment with different concentrations of optimal compound 7i by
using Annexin V-FITC and PI to stain cells and flow cytometry calcula-
tion. As shown in Fig. 4, a significant dose-dependent increase in the
h, respectively. Thus, these observations indicated that compound 7i
caused apoptosis in HCT116 cells via a concentration-dependent
manner.
In summary, based on our previous work, a series of new 2,4-
3

Q. Li et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128213
Fig. 3. Effect of compound 7i on cell cycle in HCT116 cells. Flow cytometry analysis of HCT116 cells treated by compound 7i for 24 h. (A) Control; (B) 7i, 2.5
(C) 7i, 5.0 M; (D) 7i, 10.0 M.
μM;
μ
μ
diamino-5-methyleneaminopyrimidines were designed and synthesized
through a ring-opening strategy. Among them, compound 7i demon-
strated the most promising and broad spectrum inhibition toward four
tested cancer cells (HCT116, HT-29, MCF-7, and HeLa) with IC50 values
HCT116 cells. Taken together, 7i represents a promising anticancer
agent and is worthy of further investigation.
Declaration of Competing Interest
of 4.93, 5.57, 8.84, and 14.16 μM, respectively, which are approxi-
mately 4.9-, 2.9-, 2.1-, and 3.0-fold over than that of 5-FU. Additionally,
further mechanistic studies revealed that compound 7i could
concentration-dependently induce cell cycle arrest and apoptosis in
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4

Q. Li et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128213
Fig. 4. Flow cytometry analysisof apoptosis induction in HCT116 cells after treatment with indicated concentrations of 7i (0, 2.5, 5.0, and 10.0 μM) for 24 h. The
lower left quadrant represents live cells, the lower right is for early apoptotic cells, upper right is for late apoptotic cells, and the upper left represents cells necrosis.
[4] Rasapalli S, Murphy ZF, Sammeta VR, Golen JA, Weig AW, Melander RJ, Melander
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This present research was supported by the Guangdong Basi-
c and Applied Basic Research Foundation, China (No. 2018B0303110
67), and the Special Funds for the Cultivation of Guangdong College
Students’ Scientific and Technological Innovation (“Climbing Program”
Special Funds) (No. pdjh2020b0117).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128213.
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