Design, synthesis and antiproliferative activity of novel substituted 2-amino-7,8-dihydropteridin-6(5H)-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.07.076

Based on our previous work, a series of novel 2-amino-7,8-dihydropteridin-6(5H)-one derivatives were designed and synthesized via a ring-closing strategy. Biological evaluation with four human cancer cell lines (BT549, T47D, MDA-MB-468, and MDA-MB-231) showed that most of these compounds possessed moderate to potent antiproliferative activities. The most promising compound 8-benzyl-2-(phenethylamino)-7,8-dihydropteridin-6(5H)-one (6q) possessing IC₅₀ values of 7.75, 6.37, and 10.73 μM against MDA-MB-468, T47D, and BT549, respectively, which were 49, 11, and 8 folds more active than the positive control fluorouracil. Moreover, fluorescence-activated cell sorting analysis revealed that compound 6q displayed a significant effect on G1 cell-cycle arrest in a concentration-dependent manner in T47D cells. The initial structure–activity relationship studies indicated that linker-length of amine chain in C-2 position of pyrimidine ring played a crucial role in modulating the antitumor activity, which could be of help in the rational design of dihydropteridin-6(5H)-ones as novel anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2017.07.076

Accepted Manuscript
Design, synthesis and antiproliferative activity of novel substituted 2-ami-
no-7,8-dihydropteridin-6(5H)-one derivatives
Qiu Li, Hai-Kui Yang, Qi Sun, Wen-Wei You, Pei-Liang Zhao
PII:
S0960-894X(17)30781-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.076
BMCL 25189
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2017
20 July 2017
28 July 2017
Please cite this article as: Li, Q., Yang, H-K., Sun, Q., You, W-W., Zhao, P-L., Design, synthesis and antiproliferative
activity of novel substituted 2-amino-7,8-dihydropteridin-6(5H)-one derivatives, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.076
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Design, synthesis and antiproliferative activity of novel substituted
2-amino-7,8-dihydropteridin-6(5H)-one derivatives
Qiu Li #, Hai-Kui Yang #, Qi Sun, Wen-Wei You, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science,
Southern Medical University, Guangzhou 510515, P.R.China
ABSTRACT: Based on our previous work, a series of novel 2-amino-7,8-dihydropteridin-
6(5H)-one derivatives were designed and synthesized via a ring-closing strategy. Biological
evaluation with four human cancer cell lines (BT549, T47D, MDA-MB-468, and MDA-MB-231)
showed that most of these compounds possessed moderate to potent antiproliferative activities. The
most promising compound 8-benzyl-2-(phenethylamino)-7,8-dihydropteridin-6(5H)-one (6q)
possessing IC50 values of 7.75, 6.37, and 10.73 µM against MDA-MB-468, T47D, and BT549,
respectively, which were 49, 11, and 8 folds more active than the positive control fluorouracil.
Moreover, fluorescence-activated cell sorting analysis revealed that compound 6q displayed a
significant effect on G1 cell-cycle arrest in a concentration-dependent manner in T47D cells. The
initial structure–activity relationship studies indicated that linker-length of amine chain in C-2
position of pyrimidine ring played a crucial role in modulating the antitumor activity, which could
be of help in the rational design of dihydropteridin-6(5H)-ones as novel anticancer drugs.
Keywords: Dihydropteridinones; Synthesis; Antiproliferative activity.
——————————
∗ Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao).
# These authors contributed equally to this work.
1

Over the past few years, dihydropteridinones have attracted enormous attention and constitute
important biological and pharmaceutical compounds which are being increasingly studied in
medicinal research.1-5 Among these novel biologically active molecules, 7,8-dihydropteridin
-6(5H)-one scaffold has been named a privileged heterocyclic ring system.6-10 For example,
2-butoxy-7,8-dihydropteridin-6(5H)-one analog GS-9620 (1), shown in Fig. 1, has been emerged as
an orally administered agonist of Toll-like receptor (TLR) 7 currently being evaluated in clinical
studies for the treatment of chronic HBV and HIV patients. 11-13 Sun and coworkers reported that
2-amino analog (2) has been identified as a potent and selective inhibitor of Hsp90 with an IC50 of
0.88 µM.14 In addition, 2-imidazole analog (3) and hexahydroazepino[2,1-h]pteridin-6(5H)-
one (4) has been reported as potential therapeutic agents for Parkinson’s and Alzheimer's disease,
respectively.15, 16
F
Cl
H
N
NH2
N
H
N
O
N
N
N
O
O
N
N
N
N
N
N
O
N
4
H2N
N
N
N
N
O
N
N
O
N
H
N
N
N
N
O
1 GS9620
3
2
Fig. 1. Examples of 7,8-dihydropteridin-6(5H)-one scaffold found in biologically active compounds
In previous studies, our research group designed a 2,4-diaminopyrimidine scaffold by
introducing an ester alkyl amine as a flexible chain, and resulted in identification of a series of
novel alkyl aminopyrimidine derivatives 5, (Fig. 2) with favorable antiproliferative activities.17
Based on them and as a part of our continuing effort on the development of nitrogen-containing
heterocycles as novel antitumor substances,18-26 we designed
a
series of novel
2-amino-7,8-dihydropteridin-6(5H)-one derivatives via a ring-closing strategy which has been
assumed a powerful tool in molecular modification and drug design.27-29 Herein, we described the
detailed synthetic routes, antiproliferative activity screening, and structure-activity relationships of
these compounds 6a-t.
O
H
N
N
O
O
N
N
Ring-closing Strategy
R3
O
n
n
HN
R1
R1
N
H
N
N
N
N
R2
R2
O
5
Our previous work
6a~t
Newly designed compounds
2

Fig. 2. Design strategy of the title compounds 6a-t.
The synthesis strategy of target compounds 6a-t was performed as outlined in Scheme 1.
Commercially available 2,4-dichloro-5-nitropyrimidine 7 was used as the starting point to
synthesize the desired 2-amino-7,8- dihydropteridin-6(5H)-one core structure. According to our
recently reported approach,17 the key intermediate 2,4-disubstituted aminepyrimidine derivatives 10
was synthesized via a two-step nucleophilic substitution reaction at C-4 and C-2 position of
pyrimidine ring, respectively. Subsequently, the reduction of the nitro group of the intermediate 10
was performed using iron and glacial acetic acid leading to an in situ intramolecular cyclization
affording the desired compounds 6a-t in moderate to excellent isolated yields ranging from 60% to
94%. The structures of target compounds 6a-t were characterized with spectroscopic techniques
including 1H NMR, 13C NMR and HRMS, and the spectral data agree with the proposed structures.
Scheme 1 Synthesis of the target compounds 6a-t.
The synthesized compounds 6a-t were evaluated for their in vitro antiproliferative activities
against four human cancer cell lines including BT549, T47D, MDA-MB-468, and MDA-MB-231
using the MTT assay.25 The assay results expressed as IC50 (µM) were summarized in Table 1 and
compared with the inhibitory activities of two reference compounds, Fluorouracil, an effective
anticancer agent, and the most active compound 5a which our previously reported. Here, the IC50
value represents the concentration of a compound resulting in 50% inhibition of cell growth after 48
h incubation, and is the average of three independent experiments.
3

Table 1 Cytotoxic activities of compounds 6a-t against human tumor cells.
H
N
O
N
n
N
H
N
N
R1
R2
6a-t
In vitro cytotoxicity IC50(µM) a
Comp.
n
R1
R2
BT549
T47D
MDA-MB-468
MDA-MB-231
0
0
0
0
0
0
0
0
0
1
2
0
0
0
0
1
2
H
H
Ph
4-FPh
>100
92.38
17.72
95.03
>100
85.35
>100
>100
>100
>100
>100
6a
6b
6c
6d
6e
6f
H
4-ClPh
4-MeOPh
3-MeOPh
Ph
29.32
89.20
>100
15.00
>100
41.65
>100
H
H
>100
93.24
>100
>100
>100
>100
CH3
CH3
CH3
CH3
CH3
CH3
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
4-ClPh
3-MeOPh
3,4,5-triMeOPh
Ph
>100
6g
6h
6i
91.05
>100
36.03
>100
31.33
>100
>100
45.23
78.83
41.23
11.87
>100
56.24
89.73
23.23
15.21
>100
66.08
94.39
56.23
20.58
>100
46.19
91.97
34.23
36.95
>100
6j
Ph
6k
6l
Ph
4-ClPh
3-MeOPh
3,4,5-triMeOPh
3-MeOPh
Ph
6m
6n
6o
6p
6q
81.14
12.84
15.64
10.73
78.71
8.76
40.41
>100
99.89
10.60
46.98
22.43
15.26
6.37
23.18
7.75
N
N
0
PhCH2
>100
6r
98.26
78.96
98.28
N
SO2CH3
0
1
PhCH2
PhCH2
>100
>100
>100
>100
>100
>100
6s
6t
97.36
CO2CH2CH3
>100
O2N
N
O
Ph
13.69
6.59
6.26
24.25
5a
N
N
N
H
O
Ph
fluorouracil
98.24
171.06
95.69
53.47
a IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients
of variation were <10%.
As shown in Table 1, we initially synthesized a series of compounds bearing a hydrogen as R1
group (6a-e) to examine the influence of various R2 groups for antiproliferative activities.
4

Unfortunately, the results indicated that most of the compounds were ineffective (IC50 > 100 µM)
on MDA-MB-468 and MDA-MB-231 cell lines. In contrast, compared with fluorouracil, it is clear
that nearly all synthesized compounds exhibit higher antiproliferative activities against BT549 and
T47D cell lines, which encouraged us to further explore the structural modification for the purpose
of finding novel lead compounds with comparable potent activities. However, replacement of the
hydrogens (R1) with methyl groups led to a dramatic decrease in the antiproliferative activity (6a,
6c, 6e versus 6f, 6g, 6h, respectively). Most interestingly, carefully investigations on the
structure–activity relationship of the target compounds with methyl group at the R1 position (6f-k)
suggested that antiproliferative activity may be gradually improved with increasing linker-length of
amine moiety (6f versus 6j, 6k), which indicated that lipophilicity of the molecule had a profound
effect on the antiproliferative activities.
Surprisingly, compounds (6l-q) generated by replacing the hydrogens (R1) with bulk benzyl
groups, displayed much stronger antiproliferative activity than fluorouracil, except for the
compound 6m. It should be noted that the same tendency was observed in subseries of 6l-t that
inhibitory potency was improved with extending linker-length of substituted amine groups in C-2
position of pyrimidine ring. Moreover, inserting two carbons in the amine chain produced the most
promising compound 6q with the IC50 values of 7.75, 6.37, and 10.73 µM against MDA-MB-468,
T47D, and BT549, respectively, which indicated that this compound was 49, 11, and 8 folds more
active than the positive control fluorouracil. The following modifications based on 6l-q were mainly
carried out by changing the phenyl units in the tail of amine moiety (R2) to alkyl groups, such as
piperazine, piperidine, and ethyl acetate. Unfortunately, most of the obtained compounds resulted
either in a steep loss in cell proliferation inhibitory (6r, 6s) or were entirely inactive (6t).
To study the effect of the target compounds on the cell cycle progression,
fluorescence-activated cell sorting analysis (FACS) was performed. The representative compound
6q which exhibited most promising antiproliferative activities was selected and tested against T47D
cell lines at given concentrations (3, 6, 12 µM). As shown in Fig. 3 and Table 2, the G1 peak
significantly increased from 59.67% to 61.25% (3 µM), 72.70% (6 µM), and 74.68% (12 µM) after
12 h of incubation with the compound. These cell cycle analysis results revealed that the compound
arrested the cell cycle at G1 phase in a concentration-dependent manner when compared to
untreated control cells.
5

A
B
C
D
Fig. 3. Effect of compound 6q on cell cycle and apoptosis in T47D cells. Flow cytometry analysis of T47D cells
treated with 6q for 48 h. (A) Control; (B) 6q, 3 µM; (C) 6q, 6 µM; (D) 6q, 12 µM.
Table 2 Effect of compound 6q on cell cycle distribution in T47D cells.
Concentration G0/G1(%)
S(%)
21.82
18.98
10.80
4.34
G2/M(%)
18.51
0µM
3µM
59.67
61.25
72.70
74.68
19.76
6µM
16.50
12µM
20.98
In conclusion, starting with our previously reported antiproliferative compounds, we developed
a novel series of 2-amino-7,8-dihydropteridin-6(5H)-one derivatives via a ring-closing strategy.
Bioassay indicated that most of these compounds possessed moderate to potent antiproliferative
6

activities. Particularly, compound 6q possessing IC50 values of 7.75, 6.37, and 10.73 µM against
MDA-MB-468, T47D, and BT549, respectively, which were 49, 11, and 8 folds more active than
the positive control fluorouracil. Furthermore, flow-activated cell sorting analysis revealed that
compound 6q arrested the cell cycle of T47D cells in the G1 phase with a concentration-dependent
effect. The initial structure–activity relationship studies indicated that linker-length of amine chain
in C-2 position of pyrimidine ring played a crucial role in modulating the antitumor activity, which
could be of help in the rational design of dihydropteridin-6(5H)-ones as novel anticancer drugs.
Further research on the mechanisms of these compounds and modification is underway.
Acknowledgments
This work was supported by National Natural Science Foundation of China (21372113 and
21102069), and the project of the Outstanding Young Teachers in Guangdong Province.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.
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9

Design, synthesis and antiproliferative activity of novel substituted
2-amino-7,8-dihydropteridin-6(5H)-one derivatives
Qiu Li #, Hai-Kui Yang #, Qi Sun, Wen-Wei You, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
O
H
N
N
O
O
N
N
Ring-closing Strategy
R3
O
n
n
HN
R1
R1
N
H
N
N
N
N
R2
R2
O
5
Our previous work
6a~t
Newly designed compounds
10