Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.10.037

Pteridines are an important class of heterocyclic compounds with diverse biological activities. Here, we report a series of pteridin-7(8H)-one derivatives and their antiproliferative activities toward MKN-45, MGC-803, EC-109, and H1650. Structure-activity relationship studies showed that compound 12 exerted the most potent antiproliferative activity against MKN-45 and MGC-803 with the IC₅₀ values of 4.32 and 7.01 μM, respectively. Besides, compound 12 induced morphological changes and apoptosis of MKN-45 cells, increased expression of Bax, down-regulated expression of Bcl-2 and caused cleavage of caspase-3/9. Additionally, we first reported the construction of the novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and a chiral carbon center established.

Full text of DOI:10.1016/j.ejmech.2017.10.037

Accepted Manuscript
Synthesis and preliminary antiproliferative activity of new pteridin-7(8H)-one
derivatives
Zhong-Hua Li, Tao-Qian Zhao, Xue-Qi Liu, Bing Zhao, Chao Wang, Peng-Fei Geng,
Ya-Quan Cao, Dong-Jun Fu, Li-Ping Jiang, Bin Yu, Hong-Min Liu
PII:
S0223-5234(17)30837-1
10.1016/j.ejmech.2017.10.037
EJMECH 9829
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 June 2017
Revised Date: 11 October 2017
Accepted Date: 13 October 2017
Please cite this article as: Z.-H. Li, T.-Q. Zhao, X.-Q. Liu, B. Zhao, C. Wang, P.-F. Geng, Y.-Q.
Cao, D.-J. Fu, L.-P. Jiang, B. Yu, H.-M. Liu, Synthesis and preliminary antiproliferative activity of
new pteridin-7(8H)-one derivatives, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.037.
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ACCEPTED MANUSCRIPT
Graphic abstract
Pteridin-7(8H)-ones were synthesized and evaluated for their antiproliferative activity.
Compound 12 displayed acceptable growth inhibition, induced apoptosis of MKN-45
cells. Besides, a novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was
first prepared.

ACCEPTED MANUSCRIPT
Synthesis and preliminary antiproliferative activity of new
pteridin-7(8H)-one derivatives
Zhong-Hua Li a, b, c, d, 1,Tao-Qian Zhao a, b, c, d, 1, Xue-Qi Liu a, b, c, d, Bing Zhao a, b, c, d, Chao
Wang a, b, c, d, Peng-Fei Geng a, b, c, d, Ya-Quan Cao a, b, c, d, Dong-Jun Fu a, b, c, d
Li-Ping Jiang e, Bin Yu a, b, c, d,*, Hong-Min Liu a, b, c, d,
,
*
a
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan
b
Province; Key Laboratory of Technology of Drug Preparation (Zhengzhou University),
c
Ministry of Education of China; Key Laboratory of Henan Province for Drug Quality and
d
Evaluation; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
e
PR China; School of Basic Medical Science
，Central South University, Changsha 410013,
PR China
*Corresponding Authors:
Prof. Hong-Min Liu & Dr. Bin Yu
Zhengzhou University, Zhengzhou, 450001, China
E-mail: liuhm@zzu.edu.cn (Hong-Min Liu) & zzuyubin@hotmail.com (Bin Yu)
ABSTRACT: Pteridines are an important class of heterocyclic compounds with
diverse biological activities. Here, we report a series of pteridin-7(8H)-one derivatives
and their antiproliferative activities toward MKN-45, MGC-803, EC-109, and H1650.
Structure-activity relationship studies showed that compound 12 exerted the most
potent antiproliferative activity against MKN-45 and MGC-803 with the IC50 values
of 4.32 and 7.01 µM, respectively. Besides, compound 12 induced morphological
changes and apoptosis of MKN-45 cells, increased expression of Bax, down-regulated
expression of Bcl-2 and caused cleavage of caspase-3/9. Additionally, we first
reported the construction of the novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate
scaffold through the competitive 5-endo cyclization reaction with two C-N bonds and
a chiral carbon center established.
Keywords: Pteridin-7(8H)-one, Antiproliferative activity, Apoptosis, Cell cycle arrest
1

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1. Introduction
Pteridines are an important class of bicyclic N-heteroarenes, which have received
considerable attention from medicinal community due to their diverse biological
activities, such as antimicrobial [1], antiallergic [2], immunosuppressive [3],
anti-inflammatory [4, 5], antibacterial [6, 7], and anticancer activities [8, 9]. Some
biologically active natural products (e.g. folic acid, etc.) are also found to possess
pteridine scaffolds. As shown in Fig. 1, folic acid (also named as vitamin B9 or
vitamin M) is necessary for human health and is involved in DNA synthesis, DNA
repair and DNA methylation [10]. Antifolate methotrexate (MTX) is a chemotherapy
agent and immune system suppressant for treatment of cancer and autoimmune
diseases . Moreover, pteridine analogs are well known to act upon a wide range of
targets for therapeutic potential [4]. Compound A, as a selective PI3K/mTOR dual
inhibitor, is highly efficacious in mouse xenograft model bearing glioma cell line U87
[11]. Compound B has been proved to be able to inhibit EGFRwt and EGFRT790M/L858R
potently and exerts remarkable in vivo growth inhibition of human non-small cell lung
cancers (NSCLC) [12]. Compound C is a pteridinone-based TLR7 (Toll-like receptor
7) agonist, which is currently in clinical evaluation for the treatment of chronic HBV
(hepatitis B) infection [13] (Fig. 1). The diverse bioactivities and natural prevalence
make pteridines and their structural analogs promising bicyclic scaffolds to develop
new agents for the treatment of different diseases.
Following our previous work on the synthesis of new bicyclic heterocycles with
anticancer potentials [14-17], we herein report the synthesis of a series of new
pteridin-7(8H)-one derivatives through cascade reactions, their antiproliferative
activity and preliminary mechanisms of inducing cancer cell death. Also, we first
report the synthesis of new 8,9-dihydro-7H-purine-8-carboxylate via the 5-endo
cyclization.
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Fig. 1. Biologically active pteridine derivatives.
2. Results and discussion
2.1 Chemistry
The general synthetic route is illustrated in Scheme 1. The intermediate derivatives
2a-h were synthesized by condensation of commercially available pyrimidine analogs
1a-c with appropriate primary amines in the presence of N,N-diisopropylethylamine
(DIPEA) in DMF. The obtained intermediates 2a-h reacted with ethyl glyoxalate in
AcOH/EtOH under heating to afford pteridin-7(8H)-one skeletons 3a-h [11], which
then reacted with various amines in the presence of TEA in a mixed solvent of ethanol
and DMF, generating the target compounds 4-21.
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O
Cl
Cl
H
Cl
N
OEt
N
N
NH2
R2CH2NH2
NH2
Cl
N
N
O
N
R1
N
O
N
N
H
R2
AcOH/ethanol (1 : 1)
100 oC, 2h
R1
R1
DMF, DIPEA,
100 oC, 4 h
R2
1a-c
3a-h
2a-h
1a, R1 = propyl-S-
1b, R1 = Me-
1c, R1 = H-
2a-3a, R1 = propyl-S-, R2 =Ph-
2b-3b, R1 = propyl-S-, R2 =PhC2H4-
2c-3c, R1 = Me-, R2 =Ph-
2d-3d, R1 = H-, R2 =Ph-
2e-3e, R1 = propyl-S-, R2 =
R
R'
N
N
N
N
2f-3f, R1 = propyl-S-, R2 =
R'RNH/TEA
S
R1
N
O
Ethanol/DMF (3 : 1)
reflux, 3h
2g-3g, R1 = propyl-S-, R2 =
R2
4-21
O
2h-3h, R1 = propyl-S-, R2 =
Scheme 1. Synthesis of pteridin-7(8H)-one derivatives.
Interestingly, compound 3h' was also observed in the conversion from compound 2h
to compound 3h. Compound 3h' features novel
a
8,9-dihydro-7H-purine-8-carboxylate scaffold. Both compounds were formed through
the unstable imine intermediate A. For the synthesis of compound 3h, it involved the
6-exo cyclization (Scheme 2, path b), generating one C-N bond, one C=N bond, and
the pteridin-7(8H)-onescaffold. While compound 3h' was competitively formed via
the 5-endo cyclization (Scheme 2, path a), accompanying by the formation of two
C-N bonds, a chiral center, and the 8,9-dihydro-7H-purine-8-carboxylate architecture.
To the best of our knowledge, this is the first report on the rapid construction of the
new 8,9-dihydro-7H-purine-8-carboxylate framework, which also possesses
pluripotent groups (e.g. –COOEt, Cl) for further structural derivations. Besides, the
C-N single bond of compound 3h' could be potentially oxidized to aromatic bicyclic
N-heteroarene.
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Scheme 2. Proposed reaction mechanism for the synthesis of compounds 3h and 3h'.
Compound 3h' was then characterized by 2D NMR spectra (see supporting
information for details). Intriguingly, the methylene protons (highlighted in red)
appeared at 3.99 and 3.17 ppm, respectively. The unique intramolecular H-bond
interactions and the adjacent chiral center may be responsible for the remarkably
different chemical shifts of the methylene protons.
2.2 Evaluation of biological activity
2.2.1 Antiproliferative activity
Compounds 4-21 were evaluated for their antiproliferative activity against MKN-45
and MGC803 (human gastric cancer cell lines), EC-109 (human esophageal cancer
cell line), and H1650 (human lung cancer cell line), by using the MTT assay, and
5-fluorouracil (5-FU) was employed as the reference drug [18]. The preliminary data
are summarized in Tables 1-3.
As shown in Table 1, compounds 4-6 with different aniline substituents exerted
moderate antiproliferative activity against the tested cancer cell lines, the substituents
attached had certain effect on the activity, especially for MKN-45 and MGC-803 cells.
Compound 6 with the 3,4,5-trimethoxyl group was more potent than compound 4 with
the IC50 values of 14.25 and 15.03 µM, respectively against MKN-45 and MGC-803
cells. Compounds 7, 8, 13 and 14 with the aliphatic amine substituents showed
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decreased inhibitory activity against the tested cancer cell lines. Compound 12 was
significantly more potent than compounds 9-10, highlighting the importance of
nitrogen atom of cyclic amine groups for the activity. Compound 11 with a benzyl
group attached to the piperazine nitrogen atom showed decreased activity compared
to compound 12. Compound 12 displayed good and broad-spectrum antiproliferative
activities with IC50 values less than 10 µM, comparable to that of 5-FU.
Table 1
Inhibitory activity of compounds 4-14 against the tested cancer cell lines
IC50 (µM)a
Compound
R'RN-
MKN-45
MGC-803
EC-109
H1650
>64
38.60±3.75 36.60±4.69
14.65±1.80 19.35±0.56
19.03±1.38
4
>64
51.84±3.86
36.94±0.96
5
6
14.25±1.42 15.03±1.86
38.67±1.56
>64
46.64±2.56
>64
>64
>64
7
8
21.02±1.16 30.40±2.54
36.04±0.98
>64
>64
49.64±0.86
34.71±1.43
>64
>64
>64
>64
54.80±2.43
>64
9
10
11
15.43±2.47 18.71±5.69
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4.32±1.87
7.01±3.61
>64
9.85±2.81
>64
9.92±1.41
>64
12
13
39.23±4.72
23.26±1.86 43.74±1.80
8.89±1.65 7.52±0.98
29.71±1.65
6.21±0.75
>64
14
5-Fu
-
14.25±2.73
a Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs
of three independent experiments.
Next, the influence of R2 substituents on the activity was also investigated. As shown
in Table 2, compounds 15-19 generally displayed decreased or comparable activities
toward the tested cancer cells, regardless of the patterns of R2 group.
Table 2
Inhibitory activity of compounds 15-19 against the tested cancer cell lines
IC50 (µM)a
Compound
R2
MKN-45
MGC-803
EC-109
H1650
>64
9.97±0.97
13.75±3.81
18.28±2.95
15
11.32±1.57
11.56±6.50
16.41±3.16
>64
19.69±6.07
24.04±4.88
>64
16
17
54.91±6.08
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17.92±2.82
53.67±5.66
>64
51.81±3.21
18
16.83±5.65
8.89±1.65
17.17±2.35
7.52±0.98
37.48±4.62
6.21±0.75
45.43±2.90
14.25±2.73
19
5-Fu
-
a Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs
of three independent experiments.
However, the replacement of propylthio group with methyl (compound 20) or
hydrogen (compound 21) led to the loss of antiproliferative activity against the tested
cancer cell lines, significantly less potent than 5-FU and compound 12 (Table 3).
Table 3
Inhibitory activity of compounds 20-21 against the tested cancer cell lines
IC50 (µM)a
Compound
R1
MKN-45
4.32±1.87
>64
MGC-803
7.01±3.61
>64
EC-109
9.85±2.81
38.98±0.99
41.66±5.02
6.21±0.75
H1650
24.91±4.41
>64
propyl-S-
12
20
Me-
H
>64
>64
>64
21
5-Fu
-
8.89±1.65
7.52±0.98
14.25±2.73
a Inhibitory activity was assayed by exposure for 48 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs
of three independent experiments.
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2.2.2 Cell apoptosis of MKN-45 and possible mechanism involved
The acceptable antiproliferative activity of compound 12 promoted us to investigate
its effect on the apoptosis and morphological changes. Hochest 33258 staining was
performed to investigate morphological changes of MKN-45 cells [19]. After 24 h
incubation with compound 12 at indicated concentrations, characteristic apoptotic
morphological changes were observed, including cell rounding, chromatin shrinkage
and formation of apoptotic bodies (Fig. 2A). Notably, this phenomena were more
remarkable at higher concentrations. To further explore the effect of compound 12 on
cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI
double staining and analyzed with flow-cytometry calculation [20]. Treatment of
MKN-45 cells with compound 12 resulted in a concentration-dependent apoptosis
increase (Fig. 2B/2C).
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Fig. 2. Compound 12 induced apoptosis of MKN-45 cells. (A) Apoptosis analysis with Hoechst
33258 staining; (B) Apoptosis effect on MKN-45 cell line induced by compound 12 for 24 h using
Annexin V-FITC/PI double staining and flow-cytometry calculation. The lower left quadrant
represents live cells, the lower right is for early/primary apoptotic cells, upper right is for
late/secondary apoptotic cells, and the upper left represents cells damaged during the procedure;
(C) Quantitative analysis of apoptotic cells. The experiments were performed three times, and a
representative experiment is shown.
Next, the western blot analysis was performed to examine the expression of
apoptosis-related proteins. As shown in Fig. 3A, treatment of MKN-45 cells with
compound 12 increased expression of Bax in a concentration-dependent manner (Fig.
3B). Bax was able to activate the caspases, and promoted the release of cytochrome c
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and other pro-apoptotic factors from the mitochondria [21]. Meanwhile, the
expression of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 3C). As shown
in Fig. 3D and 3E, treatment of compound 12 also concentration-dependently caused
the cleavage of caspases-3 and caspases-9, which then inducing cell apoptosis. These
results indicated that compound 12 may induce MKN-45 apoptosis through the
intrinsic apoptotic pathway.
Fig. 3. Expression changes of apoptosis-related proteins induced by compound 12. (A) Compound
12 induced expression changes of Bax, Bcl-2 and caspase family members in MKN-45 cells; (B-E)
Statistical analysis of expression levels of Bax, Bcl-2 and cleaved-caspased 9/3.
2.2.3 Cell cycle analysis
The effect of compound 12 on the cell cycle was also evaluated. After treatment of
MKN-45 cells with compound 12 for 24 h at indicated concentrations (0, 2, 4, 8 µM),
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the percentage of cells in G2/M phase were 1.62%, 16.6%, 29.2% and 40.8%,
respectively (Fig. 4), suggesting that compound 12 caused an obvious G2/M arrest in
a concentration-dependent manner with concomitant decrease in terms of the number
of cells in other phases of the cell cycle.
Fig. 4. Effect of compound 12 on the cell cycle distribution of MKN-45 cells. Cells were treated
with indicated concentrations of compound 12 (0, 2, 4, 8 µM) for 24 h. Then the cells were fixed
and stained with PI to analyze DNA content by flow cytometry. The experiments were performed
three times and a representative experiment is shown.
3. Conclusions
In summary, a new series of pteridin-7(8H)-one derivatives were prepared and
evaluated for their antiproliferative activity. Among them, compound 12 exhibited the
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most potent and broad-spectrum inhibition against the tested cancer cells (MKN-45,
MGC803, EC-109 and H1650) and displayed comparable activity with the anticancer
drug 5-FU. Besides, compound 12 induced morphological changes and apoptosis of
MKN-45 cells in a concentration-dependent manner, increased expression of
pro-apoptotic protein Bax, down-regulated expression of anti-apoptotic protein Bcl-2
and caused cleavage of caspase-3/9, indicating that compound 12 induced apoptosis
via the intrinsic apoptotic pathway. The G2/M phase arrest induced by compound 12
was also observed. Additionally, we first reported the construction of the novel
bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold through the competitive
5-endo cyclization reaction with two C-N bonds and a chiral carbon center established.
This scaffold contains several pluripotent handles, which allows for further structural
modifications for identifying potential bioactive molecules.
4. Experimental section
4.1 General
Reagents and solvents were purchased from commercial sources and were used
without further purification. Melting points were determined on an X-5 micromelting
1
13
apparatus and are uncorrected. H NMR and C NMR spectra were recorded on a
Bruker (DPX-400) spectrometer, operating at 400 and 100 MHz, respectively. High
resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-Tof of
Micromass spectrometer by electrospray ionizaton (ESI).
4.2 General procedure for the synthesis of compounds 3h', 4-21.
A solution of 2a (1eq) with ethyl glyoxalate (50 wt. % in toluene, 1.2 eq) in a mixed
solvent of AcOH/ethanol (1 : 1) was heated to 100 oC for 2 h. After the completion of
the reaction monitored by TLC (PE/EA = 4 : 1), the purification was conducted by
flash column chromatography on silica gel using PE/EA (4 : 1) as eluent to afford 3a.
As a representative example, the new compound 3h' was separated and characterized
by NMR. Compounds 4 can be readily obtained by refluxing 3a (1eq), aniline (1.2 eq)
and triethylamine (1.2 eq) in the mixed solvent of ethanol/DMF (3 : 1) for 3 h,
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following by purification on flash column chromatography on silica gel. This
procedure was also applied to preparation of compounds 5-21.
4.2.1 Ethyl (R)-6-chloro-9-(cyclohexylmethyl)-2-(propylthio)-8,9-dihydro-7H-purine-
8-carboxylate (3h')
Pale yellow solid, yield 40%. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 5.10 (s,
1H), 3.96-4.01 (m, 1H), 3.60-3.66 (m, 2H), 3.5-3.20 (m, 1H), 3.04-3.11 (m, 1H),
2.93-3.00 (m, 1H), 1.61-1.73 (m, 8H), 1.14-1.20 (m, 3H), 1.09 (t, J = 7.0 Hz, 3H),
0.98 (t, J = 7.3 Hz, 3H), 0.90-0.96 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.3,
160.2, 150.0, 141.1, 112.2, 85.9, 64.4, 52.2, 36.1, 32.3, 30.1, 30.1, 26.0, 25.3, 25.3,
22.6, 14.9, 13.3. HR-MS (ESI): Calcd. C18H27ClN4O2S, [M+H]+m/z: 399.1621, found:
399.1623.
4.2.2 8-Benzyl-4-(phenylamino)-2-(propylthio)pteridin-7(8H)-one (4)
Pink solid, yield 75%. 1H NMR (400 MHz, Chloroform-d) δ 8.42 (s, 1H), 7.98 (s, 1H),
7.75 (m, 1H), 7.49-7.51 (m, 2H), 7.36-7.40 (m, 2H), 7.23-7.34 (m, 3H), 7.12-7.16 (m,
1H), 5.51 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H), 1.73-1.83 (m, 2H), 1.04 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, Chloroform-d) δ 173.50, 156.99, 155.61, 147.37, 144.76,
137.85, 135.89, 129.01, 128.51, 127.85, 124.11, 120.41, 110.37, 43.77, 33.43, 22.99,
13.61.HR-MS (ESI): Calcd. C22H21N5OS, [M+K]+m/z: 442.1104, found: 442.1102.
4.2.3 8-Benzyl-4-((2-methoxyphenyl)amino)-2-(propylthio)pteridin-7(8H)-one (5)
1
Yellow solid, yield 81%. H NMR (400 MHz, Chloroform-d) δ 9.15 (s, 1H),
8.62-8.64 (m, 1H), 8.02 (s, 1H), 7.49 (m, 2H), 7.27-7.33 (m, 2H), 6.93-7.10 (m, 3H),
5.52 (s, 2H), 3.97 (s, 3H), 3.15 (t, J = 7.4 Hz, 2H), 1.75-1.83 (m, 2H), 1.05 (t, J = 7.4
Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 173.36, 157.03, 155.39, 148.79,
147.26, 144.77, 135.96, 128.97, 128.49, 127.80, 127.67, 123.54, 120.78, 119.99,
110.99, 110.12, 55.90, 43.74, 33.43, 23.00, 13.63.HR-MS (ESI): Calcd. C23H23N5O2S,
[M+Na]+m/z: 456.1470, found: 456.1472.
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4.2.4 8-Benzyl-2-(propylthio)-4-((3,4,5-trimethoxyphenyl)amino)pteridin-7(8H)-one
(6)
1
Yellow solid, yield 86%. H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.99 (s,
1H), 7.47-7.49 (m, 2H), 7.28-7.32 (m, 3H), 7.07 (s, 2H), 5.53 (s, 2H), 3.90 (s, 6H),
3.85 (s, 3H), 3.15 (t, J = 7.3 Hz, 2H), 1.72-1.77 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H). 13C
NMR (100 MHz, Chloroform-d) δ 173.46, 156.97, 155.48, 153.36, 147.30, 144.76,
135.84, 134.77, 133.88, 128.80, 128.52, 127.85, 110.35, 98.32, 61.02, 56.21, 43.83,
33.40, 22.56, 13.49. HR-MS (ESI): Calcd. C25H27N5O4S, [M+Na]+m/z: 516.1681,
found: 516.1682.
4.2.5 8-Benzyl-4-(benzylamino)-2-(propylthio)pteridin-7(8H)-one (7)
Yellow solid, yield 68%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.48 (m,
2H), 7.35 (m, 4H), 7.26-7.31 (m, 4H), 6.78 (t, J = 5.6 Hz, 1H), 5.49 (s, 2H), 4.77 (d, J
= 5.9 Hz, 2H), 3.09 (t, J = 7.3 Hz, 2H), 1.69-1.78 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H).
13C NMR (100 MHz, Chloroform-d) δ 173.22, 158.01, 157.14, 147.19, 144.21,
137.88, 136.05, 128.92, 128.78, 128.46, 127.76, 127.70, 110.33, 44.80, 43.75, 33.39,
22.88, 13.58. HR-MS (ESI): Calcd. C23H23N5OS, [M+Na]+m/z: 440.1521, found:
440.1524.
4.2.6 8-Benzyl-4-((cyclohexylmethyl)amino)-2-(propylthio)pteridin-7(8H)-one (8)
White solid, yield 76%. 1H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.46-7.49
(m, 2H), 7.27-7.31 (m, 2H), 7.22-7.25 (m, 1H), 6.57 (t, J = 6.1 Hz, 1H), 5.48 (s, 2H),
3.42 (t, J = 6.5 Hz, 2H), 3.09 (t, J = 7.4 Hz, 2H), 1.62-1.80 (m, 8H), 1.16-1.30 (m,
3H), 0.96-1.05 (m, 5H). 13C NMR (100 MHz, Chloroform-d) δ 173.05, 158.35,
157.20, 147.03, 143.84, 136.12, 128.94, 128.43, 127.71, 110.35, 47.04, 43.69, 37.99,
33.37, 30.88, 26.37, 25.83, 23.01, 13.62. HR-MS (ESI): Calcd. C23H29N5OS,
[M+Na]+m/z: 446.1991, found: 446.1992.
4.2.7 8-Benzyl-4-morpholino-2-(propylthio)pteridin-7(8H)-one (9)
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Yellow solid, yield 79%. 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.45 (m,
2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.53 (s, 2H), 4.27-4.30 (m, 4H), 3.80 (t, J = 4.7 Hz,
13
4H), 3.05 (t, J = 7.3 Hz, 2H), 1.68-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). C NMR
(100 MHz, Chloroform-d) δ 171.09, 157.27, 156.17, 149.70, 141.83, 136.15, 128.73,
128.43, 127.66, 112.36, 67.09, 47.94, 44.09, 33.29, 22.85, 13.56. HR-MS (ESI):
Calcd. C20H24N5O2S, [M+Na]+m/z: 420.1470, found: 420.1471.
4.2.8 8-Benzyl-2-(propylthio)-4-thiomorpholinopteridin-7(8H)-one (10)
1
White solid, yield 81%. H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.46 (m,
2H), 7.24-7.31 (m, 3H), 5.52 (s, 2H), 4.49 (t, J = 4.9 Hz, 4H), 3.05 (t, J = 7.3 Hz, 2H),
13
2.76 (t, J = 5.0 Hz, 4H), 1.70-1.76 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H). C NMR (100
MHz, Chloroform-d) δ 171.06, 157.23, 156.13, 149.80, 141.94, 136.17, 128.82,
128.42, 127.68, 112.34, 50.70, 44.09, 33.31, 27.62, 22.91, 13.58. HR-MS (ESI):
Calcd. C20H23N5OS2, [M+Na]+m/z: 436.1242, found: 436.1245.
4.2.9 8-Benzyl-4-(4-benzylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (11)
1
White solid, yield 72%. H NMR (400 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.45 (m,
2H), 7.23-7.34 (m, 8H), 5.51 (s, 2H), 4.28 (m, 4H), 3.54 (s, 2H), 3.04 (t, J = 7.3 Hz,
13
2H), 2.56 (t, J = 5.0 Hz, 4H), 1.67-1.76 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). C NMR
(100 MHz, Chloroform-d) δ 170.88, 157.15, 156.22, 149.68, 141.48, 137.60, 136.25,
129.21, 128.79, 128.40, 128.33, 127.62, 127.27, 112.35, 62.96, 53.31, 47.44, 44.04,
33.28, 22.89, 13.56. HR-MS (ESI): Calcd. C27H30N6OS, [M+H]+m/z: 487.2280,
found:487.2281.
4.2.10 8-Benzyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (12)
White solid, yield 82%. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.44-7.46
(m, 2H), 7.28-7.31 (m, 1H), 7.22-7.26 (m, 2H), 5.52 (s, 2H), 4.30 (m, 4H), 3.05 (t, J =
7.2 Hz, 2H), 2.54 (t, J = 4.9 Hz, 4H), 2.34 (s, 3H), 1.68-1.77 (m, 2H), 1.00 (t, J = 7.4
13
Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 169.64, 156.60, 155.33, 149.36, 141.68,
16

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136.17, 128.19, 127.33, 127.06, 111.54, 54.65, 46.89, 45.50, 43.49, 32.45, 22.48,
13.18. HR-MS (ESI): Calcd. C21H26N6OS, [M+H]+m/z: 411.1967, found: 411.1968.
4.2.11 8-Benzyl-4-(propylamino)-2-(propylthio)pteridin-7(8H)-one (13)
Yellow solid, yield 85%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (t, J = 6.0 Hz, 1H),
7.96 (s, 1H), 7.24-7.31 (m, 5H), 5.35 (s, 2H), 3.38-3.43 (m, 2H), 2.99 (t, J = 7.2 Hz,
2H), 1.54-1.66 (m, 2H), 0.86-0.92 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.85,
153.02, 151.93, 141.78, 138.66, 130.86, 123.64, 123.17, 122.44, 105.10, 38.44, 37.37,
28.12, 17.71, 17.54, 8.32, 6.16. HR-MS (ESI): Calcd. C19H23N5OS, [M+Na]+m/z:
392.1521, found:392.1522.
4.2.12 8-Benzyl-4-(cyclopropylamino)-2-(propylthio)pteridin-7(8H)-one (14)
1
Yellow solid, yield 68%. H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 4.5 Hz, 1H),
7.95 (s, 1H), 7.22-7.32 (m, 5H), 5.36 (s, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.96 (m, 1H),
13
1.58-1.67 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H), 0.68-0.74 (m, 4H). C NMR (100 MHz,
DMSO-d6) δ 171.37, 158.96, 156.27, 146.85, 143.68, 136.24, 128.30, 127.30, 127.13,
110.00, 43.14, 32.51, 24.04, 22.63, 13.24, 5.98. HR-MS (ESI): Calcd. C19H21N5OS,
[M+Na]+m/z: 390.1365, found: 390.1366.
4.2.13 4-(4-Methylpiperazin-1-yl)-8-(3-phenylpropyl)-2-(propylthio)pteridin-7(8H)-
one (15)
Yellow solid, yield 63%. 1H NMR (400 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.28 (m,
1H), 7.25 (m, 1H), 7.15-7.21 (m, 3H), 4.35-4.39 (m, 2H), 4.31-4.32 (m, 4H), 3.03 (t, J
= 7.2 Hz, 2H), 2.73 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 5.1 Hz, 4H), 2.36 (s, 3H),
2.03-2.08 (m, 2H), 1.74-1.79 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 170.96, 157.26, 156.20, 149.64, 141.53, 141.08, 128.35, 128.23,
125.95, 112.34, 54.96, 46.94, 45.58, 41.08, 33.34, 33.32, 28.72, 22.92, 13.56. HR-MS
(ESI): Calcd. C23H30N6OS, [M+H]+m/z: 439.2280, found: 439.2281.
4.2.14 4-(4-Methylpiperazin-1-yl)-2-(propylthio)-8-(thiophen-2-ylmethyl)pteridin-
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7(8H)-one (16)
1
Yellow waxy solid, yield 78%. H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H),
7.34-7.36 (m, 1H), 7.14 (d, J = 3.5 Hz, 1H), 6.92-6.94 (m, 1H), 5.53 (s, 2H),
4.21-4.23 (m, 4H), 3.12 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 5.0 Hz, 4H), 2.23 (s, 3H),
13
1.70-1.77 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, Chloroform-d) δ
170.97, 157.15, 155.63, 149.19, 141.53, 137.29, 128.87, 126.39, 126.10, 112.28,
55.14, 47.15, 45.81, 38.42, 33.34, 22.88, 13.62. HR-MS (ESI): Calcd. C19H24N6OS2,
[M+H]+m/z: 417.1531, found: 417.1532.
4.2.15 8-(Furan-2-ylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-
one (17)
Yellow waxy solid, yield 73%. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.55 (d,
J = 1.7 Hz, 1H), 6.39 (m, 1H), 6.29 (d, J = 3.2 Hz, 1H), 5.38 (s, 2H), 4.18 (m, 4H),
3.04 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H), 1.63-1.73 (m, 2H), 0.95
13
(t, J = 7.3 Hz, 3H). C NMR (100 MHz, Chloroform-d) δ 171.04, 157.20, 155.80,
149.37, 142.06, 141.56, 112.27, 110.41, 109.42, 55.06, 47.08, 45.72, 37.04, 33.33,
22.94, 13.58. HR-MS (ESI): Calcd. C19H24N6O2S, [M+H]+m/z: 401.1760, found:
401.1762.
4.2.16 8-(Cyclohexylmethyl)-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-
one (18)
White solid, m. p. 146-147 oC, yield 72%. 1H NMR (400 MHz, Chloroform-d) δ 7.88
(s, 1H), 4.29 (m, 4H), 4.19 (d, J = 7.3 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H), 2.53 (t, J =
5.1 Hz, 4H), 2.34 (s, 3H), 1.93-1.97 (m, 1H), 1.78-1.84 (m, 2H), 1.69-1.71 (m, 2H),
13
1.60 (m, 3H), 1.11-1.17 (m, 5H), 1.06 (t, J = 7.3 Hz, 3H). C NMR (100 MHz,
Chloroform-d) δ 170.64, 165.81, 157.41, 156.62, 150.00, 141.57, 112.36, 55.30,
47.35, 46.85, 45.99, 36.46, 33.32, 30.80, 26.29, 25.84, 23.13, 13.63. HR-MS (ESI):
Calcd. C21H32N6OS, [M+H]+m/z: 417.2437, found: 417.2438.
4.2.17 8-Isobutyl-4-(4-methylpiperazin-1-yl)-2-(propylthio)pteridin-7(8H)-one (19)
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1
Yellow waxy solid, yield 65%. H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 4.17
(m, 4H), 4.03 (d, J = 3.6 Hz, 2H), 3.04 (t, J = 7.4 Hz, 2H), 2.44 (t, J = 5.0 Hz, 4H),
2.15-2.21 (m, 4H), 1.67-1.74 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H), 0.87 (d, J = 6.7 Hz,
13
6H). C NMR (100 MHz, Chloroform-d) δ 174.65, 170.70, 157.39, 156.56, 149.96,
141.69, 127.83, 125.77, 112.31, 55.01, 47.90, 47.03, 45.65, 33.29, 27.14, 23.05, 20.14,
13.60. HR-MS (ESI): Calcd. C18H28N6OS, [M+H]+m/z: 377.2124, found: 377.2125.
4.2.18 8-Benzyl-2-methyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (20)
Yellow solid, m. p. 142-143 oC, yield 61%. 1H NMR (400 MHz, Chloroform-d) δ 7.96
(s, 1H), 7.53-7.55 (m, 2H), 7.29 (m, 2H), 7.23 (m, 1H), 5.56 (s, 2H), 4.30 (m, 4H),
2.52 (m, 7H), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.86, 157.55, 155.35,
149.54, 142.96, 136.41, 128.18, 127.99, 127.13, 111.94, 54.72, 46.82, 45.52, 43.20,
26.18. HR-MS (ESI): Calcd. C19H22N6O, [M+H]+m/z: 351.1933, found: 351.1932.
4.2.19 8-Benzyl-4-(4-methylpiperazin-1-yl)pteridin-7(8H)-one (21)
White solid, m. p. 141-142 oC, yield 73%. 1H NMR (400 MHz, Chloroform-d) δ 8.39
(s, 1H), 8.04 (s, 1H), 7.50-7.52 (m, 2H), 7.29 (m,2H), 7.23 (m, 1H), 5.57 (s, 2H), 4.32
(m, 4H), 2.55 (m, 4H), 2.35 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 157.62,
156.52, 155.30, 149.49, 144.21, 136.22, 128.22, 127.63, 127.12, 113.55, 54.69, 46.99,
45.48, 43.40. HR-MS (ESI): Calcd. C18H20N6O, [M+H]+m/z: 337.1777, found:
337.1777.
4.3 Antiproliferative activity assays
Exponentially growing cells were seeded into 96-well plates at a concentration of
3,000 cells per well. After 24 h of incubation, the culture medium was removed and
fresh medium containing various concentrations of the candidate compounds was
added to each well. The cells were then incubated for 48 h, thereafter MTT assays
were performed and cell viability was assessed at 570 nm by a microplate reader
(Biotech, Shanghai, China).
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4.4 Hoechst 33258 staining
MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated overnight
for adherent and treated with compound 12 at different concentration for 24 h, and
underwent Hoechst 33258 staining for 30 min in the dark. The cells were observed
under a Nikon Eclipse TE 2000-S fluorescence microscope (Nikon, Japan).
4.5 Cell apoptosis assay
MKN-45 cells were seeded into a 6-well plate (2×105/well) and incubated for 24 h.
Then the cells were treated with different concentrations of the tested compound 12
for 24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit
(Biovision) was used according to the manufacturer’s protocol. The cells were
analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific,
MA).
4.6 Western blot analysis
MKN-45 cells were treated with different concentrations of compound 12 for 24 h,
the cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail
o
for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4 C. After the
collection of supernatant, the protein concentration was detected using a bicinchoninic
acid assay kit (Beyotmie Biotechnology, Haimen, China). After added with loading
o
buffer, cell lyses were boiled for 10 min at 100 C for SDS- polyacrylamide gel
electrophoresis (PAGE). Proteins were transferred to nitrocellulose (NC) membranes.
Then the membranes were blocked with 5% skim milk at room temperature for 2 h,
and then incubated overnight at 4o C with primary antibodies. After washing the
membrane with the secondary antibody (1: 5000) at room temperature for 2 h. Finally,
the blots were washed in TBST/TBS. The antibody-reactive were revealed by
enhanced chemiluminescence (ECL) and exposed on Kodak radiographic film.
Acknowledgement
This work was supported by National Key Research Programs of Proteins (No.
20

ACCEPTED MANUSCRIPT
2016YFA0501800); National Natural Science Foundation of China (Project No.
81430085, 81703326 and 21372206); Key Research Program of Henan Province (No.
1611003110100); the Starting Grant of Zhengzhou University (No. 32210533).
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Highlights
ò The pteridin-7(8H)-one derivatives showed potent inhibition against the cancer
cells.
ò Compound 12 exerted the most potent and broad-spectrum antiproliferative
activity.
ò
Compound 12 induced the apoptosis and G2/M arrest of MKN-45 cells.
ò A novel bicyclic 8,9-dihydro-7H-purine-8-carboxylate scaffold was constructed.