Design, synthesis and antiproliferative activity of thiazolo[5,4-d]pyrimidine derivatives through the atom replacement strategy

Article abstract of DOI:10.1016/j.ejmech.2017.07.039

A series of thiazolo[5,4-d]pyrimidine derivatives were designed through the atom replacement strategy based on biologically validated scaffolds and then evaluated for their antiproliferative activities on cancer cell lines. The structure-activity relationship studies were conducted, leading to the identification of compound 22, which exhibited good antiproliferative activity against HGC-27 with an IC₅₀ value of 1.22 μM and low toxicity against GES-1 cells. Mechanistic studies showed that compound 22 inhibited the colony formation and migration of HGC-27 as well as induced apoptosis. The western blot experiments proved that compound 22 up-regulated expression of Bax, down-regulated expression levels of Bcl-2 and cleaved caspased-3/9. These findings indicate that compound 22 may serve as a template for designing new agents for the treatment of human gastric cancers. The atom replacement strategy could be viable strategy for designing new anticancer drugs and may find its applications in drug design.

Full text of DOI:10.1016/j.ejmech.2017.07.039

Accepted Manuscript
Design, synthesis and antiproliferative activity of thiazolo[5,4-d]pyrimidine derivatives
through the atom replacement strategy
Zhong-Hua Li, Xue-Qi Liu, Peng-Fei Geng, Ji Zhang, Jin-Lian Ma, Bo Wang, Tao-
Qian Zhao, Bing Zhao, Xin-Hui Zhang, Bin Yu, Hong-Min Liu
PII:
S0223-5234(17)30561-5
10.1016/j.ejmech.2017.07.039
EJMECH 9602
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 March 2017
Revised Date: 2 July 2017
Accepted Date: 20 July 2017
Please cite this article as: Z.-H. Li, X.-Q. Liu, P.-F. Geng, J. Zhang, J.-L. Ma, B. Wang, T.-Q.
Zhao, B. Zhao, X.-H. Zhang, B. Yu, H.-M. Liu, Design, synthesis and antiproliferative activity of
thiazolo[5,4-d]pyrimidine derivatives through the atom replacement strategy, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.039.
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ACCEPTED MANUSCRIPT
Graphic abstract

ACCEPTED MANUSCRIPT
Design, synthesis and antiproliferative activity of thiazolo[5,4-d]pyrimidine
derivatives through the atom replacement strategy
Zhong-Hua Li 1, Xue-Qi Liu 1, Peng-Fei Geng, Ji Zhang, Jin-Lian Ma, Bo Wang,
Tao-Qian Zhao, Bing Zhao, Xin-Hui Zhang,
Bin Yu*, Hong-Min Liu*
Key Laboratory of Advanced Drug Preparation Technologies (Zhengzhou University),
Ministry of Education; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou
450001, PR China; Collaborative Innovation Center of New Drug Research and Safety
Evaluation, Henan Province; Key Laboratory of Henan Province for Drug Quality and
Evaluation
Corresponding Authors: Prof. Hong-Min Liu & Dr. Bin Yu
Zhengzhou University, Zhengzhou, 450001, PR China
E-mail: liuhm@zzu.edu.cn (Hong-Min Liu) & zzuyubin@hotmail.com (Bin Yu)
ABSTRACT:
A series of thiazolo[5,4-d]pyrimidine derivatives were designed through the atom
replacement strategy based on biologically validated scaffolds and then evaluated for
their antiproliferative activities on cancer cell lines. The structure-activity relationship
studies were conducted, leading to the identification of compound 22, which exhibited
good antiproliferative activity against HGC-27 with an IC50 value of 1.22 µM and low
toxicity against GES-1 cells. Mechanistic studies showed that compound 22 inhibited
the colony formation and migration of HGC-27 as well as induced apoptosis. The
western blot experiments proved that compound 22 up-regulated expression of Bax,
down-regulated expression levels of Bcl-2 and cleaved caspased-3/9. These findings
indicate that compound 22 may serve as a template for designing new agents for the
treatment of human gastric cancers. The atom replacement strategy could be viable
strategy for designing new anticancer drugs and may find its applications in drug
design.
KEYWORDS: Thiazolo[5,4-d]pyrimidine, Antiproliferative activity, Apoptosis,
Atom replacement
1

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INTRODUCTION:
Drug repurposing (also termed as drug repositioning) has emerged as a valuable
strategy for identifying new applications of old drugs [1, 2]. For example, thalidomide,
as an anticonvulsive drug, is now used for the treatment of erythema nodosum leprosy
and some cancers [3]. The antifungal drug itraconazole has advanced into several
phase II clinical studies for the treatment of cancers [4]. Most recently, the Remya
group designed a new series of 1-phenylpyrazole analogs as potent antitubercular
agents based on the scaffold of anti-obesity drug rimonabant [5]. As part of our
ongoing efforts toward identifying new anticancer agents [6-10], we screened our
in-house small-molecule library against some cancer cell lines using the MTT
(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide) assay. Intriguingly,
we found that compound A (Fig. 1) as a TRPV1 (vanilloid receptor 1) antagonist [11],
showed acceptable antiproliferative activities against several cancer cell lines. Based
on this scaffold, we have synthesized a series of amine substituted thiazole-pyrimidine
derivatives and preliminarily evaluated their antiproliferative activities towards
HGC-27 cell line[12]. Among them, compound B inhibited growth of HGC-27 cells
with an IC50 value of about 20 µM. And compound 8 derived from compound B with
the NH group replaced with the S atom (termed as the atom replacement strategy)
showed selective inhibition toward gastric cancer cells HGC-27 and MGC-803 (IC50
= 8.87 and 9.33 µM, respectively). This finding promoted us to design new
compounds targeting gastric cancer cells based on the scaffold of
thiazolo[5,4-d]pyrimidine, finally leading to the identification of the most potent
compound 22, which inhibited growth of HGC-27 cell line with an IC50 values of 1.22
µM and exhibited around 24-fold selectivity between HGC-27 and normal gastric
epithelial cell GES-1 (Fig. 1).
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Fig. 1. Design of new compounds targeting HGC-27 cells based on the drug repurposing and atom
replacement strategies.
2. Results and discussion
2.1 Chemistry
The general synthetic route was illustrated in Scheme 1. The intermediate derivatives
5a~b were synthesized following the previously reported procedure [10]. Then
isothiocyanate analogues reacted with 5a~b in the presence of cesium carbonate in
acetonitrile to give the key active intermediates 6a~e [13]. The target compounds 7-23
were readily obtained through the NaI-catalyzed nucleophilic substitution reactions
between mercapto analogues and compounds 6a-e under alkaline conditions.
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Scheme 1. Synthesis of thiazolo[5,4-d]pyrimidine derivatives. Reagent and conditions: (a) Alkyl
o
bromide, TEA, methanol, reflux, 2h; (b) Fuming nitric acid, AcOH, 25~45 C, 1 h; (c) POCl3,
DMA, reflux, 2 h; (d) Fe, AcOH, methanol, rt~reflux; (e) R2SCN, Cs2CO3, acetonitrile, rt,
overnight; (f) TEA, NaI, mercapto analogues, isopropanol, reflux, 6 h.
2.2 Evaluation of biological activity
2.2.1 Antiproliferative activity
All target compounds were evaluated for their antiproliferative activities against
human gastric cancer lines (MGC-803 and HGC-27), human breast cancer cell line
(MCF-7) and human esophageal cancer cell line (EC-109) using the MTT assay, and
5-fluorouracil (5-FU) was employed as the reference drug. The preliminary
antiproliferative results are summarized in Table 1.
Generally, the synthesized compounds showed moderate to good antiproliferative
activities against the tested cancer cell lines. As for EC-109 cells, most of the
compounds showed weak inhibition (IC50 > 20 µM). It should be noted that compound
22 inhibited growth of EC-109 cells potently with an IC50 value of 2.69 µM,
significantly much more potent than compound 7 (IC50 = 25.03 µM) and 5-FU (IC50 =
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5.01 µM), while compound 23 was about 4-fold less potent (IC50 = 10.55 µM) than
compound 22, highlighting the importance of substituents attached to the
thiazolo[5,4-d]pyrimidine scaffold for the activity. For MCF-7 cells, a similar trend
was observed. Only compounds 12 and 22 inhibited the growth of MCF-7 cells with
the IC50 values less than 10 µM, comparable to that of 5-FU.
Interestingly, this series of compounds exhibited improved potency against human
gastric cancer lines MGC-803 and HGC-27 and certain selectivity to HGC-27 over
MGC-803. For MGC-803 cells, some compounds showed comparable activity with
5-FU. Compound 7 showed the best inhibitory effect against MGC-803 cells with an
IC50 value of 4.73 µM. Of note, this series of compounds displayed improved
inhibition against HGC-27 cells, compounds 7, 11, 14, and 22 inhibited HGC-27 cells
potently with the IC50 values less than 5 µM. Among these compounds, compound 22
showed the best inhibition (IC50 = 1.22 µM), about 9-fold more potent than 5-FU.
Besides, compound 22 also showed good inhibition against EC-109 cells with an IC50
values of 2.69 µM.
Table 1. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives against
four cancer cell lines.
IC50 (µM)a
NO.
7
R1
R2
Ph
Ph
R3
MGC-803
4.73±0.67
HGC-27
MCF-7
EC-109
Propyl
Propyl
4.65±1.02
>64
25.03±1.59
9.33±1.97
9.82±0.99
8.87±0.94
9.55±2.13
44.23±2.88
13.22±2.45
19.55±2.69
49.55±3.20
8
Propyl
Propyl
Propyl
Ph
Ph
Ph
9
14.16±2.15
23.41±3.36
12.21±1.94
2.21±0.34
>64
>64
24.32±1.55
>64
10
11
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Propyl
Propyl
Propyl
Ph
Ph
Ph
10.66±1.02
12.85±1.10
30.02±2.47
5.07±0.70
8.55±2.65
>64
>64
>64
>64
12
13
14
>64
4.03±0.60
>64
Propyl
Propyl
Propyl
Ph
18.53±1.26
46.21±5.23
>64
6.51±0.81
14.51±1.63
>64
>64
>64
15
16
17
>64
>64
22.65±1.35
>64
Propyl
Propyl
Propyl
11.22±1.87
10.22±2.36
>64
12.19±1.08
18.19±1.26
7.28±0.86
>64
>64
>64
>64
18
19
20
11.58±2.00
34.25±3.35
3,4,5-triMeO-
Ph
Propyl
Bn
>64
6.03±0.78
1.22±0.18
>64
23.87±0.99
2.69±0.64
21
22
Ph
12.21±1.91
8.55±0.55
Bn
-
Ph
-
>64
18.88±1.27
11.02±2.28
38.66±3.99
7.21±0.91
10.55±2.11
5.01±0.72
23
5-FU
-
6.89±1.33
a
Inhibitory activity was assayed by exposure for 72 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.
The excellent antiproliferative activity of compound 22 promoted us to explore its
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underlying toxicity against normal cells. Compound 22 was then subjected to the
antiproliferative evaluation toward normal human gastric epithelialcells GES-1 by the
MTT assay. As shown in Table 2, compound 22 inhibited GES-1 potently (IC50 =
29.36 µM), showing 24-fold selectivity between HGC-27 and GES-1. In contrast,
5-FU displayed no selectivity between HGC-27 and GES-1 with the IC50 values of
11.02 and 8.59 µM, respectively. These findings indicate that compound 22 may serve
as a template for designing new agents for the treatment of human gastric cancers.
The atom replacement strategy could be viable for designing new anticancer drugs.
Table 2. Inhibitory results of compound 22 against normal cells GES-1.
IC50 (µM)a
Com
R1
R2
R3N
SIb
p
HGC-27
1.22±0.18
11.02±2.28
GES-1
Bn
-
Ph
-
29.36±3.69
8.59±1.38
24.06
0.78
22
5-FU
-
a
Inhibitory activity was assayed by exposure for 72 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.
bThe selectivity index (SI) was calculated as IC50 (GES-1) / IC50 (HGC-27).
2.2.2 Clone assay and cell migration
The excellent antiproliferative activity of compound 22 against HGC-27 promoted us
to investigate the effect on colony formation and cell migration. The clone formation
of cancer cells represents an indirect estimation of neoplastic transformation [14]. As
shown in Fig. 2A and 2B, treatment of HGC-27 cells with compound 22 led to fewer
and smaller colonies compared to the control with an inhibition rate of 79% at 4 µM.
In addition, the cell migration ability was also initially evaluated by the wound
healing assay. As shown in Fig. 2C, treatment of HGC-27 cells with compound 22 at
indicated concentration markedly suppressed the wound healing in
concentration-dependent manner.
a
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Fig. 2. (A) Representative images of HGC-27 cells colonies after treatment with various
concentrations for 9 days; (B) Quantitative analysis of the colony formation inhibition rate; (C)
Wound healing assay.
2.2.3 Cell apoptosis and possible mechanism involved
Apoptosis is typically characterized by distinctive cell morphological changes [15].
Compound 22 was chosen for evaluating its ability of inducing apoptosis. Hochest
33258 staining was performed to investigate the effect on morphological changes of
HGC-27 cells [16]. After 24 h incubation with compound 22 at indicated
concentrations, characteristic apoptotic morphological changes were observed,
especially at high concentrations, including cell rounding, chromatin shrinkage and
formation of apoptotic bodies (Fig. 3A). To further explore the effect of compound 22
on cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI
double staining and analyzed with high content [17]. Treatment of HGC-27 cells with
compound 22 induced apoptosis in a concentration-dependent manner (Fig. 3B), the
percentage of apoptotic cells was 10.01%, 31.8%, and 67.9%, respectively, compared
to the control (1.8%) (Fig. 3C).
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Fig. 3. Compound 22 induced apoptosis of HGC-27 cells. (A) Apoptosis analysis with
Hoechst-33258 staining; (B, C) Quantitative analysis of apoptotic cells using Annexin V-FITC/PI
double staining and high content analysis. Data are the mean ± SD. All experiments were carried
out at least three times.
Next, the Western blot analysis was performed to examine the expression of
apoptosis-related proteins. As shown in Fig. 4A, treatment of HGC-27 cells with
compound 22 resulted in an increased expression of Bax in a concentration-dependent
manner (Fig. 4B). Bax was able to activate the caspases, and promoted the release of
cytochrome c and other pro-apoptotic factors from the mitochodria [18]. Meanwhile,
the expression levels of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 4C).
As shown in Fig. 4D and 4E, treatment of HGC-27 cells with compound 22
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concentration-dependently activatedcaspase-3 and caspase-9. These results indicated
that compound 22 may induce HGC-27 apoptosis through the intrinsic apoptotic
pathway.
Fig. 4. Expression changes of apoptosis-related proteins induced by compound 22. (A) Compound
22 induced expression changes of Bax, Bcl-2 and caspase family members in HGC-27 cells; (B-E)
Statistical analysis of expression levels of Bax, Bcl-2 and cleaved caspase -3/-9.
3. Conclusions
In summary, a new series of thiazolo[5,4-d]pyrimidine derivatives were designed
based on previously identified scaffolds through the atom replacement strategy and
then evaluated for their antiproliferative activity. Among them, compound 22
exhibited good inhibition against HGC-27 and was less toxic to GES-1, indicating a
good selectivity to cancer cells over normal cells. Further mechanistic investigations
showed that compound 22 can inhibit the cell colony formation and migration, induce
apoptosis through the intrinsic apoptotic pathway. These findings indicate that
compound 22 may serve as a template for designing new agents for the treatment of
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human gastric cancers. The atom replacement strategy could be viable for designing
new anticancer drugs.
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
apparatus and are uncorrected. HNMR and 13CNMR spectra were recorded on a
Bruker 400 MHz and 100 MHz spectrometer respectively. High resolution mass
spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionizaton (ESI).
4.2 General procedure for the synthesis of compounds 7-23
The mixture of 6b (1 eq), appropriate thiophenol or mercapto heterocyclic analogues
(1.5 eq), triethylamine (2 eq) and catalytic amount of sodium iodide was refluxed in
isopropanol for 6 h and monitored by thin layer chromatography (TLC) (petroleum
(PE) / ethyl acetate (EA) = 4:1 ~ 1:1, v/v). After the completion of the reaction, the
reaction mixture was cooled to room temperature and diluted with ethyl acetate, then
washed with water for three times. The organic phase was dried with anhydride
sodium sulfate and concentrated under reduced vacuum. The residue was purified by
flash column chromatography (PE/EA = 4:1 ~ 1:1) to give the target products.
4.2.1 N-phenyl-5-(propylthio)-7-(p-tolylthio)thiazolo[5,4-d]pyrimidin-2-amine (7)
o
1
White solid, Mp 203~204 C. H NMR (400 MHz, DMSO-d6) δ 11.11 (br, 1H),
7.76-7.79 (m, 2H), 7.50-7.52 (m, 2H), 7.38-7.42 (m, 2H), 7.31-7.33 (m, 2H),
7.07-7.11 (m, 1H), 2.64-2.68 (t, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.32-1.37 (m, 2H),
0.73-0.77 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.0, 160.2, 158.8,
155.7, 139.9, 139.3, 136.7, 135.6, 130.0, 129.9, 129.1, 128.1, 123.5, 122.9, 118.2,
32.3, 22.3, 20.9, 13.0. HR-MS (ESI): Calcd. C21H20N4S3, [M+H]+m/z: 425.0928,
found: 425.0929.
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4.2.2 7-((4-Chlorophenyl)thio)-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-2-
amine (8)
o
1
White solid, Mp 217~219 C. H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H),
7.74-7.77 (m, 2H), 7.65-7.68 (m, 2H), 7.57-7.59 (m, 2H), 7.39-7.43 (m, 2H),
7.08-7.12 (m, 1H), 2.66-2.69 (t, J = 7.2 Hz, 2H), 1.33-1.39 (m, 2H), 0.76-0.80 (t, J =
7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.1, 160.5, 158.9, 154.7, 139.7,
137.4, 136.8, 134.7, 129.3, 129.1, 126.2, 123.0, 118.2, 32.4, 22.2, 13.1. HR-MS (ESI):
Calcd. C20H17ClN4S3, [M+H]+m/z: 445.0382, found: 445.0388.
4.2.3 N-phenyl-5-(propylthio)-7-(pyrimidin-2-ylthio)thiazolo[5,4-d]pyrimidin-2-
amine (9)
o
1
Pale yellow solid, Mp 88~91 C. H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H),
8.75-8.77 (m, 2H), 7.37-7.41 (m, 3H), 7.24-7.28 (m, 2H), 7.02-7.06 (m, 1H),
13
3.00-3.03 (t, J = 7.2 Hz, 2H), 1.61-1.66 (m, 2H), 0.92-0.96 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, DMSO-d6) δ 167.5, 163.1, 162.2, 158.4, 158.3, 150.8, 140.7, 139.4,
128.9, 123.1, 119.4, 118.1, 32.5, 22.0, 13.2. HR-MS (ESI): Calcd. C18H16N6S3,
[M+Na]+m/z: 435.0496, found: 435.0502.
4.2.4 7-(Benzo[d]thiazol-2-ylthio)-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-
2-amine (10)
o
1
White solid, Mp 156~158 C. H NMR (400 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.19
(m, 1H), 8.08 (m, 1H), 7.75 (m, 2H, ), 7.50-7.60 (m, 2H), 7.35-7.39 (m, 2H),
7.07-7.11 (m, 1H), 2.85-2.89 (t, J = 7.4 Hz, 2H), 1.42-1.48 (m, 2H), 0.70-0.74 (t, J =
7.2 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 162.3, 161.8, 159.4, 157.4, 151.9, 150.3,
139.6, 137.4, 136.7, 129.1, 126.6, 125.8, 123.2, 122.6, 122.0, 118.4, 32.4, 22.1, 12.9.
HR-MS (ESI): Calcd. C21H17N5S4, [M+Na]+m/z: 490.0264, found: 490.0266.
4.2.5 N-phenyl-5-(propylthio)-7-(thiazol-2-ylthio)thiazolo[5,4-d]pyrimidin-2-amine
(11)
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Pale yellow solid, Mp 185~187 oC. 1H NMR (400 MHz, CDCl3) δ 7.98 (m, 1H), 7.74
(br, 1H), 7.61 (m, 1H), 7.48-7.50 (m, 2H), 7.38-7.42 (m, 2H), 7.17-7.21 (m, 1H),
13
2.86-2.90 (t, J = 7.3 Hz, 2H), 1.56-1.61 (m, 2H), 0.92-0.96 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, CDCl3) δ 164.0, 161.2, 160.9, 154.7, 153.6, 143.5, 138.6, 137.0,
129.7, 125.1, 124.3, 120.3, 33.3, 22.6, 13.5. HR-MS (ESI): Calcd. C17H15N5S4,
[M+H]+m/z: 418.0289, found: 418.0286.
4.2.6 7-((1,3,4-thiadiazol-2-yl)thio)-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-
2-amine (12)
o
1
Pale yellow solid, Mp 229~231 C. H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H),
9.88 (s, 1H), 7.72-7.74 (m, 2H), 7.40-7.44 (m, 2H), 7.10-7.14 (m, 1H), 2.94-2.97 (t, J
= 7.2 Hz, 2H), 1.54-1.59 (m, 2H), 0.90-0.94 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 162.4, 161.4, 159.6, 157.9, 156.8, 149.2, 139.5, 137.2, 129.2, 123.3,
118.4, 32.5, 22.0, 13.2. HR-MS (ESI): Calcd. C16H14N6S4, [M+Na]+m/z: 441.0060,
found: 441.0060.
4.2.7 7-((5-Amino-1,3,4-thiadiazol-2-yl)thio)-N-phenyl-5-(propylthio)thiazolo[5,4-d]
pyrimidin-2-amine (13)
o
1
Off-white solid, Mp 273~275 C. H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H),
7.80 (m, 2H), 7.74-7.77 (s, 2H), 7.40-7.44 (m, 2H), 7.08-7.12 (m, 1H), 2.91-2.95 (t, J
= 7.2 Hz, 2H), 1.53-1.58 (m, 2H), 0.91-0.94 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 173.2, 162.3, 161.3, 159.4, 152.1, 140.7, 139.7, 137.0, 129.1, 123.1,
118.3, 32.6, 22.3, 13.1. HR-MS (ESI): Calcd. C16H15N7S4, [M+Na]+m/z: 456.0169,
found: 456.0173.
4.2.8 7-((1-Methyl-1H-imidazol-2-yl)thio)-N-phenyl-5-(propylthio)thiazolo[5,4-d]
pyrimidin-2-amine (14)
o
1
Pale yellow solid, Mp 230~231 C. H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H),
7.74-7.76 (m, 2H), 7.59 (m, 1H), 7.41-7.45 (m, 2H), 7.17 (m, 1H), 7.10-7.14 (m, 1H),
3.65 (s, 3H), 2.68-2.71 (t, J = 7.2 Hz, 2H), 1.41-1.46 (m, 2H), 0.85-0.89 (t, J = 7.3 Hz,
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3H). 13C NMR (100 MHz, DMSO-d6) δ 162.4, 160.9, 159.3, 153.9, 139.6, 137.0,
132.1, 129.9, 129.2, 125.7, 123.2, 118.4, 33.5, 32.4, 22.1, 13.2. HR-MS (ESI): Calcd.
C18H18N6S3, [M+H]+m/z: 415.0833, found: 415.0834.
4.2.9 7-((1-(2-(Dimethylamino)ethyl)-1H-tetrazol-5-yl)thio)-N-phenyl-5-(propylthio)
thiazolo[5,4-d]pyrimidin-2-amine (15)
o
1
Yellow solid, Mp 187~188 C. H NMR (400 MHz, DMSO-d6) δ 11.19 (br, 1H),
7.71-7.74 (m, 2H), 7.41-7.45 (m, 2H), 7.11-7.15 (m, 1H), 4.52-4.55 (t, J = 6.1 Hz,
2H), 2.65-2.71 (m, 4H), 2.10 (s, 6H), 1.38-1.43 (m, 2H), 0.84-0.88 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, DMSO-d6) δ 162.3, 161.9, 159.7, 149.8, 146.6, 139.5, 137.6,
129.2, 123.4, 118.5, 57.5, 46.0, 44.9, 32.3, 22.0, 13.0. HR-MS (ESI): Calcd.
C19H23N9S3, [M+H]+m/z: 474.1317, found: 474.1315.
4.2.10 N-(naphthalen-1-yl)-5-(propylthio)-7-(p-tolylthio)thiazolo[5,4-d]pyrimidin-2-
amine (16)
o
1
Pale yellow solid, Mp 208~210 C. H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H),
8.24-8.27 (m, 1H), 8.17 (m, 1H), 8.00 (m, 1H), 7.82-7.85 (m, 1H), 7.60-7.63 (m, 3H),
7.50 (m, 2H), 7.32 (m, 2H), 2.62-2.66 (t, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.30-1.36 (m,
13
2H), 0.72-0.76 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, DMSO-d6) δ 162.6, 162.3,
160.7, 156.1, 139.9, 137.2, 136.2, 135.5, 134.4, 130.4, 128.9, 127.4, 127.0, 126.8,
126.4, 126.0, 123.9, 122.6, 120.1, 32.8, 22.8, 21.4, 13.5. HR-MS (ESI): Calcd.
C25H22N4S3, [M+H]+m/z: 475.1085, found: 475.1082.
4.2.11 7-((1,3,4-Thiadiazol-2-yl)thio)-N-(naphthalen-1-yl)-5-(propylthio)thiazolo
[5,4-d]pyrimidin-2-amine (17)
o
1
Pale yellow solid, Mp 160~162 C. H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H),
9.89 (s, 1H), 8.25 (m, 1H), 8.11-8.14 (m, 1H), 8.00-8.03 (m, 1H), 7.86 (m, 1H),
7.59-7.63 (m, 3H), 2.92-2.96 (t, J = 7.2 Hz, 2H), 1.52-1.57 (m, 2H), 0.89-0.92 (t, J =
7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.6, 162.1, 158.6, 157.2, 149.6,
14

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137.7, 135.2, 134.4, 128.9, 127.4, 127.0, 126.9, 126.4, 122.6, 120.4, 33.0, 22.6, 13.6.
HR-MS (ESI): Calcd. C20H16N6S4, [M+Na]+m/z: 491.0217, found: 491.0217.
4.2.12 5-(Propylthio)-N-(pyridin-2-yl)-7-(pyrimidin-2-ylthio)thiazolo[5,4-d]pyrimid
in-2-amine (18)
o
1
Pale yellow solid, Mp 225~228 C. H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H),
8.75 (m, 2H), 8.38 (m, 1H), 7.78-7.83 (m, 1H), 7.43 (m, 1H), 7.07-7.13 (m, 2H),
13
2.93-2.97 (t, J = 7.2 Hz, 2H), 1.54-1.62 (m, 2H), 0.88-0.92 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, DMSO-d6) δ 167.7, 164.1, 162.7, 159.1, 157.4, 151.6, 151.0, 146.9,
139.3, 138.6, 120.0, 118.4, 112.0, 33.0, 22.6, 13.7. HR-MS (ESI): Calcd. C17H15N7S3,
[M+Na]+m/z: 436.0449, found: 436.0450.
4.2.13 7-((1,3,4-Thiadiazol-2-yl)thio)-5-(propylthio)-N-(pyridin-2-yl)thiazolo[5,4-d]
pyrimidin-2-amine (19)
o
1
Yellow solid, Mp 249~250 C. H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 9.91
(s, 1H), 8.38 (m, 1H), 7.80-7.84 (m, 1H), 7.15 (m, 1H), 7.07-7.10 (m, 1H), 2.96-3.00
13
(t, J = 7.2 Hz, 2H), 1.55-1.61 (m, 2H), 0.91-0.95 (t, J = 7.3 Hz, 3H). C NMR (100
MHz, DMSO-d6) δ 162.8, 162.5, 158.6, 158.1, 157.1, 150.9, 146.9, 139.3, 135.0,
118.4, 112.1, 33.0, 22.6, 13.7. HR-MS (ESI): Calcd. C15H13N7S4, [M+Na]+m/z:
442.0013, found: 442.0016.
4.2.14 5-(Propylthio)-N-(pyridin-2-yl)-7-(p-tolylthio)thiazolo[5,4-d]pyrimidin-2-
amine (20)
o
1
White solid, Mp 242~244 C. H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H),
8.37-8.39 (m, 1H), 7.79-7.83 (m, 1H), 7.52 (m, 2H), 7.33 (m, 2H), 7.15 (m, 1H),
7.05-7.08 (m, 1H), 2.64-2.68 (t, J = 7.2 Hz, 2H), 2.38 (s, 3H), 1.30-1.39 (m, 2H),
0.73-0.77 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.6, 161.2, 157.3,
157.1, 151.2, 147.0, 140.0, 139.2, 136.3, 134.6, 130.4, 123.8, 118.1, 111.9, 32.8, 22.8,
21.4, 13.5. HR-MS (ESI): Calcd. C20H19N5S3, [M+Na]+m/z: 448.0700, found:
448.0703.
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4.2.15 5-(Propylthio)-7-(p-tolylthio)-N-(3,4,5-trimethoxyphenyl)thiazolo[5,4-d]
pyrimidin-2-amine (21)
o
1
White solid, Mp 188~190 C. H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 7.50
(m, 2H), 7.32 (m, 2H), 7.22 (s, 2H), 3.84 (s, 6H), 3.66 (s, 3H), 2.62-2.65 (t, J = 7.2 Hz,
13
2H), 2.38 (s, 3H), 1.30-1.35 (m, 2H), 0.72-0.75 (t, J = 7.3 Hz, 3H). C NMR (100
MHz, DMSO-d6) δ 162.6, 160.4, 159.2, 156.2, 153.4, 139.9, 137.0, 136.4, 136.3,
133.6, 130.4, 123.8, 96.7, 60.6, 56.2, 32.8, 22.8, 21.4, 13.5. HR-MS (ESI): Calcd.
C24H26N4O3S3, [M+Na]+m/z: 537.1065, found: 537.1064.
4.2.16 5-(Benzylthio)-N-phenyl-7-(p-tolylthio)thiazolo[5,4-d]pyrimidin-2-amine (22)
o
1
White solid, Mp 190~193 C. H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 7.78
(m, 2H), 7.54 (m, 2H), 7.38-7.42 (m, 2H), 7.28 (m, 2H), 7.23 (m, 3H), 7.07-7.11 (m,
1H), 7.03 (m, 2H), 4.04 (s, 2H), 2.28 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
161.9, 160.8, 159.4, 156.1, 140.3, 140.0, 138.3, 137.4, 136.0, 130.4, 129.6, 129.1,
128.7, 127.4, 124.0, 123.5, 118.7, 34.8, 21.3. HR-MS (ESI): Calcd. C25H20N4S3,
[M+Na]+m/z: 495.0748, found: 495.0749.
4.2.17 7-((1,3,4-Thiadiazol-2-yl)thio)-5-(benzylthio)-N-phenylthiazolo[5,4-d]pyrimi
din-2-amine (23)
o
1
Yellow solid, Mp 246~248 C. H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 9.83
(s, 1H), 7.76 (m, 2H), 7.40-7.44 (m, 2H), 7.25-7.35 (m, 5H), 7.10-7.14 (m, 1H), 4.32
13
(s, 2H). C NMR (100 MHz, DMSO-d6) δ 162.2, 162.0, 160.3, 158.3, 157.5, 149.5,
140.0, 138.0, 137.5, 129.7, 129.4, 128.9, 127.7, 123.9, 118.9, 35.3. HR-MS (ESI):
Calcd. C20H14N6S4, [M+Na]+m/z: 489.0060, found: 489.0059.
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
16

ACCEPTED MANUSCRIPT
added to each well. The cells were then incubated for 72 h, thereafter MTT assays
were performed and cell viability was assessed at 570 nm by a microplate reader
(Biotech, Shanghai, China).
4.4 Clonogenicity assay
HGC-27 cells (1000 cells/well) were seeded in a 6-well plate and incubated for 24 h,
then the media were replaced with fresh mediacontaining different concentrations of
compound 22. After 9 days of treatment, the cells were washed twice with PBS, fixed
with 4% paraformaldehyde, and colonies were visualized using 0.1% crystal violet
staining. The cells were imaged, and the number of colonies were quantified by Image
J software (Developed by National Institutes of Health). A group of >10 cells was
defined as one colony. Inhibition rate = (1-number oftreatment/number of control) *
100%. All experiments were performed in triplicate.
4.5 Hoechst 33258 staining
HGC-27 cells were seeded into a 6-well plate (2×105/well) and incubated overnight
for adherent and treated with compound 22 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).
4.6 Wound healing assay
HGC-27 cells were placed in a 24-well plate, and the cell surface was scratched using
a 10 µL pipet tip. Then the cells were treated with compound 22 with different
concentrations followed by indicated time incubation and photographed on an
inverted microsope.
4.7 Cell apoptosis assay
HGC-27 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 22
for 24 h. Thereafter, the cells were collected and the Annexin-V-FITC/PI apoptosis kit
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(Biovision) was used according to the manufacturer’s protocol. The cells were
analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific,
MA).
4.8 Western blot analysis
HGC-27 cells were treated with different concentrations of compound 22 for 24 h, the
cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail for
o
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
bicinchonininc acid assay kit (Beyotmie Biotechnology, Haimen, China). After added
o
with loading 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
o
milk at room temperature for 2 h, and then incubated overnight at 4 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
(2016YFA0501800); National Natural Science Foundation of China (Project No.
81430085 and No. 21372206 for H.-M.L.); Ph.D Educational Award from Ministry of
Education (No. 20134101130001, for H.M.L.); the Starting Grant of Zhengzhou
University (No. 32210533).
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Highlights
ò The thiazolo[5,4-d]pyrimidine derivatives showed moderate to good growth
inhibition against the tested cancer cells.
ò Among them, compound 22 exerted the most antiproliferative activity against
HGC-27 cell line.
ò Compound 22 inhibited the cell colony formation and migration of HGC-27.
ò Compound 22 induced the apoptosis of HGC-27 cells, and led to the expression
changes of key proteins related to apoptosis.
ò The atom replacement strategy would be viable for designing new anticancer
agents.