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Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy
Accepted Manuscript Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy Zhong-Hua Li, Ji Zhang, Xue-Qi Liu, Peng-Fei Geng, Jin-Lian Ma, Bo Wang, TaoQian Zhao, Bing Zhao, Hao-Ming Wei, Chao Wang, Dong-Jun Fu, Bin Yu, Hong-Min Liu PII:
S0223-5234(17)30322-7
DOI:
10.1016/j.ejmech.2017.04.056
Reference:
EJMECH 9407
To appear in:
European Journal of Medicinal Chemistry
Received Date: 26 February 2017 Revised Date:
1 April 2017
Accepted Date: 20 April 2017
Please cite this article as: Z.-H. Li, J. Zhang, X.-Q. Liu, P.-F. Geng, J.-L. Ma, B. Wang, T.-Q. Zhao, B. Zhao, H.-M. Wei, C. Wang, D.-J. Fu, B. Yu, H.-M. Liu, Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphic abstract
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A series of thiazolo[5,4-d]pyrimidine derivatives were designed through the drug repurposing strategy. Compound 24 displayed high potency and selectivity against MGC-803 cells, and induced the apoptosis of MGC-803 cells.
ACCEPTED MANUSCRIPT Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy Zhong-Hua Li, Ji Zhang, Xue-Qi Liu, Peng-Fei Geng, Jin-Lian Ma, Bo Wang, Tao-Qian Zhao, Bing Zhao, Hao-Ming Wei, Chao Wang, Dong-Jun Fu, Bin Yu*, Hong-Min Liu*
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Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province; Key Laboratory of Technology of Drug Preparation (Zhengzhou University), Ministry of Education of China; Key Laboratory of Henan Province for Drug Quality and Evaluation; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China
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Corresponding Author: Prof. Hong-Min Liu& Dr. Bin Yu
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Zhengzhou University, Zhengzhou, 450001, PR China
E-mail: [email protected] (Hong-Min Liu) & [email protected](Bin Yu)
ABSTRACT: A series of thiazolo[5,4-d]pyrimidine derivatives were synthesized and evaluated for their antiproliferative activities on three cancer cell lines. The
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structure-activity relationship studies were conducted through the variation in the three regions of the thiazolo-pyrimidine core. Substitution with morpholine led to compound 24, which exerted the most potent antiproliferative activity as well as good
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selectivity between cancer and normal cells (IC50 values of 1.03 µM against MGC803 and 38.95µM against GES-1). In addition, compound 24 inhibited the colony
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formation and migration of MGC803 as well as induced apoptosis. Western blot experiments indicated the expression changes of apoptosis-related proteins, including up-regulation of Bax and caspase-3/9, as well as down-regulation of Bcl-2.
Keywords: Thiazolo[5,4-d]pyrimidine, Antiproliferative activity, Apoptosis, Drug repurposing
INTRODUCTION: Drug repurposing (also termed as drug repositioning), referring to the application of
ACCEPTED MANUSCRIPT known drugs for new indications, has emerged as a valuable strategy for drug discovery research, making the process of drug discovery lower risky, more probability and less time consuming [1, 2]. For example, thalidomide, as an anticonvulsive drug, is now used for the treatment of erythema nodosum leprosy and
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certain cancers [3]. The antifungal drug itraconazole has advanced into several phase II clinical studies for the treatment of cancer [4]. Most recently, Remya group designed a new series of 1-phenylpyrazole analogs as potent antitubercular agents
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based on the scaffold of anti-obesity drug rimonabant [5].
As part of our ongoing efforts toward identifying new anticancer agents [6-10], we
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screened our in-house small-molecule library against MGC-803 cells using the MTT assay. Intriguingly, we found that compound A (Fig. 1) as a TRPV1 (vanilloid receptor 1) antagonist [11], showed acceptable antiproliferative activity against MGC-803 cells. Besides, the fused thiazolo[5,4-d]pyrimidine skeleton has recently received considerable attention from medicinal community due to their diverse
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biological activities such as anti-tumor [12, 13], antimicrobial [14], anti-inflammatory [15], antinociceptive [16] and human cytomegalovirus inhibitory [17]. Therefore, we speculate that the thiazolo[5,4-d]pyrimidine scaffold (with suitable substituents) could
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be used as a template for designing new anticancer agents. In this work, we designed a new series of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents based on the drug repurposing strategy, and evaluated their preliminary modes of
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action.
Fig. 1. Design of new antiproliferative compounds based on the drug repurposing strategy.
2. Results and discussion 2.1 Chemistry
ACCEPTED MANUSCRIPT The general synthetic route was illustrated in Scheme 1. The intermediate derivatives 5a~b were synthesized following the previously reported procedure [10]. For the synthesis of 8a~d, arylamines reacted with carbon disulphide in the presence of triethylamine to produce dithiocarbamic acid salts 7a-d, which then reacted with
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triphosgene to give isothiocyanates 8a~d. The obtained isothiocyanate derivatives reacted with 5a~b under alkaline conditions (cesium carbonate) in acetonitrile to give the key active intermediates 6a~e [18]. Compounds 9-12 were prepared from compounds 6a-e and various arylamines in the presence of p-TsOH [18]. Finally, the
and triethylamine in isopropanol. HO
OH N
a
N
N
HO
b
N
SH
S
1
2a~b
NO2
NO2
OH
OH
N
c
N
S
R1
Cl
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HO
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target compounds 13-24 was readily obtained by refluxing amine derivatives with 6
Cl
N
d
N S
R1
R1
4a~b
3a~b
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f
NH2 Cl N
N
e
N
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R1 5a~b
N
S
N
S
R1
9~12
2a~5a R1 = Propyl 2b~5b R1 = Bn
6a 6b 6c 6d 6e
R1 = BnR1 = Propyl R1 = Propyl R1 = Propyl R1 = Propyl
aromatic terminal or cyclopropyl
N
HN
R2
S
R2
N
Cl
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Cl
H N
NH
S
N
S
R1
HN g
H N R2
6a~e
N S
R2 = Ph R2 = Ph R2 = 2-Cl-Ph R2 = 2-Pyridyl R2 = Naphthyl
N N
S
R1
13~17 Y
Y = N, O, S
N g
H N R2
N S
N N
S
R1
18~24 S Arylamine
h
Aryl NH 7a-d
SH
TEA
7a, 8a Aryl = Ph
i Aryl N C S 8a-d
7b, 8b Aryl = 2-Cl-Ph 7c, 8c Aryl = 2-Pyridyl 7d, 8d Aryl = Naphthyl
ACCEPTED MANUSCRIPT Scheme 1. Synthesis of thiazolo[5,4-d]pyrimidine derivatives. Reagent and conditions: (a) Alkyl bromide, TEA, methanol, reflux, 2 h; (b) Fuming nitric acid, AcOH, 25~45 oC, 1 h; (c) POCl3, DMA, reflux, 2 h; (d) Fe, AcOH, methanol, rt~reflux; (e) R2SCN, Cs2CO3, acetonitrile, rt, overnight; (f) TsOH, toluene, reflux, 6 h; (g) TEA, isopropanol, reflux, 6 h; (h) TEA, THF, CS2, rt,
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overnight; (i) Chlorofrom, triphosgene, rt, overnight.
2.2 Evaluation of biological activity 2.2.1 Antiproliferative activity
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All final compounds were evaluated for their antiproliferative activities against three cancer lines, namely, H1650 (human lung cancer line), HGC27 and MGC803 (human
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gastric cancer lines) by using the MTT assay, and 5-fluorouracil (5-Fu) was employed as the reference drug [19]. All the compounds were divided into three types based on the substituents (R3) attached. The antiproliferative results of preliminary evaluation were summarized in Tables 1-3.
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As shown in Table 1, compounds 9-12 displayed moderate cytotoxic activities against the tested cancer cell lines with IC50 values ranging from 12 to 35 µM regardless of the nature of groups attached on the phenyl ring. One exception is compound 12,
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which showed no activity toward MGC803 with IC50 above 64 µM.
Table 1. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with
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aromatic amine substituents against three cancer cell lines.
Comp
R
1
R
2
IC50 (µM)a
3
RN MGC803
H1650
HGC27
9
Propyl
Ph
24.42±0.57
12.12±1.08
35.28±1.54
10
Propyl
Ph
17.42±0.87
14.76±1.16
13.12±1.11
11
Propyl
Ph
12.49±0.39
11.99±1.07
20.31±1.31
12
Propyl
Ph
>64
20.65±1.31
30.88±1.49
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5-Fu a
-
-
6.56±0.31
12.52±1.53
8.22±0.98
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.
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Next, we introduced aliphatic amine groups to the scaffold, as shown in Table 2, compound 13 with the terminal indole group presented improved antiproliferative activities against both MGC803 and H1650 cell lines with IC50 values less than 10 µM. Compound 14 with para-OH benzyl group showed comparable activity toward
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H1650 with compound 13. Compound 15 with terminal thiophene group displayed moderate antiproliferative activities against the tested cancer cell lines, albeit with
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slightly decreased activity against MGC803 and H1650. In contrast, compounds 16 and 17 showed weak antiproliferative activities against the tested cancer cell lines.
Table 2. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with
Propyl
14
Propyl
15
Propyl
a
IC50 (µM)a
R3 N
MGC803
H1650
HGC27
6.99±0.84
9.62±0.98
16.19±1.20
Ph
21.86±0.27
9.23±0.96
17.31±1.23
Ph
11.30±1.05
10.01±1.00
17.74±1.24
Ph
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13
R2
EP
R1
Comp
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aliphatic amine substituents against three cancer cell lines.
16
Propyl
Ph
>64
26.10±1.41
27.98±1.44
17
Propyl
Ph
>64
41.73±1.06
32.35±1.51
5-Fu
-
-
6.56±0.31
12.52±1.53
8.22±0.98
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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 acceptable antiproliferative activities of compounds 13 and 14 against MGC803
ACCEPTED MANUSCRIPT and H1650 cells promoted us to investigate the effect of cyclic amine substituents on the activity (Table 3). In general, this series of compounds showed improved activity compared with compounds 9-17. Compound 19 with the morphine substituent (IC50 < 10 µM) was much more potent than compounds 18 and 20, showing the importance of
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the oxygen atom of the morphine ring for the antiproliferative activity against the tested cancer cell lines. Keeping the morphine ring unchanged, we next explored the effect of other aromatic rings (R2) on the activity. Evidently, 2-chloro phenyl (21), pyridyl (22), and naphthyl (23) groups were generally less preferred for the activity.
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However, for MGC-803 cells, compound 21 showed slightly better activity than compound 19. Interestingly, replacement of propyl group with the benzyl group (R1)
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led to a significant increase in the activity. Compound 24 exhibited excellent inhibition against all the tested cancer cell lines at low micromolar levels, showing that the steric hydrophobic group may be preferred for improving the activity against the human gastric and lung cancer lines. This finding may provide directions for
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further structural modifications for designing new anticancer agents.
Table 3. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with cyclic aliphatic amine substituents against three cancer cell lines.
Propyl
R2
Ph
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18
R1
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Comp
IC50 (µM)a
R3 N
MGC803
H1650
HGC27
12.33±2.16
12.88±1.11
28.47±1.45
19
Propyl
Ph
5.81±0.45
4.18±0.62
9.51±1.52
20
Propyl
Ph
18.77±0.95
27.02±1.43
>64
21
Propyl
2-Cl-Ph
3.40±0.53
21.73±1.66
11.32±1.05
22
Propyl
9.35±0.97
7.73±1.01
8.84±0.94
23
Propyl
17.68±0.88
9.73±1.11
21.76±1.50
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a
24
Bn-
Ph
5-Fu
-
-
-
1.03±0.26
2.10±0.61
1.23±0.09
6.56±0.31
12.52±1.53
8.22±0.98
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
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experiments.
In order to investigate the possible toxicity of such series of compounds, the antiproliferative activity of the most potent compound 24 (IC50= 1.03 µM against MGC803) was examined against GES-1 (normal human gastric epithelial cell line).
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As shown in Table 4, compound 24 inhibited GES-1 with an IC50 value of 38.95 µM,
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showing around 38-fold selectivity to MGC803 cells over GSE-1 cells. Table 4. Inhibitory results of compound 24 against normal cells GES-1.
Comp
24
Bn-
R2
IC50 (µM)a
R3 N
Ph
SIb
MGC803
GES-1
1.03±0.26
38.95±1.67
37.8
Inhibitory activity was assayed by exposure for 72 h to substance and expressed as concentration required to
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a
R1
inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments. b
The selectivity index (SI) was calculated as IC50 (GES-1) / IC50 (MGC803).
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2.2.2 Clone assay and cell migration
The excellent cytotoxic activity of compound 24 against MGC803 promoted us to
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investigate the effect on colony formation and cell migration. The clone formation of cancer cells represents an indirect estimation of neoplastic transformation [20]. As shown in Fig. 2A and 2B, treatment of MGC803 cells with compound 24 led to fewer and smaller colonies compared to the control with an inhibition rate of 86% at 3 µM. In addition, the cell migration ability was also initially evaluated by the wound healing assay. As shown in Fig. 2C, treatment of MGC-803 cells with compound 24 at indicated concentration markedly suppressed the wound healing in a time-dependent manner.
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Fig. 2. (A) Representative images of MGC803 cells colonies after treatment with various concentrations for 9 days; (B) Quantitative analysis of the colony formation inhibition rate; (C)
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Wound healing assay.
2.2.3 Cell apoptosis and possible mechanism involved Apoptosis is known as programmed cell death and is typically characterized by
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distinctive cell morphological changes [21]. Compound 24 was chosen for evaluating its ability of inducing apoptosis. Hochest 33258 staining was performed to investigate
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morphological changes of MGC803 cells [22]. After 24 h incubation with compound 24 at indicated concentrations, characteristic apoptotic morphological changes were observed, including cell rounding, chromatin shrinkage and formation of apoptotic bodies (Fig. 3A). Notably, this phenomena were more remarkable at higher concentrations. To further explore the effect of compound 24 on cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI double staining and analyzed with high content [23]. Treatment of MGC803 cells with compound 24 resulted in a concentration-dependent apoptosis increase (Fig. 3B), and the percentage of apoptotic cells was 11.5%, 43.8%, and 73.9%, respectively, compared to the
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control (2.3%) (Fig. 3C).
Fig. 3. Compound 24 induced apoptosis of MGC803 cells. (A) Apoptosis analysis with
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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
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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 MGC803 cells with compound 24 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 [24]. Meanwhile, the expression of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 4C). As shown in Fig. 4D and 4E, the expression of caspase-3 and caspase-9 also increased evidently. These
ACCEPTED MANUSCRIPT results indicated that compound 24 may induce MGC803 apoptosis through the
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activation of intrinsic apoptotic pathway.
Fig. 4. Expression changes of apoptosis-related proteins induced by compound 24. (A) Compound
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24 induced expression changes of Bax, Bcl-2 and caspase family members in MGC803 cells; (B-E)
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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 the drug repurposing strategy and further evaluated for their antiproliferative activity. Among them, compound 24 exhibited the most potent inhibition against the tested cancer cells (MGC803, H1650 and HGC27) and was less toxic to GES-1, indicating a good selectivity to cancer cells over normal cells. Further mechanism investigation showed that compound 24 can inhibit the cell colony formation and migration, induce apoptosis through intrinsic apoptotic pathway of MGC803 cells.
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4. Experimental section 4.1 General Reagents and solvents were purchased from commercial sources and were used
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without further purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. IR spectra was recorded on FT-IR (Bruker). 1HNMR 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
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Micromass Q-T of Micromass spectrometer by electrospray ionizaton (ESI).
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4.2 General procedure for the synthesis of compounds 9-12
The mixture of 6b (1eq), appropriate arylamine (2eq) and TsOH (2eq) in toluene was refluxed for 8 h and monitored by TLC (PE/EA = 4:1 ~ 2:1). 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
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with anhydride sodium sulfate and concentrated under reduced vacuum. The residue was purified by flash column chromatography (PE/EA = 4:1 ~ 2:1) to give the target product.
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4.2.1 N2,N7-diphenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-diamine (9) Pink solid, yield 65%, Mp 127~130 oC. IR (KBr) υmax: 2961, 1526, 1343, 730 cm-1. H NMR (400 MHz, Chloroform-d) δ 7.76 (m, 2H), 7.47-7.49 (m, 2H), 7.35-7.43 (m,
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1
4H), 7.16-7.19 (m, 1H), 7.09-7.12 (m, 1H), 3.13-3.16 (t, J = 7.4 Hz, 2H), 1.78-1.83 (m, 2H), 1.04-1.08 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 162.76,
159.17, 157.10, 150.16, 140.66, 139.37, 129.46, 128.78, 127.04, 123.74, 122.61, 122.32, 118.36, 32.78, 23.23, 13.78. HR-MS (ESI): Calcd. C20H19N5S2, [M+H]+m/z: 394.1160, found: 394.1162.
4.2.2 N7-(4-butylphenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7diamine (10)
ACCEPTED MANUSCRIPT Purple solid, yield 72%, Mp 163~165 oC. IR (KBr) υmax: 2956, 1576, 1350, 746 cm-1. 1
H NMR (400 MHz, Chloroform-d) δ 7.64 (m, 2H), 7.39-7.49 (m, 5H), 7.16-7.18 (m,
3H), 3.12-3.16 (t, J = 7.4 Hz, 2H), 2.59-2.63 (t, J = 7.7 Hz, 2H), 1.77-1.83 (m, 2H), 1.57-1.64 (m, 2H), 1.34-1.40 (m, 2H), 1.03-1.07 (t, J = 7.3 Hz, 3H), 0.92-0.96 (t, J = 13
C NMR (100 MHz, DMSO-d6) δ 162.79, 158.92, 156.98, 150.31,
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7.3 Hz, 3H).
140.70, 137.82, 136.90, 129.45, 128.52, 126.96, 122.51, 118.32, 34.77, 33.69, 32.81, 23.26, 22.15, 14.23, 13.79. HR-MS (ESI): Calcd. C24H27N5S2, [M+H]+m/z: 450.1786,
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found: 450.1789.
4.2.3 N7-(4-chlorophenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-
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diamine (11)
Pink solid, yield 59%, Mp 140~142 oC. IR (KBr) υmax: 3345, 2960, 1527, 1343, 819, 744 cm-1. 1H NMR (400 MHz, Chloroform-d) δ 7.71 (m, 2H), 7.47 (m, 3H), 7.40-7.44 (m, 2H), 7.32-7.34 (m, 2H), 7.17-7.20 (m, 1H), 3.11-3.15 (t, J = 7.4 Hz, 2H), 1.77-1.83 (m, 2H), 1.04-1.08 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
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162.74, 159.52, 157.17, 149.93, 140.65, 138.48, 129.48, 128.67, 127.18, 123.83, 122.69, 119.14, 118.39, 32.85, 23.09, 13.79. HR-MS (ESI): Calcd. C20H18ClN5S2, [M+H]+m/z: 428.0770, found: 428.0771.
diamine (12)
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4.2.4 N7-(4-methoxyphenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-
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Purple solid, yield 72%, Mp 158~160 oC. IR (KBr) υmax: 3396, 2956, 1505, 1346, 821, 746 cm-1. 1H NMR (400 MHz, DMSO-d6 ) δ 10.56 (s, 1H), 9.22 (s, 1H), 7.89 (m, 2H), 7.62-7.64 (m, 2H), 7.33-7.37 (m, 2H), 7.02-7.05 (m, 1H), 6.95-6.97 (m, 2H), 3.77 (s, 3H), 2.98-3.02 (t, J = 7.2 Hz, 2H), 1.62-1.68 (m, 2H), 0.92-0.96 (t, J = 7.3 Hz, 3H). 13
C NMR (100 MHz, DMSO-d6) δ 162.82, 158.78, 156.86, 156.15, 150.59, 140.76,
132.19, 129.44, 126.74, 124.56, 122.52, 118.29, 114.01, 55.69, 32.79, 23.21, 13.80. HR-MS (ESI): Calcd. C21H21N5OS2, [M+H]+m/z: 424.1266, found: 424.1264.
4.3 General procedure for the synthesis of compounds 13-24
ACCEPTED MANUSCRIPT The mixture of 6 (1 eq), appropriate amine (1.1 eq) and TEA (2eq) in isopropanol was refluxed for 6 h and monitored by TLC (PE/EA = 4:1 ~ 2:1). 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
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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 product.
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4.3.1 N7-(2-(1H-indol-2-yl)ethyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine2,7-diamine (13)
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Pale yellow solid, yield 78%, Mp 194~195 oC. IR (KBr) υmax: 3425, 2919, 1589, 1350, 741 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 10.47 (s, 1H), 7.82-7.84 (m, 2H), 7.67-7.70 (m, 2H), 7.34-7.37 (m, 3H), 7.19 (d, J = 2.3 Hz, 1H), 7.06-7.10 (m, 1H), 6.98-7.04 (m, 2H), 3.75-3.77 (m, 2H), 3.04-3.10 (m, 4H), 1.67-1.72 (m, 2H), 0.94-0.98 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 162.73, 156.75,
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156.22, 152.30, 140.45, 136.26, 128.95, 127.27, 125.89, 122.51, 121.88, 120.95, 118.44, 118.20, 117.56, 111.81, 111.37, 40.96, 32.36, 25.19, 22.65, 13.32. HR-MS
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(ESI): Calcd. C24H24N6S2, [M+H]+m/z: 461.1582, found: 461.1585.
4.3.2 4-(((2-(phenylamino)-5-(propylthio)thiazolo[5,4-d]pyrimidin-7-yl)amino) methyl)phenol (14)
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Pale yellow solid, yield 81%, Mp 160~162 oC. IR (KBr) υmax: 3433, 3415, 2916, 1557, 1352, 749 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 9.25 (s, 1H), 8.09 (t, J = 6.3 Hz, 1H), 7.83-7.85 (m, 2H), 7.31-7.35 (m, 2H), 7.15-7.17 (m, 2H), 6.99-7.03 (m, 1H), 6.69-6.71 (m, 2H), 4.58-4.60 (d, J = 6.2 Hz, 2H), 2.98-3.01 (t, J = 7.2 Hz, 2H), 1.60-1.65 (m, 2H), 0.91-0.94 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.60, 156.15, 152.15, 140.42, 131.46, 130.06, 128.93, 128.62, 128.36, 125.80, 121.87, 117.59, 114.95, 42.69, 32.31, 22.62, 13.29. HR-MS (ESI): Calcd. C21H21N5OS2, [M+H]+m/z: 424.1266, found: 424.1262.
ACCEPTED MANUSCRIPT 4.3.3 N2-phenyl-5-(propylthio)-N7-(2-(thiophen-2-yl)ethyl)thiazolo[5,4-d]pyrimidine2,7-diamine (15) Brown solid, yield 85%, Mp 123~125 oC. IR (KBr) υmax: 2959, 1541, 1342, 746, 691 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.82-7.84 (m, 2H), 7.71-7.74
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(m, 1H), 7.32-7.37 (m, 3H), 7.02-7.04 (m, 1H), 6.97-6.99 (m, 1H), 6.93-6.94 (m, 1H), 3.71-3.73 (m, 2H), 3.15-3.19 (t, J = 7.6 Hz, 2H), 3.05-3.09 (t, J = 7.2 Hz, 2H), 1.67-1.72 (m, 2H), 0.96-1.00 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
162.62, 156.30, 152.16, 141.37, 140.41, 128.93, 127.92, 127.81, 126.96, 125.90,
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125.08, 124.05, 121.93, 117.60, 41.87, 32.40, 29.31, 22.69, 13.32. HR-MS (ESI):
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Calcd. C20H21N5S3, [M+H]+m/z: 428.1037, found: 428.1039.
4.3.4 N7-(furan-2-ylmethyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7diamine (16)
Brown solid, yield 80%, Mp 143~146 oC. IR (KBr) υmax: 3439, 2959, 1531, 1350, 750, 689 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.44-7.47 (m, 3H), 7.34-7.38 (m, 3H),
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7.09-7.14 (m, 1H), 6.33 (m, 1H), 6.27 (m, 1H), 5.90-5.93 (t, J = 5.8 Hz, 1H), 4.77 (d, J = 5.8 Hz, 2H), 3.10-3.14 (t, J = 7.4 Hz, 2H), 1.75-1.80 (m, 2H), 1.02-1.05 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.98, 156.79, 153.23, 152.42, 142.27, 140.86, 129.39, 126.42, 122.39, 118.07, 110.91, 107.12, 37.30, 32.80, 23.08, 13.78.
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HR-MS (ESI): Calcd. C19H19N5OS2, [M+H]+m/z: 398.1109, found: 398.1110.
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4.3.5 N7-cyclopropyl-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-diamine (17)
Pale yellow solid, yield 89%, Mp 140~143 oC. IR (KBr) υmax: 3250, 2960, 1548, 1348, 748 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 7.82-7.84 (m, 2H), 7.68 (d, J = 3.4 Hz, 1H), 7.31-7.35 (m, 2H), 6.98-7.03 (m, 1H), 3.06-3.09 (t, J = 7.2 Hz, 2H), 2.91 (m, 1H), 1.69-1.75 (m, 2H), 0.97-1.01 (t, J = 7.3 Hz, 3H), 0.75-0.78 (m, 2H), 0.66-0.70 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.56, 155.99, 153.33, 140.39, 131.47, 128.92, 128.62, 125.91, 121.84, 117.59, 32.40, 23.70, 22.73, 13.34, 6.41. HR-MS (ESI): Calcd. C20H21N5S3, [M+H]+m/z: 358.1160, found: 358.1160.
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4.3.6
N-phenyl-5-(propylthio)-7-thiomorpholinothiazolo[5,4-d]pyrimidin-2-amine
(18) Yellow solid, yield 89%, Mp 135~137 oC. IR (KBr) υmax: 2962, 1553, 1348, 730 cm-1. H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.58-7.61 (m, 2H), 7.34-7.38 (m, 2H),
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1
7.00-7.04 (m, 1H), 4.44 (br, 4H), 3.01-3.04 (t, J = 7.2 Hz, 2H), 2.74-2.77 (m, 4H), 1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.4 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
161.89, 161.22, 154.30, 151.07, 140.20, 129.04, 125.35, 122.11, 117.50, 48.27, 32.34,
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26.38, 22.55, 13.30. HR-MS (ESI): Calcd. C18H21N5S3, [M+H]+m/z: 404.1037, found:
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404.1037.
4.3.7 7-morpholino-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-2-amine (19) Yellow solid, yield 89%, Mp 149~150 oC. IR (KBr) υmax: 2955, 1557, 1346, 746 cm-1. 1
H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.56-7.59 (m, 2H), 7.33-7.37 (m, 2H),
7.00-7.04 (m, 1H), 4.15 (br, 4H), 3.73-3.76 (m, 4H), 3.02-3.05 (t, J = 7.2 Hz, 2H), 13
C NMR (100 MHz, DMSO-d6) δ
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1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.4 Hz, 3H).
161.87, 161.03, 154.51, 151.41, 140.16, 129.06, 125.35, 122.19, 117.62, 66.08, 45.90, 32.30, 22.46, 13.26. HR-MS (ESI): Calcd. C18H21N5OS2, [M+H]+m/z: 388.1266,
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found: 388.1263.
4.3.8 7-(4-benzylpiperazin-1-yl)-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-
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2-amine (20)
Pale yellow solid, yield 82%, Mp 144~146 oC. IR (KBr) υmax: 2963, 1514, 1350, 741 cm-1. 1H NMR (400 MHz, ) δ 10.50 (s, 1H), 7.57-7.59 (m, 2H), 7.33-7.35 (m, 6H), 7.26-7.29 (m, 1H), 6.98-7.02 (m, 1H), 4.17 (m, 4H), 3.52 (s, 2H), 3.00-3.03 (t, J = 7.2Hz, 2H), 2.51 (m, 4H), 1.64-1.69 (m, 2H), 0.95-0.98 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.34, 161.47, 154.77, 151.81, 140.70, 138.32, 129.50, 129.39, 128.66, 127.47, 125.74, 122.62, 118.04, 62.41, 53.02, 45.84, 32.80, 23.01, 13.77. HR-MS (ESI): Calcd. C25H28N6S2, [M+H]+m/z: 477.1895, found: 477.1895.
ACCEPTED MANUSCRIPT 4.3.9 N-(2-chlorophenyl)-7-morpholino-5-(propylthio)thiazolo[5,4-d]pyrimidin-2amine (21) White solid, yield 79%, Mp 196~197 oC. IR (KBr) υmax: 3276, 2956, 1557, 1342, 776 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 7.99 (m, 1H), 7.34-7.36 (m,
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2H), 7.04-7.07 (m, 1H), 4.13-4.16 (m, 4H), 3.76-3.78 (t, J = 4.7 Hz, 4H), 3.02-3.05 (t, J = 7.1 Hz, 2H), 1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.3 Hz, 3H).
13
C NMR (100
MHz, DMSO-d6) δ 162.76, 161.70, 154.52, 152.08, 142.00, 133.84, 131.05, 125.55, 122.00, 117.47, 116.53, 66.58, 46.37, 32.80, 22.94, 13.76.HR-MS (ESI): Calcd.
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C18H20ClN5OS2, [M+H]+m/z: 422.0876, found: 422.0877.
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4.3.10 7-morpholino-5-(propylthio)-N-(pyridin-2-yl)thiazolo[5,4-d]pyrimidin-2-amine (22)
Pale yellow solid, yield 83%, Mp 240~241 oC. IR (KBr) υmax: 2960, 1550, 1326, 768 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.31-8.33 (m, 1H), 7.73-7.76 (m, 1H), 7.09-7.11 (m, 1H), 6.99 (m, 1H), 4.18-4.20 (m, 4H), 3.73-3.76 (t, J = 4.6 Hz, 13
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4H), 3.05 (t, J = 7.1 Hz, 2H), 1.66-1.72 (m, 2H), 0.97-1.00 (t, J = 7.3 Hz, 3H).
NMR (100 MHz, DMSO-d6) δ 161.84, 161.79, 151.94, 151.77, 151.15, 146.35, 138.34, 123.53, 116.88, 111.03, 66.10, 45.73, 32.30, 22.55, 13.27. HR-MS (ESI):
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Calcd. C17H20N6OS2, [M+H]+m/z: 389.1218, found: 389.1220.
4.3.11 7-morpholino-N-(naphthalen-1-yl)-5-(propylthio)thiazolo[5,4-d]pyrimidin-2-
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amine (23)
White solid, yield 83%, Mp 142~144 oC. IR (KBr) υmax: 2957, 1544, 1346, 764 cm-1. 1
H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.22-8.25 (m, 1H), 8.04-8.06 (m, 1H),
7.95-7.97 (m, 1H), 7.73-7.75 (m, 1H), 7.52-7.59 (m, 3H), 4.10-4.13 (m, 4H), 3.69-3.71 (t, J = 4.7 Hz, 4H), 3.01-3.05 (t, J = 7.1 Hz, 2H), 1.65-1.70 (m, 2H), 0.96-0.99 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 161.75, 161.26,
157.00, 151.40, 135.53, 133.95, 128.29, 126.40, 126.25, 125.90, 125.84, 125.24, 124.36, 122.16, 117.94, 66.06, 45.86, 32.29, 22.48, 13.25. HR-MS (ESI): Calcd. C22H23N5OS2, [M+H]+m/z: 438.1422, found: 438.1420.
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4.3.12 5-(benzylthio)-7-morpholino-N-phenylthiazolo[5,4-d]pyrimidin-2-amine (24) Pale yellow solid, yield 80%, Mp 183~185 oC. IR (KBr) υmax: 3277, 2849, 1559, 1339, 746 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 7.56-7.59 (m, 2H),
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7.40-7.43 (m, 2H), 7.34-7.38 (m, 2H), 7.29-7.33 (m, 2H), 7.21-7.26 (m, 1H), 7.00-7.05 (m, 1H), 4.36 (s, 2H), 4.16 (m, 4H), 3.71-3.74 (t, J = 4.7 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 161.34, 160.91, 154.69, 151.37, 140.14, 138.18, 129.08, 128.70, 128.33, 126.88, 125.50, 122.25, 117.67, 66.11, 45.95, 34.56. HR-MS (ESI):
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Calcd. C22H21N5OS2, [M+H]+m/z: 436.1266, found: 436.1266.
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4.4 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 72 h, thereafter MTT assays
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were performed and cell viability was assessed at 570nm by a microplate reader (Biotech, Shanghai, China).
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4.5 Clonogenicity assay
MGC803 cells (1000 cells/well) were seeded in a 6-well plate and incubated for 24 h,
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then the media were replaced with fresh media containing different concentrations of compound 24. 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 of treatment/number of control) * 100%. All experiments were performed in triplicate.
4.6 Hoechst 33258 staining MGC803 cells were seeded into a 6-well plate (2×105/well) and incubated overnight
ACCEPTED MANUSCRIPT for adherent and treated with compound 24 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).
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4.7 Wound healing assay MGC803 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 24 with different concentrations followed by indicated time incubation and photographed on an
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inverted microsope.
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4.8 Cell apoptosis assay
MGC803 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 24 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
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analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific, MA).
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4.9 Western blot analysis
MGC803 cells were treated with different concentrations of compound 24 for 24 h, the cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail
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for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4 oC. After the collection of supernatant, the protein concentration was detected using a bicinchonininc acid assay kit (Beyotmie Biotechnology, Haimen, China). After added with loading buffer, cell lyses were boiled for 10 min at 100oC for SDSpolyacrylamide
gel
electrophoresis
(PAGE).
Proteins
were
transferred
to
nitrocellulose (NC) membranes. Then the membranes were blocked with 5% skim milk at room temperature for 2h, and then incubated overnight at 4oC 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
ACCEPTED MANUSCRIPT antibody-reactive were revealed by enhanced chemiluminescence (ECL) and exposed on Kodak radiographic film.
Acknowledgement
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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
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Education (No. 20134101130001, for H.M.L.).
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Highlights The thiazolo[5,4-d]pyrimidine derivatives showed potent inhibition against the cancer cells. Compound 24 exerted the most antiproliferative activity against MGC-803 cells. Compound 24 inhibited the cell colony formation and migration of MGC803. Compound 24 induced the apoptosis of MGC-803 cells.
S0223-5234(17)30322-7
DOI:
10.1016/j.ejmech.2017.04.056
Reference:
EJMECH 9407
To appear in:
European Journal of Medicinal Chemistry
Received Date: 26 February 2017 Revised Date:
1 April 2017
Accepted Date: 20 April 2017
Please cite this article as: Z.-H. Li, J. Zhang, X.-Q. Liu, P.-F. Geng, J.-L. Ma, B. Wang, T.-Q. Zhao, B. Zhao, H.-M. Wei, C. Wang, D.-J. Fu, B. Yu, H.-M. Liu, Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Graphic abstract
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A series of thiazolo[5,4-d]pyrimidine derivatives were designed through the drug repurposing strategy. Compound 24 displayed high potency and selectivity against MGC-803 cells, and induced the apoptosis of MGC-803 cells.
ACCEPTED MANUSCRIPT Identification of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents through the drug repurposing strategy Zhong-Hua Li, Ji Zhang, Xue-Qi Liu, Peng-Fei Geng, Jin-Lian Ma, Bo Wang, Tao-Qian Zhao, Bing Zhao, Hao-Ming Wei, Chao Wang, Dong-Jun Fu, Bin Yu*, Hong-Min Liu*
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Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province; Key Laboratory of Technology of Drug Preparation (Zhengzhou University), Ministry of Education of China; Key Laboratory of Henan Province for Drug Quality and Evaluation; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China
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Corresponding Author: Prof. Hong-Min Liu& Dr. Bin Yu
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Zhengzhou University, Zhengzhou, 450001, PR China
E-mail: [email protected] (Hong-Min Liu) & [email protected](Bin Yu)
ABSTRACT: A series of thiazolo[5,4-d]pyrimidine derivatives were synthesized and evaluated for their antiproliferative activities on three cancer cell lines. The
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structure-activity relationship studies were conducted through the variation in the three regions of the thiazolo-pyrimidine core. Substitution with morpholine led to compound 24, which exerted the most potent antiproliferative activity as well as good
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selectivity between cancer and normal cells (IC50 values of 1.03 µM against MGC803 and 38.95µM against GES-1). In addition, compound 24 inhibited the colony
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formation and migration of MGC803 as well as induced apoptosis. Western blot experiments indicated the expression changes of apoptosis-related proteins, including up-regulation of Bax and caspase-3/9, as well as down-regulation of Bcl-2.
Keywords: Thiazolo[5,4-d]pyrimidine, Antiproliferative activity, Apoptosis, Drug repurposing
INTRODUCTION: Drug repurposing (also termed as drug repositioning), referring to the application of
ACCEPTED MANUSCRIPT known drugs for new indications, has emerged as a valuable strategy for drug discovery research, making the process of drug discovery lower risky, more probability and less time consuming [1, 2]. For example, thalidomide, as an anticonvulsive drug, is now used for the treatment of erythema nodosum leprosy and
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certain cancers [3]. The antifungal drug itraconazole has advanced into several phase II clinical studies for the treatment of cancer [4]. Most recently, Remya group designed a new series of 1-phenylpyrazole analogs as potent antitubercular agents
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based on the scaffold of anti-obesity drug rimonabant [5].
As part of our ongoing efforts toward identifying new anticancer agents [6-10], we
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screened our in-house small-molecule library against MGC-803 cells using the MTT assay. Intriguingly, we found that compound A (Fig. 1) as a TRPV1 (vanilloid receptor 1) antagonist [11], showed acceptable antiproliferative activity against MGC-803 cells. Besides, the fused thiazolo[5,4-d]pyrimidine skeleton has recently received considerable attention from medicinal community due to their diverse
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biological activities such as anti-tumor [12, 13], antimicrobial [14], anti-inflammatory [15], antinociceptive [16] and human cytomegalovirus inhibitory [17]. Therefore, we speculate that the thiazolo[5,4-d]pyrimidine scaffold (with suitable substituents) could
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be used as a template for designing new anticancer agents. In this work, we designed a new series of thiazolo[5,4-d]pyrimidine derivatives as potent antiproliferative agents based on the drug repurposing strategy, and evaluated their preliminary modes of
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action.
Fig. 1. Design of new antiproliferative compounds based on the drug repurposing strategy.
2. Results and discussion 2.1 Chemistry
ACCEPTED MANUSCRIPT The general synthetic route was illustrated in Scheme 1. The intermediate derivatives 5a~b were synthesized following the previously reported procedure [10]. For the synthesis of 8a~d, arylamines reacted with carbon disulphide in the presence of triethylamine to produce dithiocarbamic acid salts 7a-d, which then reacted with
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triphosgene to give isothiocyanates 8a~d. The obtained isothiocyanate derivatives reacted with 5a~b under alkaline conditions (cesium carbonate) in acetonitrile to give the key active intermediates 6a~e [18]. Compounds 9-12 were prepared from compounds 6a-e and various arylamines in the presence of p-TsOH [18]. Finally, the
and triethylamine in isopropanol. HO
OH N
a
N
N
HO
b
N
SH
S
1
2a~b
NO2
NO2
OH
OH
N
c
N
S
R1
Cl
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HO
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target compounds 13-24 was readily obtained by refluxing amine derivatives with 6
Cl
N
d
N S
R1
R1
4a~b
3a~b
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f
NH2 Cl N
N
e
N
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R1 5a~b
N
S
N
S
R1
9~12
2a~5a R1 = Propyl 2b~5b R1 = Bn
6a 6b 6c 6d 6e
R1 = BnR1 = Propyl R1 = Propyl R1 = Propyl R1 = Propyl
aromatic terminal or cyclopropyl
N
HN
R2
S
R2
N
Cl
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Cl
H N
NH
S
N
S
R1
HN g
H N R2
6a~e
N S
R2 = Ph R2 = Ph R2 = 2-Cl-Ph R2 = 2-Pyridyl R2 = Naphthyl
N N
S
R1
13~17 Y
Y = N, O, S
N g
H N R2
N S
N N
S
R1
18~24 S Arylamine
h
Aryl NH 7a-d
SH
TEA
7a, 8a Aryl = Ph
i Aryl N C S 8a-d
7b, 8b Aryl = 2-Cl-Ph 7c, 8c Aryl = 2-Pyridyl 7d, 8d Aryl = Naphthyl
ACCEPTED MANUSCRIPT Scheme 1. Synthesis of thiazolo[5,4-d]pyrimidine derivatives. Reagent and conditions: (a) Alkyl bromide, TEA, methanol, reflux, 2 h; (b) Fuming nitric acid, AcOH, 25~45 oC, 1 h; (c) POCl3, DMA, reflux, 2 h; (d) Fe, AcOH, methanol, rt~reflux; (e) R2SCN, Cs2CO3, acetonitrile, rt, overnight; (f) TsOH, toluene, reflux, 6 h; (g) TEA, isopropanol, reflux, 6 h; (h) TEA, THF, CS2, rt,
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overnight; (i) Chlorofrom, triphosgene, rt, overnight.
2.2 Evaluation of biological activity 2.2.1 Antiproliferative activity
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All final compounds were evaluated for their antiproliferative activities against three cancer lines, namely, H1650 (human lung cancer line), HGC27 and MGC803 (human
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gastric cancer lines) by using the MTT assay, and 5-fluorouracil (5-Fu) was employed as the reference drug [19]. All the compounds were divided into three types based on the substituents (R3) attached. The antiproliferative results of preliminary evaluation were summarized in Tables 1-3.
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As shown in Table 1, compounds 9-12 displayed moderate cytotoxic activities against the tested cancer cell lines with IC50 values ranging from 12 to 35 µM regardless of the nature of groups attached on the phenyl ring. One exception is compound 12,
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which showed no activity toward MGC803 with IC50 above 64 µM.
Table 1. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with
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aromatic amine substituents against three cancer cell lines.
Comp
R
1
R
2
IC50 (µM)a
3
RN MGC803
H1650
HGC27
9
Propyl
Ph
24.42±0.57
12.12±1.08
35.28±1.54
10
Propyl
Ph
17.42±0.87
14.76±1.16
13.12±1.11
11
Propyl
Ph
12.49±0.39
11.99±1.07
20.31±1.31
12
Propyl
Ph
>64
20.65±1.31
30.88±1.49
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5-Fu a
-
-
6.56±0.31
12.52±1.53
8.22±0.98
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.
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Next, we introduced aliphatic amine groups to the scaffold, as shown in Table 2, compound 13 with the terminal indole group presented improved antiproliferative activities against both MGC803 and H1650 cell lines with IC50 values less than 10 µM. Compound 14 with para-OH benzyl group showed comparable activity toward
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H1650 with compound 13. Compound 15 with terminal thiophene group displayed moderate antiproliferative activities against the tested cancer cell lines, albeit with
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slightly decreased activity against MGC803 and H1650. In contrast, compounds 16 and 17 showed weak antiproliferative activities against the tested cancer cell lines.
Table 2. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with
Propyl
14
Propyl
15
Propyl
a
IC50 (µM)a
R3 N
MGC803
H1650
HGC27
6.99±0.84
9.62±0.98
16.19±1.20
Ph
21.86±0.27
9.23±0.96
17.31±1.23
Ph
11.30±1.05
10.01±1.00
17.74±1.24
Ph
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13
R2
EP
R1
Comp
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aliphatic amine substituents against three cancer cell lines.
16
Propyl
Ph
>64
26.10±1.41
27.98±1.44
17
Propyl
Ph
>64
41.73±1.06
32.35±1.51
5-Fu
-
-
6.56±0.31
12.52±1.53
8.22±0.98
-
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 acceptable antiproliferative activities of compounds 13 and 14 against MGC803
ACCEPTED MANUSCRIPT and H1650 cells promoted us to investigate the effect of cyclic amine substituents on the activity (Table 3). In general, this series of compounds showed improved activity compared with compounds 9-17. Compound 19 with the morphine substituent (IC50 < 10 µM) was much more potent than compounds 18 and 20, showing the importance of
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the oxygen atom of the morphine ring for the antiproliferative activity against the tested cancer cell lines. Keeping the morphine ring unchanged, we next explored the effect of other aromatic rings (R2) on the activity. Evidently, 2-chloro phenyl (21), pyridyl (22), and naphthyl (23) groups were generally less preferred for the activity.
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However, for MGC-803 cells, compound 21 showed slightly better activity than compound 19. Interestingly, replacement of propyl group with the benzyl group (R1)
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led to a significant increase in the activity. Compound 24 exhibited excellent inhibition against all the tested cancer cell lines at low micromolar levels, showing that the steric hydrophobic group may be preferred for improving the activity against the human gastric and lung cancer lines. This finding may provide directions for
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further structural modifications for designing new anticancer agents.
Table 3. Antiproliferative activities of thiazolo[5,4-d]pyrimidine derivatives with cyclic aliphatic amine substituents against three cancer cell lines.
Propyl
R2
Ph
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18
R1
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Comp
IC50 (µM)a
R3 N
MGC803
H1650
HGC27
12.33±2.16
12.88±1.11
28.47±1.45
19
Propyl
Ph
5.81±0.45
4.18±0.62
9.51±1.52
20
Propyl
Ph
18.77±0.95
27.02±1.43
>64
21
Propyl
2-Cl-Ph
3.40±0.53
21.73±1.66
11.32±1.05
22
Propyl
9.35±0.97
7.73±1.01
8.84±0.94
23
Propyl
17.68±0.88
9.73±1.11
21.76±1.50
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a
24
Bn-
Ph
5-Fu
-
-
-
1.03±0.26
2.10±0.61
1.23±0.09
6.56±0.31
12.52±1.53
8.22±0.98
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
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experiments.
In order to investigate the possible toxicity of such series of compounds, the antiproliferative activity of the most potent compound 24 (IC50= 1.03 µM against MGC803) was examined against GES-1 (normal human gastric epithelial cell line).
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As shown in Table 4, compound 24 inhibited GES-1 with an IC50 value of 38.95 µM,
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showing around 38-fold selectivity to MGC803 cells over GSE-1 cells. Table 4. Inhibitory results of compound 24 against normal cells GES-1.
Comp
24
Bn-
R2
IC50 (µM)a
R3 N
Ph
SIb
MGC803
GES-1
1.03±0.26
38.95±1.67
37.8
Inhibitory activity was assayed by exposure for 72 h to substance and expressed as concentration required to
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a
R1
inhibit tumor cell proliferation by 50% (IC50). Data are presented as the means ± SDs of three independent experiments. b
The selectivity index (SI) was calculated as IC50 (GES-1) / IC50 (MGC803).
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2.2.2 Clone assay and cell migration
The excellent cytotoxic activity of compound 24 against MGC803 promoted us to
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investigate the effect on colony formation and cell migration. The clone formation of cancer cells represents an indirect estimation of neoplastic transformation [20]. As shown in Fig. 2A and 2B, treatment of MGC803 cells with compound 24 led to fewer and smaller colonies compared to the control with an inhibition rate of 86% at 3 µM. In addition, the cell migration ability was also initially evaluated by the wound healing assay. As shown in Fig. 2C, treatment of MGC-803 cells with compound 24 at indicated concentration markedly suppressed the wound healing in a time-dependent manner.
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Fig. 2. (A) Representative images of MGC803 cells colonies after treatment with various concentrations for 9 days; (B) Quantitative analysis of the colony formation inhibition rate; (C)
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Wound healing assay.
2.2.3 Cell apoptosis and possible mechanism involved Apoptosis is known as programmed cell death and is typically characterized by
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distinctive cell morphological changes [21]. Compound 24 was chosen for evaluating its ability of inducing apoptosis. Hochest 33258 staining was performed to investigate
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morphological changes of MGC803 cells [22]. After 24 h incubation with compound 24 at indicated concentrations, characteristic apoptotic morphological changes were observed, including cell rounding, chromatin shrinkage and formation of apoptotic bodies (Fig. 3A). Notably, this phenomena were more remarkable at higher concentrations. To further explore the effect of compound 24 on cell apoptosis, the apoptotic analysis was also performed with Annexin V-FITC/PI double staining and analyzed with high content [23]. Treatment of MGC803 cells with compound 24 resulted in a concentration-dependent apoptosis increase (Fig. 3B), and the percentage of apoptotic cells was 11.5%, 43.8%, and 73.9%, respectively, compared to the
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control (2.3%) (Fig. 3C).
Fig. 3. Compound 24 induced apoptosis of MGC803 cells. (A) Apoptosis analysis with
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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
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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 MGC803 cells with compound 24 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 [24]. Meanwhile, the expression of anti-apoptotic protein Bcl-2 decreased accordingly (Fig. 4C). As shown in Fig. 4D and 4E, the expression of caspase-3 and caspase-9 also increased evidently. These
ACCEPTED MANUSCRIPT results indicated that compound 24 may induce MGC803 apoptosis through the
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activation of intrinsic apoptotic pathway.
Fig. 4. Expression changes of apoptosis-related proteins induced by compound 24. (A) Compound
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24 induced expression changes of Bax, Bcl-2 and caspase family members in MGC803 cells; (B-E)
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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 the drug repurposing strategy and further evaluated for their antiproliferative activity. Among them, compound 24 exhibited the most potent inhibition against the tested cancer cells (MGC803, H1650 and HGC27) and was less toxic to GES-1, indicating a good selectivity to cancer cells over normal cells. Further mechanism investigation showed that compound 24 can inhibit the cell colony formation and migration, induce apoptosis through intrinsic apoptotic pathway of MGC803 cells.
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4. Experimental section 4.1 General Reagents and solvents were purchased from commercial sources and were used
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without further purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected. IR spectra was recorded on FT-IR (Bruker). 1HNMR 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
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Micromass Q-T of Micromass spectrometer by electrospray ionizaton (ESI).
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4.2 General procedure for the synthesis of compounds 9-12
The mixture of 6b (1eq), appropriate arylamine (2eq) and TsOH (2eq) in toluene was refluxed for 8 h and monitored by TLC (PE/EA = 4:1 ~ 2:1). 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
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with anhydride sodium sulfate and concentrated under reduced vacuum. The residue was purified by flash column chromatography (PE/EA = 4:1 ~ 2:1) to give the target product.
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4.2.1 N2,N7-diphenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-diamine (9) Pink solid, yield 65%, Mp 127~130 oC. IR (KBr) υmax: 2961, 1526, 1343, 730 cm-1. H NMR (400 MHz, Chloroform-d) δ 7.76 (m, 2H), 7.47-7.49 (m, 2H), 7.35-7.43 (m,
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1
4H), 7.16-7.19 (m, 1H), 7.09-7.12 (m, 1H), 3.13-3.16 (t, J = 7.4 Hz, 2H), 1.78-1.83 (m, 2H), 1.04-1.08 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 162.76,
159.17, 157.10, 150.16, 140.66, 139.37, 129.46, 128.78, 127.04, 123.74, 122.61, 122.32, 118.36, 32.78, 23.23, 13.78. HR-MS (ESI): Calcd. C20H19N5S2, [M+H]+m/z: 394.1160, found: 394.1162.
4.2.2 N7-(4-butylphenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7diamine (10)
ACCEPTED MANUSCRIPT Purple solid, yield 72%, Mp 163~165 oC. IR (KBr) υmax: 2956, 1576, 1350, 746 cm-1. 1
H NMR (400 MHz, Chloroform-d) δ 7.64 (m, 2H), 7.39-7.49 (m, 5H), 7.16-7.18 (m,
3H), 3.12-3.16 (t, J = 7.4 Hz, 2H), 2.59-2.63 (t, J = 7.7 Hz, 2H), 1.77-1.83 (m, 2H), 1.57-1.64 (m, 2H), 1.34-1.40 (m, 2H), 1.03-1.07 (t, J = 7.3 Hz, 3H), 0.92-0.96 (t, J = 13
C NMR (100 MHz, DMSO-d6) δ 162.79, 158.92, 156.98, 150.31,
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7.3 Hz, 3H).
140.70, 137.82, 136.90, 129.45, 128.52, 126.96, 122.51, 118.32, 34.77, 33.69, 32.81, 23.26, 22.15, 14.23, 13.79. HR-MS (ESI): Calcd. C24H27N5S2, [M+H]+m/z: 450.1786,
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found: 450.1789.
4.2.3 N7-(4-chlorophenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-
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diamine (11)
Pink solid, yield 59%, Mp 140~142 oC. IR (KBr) υmax: 3345, 2960, 1527, 1343, 819, 744 cm-1. 1H NMR (400 MHz, Chloroform-d) δ 7.71 (m, 2H), 7.47 (m, 3H), 7.40-7.44 (m, 2H), 7.32-7.34 (m, 2H), 7.17-7.20 (m, 1H), 3.11-3.15 (t, J = 7.4 Hz, 2H), 1.77-1.83 (m, 2H), 1.04-1.08 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
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162.74, 159.52, 157.17, 149.93, 140.65, 138.48, 129.48, 128.67, 127.18, 123.83, 122.69, 119.14, 118.39, 32.85, 23.09, 13.79. HR-MS (ESI): Calcd. C20H18ClN5S2, [M+H]+m/z: 428.0770, found: 428.0771.
diamine (12)
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4.2.4 N7-(4-methoxyphenyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-
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Purple solid, yield 72%, Mp 158~160 oC. IR (KBr) υmax: 3396, 2956, 1505, 1346, 821, 746 cm-1. 1H NMR (400 MHz, DMSO-d6 ) δ 10.56 (s, 1H), 9.22 (s, 1H), 7.89 (m, 2H), 7.62-7.64 (m, 2H), 7.33-7.37 (m, 2H), 7.02-7.05 (m, 1H), 6.95-6.97 (m, 2H), 3.77 (s, 3H), 2.98-3.02 (t, J = 7.2 Hz, 2H), 1.62-1.68 (m, 2H), 0.92-0.96 (t, J = 7.3 Hz, 3H). 13
C NMR (100 MHz, DMSO-d6) δ 162.82, 158.78, 156.86, 156.15, 150.59, 140.76,
132.19, 129.44, 126.74, 124.56, 122.52, 118.29, 114.01, 55.69, 32.79, 23.21, 13.80. HR-MS (ESI): Calcd. C21H21N5OS2, [M+H]+m/z: 424.1266, found: 424.1264.
4.3 General procedure for the synthesis of compounds 13-24
ACCEPTED MANUSCRIPT The mixture of 6 (1 eq), appropriate amine (1.1 eq) and TEA (2eq) in isopropanol was refluxed for 6 h and monitored by TLC (PE/EA = 4:1 ~ 2:1). 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
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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 product.
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4.3.1 N7-(2-(1H-indol-2-yl)ethyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine2,7-diamine (13)
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Pale yellow solid, yield 78%, Mp 194~195 oC. IR (KBr) υmax: 3425, 2919, 1589, 1350, 741 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 10.47 (s, 1H), 7.82-7.84 (m, 2H), 7.67-7.70 (m, 2H), 7.34-7.37 (m, 3H), 7.19 (d, J = 2.3 Hz, 1H), 7.06-7.10 (m, 1H), 6.98-7.04 (m, 2H), 3.75-3.77 (m, 2H), 3.04-3.10 (m, 4H), 1.67-1.72 (m, 2H), 0.94-0.98 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 162.73, 156.75,
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156.22, 152.30, 140.45, 136.26, 128.95, 127.27, 125.89, 122.51, 121.88, 120.95, 118.44, 118.20, 117.56, 111.81, 111.37, 40.96, 32.36, 25.19, 22.65, 13.32. HR-MS
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(ESI): Calcd. C24H24N6S2, [M+H]+m/z: 461.1582, found: 461.1585.
4.3.2 4-(((2-(phenylamino)-5-(propylthio)thiazolo[5,4-d]pyrimidin-7-yl)amino) methyl)phenol (14)
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Pale yellow solid, yield 81%, Mp 160~162 oC. IR (KBr) υmax: 3433, 3415, 2916, 1557, 1352, 749 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 9.25 (s, 1H), 8.09 (t, J = 6.3 Hz, 1H), 7.83-7.85 (m, 2H), 7.31-7.35 (m, 2H), 7.15-7.17 (m, 2H), 6.99-7.03 (m, 1H), 6.69-6.71 (m, 2H), 4.58-4.60 (d, J = 6.2 Hz, 2H), 2.98-3.01 (t, J = 7.2 Hz, 2H), 1.60-1.65 (m, 2H), 0.91-0.94 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.60, 156.15, 152.15, 140.42, 131.46, 130.06, 128.93, 128.62, 128.36, 125.80, 121.87, 117.59, 114.95, 42.69, 32.31, 22.62, 13.29. HR-MS (ESI): Calcd. C21H21N5OS2, [M+H]+m/z: 424.1266, found: 424.1262.
ACCEPTED MANUSCRIPT 4.3.3 N2-phenyl-5-(propylthio)-N7-(2-(thiophen-2-yl)ethyl)thiazolo[5,4-d]pyrimidine2,7-diamine (15) Brown solid, yield 85%, Mp 123~125 oC. IR (KBr) υmax: 2959, 1541, 1342, 746, 691 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.82-7.84 (m, 2H), 7.71-7.74
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(m, 1H), 7.32-7.37 (m, 3H), 7.02-7.04 (m, 1H), 6.97-6.99 (m, 1H), 6.93-6.94 (m, 1H), 3.71-3.73 (m, 2H), 3.15-3.19 (t, J = 7.6 Hz, 2H), 3.05-3.09 (t, J = 7.2 Hz, 2H), 1.67-1.72 (m, 2H), 0.96-1.00 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
162.62, 156.30, 152.16, 141.37, 140.41, 128.93, 127.92, 127.81, 126.96, 125.90,
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125.08, 124.05, 121.93, 117.60, 41.87, 32.40, 29.31, 22.69, 13.32. HR-MS (ESI):
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Calcd. C20H21N5S3, [M+H]+m/z: 428.1037, found: 428.1039.
4.3.4 N7-(furan-2-ylmethyl)-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7diamine (16)
Brown solid, yield 80%, Mp 143~146 oC. IR (KBr) υmax: 3439, 2959, 1531, 1350, 750, 689 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.44-7.47 (m, 3H), 7.34-7.38 (m, 3H),
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7.09-7.14 (m, 1H), 6.33 (m, 1H), 6.27 (m, 1H), 5.90-5.93 (t, J = 5.8 Hz, 1H), 4.77 (d, J = 5.8 Hz, 2H), 3.10-3.14 (t, J = 7.4 Hz, 2H), 1.75-1.80 (m, 2H), 1.02-1.05 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.98, 156.79, 153.23, 152.42, 142.27, 140.86, 129.39, 126.42, 122.39, 118.07, 110.91, 107.12, 37.30, 32.80, 23.08, 13.78.
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HR-MS (ESI): Calcd. C19H19N5OS2, [M+H]+m/z: 398.1109, found: 398.1110.
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4.3.5 N7-cyclopropyl-N2-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidine-2,7-diamine (17)
Pale yellow solid, yield 89%, Mp 140~143 oC. IR (KBr) υmax: 3250, 2960, 1548, 1348, 748 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 7.82-7.84 (m, 2H), 7.68 (d, J = 3.4 Hz, 1H), 7.31-7.35 (m, 2H), 6.98-7.03 (m, 1H), 3.06-3.09 (t, J = 7.2 Hz, 2H), 2.91 (m, 1H), 1.69-1.75 (m, 2H), 0.97-1.01 (t, J = 7.3 Hz, 3H), 0.75-0.78 (m, 2H), 0.66-0.70 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.56, 155.99, 153.33, 140.39, 131.47, 128.92, 128.62, 125.91, 121.84, 117.59, 32.40, 23.70, 22.73, 13.34, 6.41. HR-MS (ESI): Calcd. C20H21N5S3, [M+H]+m/z: 358.1160, found: 358.1160.
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4.3.6
N-phenyl-5-(propylthio)-7-thiomorpholinothiazolo[5,4-d]pyrimidin-2-amine
(18) Yellow solid, yield 89%, Mp 135~137 oC. IR (KBr) υmax: 2962, 1553, 1348, 730 cm-1. H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.58-7.61 (m, 2H), 7.34-7.38 (m, 2H),
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1
7.00-7.04 (m, 1H), 4.44 (br, 4H), 3.01-3.04 (t, J = 7.2 Hz, 2H), 2.74-2.77 (m, 4H), 1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.4 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ
161.89, 161.22, 154.30, 151.07, 140.20, 129.04, 125.35, 122.11, 117.50, 48.27, 32.34,
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26.38, 22.55, 13.30. HR-MS (ESI): Calcd. C18H21N5S3, [M+H]+m/z: 404.1037, found:
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404.1037.
4.3.7 7-morpholino-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-2-amine (19) Yellow solid, yield 89%, Mp 149~150 oC. IR (KBr) υmax: 2955, 1557, 1346, 746 cm-1. 1
H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.56-7.59 (m, 2H), 7.33-7.37 (m, 2H),
7.00-7.04 (m, 1H), 4.15 (br, 4H), 3.73-3.76 (m, 4H), 3.02-3.05 (t, J = 7.2 Hz, 2H), 13
C NMR (100 MHz, DMSO-d6) δ
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1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.4 Hz, 3H).
161.87, 161.03, 154.51, 151.41, 140.16, 129.06, 125.35, 122.19, 117.62, 66.08, 45.90, 32.30, 22.46, 13.26. HR-MS (ESI): Calcd. C18H21N5OS2, [M+H]+m/z: 388.1266,
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found: 388.1263.
4.3.8 7-(4-benzylpiperazin-1-yl)-N-phenyl-5-(propylthio)thiazolo[5,4-d]pyrimidin-
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2-amine (20)
Pale yellow solid, yield 82%, Mp 144~146 oC. IR (KBr) υmax: 2963, 1514, 1350, 741 cm-1. 1H NMR (400 MHz, ) δ 10.50 (s, 1H), 7.57-7.59 (m, 2H), 7.33-7.35 (m, 6H), 7.26-7.29 (m, 1H), 6.98-7.02 (m, 1H), 4.17 (m, 4H), 3.52 (s, 2H), 3.00-3.03 (t, J = 7.2Hz, 2H), 2.51 (m, 4H), 1.64-1.69 (m, 2H), 0.95-0.98 (t, J = 7.4 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 162.34, 161.47, 154.77, 151.81, 140.70, 138.32, 129.50, 129.39, 128.66, 127.47, 125.74, 122.62, 118.04, 62.41, 53.02, 45.84, 32.80, 23.01, 13.77. HR-MS (ESI): Calcd. C25H28N6S2, [M+H]+m/z: 477.1895, found: 477.1895.
ACCEPTED MANUSCRIPT 4.3.9 N-(2-chlorophenyl)-7-morpholino-5-(propylthio)thiazolo[5,4-d]pyrimidin-2amine (21) White solid, yield 79%, Mp 196~197 oC. IR (KBr) υmax: 3276, 2956, 1557, 1342, 776 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 7.99 (m, 1H), 7.34-7.36 (m,
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2H), 7.04-7.07 (m, 1H), 4.13-4.16 (m, 4H), 3.76-3.78 (t, J = 4.7 Hz, 4H), 3.02-3.05 (t, J = 7.1 Hz, 2H), 1.65-1.71 (m, 2H), 0.96-1.00 (t, J = 7.3 Hz, 3H).
13
C NMR (100
MHz, DMSO-d6) δ 162.76, 161.70, 154.52, 152.08, 142.00, 133.84, 131.05, 125.55, 122.00, 117.47, 116.53, 66.58, 46.37, 32.80, 22.94, 13.76.HR-MS (ESI): Calcd.
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C18H20ClN5OS2, [M+H]+m/z: 422.0876, found: 422.0877.
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4.3.10 7-morpholino-5-(propylthio)-N-(pyridin-2-yl)thiazolo[5,4-d]pyrimidin-2-amine (22)
Pale yellow solid, yield 83%, Mp 240~241 oC. IR (KBr) υmax: 2960, 1550, 1326, 768 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.31-8.33 (m, 1H), 7.73-7.76 (m, 1H), 7.09-7.11 (m, 1H), 6.99 (m, 1H), 4.18-4.20 (m, 4H), 3.73-3.76 (t, J = 4.6 Hz, 13
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4H), 3.05 (t, J = 7.1 Hz, 2H), 1.66-1.72 (m, 2H), 0.97-1.00 (t, J = 7.3 Hz, 3H).
NMR (100 MHz, DMSO-d6) δ 161.84, 161.79, 151.94, 151.77, 151.15, 146.35, 138.34, 123.53, 116.88, 111.03, 66.10, 45.73, 32.30, 22.55, 13.27. HR-MS (ESI):
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Calcd. C17H20N6OS2, [M+H]+m/z: 389.1218, found: 389.1220.
4.3.11 7-morpholino-N-(naphthalen-1-yl)-5-(propylthio)thiazolo[5,4-d]pyrimidin-2-
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amine (23)
White solid, yield 83%, Mp 142~144 oC. IR (KBr) υmax: 2957, 1544, 1346, 764 cm-1. 1
H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.22-8.25 (m, 1H), 8.04-8.06 (m, 1H),
7.95-7.97 (m, 1H), 7.73-7.75 (m, 1H), 7.52-7.59 (m, 3H), 4.10-4.13 (m, 4H), 3.69-3.71 (t, J = 4.7 Hz, 4H), 3.01-3.05 (t, J = 7.1 Hz, 2H), 1.65-1.70 (m, 2H), 0.96-0.99 (t, J = 7.3 Hz, 3H).
13
C NMR (100 MHz, DMSO-d6) δ 161.75, 161.26,
157.00, 151.40, 135.53, 133.95, 128.29, 126.40, 126.25, 125.90, 125.84, 125.24, 124.36, 122.16, 117.94, 66.06, 45.86, 32.29, 22.48, 13.25. HR-MS (ESI): Calcd. C22H23N5OS2, [M+H]+m/z: 438.1422, found: 438.1420.
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4.3.12 5-(benzylthio)-7-morpholino-N-phenylthiazolo[5,4-d]pyrimidin-2-amine (24) Pale yellow solid, yield 80%, Mp 183~185 oC. IR (KBr) υmax: 3277, 2849, 1559, 1339, 746 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 7.56-7.59 (m, 2H),
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7.40-7.43 (m, 2H), 7.34-7.38 (m, 2H), 7.29-7.33 (m, 2H), 7.21-7.26 (m, 1H), 7.00-7.05 (m, 1H), 4.36 (s, 2H), 4.16 (m, 4H), 3.71-3.74 (t, J = 4.7 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 161.34, 160.91, 154.69, 151.37, 140.14, 138.18, 129.08, 128.70, 128.33, 126.88, 125.50, 122.25, 117.67, 66.11, 45.95, 34.56. HR-MS (ESI):
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Calcd. C22H21N5OS2, [M+H]+m/z: 436.1266, found: 436.1266.
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4.4 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 72 h, thereafter MTT assays
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were performed and cell viability was assessed at 570nm by a microplate reader (Biotech, Shanghai, China).
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4.5 Clonogenicity assay
MGC803 cells (1000 cells/well) were seeded in a 6-well plate and incubated for 24 h,
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then the media were replaced with fresh media containing different concentrations of compound 24. 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 of treatment/number of control) * 100%. All experiments were performed in triplicate.
4.6 Hoechst 33258 staining MGC803 cells were seeded into a 6-well plate (2×105/well) and incubated overnight
ACCEPTED MANUSCRIPT for adherent and treated with compound 24 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).
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4.7 Wound healing assay MGC803 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 24 with different concentrations followed by indicated time incubation and photographed on an
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inverted microsope.
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4.8 Cell apoptosis assay
MGC803 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 24 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
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analyzed by high content screening system (ArrayScan XTI, Thermo Fisher Scientific, MA).
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4.9 Western blot analysis
MGC803 cells were treated with different concentrations of compound 24 for 24 h, the cells were collected, lysed in RIPA buffer contained a protease inhibitor cocktail
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for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4 oC. After the collection of supernatant, the protein concentration was detected using a bicinchonininc acid assay kit (Beyotmie Biotechnology, Haimen, China). After added with loading buffer, cell lyses were boiled for 10 min at 100oC for SDSpolyacrylamide
gel
electrophoresis
(PAGE).
Proteins
were
transferred
to
nitrocellulose (NC) membranes. Then the membranes were blocked with 5% skim milk at room temperature for 2h, and then incubated overnight at 4oC 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
ACCEPTED MANUSCRIPT antibody-reactive were revealed by enhanced chemiluminescence (ECL) and exposed on Kodak radiographic film.
Acknowledgement
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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
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Education (No. 20134101130001, for H.M.L.).
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Highlights The thiazolo[5,4-d]pyrimidine derivatives showed potent inhibition against the cancer cells. Compound 24 exerted the most antiproliferative activity against MGC-803 cells. Compound 24 inhibited the cell colony formation and migration of MGC803. Compound 24 induced the apoptosis of MGC-803 cells.