Synthesis and cytotoxic activity of chalcone analogues containing a thieno[2,3-d]pyrimidin-2-yl group as the A-ring or B-ring

Article abstract of DOI:10.1016/j.bioorg.2019.103346

Many natural or synthetic chalcones have potential anti-tumor activity. Here, we synthesized two series of chalcone analogues containing a thieno[2,3-d]pyrimidin-2-yl group and evaluated for their cytotoxic activity towards cultured human lung cancer A549 and colorectal HCT-116 cells. Among them, compound 8d was the most cytotoxic against HCT-116 cells, with an IC₅₀ value of 2.65 μM. Analyses of the phenotypic changes induced by this compound found a dose-dependent accumulation of HCT-116 cells in sub-G1 phase, indicating that compound 8d might induce apoptosis. Furthermore, we found that 8d triggered mitochondrial membrane potential depolarization, promoted reactive oxygen species formation in HCT-116 cells, and increased the percentage of early and late apoptotic cells. Finally, immunoblotting indicated that 8d increased PARP-1 and caspases 3, 7 and 9 cleavage. These data suggest that compound 8d induces apoptosis via the mitochondrial death pathway.

Full text of DOI:10.1016/j.bioorg.2019.103346

BioorganicChemistryxxx(xxxx)xxxx
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and cytotoxic activity of chalcone analogues containing a thieno
[2,3-d]pyrimidin-2-yl group as the A-ring or B-ring
⁎
Fu-Cheng Wanga,1, Bin Pengb,1, Sheng-Li Caoa, , Hong-Yun Lia, Xiao-Li Yuana, Ting-Ting Zhanga,
⁎⁎
Ruifeng Shib, Zhuqing Lib, Ji Liaob, Hailong Wangc, Jing Lic, Xingzhi Xub,
a Department of Chemistry and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048, PR China
b Guangdong Key Laboratory for Genome Stability & Disease Prevention and Carson International Cancer Center and Marshall Laboratory of Biomedical Engineering,
Shenzhen University School of Medicine, Shenzhen, Guangdong, 518060, PR China
c College of Life Science and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Many natural or synthetic chalcones have potential anti-tumor activity. Here, we synthesized two series of
chalcone analogues containing a thieno[2,3-d]pyrimidin-2-yl group and evaluated for their cytotoxic activity
towards cultured human lung cancer A549 and colorectal HCT-116 cells. Among them, compound 8d was the
most cytotoxic against HCT-116 cells, with an IC50 value of 2.65 μM. Analyses of the phenotypic changes induced
by this compound found a dose-dependent accumulation of HCT-116 cells in sub-G1 phase, indicating that
compound 8d might induce apoptosis. Furthermore, we found that 8d triggered mitochondrial membrane po-
tential depolarization, promoted reactive oxygen species formation in HCT-116 cells, and increased the per-
centage of early and late apoptotic cells. Finally, immunoblotting indicated that 8d increased PARP-1 and
caspases 3, 7 and 9 cleavage. These data suggest that compound 8d induces apoptosis via the mitochondrial
death pathway.
Thieno[2,3-d]pyrimidin-4(3H)-one
Chalcone analogue
Cytotoxicity
Apoptosis
Mitochondrial death pathway
1. Introduction
amines at the C-4 position may lead to the inhibition of protein kinases.
Abdelhaleem et al. introduced phenyl urea or a phenyl thiourea moiety
Thieno[2,3-d]pyrimidine is an important heterocycle core structure,
and its derivatives have drawn much attention in the discovery and
development of anti-tumor agents [1–3]. Thieno[2,3-d]pyrimidine de-
rivatives bearing side chains at the C-5 or C-6 positions may act as
antifolate and anti-tumor agents. Gangjee et al. synthesized 2-ami-
nothieno[2,3-d]pyrimidin-4(3H)-one derivatives bearing a benzoyl-L-
glutamic acid moiety or lipophilic side chain at the C-5 position, and
evaluated these derivatives as classical or non-classical antifolate anti-
tumor agents [4–7]. They found that compound I (Fig. 1) was an ex-
cellent dual inhibitor of human thymidylate synthase (TS,
IC50 = 54 nM) and dihydrofolate reductase (DHFR, IC50 = 19 nM) and
had GI50 values < 10 nM in eight human cancer cell lines[4]. Mod-
ification of thieno[2,3-d]pyrimidine with arylamines or heteroaromatic
into the C-4 position of thieno[2,3-d]pyrimidine to synthesize a series of
novel tetrahydrobenzothieno[2,3-d]pyrimidine urea derivatives, of
which compound II (Fig. 1) was cytotoxic towards MCF-7 cells
(IC50 = 7.10 μM). Here, the cytotoxicity seemed to correlate with the
inhibitory activity against topoisomerase II (IC50 = 9.29 μM) and vas-
cular endothelial growth factor receptor 2 (VEGFR-2) (IC50 = 0.2 μM)
[8].
While the majority of studies have focused on substitutions at the C-
4, C-5 or C-6 positions, increasing evidence indicate that thieno[2,3-d]
pyrimidine derivatives bearing various substituents at the C-2 position
exhibit cytotoxic effects on tumor cells. Amawi et al. synthesized a
series of thieno[2,3-d]pyrimidine derivatives bearing a substituted
phenyl group at the C-2 position and found that compound III (Fig. 1)
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulphoxide; 5-FU, 5-Fluorouracil; FITC, fluorescein isothiocyanate; JC-1, 5,5′,6,6′-tetrachloro-
1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;
RNS, reactive nitrogen species; ROS, reactive oxygen species; PARP-1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; PS, phosphatidylserine; STS, staur-
osporine; Δψm, mitochondrial transmembrane potential
⁎ Corresponding author.
⁎⁎ Corresponding author.
E-mail addresses: slcao@cnu.edu.cn (S.-L. Cao), xingzhi.xu@szu.edu.cn (X. Xu).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103346
Received 26 April 2019; Received in revised form 18 September 2019; Accepted 4 October 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Fu-ChengWang,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103346

F.-C. Wang, et al.
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Fig. 1. Chemical structures of thieno[2,3-d]pyrimidine derivatives (I–IV), chalcone and its synthetic analogues (V–IX).
exhibited antiproliferative activity by inducing apoptosis and mitotic
catastrophe in five cancer cell lines [9]. We recently synthesized two
series of thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate
side chain at the C-2 position, and compound IV (Fig. 1) exhibited
cytotoxicity towards A549 cells (IC50 = 4.87 μM), inducing a cell cycle
arrest at G2/M phase and activating the spindle assembly checkpoint
(SAC). We synthesized a biotinylated probe of compound IV for affinity
purification and mass spectrometric analysis of its binding proteins and
identified tubulin as a target that is associated with the SAC and directly
binds to the probe both in vivo and in vitro. Compound IV inhibited
tubulin polymerization in vitro in a dose-dependent manner and com-
peted with taxol in binding to tubulin, exerting cytotoxic activity to-
ward taxol-resistant A549 cells [10].
(IC50 = 3.36 μM). Compound IXd triggered mitochondrial potential
(MMP) loss, and increased the level of cleaved PARP-1, caspases 3, 7
and 9, inducing apoptosis via the mitochondrial death pathway [18]. In
this study, we replaced the quinazolin-2-yl group in compounds VIIIa–s
and IXa–s with a thieno[2,3-d]pyrimidin-2-yl group to synthesize
chalcone analogues 4a–p and 8a–p, respectively. We evaluated these
compounds in terms of their potential as anti-tumor agents. We also
investigated the effect of bioisosteric replacement on the cytotoxic ac-
tivity of compounds VIIIa–s and IXa–s.
2. Results and discussion
2.1. Synthesis
Chalcones (Fig. 1) are open-chain precursors for flavonoid and
isoflavonoid biosynthesis, and their analogues cover a wide spectrum of
biological activity, including anti-tumor activity [11–14]. Chalcones
consist of two benzene rings that are joined via an α,β-unsaturated
carbonyl three-carbon chain. The benzene ring adjacent to the carbonyl
group is defined as the “A-ring” and the other benzene ring is known as
the “B-ring”. To search for effective anti-tumor agents, many efforts
have been made to modify the A-ring and/or B-ring with substituents or
to replace the A-ring or B-ring with different aromatic heterocycles.
Zhang et al. synthesized 43 chalcone analogues with various sub-
stituents on the A-ring or B-ring; compound V (Fig. 1) displayed the
highest cytotoxicity toward HeLa cells (IC50 = 1.4 μM). Compound V
selectively inhibited thioredoxin reductase (TrxR) and induced reactive
oxygen species (ROS) production and apoptosis in HeLa cells [15]. Yan
et al. synthesized a series of chalcone analogues containing an indolin-
3-yl, 4-yl or 5-yl group, of which compound VI (Fig. 1) exhibited the
most potent cytotoxicity and high selectivity towards cancer cells.
Compound VI induced a G2/M arrest in A549 cells by binding the tu-
bulin colchicine’s binding site and inhibiting tubulin polymerization
[16]. Mirzaei et al. synthesized a series of indole-based chalconoids,
among which compound VII (Fig. 1) — with an IC50 value of 4.3 μg/mL
against A549 cells — was more potent than etoposide (IC50, 7.8 μg/mL).
Compound VII inhibits tubulin polymerization in a concentration-de-
pendent manner by binding to the tubulin colchicine binding site [17].
We recently synthesized several chalcone analogues VIIIa–s and IXa–s
Scheme 1 outlines the synthetic pathway of chalcone analogues
4a–p in which the thieno[2,3-d]pyrimidin-2-yl group serves as the A-
ring. Heating propionaldehyde, 2-cyanoacetamide and elemental sulfur
in the presence of triethylamine (TEA) in ethanol at 50–55 °C for 4 h
gave 2-amino-5-methylthiophene-3-carboxamide (intermediate 1) [19].
Intermediate 1 reacted with 2-oxopropanoyl chloride, prepared from
reaction of 2-oxopropanoic acid and thionyl chloride in di-
chloromethane (DCM), to give 5-methyl-2-(2-oxopropanamido)thio-
phene-3-carboxamide (intermediate 2) [20]. Intermediate 2 reacted
with an aqueous solution of sodium hydroxide in ethanol under reflux
to yield 2-acetyl-6-methylthieno[2,3-d]pyrimidin-4(3H)-one (inter-
mediate 3) [21]. In the presence of potassium hydroxide or piperidine,
reactions of intermediate 3 with various aromatic aldehydes or het-
eroaromatic aldehydes in methanol generated compounds 4a–p [22].
Scheme 2 outlines the synthetic pathway of compounds 8a–p, in
which the thieno[2,3-d]pyrimidin-2-yl group serves as the B-ring.
Stirring a mixture of ethyl cyanoacetate, propionaldehyde, elemental
sulfur and TEA in N,N-dimethylformamide (DMF) at room temperature
for 4 h generated ethyl 2-amino-5-methylthiophene-3-carboxylate (in-
termediate 5) [7]. This product reacted with excess chloroacetonitrile
in the presence of hydrogen chloride gas to yield 2-(chloromethyl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (intermediate 6). In the pre-
sence of potassium iodide, intermediate 6 was oxidized by N-methyl-
morpholine N-oxide in tetrahydrofuran (THF) to give 6-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-2-carbaldehyde (intermediate 7)
[23]. Condensation of intermediate 7 with different acetophenones or
(Fig. 1) containing
a
quinazolin-2-yl; compound IXd (R2 = 2',5'-
diOCH3C6H3) was the most potent against HCT-116 cells
2

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Scheme 1. Synthetic pathway of compounds 4a–p. Reagents and conditions: (a) S8, Et3N, EtOH, 50–55 °C, 4 h, 71%. (b) CH3COCOCl, THF, rt, overnight, 70%. (c) i.
4% NaOH, EtOH, reflux, 2 h; ii. 1 mol/L HCl, 58%. (d) i. Aldehydes (R1CHO), 30% KOH (4a–j, 4l, 4m) or piperidine (4k, 4n–p), MeOH, rt, overnight; ii. 1 mol/L HCl,
45–80%.
heterocyclylethan-1-ones under alkaline conditions yielded compounds
8a–p.
well-known anti-cancer drug that induces cell-cycle arrest in S phase;
we therefore used 5-FU as a positive control.
The final reactions producing compounds 4a–p and 8a–p were
monitored by thin-layer chromatography (TLC) or high-performance
liquid chromatography (HPLC); only one spot or peak corresponding to
the product was observed. In the 1H NMR spectra of compounds 4a–p
and 8a–p, the signal of each proton in the CeC double bond appear as a
single doublet, with a coupling constant of 15.6 or 16.2 Hz. Chalcone
analogues can exist as an E-isomer and/or Z-isomer, with the E-isomer
being thermodynamically more stable in most cases. The Z-isomer is
unstable due to the strong steric effects between the carbonyl group and
the A-ring [11,13]. The chemical analyses indicated that compounds
4a–p and 8a–p exist as geometrical E-isomers.
We found that 8/16 compounds (4b–c, 4e, 4g–j and 4l) in the 4a–p
series were active towards A549 cells (IC50 < 20 μM), three of which
(4h, 4j and 4l) exhibited promising cytotoxic activity (IC50 < 10 μM).
In addition, we found that 10/16 compounds (4a, 4e, 4g–j, 4l–m and
4o–p) were active in HCT-116 cells (IC50 < 20 μM), of which com-
pounds 4b, 4g, 4h and 4l were more active with IC50 values < 10 μM.
In this series, two compounds (4h and 4l) had IC50 values < 10 μM in
both A549 and HCT-116 cells, but they were less potent than the po-
sitive control 5-FU (Table 1).
We found that 9/16 final compounds (8a–b, 8d–h, 8j and 8m) in
the 8a–p series were active towards A549 cells (IC50 < 20 μM), and
five compounds (8d–g and 8m) had IC50 values < 10 μM. In addition,
12/16 compounds (8a–h and 8j–m) were active in HCT-116 cells
(IC50 < 20 μM), while five compounds (8b and 8d–g) had IC50
values < 10 μM. Compounds bearing a halogen or a nitro group on the
benzene ring, and compounds with a heterocycle as the A-ring (8 l–p),
were comparable to or less potent than the parent compound 8a in both
cell lines. Compounds bearing an electron-donating group (CH3O or
CH3) on the benzene ring (8b and 8d–g), however, were more active
than 8a against A549 and HCT-116 cells, with the exception of 8c being
inactive in A549 cells and comparable to 8a in HCT-116 cells.
Compounds 8d–g were potent in both A549 and HCT-116 cell lines
(IC50 < 10 μM), while 8d–f had comparable cytotoxic activity to 5-FU
(Table 2).
2.2. Cytotoxic activity
We first performed an MTS cell proliferation assay to evaluate the
cytotoxic activity of the synthesized compounds against several
common cancer cell lines (data not shown). The inhibition of cell
proliferation was determined 72 h after the cells were treated with
20 μM of the tested compounds. The compounds with ≥50% inhibition
compared to vehicle-treated cells were considered active. We found
that most tested compounds had better activity against human lung
cancer A549 and colorectal cancer HCT-116 cells than other cell lines.
We therefore measured the level of inhibited cell proliferation by these
active compounds in these two cell lines at various concentrations and
determined the IC50 (the concentration that causes 50% of cell pro-
liferation inhibition) values (Tables 1 and 2). 5-Fluorouracil (5-FU) is a
In our previous work [18], compounds within the IXa–s series
(quinazolin-2-yl group as the B-ring) were more cytotoxic than the
Scheme 2. Synthetic pathway of compounds 8a–p. Reagents and conditions: (a) S8, Et3N, DMF, rt, 4 h, 87%. (b) i. ClCH2CN, dry HCl (g), 1,4-dioxane, rt, 5 h; ii. 25%
NH3/H2O; 66%. (c) N-methylmorpholine N-oxide, KI, dry THF, reflux, 2 h, 58%. (d) i. R2COCH3, 20–50% KOH, MeOH, rt, overnight; ii. 1 mol/L HCl; 35–68%.
3

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Table 1
cytotoxic in HCT-116 than A549 cells. Moreover, and similar to the
IXa–s series, compounds bearing electron-donating group(s) were more
potent than those bearing electron-withdrawing group(s) in the 8a–p
series; however, compounds 8a–p were generally less potent than their
bioisosteric analogues IXa–s. Compound 8d (IC50 = 2.65 μM) was
slightly more cytotoxic than its counterpart (IXg, IC50 = 5.57 μM) in
HCT-116 cells, and comparable to the most potent compound (IXf,
IC50 = 3.56 μM) within the VIIIa–s series.
IC50 values for compounds 4a–p in cultured A549 and HCT-116 cells.
Compound
R1
IC50 (μM)a
A549
We selected compounds 8d–f, which had IC50 values < 5 μM against
HCT-116 cells, for cytotoxic evaluation in human breast cancer MCF-7,
MDA-MB-231, cervical cancer HeLa, colon cancer HT-29, hepatocel-
lular carcinoma HepG2 cell lines (Table 3), and normal human lung
fibroblast MRC-5, human mammary epithelial MCF-10A, and mouse
embryonic fibroblast NIH3T3 cell lines (Table 4). These three com-
pounds were active towards the tumor cell lines tested; however, they
were also cytotoxic against immortalized cell lines. As in A549 and
HCT-116 cells, compound 8d also had relatively low IC50 values in
MCF-7, MD-MBA-231, HeLa, HT-29 and HepG2 cells. We therefore took
this compound (8d) forward for further investigation into the me-
chanisms underlying cytotoxicity in cancer cells.
HCT-116
> 20
4a
4b
4c
4d
4e
4f
C6H5
> 20
4′-OCH3C6H4
2′,4′-diOCH3C6H3
2′,4′,6′-triOCH3C6H2
3′,4′,5′-triOCH3C6H2
4′-OH-3′-OCH3C6H3
4′-CH3C6H4
10.66
17.13
> 20
16.22
> 20
18.52
8.93
0.79c
8.92
0.45
1.50
1.60
3.30
> 20
> 20
17.28
> 20
9.59
1.55
4g
4h
4i
0.41
4′-BrC6H4
0.26
9.40
0.22
0.80
0.73
4′-ClC6H4
11.53
9.26
0.71
0.61
10.62
10.20
> 20
7.87
4j
4′-FC6H4
4k
4l
4′-NO2C6H4
> 20
8.66
furan-2-yl
0.50
0.39
4m
4n
4o
4p
5-FU
thiophene-2-yl
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
> 20
> 20
> 20
> 20
5.52
18.82
> 20
17.85
19.62
4.26
1.02
2.3. Compound 8d induces sub-G1 accumulation of HCT-116 cells
0.77
1.29
We first examined the effects of compound 8d on cell-cycle pro-
gression. HCT-116 cells were exposed to 8d at various concentrations
(1×, 1.5× or 2 × IC50), 5-FU (IC50), or taxol (0.10 μM) for 24 h, and
analyzed for DNA content by flow cytometry. We performed three in-
dependent experiments (Table 5 and Fig. 2A), and the results from one
representative experiment are shown (Fig. 2B).
0.31
0.19
a
The concentration that causes 50% of cell proliferation inhibition. The data
are expressed as the means
independent experiments.
SD from triplicate determination from three
In agreement with previous reports, HCT-116 cells treated with 5-
FU or taxol for 24 h arrested in S-phase or G2/M phase, respectively.
Conversely, treatment with 8d only induced a sub-G1 accumulation
with no obvious effects on the cell-cycle distribution in HCT-116 cells.
Moreover, we observed a dose-dependent increase of sub-G1 cells when
using concentrations ranging from one-to-two times of the 8d IC50
value. These results suggest that 8d might induce apoptosis in HCT-116
cells.
Table 2
IC50 values for compounds 8a–p in cultured A549 and HCT-116 cells.
Compound
R2
IC50 (μM)a
A549
2.4. Compound 8d induces apoptosis in HCT-116 cells
HCT-116
13.91
To confirm the ability of compound 8d to induce apoptosis, we
performed a biparametric cytofluorimetric analysis using Propidium
iodide (PI), which stains DNA only in dead cells, and fluorescent im-
munolabeling of the protein annexin V, which binds to phosphati-
dylserine (PS) expressed on the surface of the apoptotic cells and
fluoresces green after interacting with the labeled annexin V. Dual
Annexin V and PI staining permits discrimination between live cells,
early apoptotic cells, late apoptotic cells and necrotic cells. Here we
found that compound 8d increased early and late apoptotic cells in a
dose-dependent manner in HCT-116 cells (Fig. 3). The percentage of
Annexin V-FITC-positive cells induced by 8d at the dose of 2 × IC50 was
equivalent to that induced by STS (1 μM), an ATP-competitive kinase
inhibitor that induces apoptosis, demonstrating that 8d induces apop-
tosis in HCT-116 cells.
8a
8b
8c
8d
8e
8f
C6H5
19.48
10.58
> 20
3.94
2.59
1.30
0.87
0.81
4′-OCH3C6H4
2′,4′-diOCH3C6H3
2′,4′,6′-triOCH3C6H2
3′,4′,5′-triOCH3C6H2
4′-OH-3′-OCH3C6H3
4′-CH3C6H4
8.18
0.64
14.62
2.65
0.32
0.09
7.92
0.23
0.26
0.10
4.27
0.24
0.47
0.53
2.56
4.30
8g
8h
8i
8.06
9.06
4′-BrC6H4
18.86
> 20
11.26
> 20
> 20
8.66
3.27
16.68
> 20
17.28
19.66
15.60
12.62
> 20
> 20
> 20
4.26
0.82
4′-ClC6H4
8j
4′-FC6H4
0.61
0.74
1.02
0.43
0.66
8k
8l
4′-NO2C6H4
furan-2-yl
8m
8n
8o
8p
5-FU
thiophene-2-yl
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
0.5
> 20
> 20
> 20
5.52
0.31
0.19
a
2.5. Compound 8d induces MMP loss in HCT-116 cells
The concentration that causes 50% of cell proliferation inhibition. The data
are expressed as the means
independent experiments.
SD from triplicate determination from three
Mitochondria have an important role in inducing apoptosis in
cancer cells. Damaged mitochondria release various proteins that acti-
vate the procaspase cascade within the cells and trigger apoptosis.
Therefore, a decreased MMP (Δψm) has been implicated as an early
event in apoptotic cells [24]. JC-1 is an ideal MMP-detecting probe, as it
forms aggregates at high MMPs and monomers when the MMP reduces.
After treatment of HCT-116 cells with compound 8d at various con-
centrations (1× or 2 × IC50) for 24 h, or STS (1 μM) for 2 h, the red
fluorescence intensity (JC-1 aggregates) decreased from 85.1% to 79.6
VIIIa–s series (quinazolin-2-yl group as the A-ring), and most of the
active compounds exhibited higher cytotoxicity in HCT-116 than MCF-
7 cells. Similarly, most compounds in the 8a–p series (thieno[2,3-d]
pyrimidin-2-yl group as the B-ring) were more cytotoxic than their
counterparts in the 4a–p series (with the exception of 8h, 8j and 8l),
and compounds in both the 4a–p and 8a–p series were generally more
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Table 3
IC50 values for compounds 8d–f in MCF-7, MDA-MB-231, HeLa and HT-29 cells.
Compound
R
IC50 (μM)a
MCF-7
MDA-MB-231
HeLa
HT-29
HepG2
8d
2′,4′,6′-triOCH3
3′,4′,5′-triOCH3
4′-OH-3′-OCH3
3.87
4.13
2.28
> 20
0.26
14.86
9.91
0.46
4.94
0.59
0.75
0.40
10.18
11.33
15.65
9.99
0.15
1.22
0.43
2.85
4.06
4.43
8.66
0.35
0.29
0.17
0.33
8e
0.55
0.17
0.26
11.44
7.98
8f
10.35
10.22
0.59
0.87
5-FU
> 20
0.18
a
The concentration that causes 50% of cell proliferation inhibition. The data are expressed as the means
dependent experiments.
SD from triplicate determination from three in-
significantly higher in cells incubated with compound 8d compared to
DMSO vehicle-incubated cells (Fig. 5A), indicating that compound 8d
induces ROS formation. Meanwhile, we observed a dose-dependent
increase in mitochondrial ROS levels in HCT-116 cells by staining with
MitoSox™ (Fig. 5B). These results demonstrate that 8d significantly
increases ROS production, which might be responsible for inducing
apoptosis.
Table 4
IC50 values for compounds 8d–f in MRC-5, MCF-10A and NIH3T3 cells.
Compound
R
IC50 (μM)a
MRC-5
MCF-10A
NIH3T3
2.7. Compound 8d increases the levels of cleaved PARP-1, and caspases 3,
7 and 9 in HCT-116 cells
8d
2′,4′,6′-triOCH3
3′,4′,5′-triOCH3
4′-OH-3′-OCH3
10.23
12.10
14.88
16.85
0.37
6.03
0.84
5.41
8.56
4.21
> 20
0.33
0.42
0.07
8e
0.48
0.68
0.28
13.21
10.50
> 20
1.17
0.52
Caspases are the family of cysteinyl aspartate specific proteinases
that can be grouped into pro-apoptotic and pro-inflammatory sub-
families based on their known major functions. Pro-apoptotic caspases
are divided into initiator and executioner caspases and are mainly in-
volved in mediating the apoptotic pathway in mammalian cells.
Caspase 9 is one of the initiator caspases that induces the activation of
the executioner caspases 3 and 7, which might induce PARP-1 cleavage.
PARP-1 is a nuclear poly (ADP-ribose) polymerase involved in DNA
repair predominantly in response to environmental stress, leading to
apoptotic cell death [25].
8f
5-FU
a
The concentration that causes 50% of cell proliferation inhibition. The data
are expressed as the means
independent experiments.
SD from triplicate determination from three
or 69.6% (64.3% for STS). Consistently, the green fluorescence in-
tensity (JC-1 monomers) correspondingly increased from 13.2% to
17.1% or 28.9% (32.3% for STS) (Fig. 4A). Monomer formation ag-
gregate reduction was also observed by confocal microscopy (Fig. 4B).
These results demonstrate that compound 8d triggers apoptotic cell
death by inducing a decrease in MMP and subsequent mitochondrial
dysfunction.
To investigate the roles of caspases and PARP-1 in the cellular re-
sponse to compound 8d, we examined the cleavage levels of these
proteins in HCT-116 cells by western blotting. Treating HCT-116 cells
with compound 8d (1× or 2 × IC50) effectively induced cleavage of
caspases 3, 7, 9 and PARP-1, respectively, in a dose-dependent manner
compared with control (Fig. 6). These data demonstrate that compound
8d activates caspases 3, 7 and 9 in HCT-116 cells, and then promotes
the cleavage of PARP-1 to induce HCT-116 apoptosis via the mi-
tochondrial death pathway.
2.6. Compound 8d induces oxidative stress in HCT-116 cells
Intracellular ROS, such as hydrogen peroxide, superoxide and hy-
droxyl radicals, has a vital role in regulating cell apoptosis. Derivatives
of reduced fluorescein are used as indicators for ROS. Chemically re-
duced and acetylated forms of 2′,7′-dichlorofluorescein (DCF), such as
2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), are not fluor-
escent until the acetate groups are removed by oxidation within the
cell. Accordingly, we determined the overall intracellular ROS level in
HCT-116 cells exposed to compound 8d (1× or 2 × IC50) by staining
the cells with H2DCFDA. The oxidized DCFDA fluorescence levels were
2.8. Compound 8d does not suppress PARP-1 enzymatic activity
The poly-ADP-ribose-polymerase (PARP) family, including PARP-1
and PARP-2 enzymes, has an important role in recognizing damaged
DNA and initiating a cascade of cellular events required for various
types of DNA damage repair. PARP catalyzes PARylation by
Table 5
Effects of 8d on HCT-116 cell-cycle progression.
Compound
Sub-G1 phase
G0/G1 phase
S phase
G2/M phase
DMSO
2.24
5.86
3.77
6.50
15.39
25.51
0.12
0.19
0.23
0.20
40.01
4.65
0.77
0.15
0.33
31.17
80.12
7.11
0.78
1.56
25.25
8.89
0.38
0.79
5FU
Taxol
5.92
0.12
73.17
23.33
20.79
19.34
2.73
8d (1 × IC50
)
45.00
44.10
38.82
0.83
26.42
25.94
22.51
0.68
0.73
0.69
0.84
0.53
0.37
8d (1.5 × IC50
)
0.65
0.76
0.55
8d (2 × IC50
)
0.43
5

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Fig. 2. Effects of compound 8d on the cell-cycle distribution of HCT-116 cells. (A) HCT-116 cells were treated with 5-FU (IC50), taxol (0.10 μM), or increasing
concentrations of compound 8d (1×, 1.5× or 2 × IC50) for 24 h before being harvested and analyzed by flow cytometry. The data are expressed as the means
of three independent experiments. **P < 0.001 (Student's t-test). (B) Representative cell-cycle profiles from three independent experiments.
SD
consecutively adding ADP-ribose moieties from NAD+ to amino acid
receptor residues on target proteins. PARP inhibitors (nicotinamide
analogues) can bind to the PARP catalytic domain to inhibit PARylation
of target proteins, including PARP-1 itself [26–28].
proliferation. Compounds 8d–f with IC50 values < 5 μM in HCT-116
cells were also cytotoxic to MCF-7, MDA-MB-231, HeLa, HT-29 and
HepG2 cells and normal MCF-10A cells. Compound 8d was the most
potent compound against HCT-116 cells (IC50 = 2.65 μM), and was thus
selected for further analysis to determine the mechanisms underlying
compound 8d-induced cytotoxicity. Compound 8d had no obvious ef-
fect on the cell-cycle distribution in HCT-116 cells, but a sub-G1 peak
was observed in the cell-cycle profile, indicating that the cells under-
went apoptosis. We then confirmed that compound 8d induced apop-
tosis by Annexin V-FITC/PI dual staining, MitoSOX™ Red, JC-1 staining
assays and ROS detection. Finally, western blotting indicated that
treatment of HCT-116 cells with compound 8d caused an increase of
cleaved PARP-1 and cleaved caspases 3, 7, and 9. These results suggest
that compound 8d can induce HCT-116 cell apoptosis via the mi-
tochondrial death pathway. Replacing the quinazolin-2-yl group in its
chalcone analogues with a thieno[2,3-d]pyrimidin-2-yl group does not
alter the apoptosis induction efficacy. The molecular mechanisms as to
how compound 8d targets and induces apoptosis is under investigation
in our laboratory; we anticipate that this compound will be beneficial
We performed an in vitro PARylation assay to evaluate if our re-
presentative compounds 8d–f inhibited the PARP-1 activity. We found
that none of the compounds 8d–f exhibited inhibitory activity towards
PARP-1 PARylation, while Olaparib, a well-known PARP-1 inhibitor,
led to significant inhibition (Fig. 7). We therefore conclude that com-
pounds 8d–f do not inhibit PARP-1 enzymatic activity and are not
PARP-1 inhibitors.
3. Conclusions
We synthesized two series of chalcone analogues containing thieno
[2,3-d]pyrimidin-2-yl as the A-ring (4a–p) or B-ring (8a–p) and eval-
uated their cytotoxic activities towards cultured A549 and HCT-116 cell
lines. Compounds 8a–p were generally more active than compounds
4a–p in both cell lines, based on their IC50 values to inhibit
6

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Fig. 3. Annexin V–FITC/PI dual staining assay. Quantification of apoptotic HCT-116 cells by Annexin V-FITC/PI dual staining, following 8d and STS treatment. (A)
HCT-116 cells were treated with 8d (1× or 2 × IC50) for 24 h, while STS (1 μM) for 2 h. Cells were harvested, and apoptotic HCT-116 cells were determined by
Annexin V-FITC/PI staining and flow cytometry. Cells in the lower left quadrant (Q1-LL: Annexin V−/PI+) represent live cells; cells in the lower right quadrant (Q1-
LR: Annexin V+/PI−) represent early apoptotic cells; cells in the upper right quadrant (Q1-UR: Annexin V+/PI+) represent late apoptotic cells; cells in the upper
left quadrant (Q1-UL: Annexin V−/PI−) represent necrotic cells. (B) Percentage of apoptotic cells determined as the sum of late apoptotic cells (UR) and early
apoptosis cells (LR) from three separate experiments.
for the discovery of chalcone analogues as cancer chemotherapeutic
agents.
washed with petroleum ether (PE, 10 mL × 3) and dried in a vacuum
oven to give 2-cyanoacetamide (2.3 g, 91%, mp 116–117 °C) as a white
solid, which was used in the next step without purification.
A mixture of 2-cyanoacetamide (0.84 g, 10 mmol), propionaldehyde
(0.58 g, 10 mmol), elemental sulfur (0.32 g, 10 mmol) and triethyla-
mine (1.0 g, 10 mmol) in ethanol (30 mL) was stirred at 50–55 °C for
4 h. Partial solvent was removed by rotary evaporation, and the re-
maining solution was mixed with ethyl acetate (100 mL) and washed
with brine (100 mL × 3). The organic layer was dried over anhydrous
sodium sulfate overnight. The solvent was removed by rotary eva-
poration, and the residue was purified by column chromatography on a
silica gel (eluent: PE/ethyl acetate = 1:1) to yield intermediate 1 as a
pale yellow solid (0.95 g, 71%), mp 119–121 °C. 1H NMR (600 MHz,
DMSO‑d6) δ: 2.17 (d, J = 1.2 Hz, 3H, CH3), 6.64 (br s, 1H, 1/2 CONH2),
6.69 (d, J = 1.2 Hz, 1H, thiophene 4-H), 7.05 (br s, 3H, 1/2 CONH2,
NH2). EI-MS m/z: 156 (M+).
4. Experimental procedures
4.1. Chemistry
Melting points were determined on an XT5B microscopic melting
point apparatus and are presented uncorrected. 1H and 13C NMR
spectra were recorded on a Varian VNMRS-600 spectrometer at 600 or
150 MHz using tetramethylsilane (TMS) as an internal standard
(Abbreviation: Ph = phenyl). Mass spectra [electron impact ionization;
MS (EI)] were recorded on a GCMS-QP2010Plus (Shimadzu, Japan) gas
chromatograph/mass spectrometer. High-resolution mass spectra
[electrospray ionization; HRMS (ESI)] were recorded on a Thermo
Scientific LTQ Orbitrap Discovery (Bremen, Germany) mass spectro-
meter. Column chromatography was carried out on
a silica gel
(200–300 mesh). High performance liquid chromatography (HPLC)
analyses were performed on an Agilent 1200 Series HPLC instrument
(ZORBAX Eclipse XDB-C18 column, 150 mm × 4.6 mm, 5 μm) with a
G1314B variable-wavelength detector (VWD; λ = 238 or 280 nm; mo-
bile phase: MeOH:H2O = 7:3, v/v). The profiles are presented in the
supplementary materials. The purities of the target compounds used in
the biological studies were typically ≥95%. All the chemical reagents
used were commercially available and used without further purifica-
tion.
4.1.2. Preparation
of
5-methyl-2-(2-oxopropanamido)thiophene-3-
carboxamide (intermediate 2)
Thionyl chloride (1.3 g, 11 mmol) was added drop-wise to a solution
of 2-oxopropanoic acid (0.88 g, 10 mmol) in dry dichloromethane
(DCM, 5 mL). The mixture was stirred for 5 min, and then heated under
reflux for 2 h before the solvent was removed by rotary evaporation.
The residue was mixed with DCM (5 mL) and evaporated under reduced
pressure to dryness. The procedure was repeated twice to remove vo-
latiles. 2-Oxopropanoyl chloride was obtained as an oil (1.0 g, 94%),
which was used in the next step without further purification.
4.1.1. Preparation
of
2-amino-5-methylthiophene-3-carboxamide
A
solution of 2-amino-5-methylthiophene-3-carboxamide (1)
(intermediate 1)
(0.78 g, 5 mmol) and 2-oxopropanoyl chloride (0.64 g, 6 mmol) in dry
THF (5 mL) was stirred at room temperature overnight. The separated
solid was filtered and washed with THF (5 mL × 3). The combined
filtrate was concentrated under reduced pressure and the residue was
mixed with cold methane (10 mL × 3). The solid was collected by fil-
tration, which was purified by column chromatography on a silica gel
A mixture of ethyl cyanoacetate (3.4 g, 30 mmol) and aqueous
ammonia (15 mL) in tetrahydrofuran (THF, 10 mL) was stirred at room
temperature for 24 h. The mixture was concentrated under reduced
pressure to remove most of the solvents and the remaining solution was
kept in a refrigerator overnight. The separated solid was filtered,
7

F.-C. Wang, et al.
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Fig. 4. (A) HCT-116 cells were treated with compound 8d (1× or 2 × IC50) for 24 h, or STS (1 μM) for 2 h. Cells were harvested and the percentage of JC-1
aggregates and monomers were determined by flow cytometry. Cells in the upper right quadrant represent aggregated cells; cells in the lower right quadrant
represent monomer cells. (B) The depolarization of the MMP (Δψm) could be induced by compound 8d. HCT-116 cells were treated with compound 8d (1× or
2 × IC50) for 24 h, or STS (1 μM) for 2 h, and then stained with JC-1 for 20 min at 37 °C. The loss of red fluorescence and the gain of green-emitting monomers
indicates the disruption of the MMP (Δψm). STS served as a positive control. Representative images are shown of the corresponding fluorescent channel
(40 × magnification).
Fig. 5. Effect of compound 8d on oxidative stress in HCT-116 cells. (A) HCT-116 cells were treated with compound 8d (1× or 2 × IC50) for 24 h, or 1 mM H2O2 for
20 min. The cells were then removed from the culture medium by centrifugation and stained with H2DCFDA. After incubation at 37 °C for 30 min, the cells were
analyzed by flow cytometry. (B) HCT-116 cells were treated to compound 8d (1× or 2 × IC50) for 24 h, or 1 mM H2O2 for 20 min, and then incubated with MitoSOX™
Red. The stained cells were analyzed by flow cytometry.
8

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4.1.4. General preparation procedure for compounds 4a–p
A solution of 2-acetyl-6-methylthieno[2,3-d]pyrimidin-4(3H)-one
(3) (62 mg, 0.3 mmol) and a 30% aqueous solution of potassium hy-
droxide (0.2 mL, for 4a–j, 4l and 4m) or piperidine (0.1 mL, for 4k and
4n–p) in methanol (1 mL) was stirred for 30 min before the appropriate
aromatic aldehyde or heteroaromatic aldehyde (0.4 mmol) was added.
The reaction mixture was stirred overnight at room temperature, and
then acidified with 1 mol/L hydrochloric acid to pH 2–3. The pre-
cipitate was collected by filtration, washed with methanol and air-
dried. The crude product was re-crystallized from methanol, unless
otherwise stated.
4.1.4.1. (E)-2-(3-Phenylacryloyl)-6-methylthieno[2,3-d]pyrimidin-4(3H)-
one (4a). Yield: 61% (54 mg), yellow solid, mp 234–237 °C. HPLC
retention time (rt) = 1.892 min (100%). 1H NMR (600 MHz, DMSO‑d6)
δ: 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17 (d, J = 1.2 Hz, 1H, thieno[2,3-d]
pyrimidine 5-H), 7.36 (d, J = 15.6 Hz, 1H, COCH]), 7.61 (t,
J = 7.8 Hz, 2H, Ph 3′-H, 5′-H), 7.73 (t, J = 7.8 Hz, 1H, Ph 4′-H), 8.13
(d, J = 7.8 Hz, 2H, Ph 2′-H, 6′-H), 8.33 (d, J = 15.6 Hz, 1H, CH]),
12.82 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.74, 119.65,
124.85, 128.73 (2C), 129.03 (2C), 129.85, 133.89, 134.78, 136.64,
139.42, 149.52, 157.34, 162.88, 188.53. HRMS (ESI) m/z: calcd for
Fig. 6. Effects of compound 8d on apoptosis-associated protein expression in
HCT-116 cells. HCT-116 cells were treated with compound 8d (1× or 2 × IC50
)
for 24 h, or 1 mM STS for 4 h. Total lysates were harvested and immuno-blotted
with various antibodies, as indicated. β-actin was used as a loading control.
C
16H13N2O2S ([M+H]+): 297.0698; found: 297.0691.
4.1.4.2. (E)-2-(3-(4-Methoxyphenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4b). Yield: 47% (46 mg), yellow solid, mp
268–271 °C. HPLC rt = 1.880 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.54 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 7.12 (d,
J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 7.16 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.31 (d, J = 15.6 Hz, 1H, COCH]), 8.13 (d, J = 8.4 Hz, 2H, Ph
2′-H, 6′-H), 8.32 (d, J = 15.6 Hz, 1H, CH]), 12.75 (s, 1H, NH). 13C
NMR (150 MHz, DMSO‑d6) δ: 15.74, 55.71, 113.89, 114.34 (2C),
119.63, 129.64, 129.97, 130.44, 131.25 (2C), 134.08, 139.31,
149.67, 157.39, 163.83, 186.54. HRMS (ESI) m/z: calcd for
C
17H15N2O3S ([M+H]+): 327.0803; found: 327.0792.
4.1.4.3. (E)-2-(3-(2,4-Dimethoxyphenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4c). Yield: 70% (75 mg), yellow solid, mp
246–251 °C. HPLC rt = 1.725 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.54 (d, J = 1.8 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 3.91
(s, 3H, OCH3), 6.68 (dd, J = 9.0, 2.4 Hz, 1H, Ph 5′-H), 6.71 (d,
J = 2.4 Hz, 1H, Ph 3′-H), 7.14 (d, J = 1.8 Hz, 1H, thieno[2,3-d]
pyrimidine 5-H), 7.22 (d, J = 15.6 Hz, 1H, COCH]), 7.67 (d,
J = 9.0 Hz, 1H, Ph 6′-H), 8.03 (d, J = 15.6 Hz, 1H, CH]), 12.71 (s,
1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.72, 55.60, 56.50, 113.95,
114.08, 119.57, 119.78, 124.67, 128.03, 133.15, 134.19, 139.07,
149.56, 152.46, 153.07, 157.31, 162.99, 190.74. HRMS (ESI) m/z:
calcd for C18H17N2O4S ([M+H]+): 357.0909; found: 357.0894.
Fig. 7. Compounds 8d–f have no impact on PARylation activity of PARP-1.
293T cells were transfected with FLAG-PARP-1. Total cell lysates were har-
vested 48 h after transfection and subjected to immunoprecipitation by an anti-
FLAG antibody. The immunoprecipitated FLAG-PARP-1 was used for in vitro
PARylation.
(eluent: PE/ethyl acetate = 1:1) to give intermediate 2 as a pale yellow
solid (0.79 g, 70%), mp 211–215 °C. 1H NMR (600 MHz, DMSO‑d6) δ:
2.38 (d, J = 1.2 Hz, 3H, CH3), 2.43 (s, 3H, CH3CO), 7.16 (d, J = 1.2 Hz,
1H, thiophene 4-H), 7.54 (br s, 1H, 1/2 CONH2), 7.86 (br s, 1H, 1/2
CONH2), 12.87 (s, 1H, NH). EI-MS m/z: 226 (M+).
4.1.4.4. (E)-6-Methyl-2-(3-(2,4,6-trimethoxyphenyl)acryloyl)thieno[2,3-
d]pyrimidin-4(3H)-one (4d). Yield: 56% (65 mg), yellow solid, mp
233–236 °C (from DMF). HPLC rt = 7.787 min (99%). 1H NMR
(600 MHz, CDCl3) δ: 2.52 (d, J = 1.2 Hz, 3H, CH3), 3.74 (s, 6H,
2OCH3), 3.85 (s, 3H, OCH3), 6.33 (s, 2H, Ph 3′-H, 5′-H), 7.0 (d,
J = 15.6 Hz, 1H, COCH]), 7.12 (d, J = 1.2 Hz, 1H, thieno[2,3-d]
pyrimidine 5-H), 7.38 (d, J = 15.6 Hz, 1H, CH]), 12.61 (s, 1H, NH).
13C NMR (150 MHz, CDCl3) δ: 15.69, 55.56, 55.93 (2C), 91.10 (2C),
110.20, 119.55, 124.61, 133.96, 135.82, 139.08, 149.52, 157.23,
158.48 (2C), 162.63, 162.89, 192.04. HRMS (ESI) m/z: calcd for
4.1.3. Preparation of 2-acetyl-6-methylthieno[2,3-d]pyrimidin-4(3H)-one
(intermediate 3)
A mixture of 5-methyl-2-(2-oxopropanamido)thiophene-3-carbox-
amide (2) (0.23 g, 1 mmol) and a 4% aqueous solution of sodium hy-
droxide (4 mL) in ethanol (1 mL) was heated under reflux for 2 h. After
cooling to room temperature, the solution was acidified with 1 mol/L
hydrochloric acid to pH 4–5. The separated solid was removed by fil-
tering and the filtrate was extracted with ethyl acetate (15 mL × 3).
The combined extracts were washed with brine (15 mL × 3) and dried
over anhydrous sodium sulfate overnight. The solvent was removed by
rotary evaporation, and the residue was purified by column chroma-
tography on a silica gel (eluent: DCM/methanol = 98:2) to give inter-
mediate 3 as a pale yellow solid (0.79 g, 58%), mp > 350 °C. 1H NMR
(600 MHz, DMSO‑d6) δ: 2.33 (s, 3H, CH3CO), 2.46 (d, J = 1.2 Hz, 3H,
CH3), 7.0 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H). EI-MS m/z:
208 (M+).
C
19H19N2O5S ([M+H]+): 387.1015; found: 387.1005.
4.1.4.5. (E)-6-Methyl-2-(3-(3,4,5-trimethoxyphenyl)acryloyl)thieno[2,3-
d]pyrimidin-4(3H)-one (4e). Yield: 60% (69 mg), yellow solid,
mp > 330 °C (from DMF). HPLC rt = 1.849 min (100%). 1H NMR
(600 MHz, DMSO‑d6) δ: 2.55 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 3.91
(s, 6H, 2OCH3), 7.18 (s, 1H, thieno[2,3-d]pyrimidine 5-H), 7.35 (d,
9

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J = 15.6 Hz, 1H, COCH]), 7.42 (s, 2H, Ph 2′-H, 6′-H), 8.30 (d,
J = 15.6 Hz, 1H, CH]), 12.73 (s, 1H, NH). 13C NMR (150 MHz,
DMSO‑d6) δ: 16.14, 56.01, 56.38 (2C), 91.54 (2C), 110.65, 120.0,
125.06, 134.41, 136.26, 139.53, 149.97, 157.68, 158.93 (2C), 163.08,
163.34, 192.49. HRMS (ESI) m/z: calcd for C19H19N2O5S ([M+H]+):
387.1015; found: 387.1006.
140.56, 158.45, 163.14 (d, J = 247.05 Hz), 164.67, 168.27, 189.66.
HRMS (ESI) m/z: calcd for C16H12FN2O2S ([M+H]+): 315.0604; found:
315.0593.
4.1.4.11. (E)-6-Methyl-2-(3-(4-nitrophenyl)acryloyl)thieno[2,3-d]
pyrimidin-4(3H)-one (4k). Yield: 56% (57 mg), yellow solid, mp
284–286 °C. HPLC rt = 1.984 min (100%). 1H NMR (600 MHz,
CF3COOD) δ: 2.60 (s, 3H, CH3), 7.30 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.89 (d, J = 15.6 Hz, 1H, COCH]), 8.31 (d, J = 15.6 Hz, 1H,
CH]), 8.70 (d, J = 4.8 Hz, 2H, Ph 2′-H, 6′-H), 9.03 (d, J = 4.8 Hz, 2H,
Ph 3′-H, 5′-H). 13C NMR (150 MHz, CF3COOD) δ: 16.21, 119.52,
121.46, 124.36 (2C), 126.23, 129.97 (2C), 140.23, 144.38, 145.95,
147.52, 148.97, 161.36 (overlapped with CF3COOD), 164.38, 182.15.
HRMS (ESI) m/z: calcd for C16H12N3O4S ([M+H]+): 342.0549; found:
342.0538.
4.1.4.6. (E)-2-(3-(4-Hydroxy-3-methoxyphenyl)acryloyl)-6-methylthieno
[2,3-d]pyrimidin-4(3H)-one (4f). Yield: 69% (71 mg), yellow solid,
mp > 330 °C. HPLC rt = 8.016 min (99%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.54 (d, J = 1.2 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 6.95
(d, J = 8.4 Hz, 1H, Ph 5′-H), 7.15 (d, J = 1.2 Hz, 1H, thieno[2,3-d]
pyrimidine 5-H), 7.29 (d, J = 15.6 Hz, 1H, COCH]), 7.59 (d,
J = 2.4 Hz, 1H, Ph 2′-H), 7.76 (dd, J = 8.4, 2.4 Hz, 1H, Ph 6′-H),
8.30 (d, J = 15.6 Hz, 1H, CH]), 10.28 (s, 1H, OH), 12.72 (br s, 1H,
NH). 13C NMR (150 MHz, DMSO‑d6) δ: 16.15, 56.14, 111.94, 115.57,
120.03, 124.82, 125.12, 129.09, 130.38, 134.14, 139.60, 148.45,
150.16, 153.19, 157.82, 163.41, 186.53. HRMS (ESI) m/z: calcd for
4.1.4.12. (E)-2-(3-(Furan-2-yl)acryloyl)-6-methylthieno[2,3-d]pyrimidin-
4(3H)-one (4l). Yield: 80% (69 mg), yellow solid, mp 294–296 °C.
HPLC rt = 3.837 min (95%). 1H NMR (600 MHz, DMSO‑d6) δ: 2.55
(d, J = 1.2 Hz, 3H, CH3), 6.84 (dd, J = 3.6, 1.2 Hz, 1H, furan 4′-H),
7.16 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.38 (d,
J = 15.6 Hz, 1H, COCH]), 7.83 (d, J = 3.6 Hz, 1H, furan 3′-H), 8.08
(d, J = 15.6 Hz, 1H, CH]), 8.15 (d, J = 1.2 Hz, 1H, furan 5′-H), 12.74
(s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.91, 113.52, 116.21,
119.05, 120.12, 127.27, 131.41, 142.03, 147.21, 147.72, 150.97,
156.68, 160.85, 181.41. HRMS (ESI) m/z: calcd for C14H11N2O3S ([M
+H]+): 287.0490; found: 287.0482.
C
17H15N2O4S ([M+H]+): 343.0753; found: 343.0744.
4.1.4.7. (E)-2-(3-(4-Methylphenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4g). Yield: 64% (60 mg), yellow solid, mp
264–267 °C. HPLC rt = 1.865 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.42 (s, 3H, CH3), 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17
(d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.33 (d, J = 15.6 Hz,
1H, COCH]), 7.42 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 8.05 (d,
J = 8.4 Hz, 2H, Ph 2′-H, 6′-H), 8.32 (d, J = 15.6 Hz, 1H, CH]),
12.77 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.74, 21.26,
119.63, 124.80, 128.88 (2C), 129.61 (2C), 129.87, 134.19, 134.49,
139.38, 144.56, 149.57, 157.36, 162.92, 187.85. HRMS (ESI) m/z:
calcd for C17H15N2O2S ([M+H]+): 311.0854; found: 311.0841.
4.1.4.13. (E)-6-Methyl-2-(3-(thiophen-2-yl)acryloyl)thieno[2,3-d]
pyrimidin-4(3H)-one (4m). Yield: 61% (55 mg), yellow solid, mp
308–311 °C. HPLC rt = 6.879 min (99%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.59 (d, J = 1.2 Hz, 3H, CH3), 7.24 (dd, J = 4.8, 3.6 Hz,
1H, thiophene 4′-H), 7.25 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine
5-H), 7.68 (d, J = 15.6 Hz, 1H, COCH]), 7.75 (d, J = 3.6 Hz, 1H,
thiophene 3′-H), 7.88 (d, J = 4.8 Hz, 1H, thiophene 5′-H), 8.07 (d,
J = 15.6 Hz, 1H, CH]), 12.49 (s, 1H, NH). 13C NMR (150 MHz,
DMSO‑d6) δ: 15.92, 117.66, 120.13, 127.32, 129.14, 131.68, 134.62,
138.32, 139.55, 142.03, 147.72, 156.69, 160.82, 181.36. HRMS (ESI)
m/z: calcd for C14H11N2O2S2 ([M+H]+): 303.0262; found: 303.0251.
4.1.4.8. (E)-2-(3-(4-Bromophenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4h). Yield: 61% (69 mg), yellow solid,
mp > 330 °C. HPLC rt = 5.571 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.59 (d, J = 1.2 Hz, 3H, CH3), 7.26 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.68 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H),
7.83 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 7.86 (d, J = 16.2 Hz, 1H,
COCH]), 8.03 (d, J = 16.2 Hz, 1H, CH]), 12.54 (s, 1H, NH). 13C NMR
(150 MHz, DMSO‑d6) δ: 15.92, 120.15, 120.20, 124.78, 127.41, 130.98
(2C), 132.09 (2C), 133.39, 142.18, 144.18, 147.66, 156.70, 160.77,
181.95. HRMS (ESI) m/z: calcd for C16H12BrN2O2S ([M+H]+):
374.9803, 376.9782; found: 374.9795, 376.9765.
4.1.4.14. (E)-6-Methyl-2-(3-(pyridin-2-yl)acryloyl)thieno[2,3-d]
pyrimidin-4(3H)-one (4n). Yield: 56% (50 mg), yellow solid,
mp > 330 °C. HPLC rt = 3.272 min (95%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.60 (d, J = 1.2 Hz, 3H, CH3), 7.27 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.48 (ddd, J = 7.8, 4.8, 1.2 Hz, 1H,
pyridine 4′-H), 7.85 (d, J = 7.8 Hz, 1H, pyridine 6′-H), 7.87 (d,
J = 15.6 Hz, 1H, COCH]), 7.92 (td, J = 7.8, 1.8 Hz, 1H, pyridine 5′-
H), 8.41 (d, J = 15.6 Hz, 1H, CH]), 8.73 (d, J = 4.8 Hz, 1H, pyridine
3′-H), 12.57 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.94,
120.20, 122.80, 125.38, 126.29, 127.45, 137.42, 142.33, 143.86,
147.67, 150.27, 152.08, 156.73, 160.80, 182.31. HRMS (ESI) m/z:
calcd for C15H12N3O2S ([M+H]+): 298.0650; found: 298.0641.
4.1.4.9. (E)-2-(3-(4-Chlorophenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4i). Yield: 57% (57 mg), yellow solid, mp
220–223 °C. HPLC rt = 1.844 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.58 (d, J = 1.2 Hz, 3H, CH3), 7.25 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.67 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H),
7.83 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 7.85 (d, J = 16.2 Hz, 1H,
COCH]), 8.03 (d, J = 16.2 Hz, 1H, CH]), 12.53 (s, 1H, NH). 13C NMR
(150 MHz, DMSO‑d6) δ: 15.92, 120.14, 120.17, 124.77, 127.40, 130.96
(2C), 132.07 (2C), 133.38, 142.16, 144.16, 147.64, 156.69, 160.75,
181.93. HRMS (ESI) m/z: calcd for C16H12ClN2O2S ([M+H]+):
331.0308, 333.0279; found: 331.0302, 333.0268.
4.1.4.15. (E)-6-Methyl-2-(3-(pyridin-3-yl)acryloyl)thieno[2,3-d]
pyrimidin-4(3H)-one (4o). Yield: 76% (68 mg), yellow solid, mp
221–224 °C. HPLC rt = 1.821 min (99%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.59 (s, 3H, CH3), 7.27 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.51 (dd, J = 7.8, 4.8 Hz, 1H, pyridine 5′-H), 7.92 (d,
J = 16.2 Hz, 1H, COCH]), 8.13 (d, J = 16.2 Hz, 1H, CH]), 8.36 (d,
J = 7.8 Hz, 1H, pyridine 4′-H), 8.65 (d, J = 4.8 Hz, 1H, pyridine 6′-H),
9.0 (s, 1H, pyridine 2′-H), 12.58 (s, 1H, NH). 13C NMR (150 MHz,
CF3COOD) δ: 16.26, 120.98, 126.05, 128.15, 128.76, 130.09, 134.80,
138.84, 143.17, 143.61, 145.53, 147.94, 157.54, 161.63, 182.67.
4.1.4.10. (E)-2-(3-(4-Fluorophenyl)acryloyl)-6-methylthieno[2,3-d]
pyrimidin-4(3H)-one (4j). Yield: 45% (42 mg), yellow solid, mp
217–219 °C. HPLC rt = 3.511 min (98%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.55 (s, 3H, CH3), 7.17 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.34 (d, J = 15.6 Hz, 1H, COCH]), 7.44 (t, J = 8.4 Hz, 2H, Ph 3′-
H, 5′-H), 8.23 (dd, J = 8.4, 6.0 Hz, 2H, Ph 2′-H, 6′-H), 8.31 (d,
J = 15.6 Hz, 1H, CH]), 12.77 (s, 1H, NH). 13C NMR (150 MHz,
DMSO‑d6) δ: 15.92, 115.96 (d, J = 21.6 Hz, 2C), 119.87, 123.86 (d,
J = 1.8 Hz), 125.12, 130.70 (d, J = 8.55 Hz, 2C), 131.65, 132.71,
HRMS (ESI) m/z: calcd for
C
15H12N3O2S ([M+H]+): 298.0650;
found: 298.0642.
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4.1.4.16. (E)-6-Methyl-2-(3-(pyridin-4-yl)acryloyl)thieno[2,3-d]
pyrimidin-4(3H)-one (4p). Yield: 80% (71 mg), yellow solid, mp
221–224 °C. HPLC rt = 1.720 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.60 (d, J = 1.2 Hz, 3H, CH3), 7.27 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.81 (d, J = 6.0 Hz, 2H, pyridine 2′-H,
6′-H), 7.83 (d, J = 16.2 Hz, 1H, COCH]), 8.19 (d, J = 16.2 Hz, 1H,
CH]), 8.70 (d, J = 6.0 Hz, 2H, pyridine 3′-H, 5′-H), 12.63 (s, 1H). 13C
NMR (150 MHz, CF3COOD) δ: 16.06, 120.56, 126.16 (2C), 128.14,
129.53, 139.01, 142.69 (2C), 143.17, 147.48, 151.01, 156.94, 160.86,
182.10. HRMS (ESI) m/z: calcd for C15H12N3O2S ([M+H]+): 298.0650;
found: 298.0642.
114.32 (2C), 119.61, 124.74, 129.63, 129.95, 131.23 (2C), 134.06,
139.28, 149.66, 157.37, 162.94, 163.82, 186.52. HRMS (ESI) m/z:
calcd for C17H15N2O3S ([M+H]+): 327.0803; found: 327.0794.
4.1.7.3. (E)-2-(3-(2,4-Dimethoxyphenyl)-3-oxoprop-1-en-1-yl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (8c). Yield: 51% (54 mg),
yellow solid, mp 220–223 °C. HPLC rt = 1.812 min (100%). 1H NMR
(600 MHz, DMSO‑d6) δ: 2.54 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 6.67 (d, J = 9.0 Hz, 1H, Ph 5′-H), 6.71 (s, 1H, Ph 3′-H), 7.14
(s, 1H, thieno[2,3-d]pyrimidine 5-H), 7.22 (d, J = 15.6 Hz, 1H,
COCH]), 7.67 (d, J = 9.0 Hz, 1H, Ph 6′-H), 8.03 (d, J = 15.6 Hz, 1H,
CH]), 12.71 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.71,
55.73, 56.17, 98.64, 106.42, 119.53, 120.40, 124.52, 131.90, 132.42,
134.86, 138.81, 149.84, 157.38, 160.81, 163.12, 164.75, 188.16.
4.1.5. Preparation of ethyl 2-amino-5-methylthiophene-3-carboxylate
(intermediate 5) and 2-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-
4(3H)-one (intermediate 6)
HRMS (ESI) m/z: calcd for
C
18H17N2O4S ([M+H]+): 357.0909;
Intermediates 5 and 6 were prepared according to the procedures
reported by Yang et al. [10].
found: 357.0899.
4.1.7.4. (E)-2-(3-(2,4,6-Trimethoxyphenyl)-3-oxoprop-1-en-1-yl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (8d). Yield: 35% (41 mg),
yellow solid, mp 233–237 °C (from DMF). HPLC rt = 2.446 min
(98%). 1H NMR (600 MHz, DMSO‑d6) δ: 2.52 (d, J = 1.2 Hz, 3H,
CH3), 3.74 (s, 6H, 2OCH3), 3.85 (s, 3H, OCH3), 6.33 (s, 2H, Ph 3′-H,
5′-H), 7.0 (d, J = 16.2 Hz, 1H, COCH]), 7.13 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.38 (d, J = 16.2 Hz, 1H, CH]), 12.61
(s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.69, 55.56, 55.93 (2C),
91.10 (2C), 110.20, 119.55, 124.61, 133.96, 135.82, 139.08, 149.52,
157.23, 158.48 (2C), 162.63, 162.89, 192.04. HRMS (ESI) m/z: calcd
for C19H19N2O5S ([M+H]+): 387.1015; found: 387.1005.
4.1.6. Preparation of 6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-
2-carbaldehyde (intermediate 7)
A
mixture of 2-(chloromethyl)-6-methylthieno[2,3-d]pyrimidin-
4(3H)-one (6) (1.1 g, 5 mmol), N-methylmorpholine N-oxide (1.2 g,
10 mmol) and potassium iodide (1.1 g, 6.5 mmol) in dry THF (10 mL)
was heated under reflux for 2 h. After cooling to ambient temperature,
the solid was removed by filtering and washed with THF (10 mL). The
combined filtrate and washes were concentrated in vacuo and the re-
sidue was purified by column chromatography on a silica gel (eluent:
DCM/methanol = 98:2) to give intermediate 7 as a pale white solid
(0.56 g, 58%), mp 190–192 °C. 1H NMR (600 MHz, DMSO‑d6) δ: 2.57
(d, J = 1.2 Hz, 3H, CH3), 7.24 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyr-
imidine 5-H), 9.50 (s, 1H, CHO), 12.80 (br s, 1H, NH). EI-MS m/z: 194
(M+).
4.1.7.5. (E)-2-(3-(3,4,5-Trimethoxyphenyl)-3-oxoprop-1-en-1-yl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (8e). Yield: 52% (60 mg),
yellow solid, mp 211–213 °C (from DMF). HPLC rt = 8.137 min
(100%). 1H NMR (600 MHz, DMSO‑d6) δ: 2.55 (d, J = 1.2 Hz, 3H,
CH3), 3.79 (s, 3H, OCH3), 3.91 (s, 6H, 2OCH3), 7.18 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.34 (d, J = 15.6 Hz, 1H, COCH]), 7.42
(s, 2H, Ph 2′-H, 6′-H), 8.30 (d, J = 15.6 Hz, 1H, CH]), 12.73 (s, 1H,
NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.74, 56.28 (2C), 60.25,
106.50 (2C), 119.64, 124.82, 129.75, 131.96, 134.86, 139.46,
142.67, 149.53, 153.01 (2C), 157.36, 162.89, 187.31. HRMS (ESI) m/
z: calcd for C19H19N2O5S ([M+H]+): 387.1015; found: 387.1005.
4.1.7. General preparation procedure for compounds 8a–p
A mixture of the appropriate acetophenone or heterocyclylethan-1-
one (0.55 mmol) and an aqueous solution of potassium hydroxide
(84 mg, 1.5 mmol) (15% for 8k and 8n–p, 30% for 8a–e and 8g–j, or
50% for 8f, 8l and 8m) in methanol (2 mL) was stirred for 30 min be-
fore 6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-2-carbalde-
hyde (7) (97 mg, 0.5 mmol) was added. The reaction mixture was
stirred overnight at room temperature, and then acidified with 1 mol/L
hydrochloric acid to pH 2–3. The separated solid was collected by fil-
tering, washed with methanol and air-dried. The crude product was re-
crystallized from methanol, unless otherwise stated.
4.1.7.6. (E)-2-(3-(4-Hydroxy-3-methoxyphenyl)-3-oxoprop-1-en-1-yl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (8f). Yield: 47% (48 mg),
yellow solid, mp 256–260 °C. HPLC rt = 19.281 min (100%). 1H NMR
(600 MHz, DMSO‑d6) δ: 2.54 (d, J = 1.2 Hz, 3H, CH3), 3.88 (s, 3H,
OCH3), 6.95 (d, J = 8.4 Hz, 1H, Ph 5′-H), 7.15 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.29 (d, J = 15.6 Hz, 1H, COCH]), 7.58
(d, J = 1.8 Hz, 1H, Ph 2′-H), 7.76 (dd, J = 8.4, 1.8 Hz, 1H, Ph 6′-H),
8.30 (d, J = 15.6 Hz, 1H, CH]), 10.27 (s, 1H, OH), 12.72 (s, 1H, NH).
13C NMR (150 MHz, DMSO‑d6) δ: 15.71, 55.70, 111.50, 115.13, 119.59,
124.38, 124.68, 128.65, 129.95, 133.69, 139.15, 148.01, 149.72,
152.75, 157.37, 162.97, 186.09. HRMS (ESI) m/z: calcd for
4.1.7.1. (E)-6-Methyl-2-(3-oxo-3-phenylprop-1-en-1-yl)thieno[2,3-d]
pyrimidin-4(3H)-one (8a). Yield: 47% (42 mg), yellow solid, mp
200–205 °C. HPLC rt = 2.452 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.36 (d, J = 15.6 Hz, 1H, COCH]), 7.62
(t, J = 7.8 Hz, 2H, Ph 3′-H, 5′-H), 7.73 (t, J = 7.8 Hz, 1H, Ph 4′-H), 8.14
(d, J = 7.8 Hz, 2H, Ph 2′-H, 6′-H), 8.33 (d, J = 15.6 Hz, 1H, CH]),
12.81 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.74, 119.65,
124.85, 128.73 (2C), 129.03 (2C), 129.85, 133.89, 134.78, 136.64,
139.42, 149.52, 157.34, 162.88, 188.54. HRMS (ESI) m/z: calcd for
C
17H15N2O4S ([M+H]+): 343.0753; found: 343.0745.
4.1.7.7. (E)-2-(3-(4-Methylphenyl)-3-oxoprop-1-en-1-yl)-6-methylthieno
[2,3-d]pyrimidin-4(3H)-one (8g). Yield: 60% (56 mg), yellow solid, mp
248–249 °C. HPLC rt = 2.846 min (95%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.42 (s, 3H, CH3), 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17
(d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.33 (d, J = 15.6 Hz,
1H, COCH]), 7.42 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 8.04 (d,
J = 8.4 Hz, 2H, Ph 2′-H, 6′-H), 8.32 (d, J = 15.6 Hz, 1H, CH]),
12.77 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.75, 21.27,
119.64, 124.81, 128.89 (2C), 129.61 (2C), 129.87, 134.20, 134.49,
139.39, 144.57, 149.58, 157.36, 162.92, 187.85. HRMS (ESI) m/z:
calcd for C17H15N2O2S ([M+H]+): 311.0854; found: 311.0845.
C
16H13N2O2S ([M+H]+): 297.0698; found: 297.0688.
4.1.7.2. (E)-2-(3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl)-6-
methylthieno[2,3-d]pyrimidin-4(3H)-one (8b). Yield: 46% (45 mg),
yellow solid, mp 245–248 °C. HPLC rt = 7.416 min (99%). 1H NMR
(600 MHz, DMSO‑d6) δ: 2.55 (d, J = 0.6 Hz, 3H, CH3), 3.89 (s, 3H,
OCH3), 7.13 (d, J = 9.0 Hz, 2H, Ph 3′-H, 5′-H), 7.16 (d, J = 0.6 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.31 (d, J = 15.6 Hz, 1H, COCH]), 8.14
(d, J = 9.0 Hz, 2H, Ph 2′-H, 6′-H), 8.33 (d, J = 15.6 Hz, 1H, CH]),
12.75 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.73, 55.69,
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4.1.7.8. (E)-2-(3-(4-Bromophenyl)-3-oxoprop-1-en-1-yl)-6-methylthieno
[2,3-d]pyrimidin-4(3H)-one (8h). Yield: 68% (76 mg), yellow solid, mp
230–234 °C. HPLC rt = 9.798 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.55 (s, 3H, CH3), 7.17 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.35 (d, J = 15.6 Hz, 1H, COCH]), 7.83 (d, J = 8.4 Hz, 2H, Ph
3′-H, 5′-H), 8.06 (d, J = 8.4 Hz, 2H, Ph 2′-H, 6′-H), 8.28 (d,
J = 15.6 Hz, 1H, CH]), 12.78 (s, 1H, NH). 13C NMR (150 MHz,
DMSO‑d6) δ: 15.75, 119.66, 124.90, 128.15, 129.45, 130.70 (2C),
132.11 (2C), 135.09, 135.63, 139.55, 149.41, 157.32, 162.86, 187.67.
HRMS (ESI) m/z: calcd for C16H12BrN2O2S ([M+H]+): 374.9803,
376.9782; found: 374.9797, 376.9770.
(100%). 1H NMR (600 MHz, DMSO‑d6) δ: 2.55 (d, J = 1.2 Hz, 3H,
CH3), 7.17 (d, J = 1.2 Hz, 1H, thieno[2,3-d]pyrimidine 5-H), 7.35 (dd,
J = 4.8, 3.6 Hz, 1H, thiophene 4′-H), 7.35 (d, J = 15.6 Hz, 1H,
COCH]), 8.17 (dd, J = 4.8, 1.2 Hz, 1H, thiophene 3′-H), 8.22 (d,
J = 15.6 Hz, 1H, CH]), 8.28 (dd, J = 4.2, 1.2 Hz, 1H, thiophene 5′-H),
12.73 (s, 1H, NH). 13C NMR (150 MHz, DMSO‑d6) δ: 15.75, 119.64,
124.86, 129.26, 129.71, 134.09, 134.82, 136.96, 139.47, 144.47,
149.36, 157.33, 162.88, 180.70. HRMS (ESI) m/z: calcd for
C
14H11N2O2S2 ([M+H]+): 303.0262; found: 303.0253.
4.1.7.14. (E)-6-Methyl-2-(3-oxo-3-(pyridin-2-yl)prop-1-en-1-yl)thieno
[2,3-d]pyrimidin-4(3H)-one (8n). Yield: 67% (60 mg), yellow solid, mp
206–209 °C. HPLC rt = 1.715 min (100%). 1H NMR (600 MHz,
DMSO‑d6/CF3COOD = 2:1) δ: 2.55 (s, 3H, CH3), 7.16 (s, 1H, thieno
[2,3-d]pyrimidine 5-H), 7.52 (d, J = 15.6 Hz, 1H, COCH]), 7.75 (t,
J = 4.8 Hz, 1H, pyridine 5′-H), 8.09 (t, J = 7.2 Hz, 1H, pyridine 4′-H),
8.13 (d, J = 7.2 Hz, 1H, pyridine 3′-H), 8.75 (d, J = 15.6 Hz, 1H,
CH]), 8.84 (d, J = 3.0 Hz, 1H, pyridine 6′-H), 12.84 (br s, 1H, NH).
13C NMR (150 MHz, DMSO‑d6/CF3COOD) δ: 16.07, 113.78, 116.75,
119.22, 120.53, 127.86, 131.92, 142.50, 147.43, 148.04, 151.58,
157.12, 158.31 (overlapped with CF3COOD), 161.51, 181.95. HRMS
(ESI) m/z: calcd for C15H12N3O2S ([M+H]+): 298.0650; found:
298.0642.
4.1.7.9. (E)-2-(3-(4-Chlorophenyl)-3-oxoprop-1-en-1-yl)-6-methylthieno
[2,3-d]pyrimidin-4(3H)-one (8i). Yield: 39% (39 mg), yellow solid, mp
236–240 °C. HPLC rt = 5.528 min (100%). 1H NMR (600 MHz,
CF3COOD) δ: 2.55 (s 3H, CH3), 7.17 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.35 (d, J = 15.6 Hz, 1H, COCH]), 7.68 (d, J = 9.0 Hz, 2H, Ph
3′-H, 5′-H), 8.15 (d, J = 9.0 Hz, 2H, Ph 2′-H, 6′-H), 8.30 (d,
J = 15.6 Hz, 1H, CH]), 12.78 (s, 1H, NH). 13C NMR (150 MHz,
CF3COOD) δ: 13.88, 119.21, 125.03, 128.78, 129.46 (2C), 130.60
(2C), 133.15, 134.59, 143.61, 146.53, 149.35, 158.32, 162.10
(overlapped with CF3COOD), 189.35. HRMS (ESI) m/z: calcd for
C
16H12ClN2O2S ([M+H]+): 331.0308, 333.0279; found: 331.0301,
333.0268.
4.1.7.15. (E)-6-Methyl-2-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)thieno
[2,3-d]pyrimidin-4(3H)-one (8o). Yield: 59% (53 mg), yellow solid, mp
239–241 °C. HPLC rt = 1.639 min (95%). 1H NMR (600 MHz,
CF3COOD) δ: 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.37 (d, J = 15.6 Hz, 1H, COCH]), 7.64
(ddd, J = 7.8, 4.8, 0.6 Hz, 1H, pyridine 5′-H), 8.30 (d, J = 15.6 Hz, 1H,
CH]), 8.45 (dt, J = 7.8, 1.8 Hz, 1H, pyridine 4′-H), 8.86 (dd, J = 4.8,
1.8 Hz, 1H, pyridine 6′-H), 9.28 (d, J = 1.8 Hz, 1H, pyridine 2′-H),
12.78 (br s, 1H, NH). 13C NMR (150 MHz, CF3COOD) δ: 14.26, 118.87,
124.91, 127.66, 127.82, 135.46, 136.75, 141.89, 142.28, 144.60,
145.23, 148.21, 158.78, 163.93, 184.47. HRMS (ESI) m/z: calcd for
4.1.7.10. (E)-2-(3-(4-Fluorophenyl)-3-oxoprop-1-en-1-yl)-6-methylthieno
[2,3-d]pyrimidin-4(3H)-one (8j). Yield: 57% (54 mg), yellow solid, mp
243–246 °C. HPLC rt = 1.915 min (100%). 1H NMR (600 MHz,
CF3COOD) δ: 2.55 (d, J = 1.2 Hz, 3H, CH3), 7.17 (d, J = 1.2 Hz, 1H,
thieno[2,3-d]pyrimidine 5-H), 7.34 (d, J = 15.6 Hz, 1H, COCH]), 7.44
(t, J = 9.0 Hz, 2H, Ph 3′-H, 5′-H), 8.23 (dd, J = 9.0, 5.4 Hz, 2H, Ph 2′-H,
6′-H), 8.31 (d, J = 15.6 Hz, 1H, CH]), 12.77 (s, 1H, NH). 13C NMR
(150 MHz, CF3COOD) δ: 13.86, 116.35 (d, J = 22.65 Hz, 2C), 119.24,
125.07, 128.31 (d, J = 3.75 Hz), 131.30, 132.54 (d, J = 10.35 Hz, 2C),
135.01, 146.55, 149.46, 158.17, 162.08 (overlapped with CF3COOD),
167.98 (d, J = 259.8 Hz), 188.84. HRMS (ESI) m/z: calcd for
16H12FN2O2S ([M+H]+): 315.0604; found: 315.0594.
C
15H12N3O2S ([M+H]+): 298.0650; found: 298.0641.
C
4.1.7.11. (E)-6-Methyl-2-(3-(4-nitrophenyl)-3-oxoprop-1-en-1-yl)thieno
[2,3-d]pyrimidin-4(3H)-one (8 k). Yield: 41% (42 mg), yellow solid, mp
236–238 °C. HPLC rt = 1.778 min (100%). 1H NMR (600 MHz,
DMSO‑d6) δ: 2.50 (s, 3H, CH3), 7.04 (s, 1H, thieno[2,3-d]pyrimidine
5-H), 7.33 (d, J = 15.6 Hz, 1H, COCH]), 7.66 (d, J = 8.4 Hz, 2H, Ph
2′-H, 6′-H), 8.11 (d, J = 8.4 Hz, 2H, Ph 3′-H, 5′-H), 8.16 (d,
J = 15.6 Hz, 1H, CH]), 12.79 (s, 1H, NH). 13C NMR (150 MHz,
DMSO‑d6) δ: 15.50, 119.0, 123.51, 123.83 (2C), 124.03, 129.51 (2C),
130.11, 136.91, 141.29, 149.94, 157.49, 158.02, 162.90, 196.91.
HRMS (ESI) m/z: calcd for C16H12N3O4S ([M+H]+): 342.0549;
found: 342.0540.
4.1.7.16. (E)-6-Methyl-2-(3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl)thieno
[2,3-d]pyrimidin-4(3H)-one (8p). Yield: 66% (59 mg), yellow solid, mp
238–239 °C. HPLC rt = 2.515 min (95%). 1H NMR (600 MHz,
DMSO‑d6/CF3COOD = 2:1) δ: 2.55 (s, 3H, CH3), 7.17 (d, J = 1.2 Hz,
1H, thieno[2,3-d]pyrimidine 5-H), 7.38 (d, J = 15.6 Hz, 1H, COCH]),
7.96 (dd, J = 4.8, 1.8 Hz, 2H, pyridine 3′-H, 5′-H), 8.22 (d, J = 15.6 Hz,
1H, CH]), 8.88 (dd, J = 4.8, 1.8 Hz, 2H, pyridine 2′-H, 6′-H), 12.79 (s,
1H, NH). 13C NMR (150 MHz, DMSO‑d6/CF3COOD) δ: 15.95, 120.21,
125.61 (2C), 125.86, 129.07 (2C), 137.67, 140.60, 144.94, 149.41,
149.66, 157.79, 163.39, 187.38. HRMS (ESI) m/z: calcd for
C
15H12N3O2S ([M+H]+): 298.0650; found: 298.0642.
4.1.7.12. (E)-2-(3-(Furan-2-yl)-3-oxoprop-1-en-1-yl)-6-methylthieno[2,3-
d]pyrimidin-4(3H)-one (8l). Yield: 60% (51 mg), yellow solid, mp
255–259 °C (eluent: DCM/methanol = 98:2). HPLC rt = 10.0 min
(100%). 1H NMR (600 MHz, DMSO‑d6) δ: 2.55 (d, J = 1.2 Hz, 3H,
CH3), 6.84 (dd, J = 3.6, 1.8 Hz, 1H, furan 4′-H), 7.17 (d, J = 1.2 Hz,
1H, thieno[2,3-d]pyrimidine 5-H), 7.39 (d, J = 15.6 Hz, 1H, COCH]),
7.83 (d, J = 3.6 Hz, 1H, furan 3′-H), 8.08 (d, J = 15.6 Hz, 1H, CH]),
8.15 (d, J = 1.8 Hz, 1H, furan 5′-H), 12.74 (s, 1H, NH). 13C NMR
(150 MHz, DMSO‑d6) δ: 15.73, 113.20, 119.61, 120.97, 124.82, 129.75,
133.73, 139.37, 149.39, 152.43, 157.30, 162.27, 162.89, 175.47.
4.2. Biological assays
4.2.1. Cell lines and culture conditions
All the cell lines were purchased from the American Type Culture
Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium
(DMEM; Hyclone) supplemented with 10% fetal bovine serum (FBS;
PAN) and standard antibiotics (Hyclone), at 37 °C in a 5% CO2 in-
cubator.
HRMS (ESI) m/z: calcd for
C
14H11N2O3S ([M+H]+): 287.0490;
4.2.2. Cell proliferation assay
found: 287.0481.
A cell proliferation assay was performed as previously described
[29]. Cell viability was tested using a CellTiter 96® AQueous One So-
lution Cell Proliferation Assay kit (Promega), based on the use of MTS.
The data were obtained from triplicate wells in three independent ex-
periments.
4.1.7.13. (E)-6-Methyl-2-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)thieno
[2,3-d]pyrimidin-4(3H)-one (8m). Yield: 66% (60 mg), yellow solid, mp
201–204 °C (from DMF/H2O = 3:1, v/v). HPLC rt = 15.923 min
12

F.-C. Wang, et al.
BioorganicChemistryxxx(xxxx)xxxx
4.2.3. Determination of cell-cycle distribution in HCT-116 cells
[18]. Antibodies against PARP-1 (A301-376A) and β-actin (A5441)
were purchased from Bethyl Laboratories and Sigma, respectively.
Antibodies against caspase 3 (9665), caspase 7 (12827) and caspase 9
(9508), cleaved caspase 3 (9664), cleaved caspase 7 (8438) and cleaved
caspase 9 (7237) were purchased from Cell Signaling Technology.
Briefly, protein samples were separated on a 10% SDS-polyacrylamide
gel and transferred onto a polyvinylidene difluoride membrane. After
blocking, the membrane was incubated in 0.05% PBST containing 5%
bovine serum albumin for 30 min at room temperature and incubated
with the primary antibody (1:2000) overnight at 4 °C. The membrane
was then washed with PBST, incubated with HRP-conjugated Goat Anti-
Mouse IgG (1:1000, 115-035-116) or Donkey Anti-Rabbit (1:1000, 711-
035-152) secondary antibodies (Jackson ImmunoResearch) at room
temperature for 1 h. The immunoblots were visualized by ImageQuant™
LAS 4000 (GE Healthcare Life Sciences).
Cell-cycle profiles were determined by propidium iodide (PI)
staining and fluorescence-activated cell sorting as previously described
[18]. HCT-116 cells were incubated with compound 8d (1×, 1.5 × or
2 × IC50), 5-FU (IC50) or Taxol (0.1 μM) for 24 h. The cells were then
harvested by trypsinization, washed twice with PBS, fixed in 75% ice-
cold ethanol and then stained with PI at 37 °C for 30 min. The stained
cells were analyzed for DNA content using a Beckman Coulter Cyto-
FLEX flow cytometer (Beckman Coulter Diagnostics, USA). The cell-
cycle distribution was analyzed using FlowJo software.
4.2.4. Annexin V-FITC/PI dual staining assay
An Annexin V-FITC/PI dual staining assay was used to determine
the percentage of apoptotic cells as described previously [30]. HCT-116
cells (2 × 106/mL per well) were plated in 6-well culture plates and
allowed to grow for 24 h. The cells were exposed to compound 8d (1×
or 2 × IC50) for 24 h, or STS (1 μM) for 2 h, and harvested with trypsin.
The cells were washed twice with ice-cold PBS and incubated in the
dark at room temperature in 100 mL 1 × binding buffer containing
1 mL Annexin VFITC and 12.5 mL PI. After 15 min incubation, the cells
4.2.9. In vitro PARylation assay
In vitro PARylation assay was performed as previously described
[31]. FLAG tagged PARP-1 was transfected into 293 T cells for 48 h,
then harvested and lysed in NETN buffer (20 mM Tris·HCl pH 8.0,
150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 1% protease inhibitor cock-
tail) before immunoprecipitation with an anti-FLAG antibody. After
overnight incubation and extended washing with NETN buffer, FLAG-
PARP-1 was subjected to in vitro PARylation at 37 °C for 60 min in a
reaction buffer (50 mM Tris·HCl pH 8.0, 25 mM MgCl2, 50 mM NaCl)
supplemented with 200 μM NAD+. The reaction was terminated by
6 × SDS-PAGE sample buffer and then analyzed by immunoblotting.
were analyzed for the percentage undergoing apoptosis using
a
Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter Diag-
nostics, USA).
4.2.5. Mitochondrial membrane potential monitored by JC-1 staining
A mitochondrial membrane potential assay was performed as pre-
viously described [18]. HCT-116 cells (107/mL per well) were seeded in
a 6-well plate and treated with compound 8d (1× or 2 × IC50) for 24 h,
or STS (1 μM) for 2 h. The cells were then washed with PBS and added
to fresh medium (1 mL) containing the JC-1 (5,5′,6,6′-tetrachloro-
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine
iodide,
Beyotime,
C2006) molecular probe (1 mL) and incubated at 37 °C for 20 min. The
cells were then washed twice with PBS and JC-1 aggregates (red
fluorescence) and JC-1 monomers (green fluorescence) were visualized
under a Nikon fluorescence microscope (TE2000-U, Nikon Inc., Japan)
at 40 × magnification, and an excitation wavelength of 488 nm and an
emission wavelength of 550 nm. The fluorescence intensity was de-
termined on a Beckman Coulter CytoFLEX flow cytometer (Beckman
Coulter Diagnostics, USA).
This work was supported by the 973 Project (Grant No.
2013CB911002), National Natural Science Foundation of China (Grant
No. 31761133012 and 31530016), and the Shenzhen Science and
Technology
Innovation
Commission
(Grant
No.
JCYJ20170412113009742 and JCYJ20180307182213033). The au-
thors would like to thank Dr. Jessica Tamanini (Shenzhen University
Health Science Centre) for proof-reading the manuscript prior to sub-
mission.
4.2.6. Reactive oxygen species (ROS) detection
Shortly before performing the experiments, H2DCFDA was recon-
stituted to make a concentrated stock solution and then kept tightly
sealed until further use. HCT-116 cells (3 × 105/mL per well) were
cultured in a 6-well plate and treated with compound 8d (1 × or
2 × IC50) for 24 h, or 1 mM H2O2 for 20 min. Cells were removed from
the culture medium by centrifugation, then washed and resuspended in
pre-warmed PBS containing H2DCFDA. After incubation at 37 °C for
15 min, the cells were determined for fluorescence intensity using a
Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter
Diagnostics, USA).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103346.
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