Synthesis and evaluation of chalcone analogues containing a 4-oxoquinazolin-2-yl group as potential anti-tumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.11.034

The chalcone motif can be found in many molecules that contribute to essential biological processes, and many chalcone-containing compounds exhibit potent anti-cancer activity. Here, we synthesized two series of chalcone analogues (3a?s and 6a?s) based on substituting the chalcone B-ring or A-ring with a 4-oxoquinazolin-2-yl group, and then evaluated them for cytotoxic activity in human colorectal HCT-116 and breast cancer MCF-7 cell lines. Compounds 3a?s (in which a 4-oxoquinazolin-2-yl group functioned as the B-ring) were markedly more cytotoxic than compounds 6a?s (in which 4-oxoquinazolin-2-yl group functioned as the A-ring), based on their IC₅₀ values to inhibit proliferation. Compound 3f was found as the most potent among 38 analogues and the mechanism of its cytotoxicity was investigated. Flow cytometry indicated that HCT-116 cells treated with compound 3f resulted in a dose-dependent accumulation of cells in the sub-G1 phase, which is representative of apoptotic cells. Subsequent assays (including Annexin V-FITC/PI, AO-EB, MitoSOX Red and JC-1 staining) confirmed that 3f exposure induced apoptosis in HCT-116 cells. Immunoblotting analysis indicated that cellular exposure to 3f increased the cleavage of PARP1 and caspases 3, 7, and 9. Taken together, this novel chalcone analogue has a cytotoxic effect on cultured cancer cell-lines that is likely mediated by inducing apoptosis via the mitochondrial death pathway.

Full text of DOI:10.1016/j.ejmech.2018.11.034

Accepted Manuscript
Synthesis and evaluation of chalcone analogues containing a 4-oxoquinazolin-2-yl
group as potential anti-tumor agents
Xue Han, Bin Peng, Bei-Bei Xiao, Sheng-Li Cao, Chao-Rui Yang, Wen-Zhu Wang,
Fu-Cheng Wang, Hong-Yun Li, Xiao-Li Yuan, Ruifeng Shi, Ji Liao, Hailong Wang,
Jing Li, Xingzhi Xu
PII:
S0223-5234(18)30993-0
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.034
EJMECH 10891
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 September 2018
Revised Date: 11 November 2018
Accepted Date: 14 November 2018
Please cite this article as: X. Han, B. Peng, B.-B. Xiao, S.-L. Cao, C.-R. Yang, W.-Z. Wang, F.-C.
Wang, H.-Y. Li, X.-L. Yuan, R. Shi, J. Liao, H. Wang, J. Li, X. Xu, Synthesis and evaluation of chalcone
analogues containing a 4-oxoquinazolin-2-yl group as potential anti-tumor agents, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.034.
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ACCEPTED MANUSCRIPT
Synthesis
and
evaluation
of
chalcone
analogues
containing
a
4-oxoquinazolin-2-yl group as potential anti-tumor agents
Xue Hana,1, Bin Pengb,1, Bei-Bei Xiaoa,1, Sheng-Li Caoa,*, Chao-Rui Yanga, Wen-Zhu Wanga, Fu-Cheng Wanga, Hong-Yun Lia,
Xiao-Li Yuana, Ruifeng Shib, Ji Liaoc, Hailong Wangc, Jing Lic, Xingzhi Xub,**
aDepartment of Chemistry and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048,
PR China
bShenzhen University School of Medicine and Guangdong Key Laboratory for Genome Stability & Disease Prevention, Shenzhen,
Guangdong 518060, PR China
cCollege of Life Science and Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048, PR
China

ACCEPTED MANUSCRIPT
Synthesis
and
evaluation
of
chalcone
analogues
containing
a
4-oxoquinazolin-2-yl group as potential anti-tumor agents
Xue Hana,1, Bin Pengb,1, Bei-Bei Xiaoa,1, Sheng-Li Caoa,*, Chao-Rui Yanga, Wen-Zhu Wanga, Fu-Cheng
Wanga, Hong-Yun Lia, Xiao-Li Yuana, Ruifeng Shib, Ji Liaoc, 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
bShenzhen University School of Medicine and Guangdong Key Laboratory for Genome Stability &
Disease Prevention, Shenzhen, Guangdong 518060, PR China
cCollege of Life Science and Beijing Key Laboratory of DNA Damage Response, Capital Normal
University, Beijing 100048, PR China
Abbreviations: AO−EB, acridine orange−ethidium bromide; 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; PARP1, poly(ADP-ribose) polymerase
1; PI, propidium iodide; PS, phosphatidylserine; STS, staurosporine; ∆ψm, mitochondrial
transmembrane potential.
* Corresponding author.
** Corresponding author
E-mail address: slcao@cnu.edu.cn (S.-L. Cao); xingzhi.xu@szu.edu.cn (X. Xu).
1 These authors contributed equally to this work.
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ABSTRACT:
The chalcone motif can be found in many molecules that contribute to essential biological
processes, and many chalcone-containing compounds exhibit potent anti-cancer activity. Here, we
synthesized two series of chalcone analogues (3a−s and 6a−s) based on substituting the chalcone
B-ring or A-ring with a 4-oxoquinazolin-2-yl group, and then evaluated them for cytotoxic activity in
human colorectal HCT-116 and breast cancer MCF-7 cell lines. Compounds 3a−s (in which a
4-oxoquinazolin-2-yl group functioned as the B-ring) were markedly more cytotoxic than compounds
6a−s (in which 4-oxoquinazolin-2-yl group functioned as the A-ring), based on their IC50 values to
inhibit proliferation. Compound 3f was found as the most potent among 38 analogues and the
mechanism of its cytotoxicity was investigated. Flow cytometry indicated that HCT-116 cells treated
with compound 3f resulted in a dose-dependent accumulation of cells in the sub-G1 phase, which is
representative of apoptotic cells. Subsequent assays (including Annexin V-FITC/PI, AO-EB,
MitoSOXTM Red and JC-1 staining) confirmed that 3f exposure induced apoptosis in HCT-116 cells.
Immunoblotting analysis indicated that cellular exposure to 3f increased the cleavage of PARP1 and
caspases 3, 7, and 9. Taken together, this novel chalcone analogue has a cytotoxic effect on cultured
cancer cell-lines that is likely mediated by inducing apoptosis via the mitochondrial death pathway.
Keywords: Quinazolin-4(3H)-one; chalcone analogue; cytotoxicity; apoptosis; mitochondrial death
pathway
1. Introduction
Chalcones (1,3-diaryl-2-propen-1-ones) are natural aromatic ketones predominantly found in
plants and are essential precursors for flavonoid and isoflavonoid biosynthesis. Chalcones consist of
two aromatic rings (A and B rings) joined via a three-carbon (C) α,β-unsaturated carbonyl system (Fig.
1); the A-ring lies adjacent to the carbonyl group and the B-ring lies adjacent to the C=C double bond
[1-3]. Chalcones and their synthetic analogues have attracted much attention due to their anti-cancer,
anti-malarial, anti-microbial, anti-viral, and anti-inflammatory activities [3,4], and have guided the
design and discovery of many anti-cancer agents [5-7].
Recent attempts to develop anti-cancer chalcone analogues have been based on the addition of
various substituents to the A-ring and/or B-ring, or the replacement of these rings with heteroaryl
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groups [3-9]. Introducing an imidazolone group onto the A-ring produced a chalcone analogue (Fig. 1,
I; R = 3,4,5-triOCH3, R1 = Phenyl) with anti-proliferative activity towards cultured MCF-7, HT-29 and
HCT-116 cancer-cell lines (IC50 = 0.31, 0.66 and 0.33 µM, respectively) [8]. Exposure of MCF-7 cells
to a high concentration (10 µM) of this compound arrested the cell cycle in G2/M phase. Replacing the
B-ring with an indole ring also generated a chalcone analogue with potent anti-proliferative activity
against cultured cancer cell lines (Fig. 1, II; IC50 = 3−9 nM) [9]. Further investigation revealed that this
compound inhibited tubulin polymerization to arrest the cell cycle at G2/M phase, thus inducing
apoptosis together with a decrease in mitochondrial membrane potential.
Quinazoline is an important heterocycle, and compounds derived from its C5 or C6 position have
been the main source of antifolates used in cancer chemotherapy [10,11]. Raltitrexed, a
2-methylquinazolin-4(3H)-one analogue of folate, has been available in a number of countries for the
first-line treatment of colorectal cancer [12]. AG337 is a 2-aminoquinazolin-4(3H)-one derivative
bearing a lipophilic side chain at the C5 position, which had entered phase II/III trial in patients with
advanced hepatocellular carcinoma [13]. Derived from the C6 position of 2,4-diaminoquinazoline,
trimetrexate have been reached clinical trials for treatment of solid tumors such as colorectal cancer,
gastric cancer and osteosarcoma [14,15]. Many quinazolin-4(3H)-ones with substituents at the
C2-position also exhibit anti-cancer activity [16,17]. Ispinesib is a quinazoline-based drug that inhibits
the kinesin spindle protein and prevents spindle assembly and mitosis. This inhibitor reached phase II
clinical trials in patients with metastatic hepatocellular carcinoma, but despite being well-tolerated, no
conclusive benefit to ispinesib monotherapy was reported [18,19]. Another study synthesized 30
derivatives of 2-phenylquinazolin-4(3H)-one and upon testing for cytotoxic activity in human cancer
cell lines found that one (Fig. 1, III) could induce G0/G1 phase arrest in HeLa cells [17]. Recently, we
incorporated dithiocarbamate into the 2-position of quinazolin-4(3H)-one and found that one of the
derivatives (Fig. 1, IVc) inhibited HT29 cell proliferation and arrested the cell cycle at G2/M phase
[20]. Cellular exposure to this compound promoted tubulin polymerization and subsequent activation
of the spindle assembly checkpoint [20].
Apoptosis is a cell death process accompanied by the activation of caspase proteases [21]. The
mitochondrial (the intrinsic) pathway and the death receptor (DR) (the extrinsic) pathway are two
major mechanisms triggering apoptotic cell death. DR-induced apoptosis requires an interaction
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between a classical ligand (e.g., tumor necrosis factor (TNF)) and its cell surface receptor (e.g., TNF
receptor (TNFR)) and is essential for immune system function and homeostasis. On the other hand,
both excessive environmental stress (e.g., induction by genotoxic agents) and endogenous cellular
stress (e.g., induction by reactive oxygen species (ROS) and unfolded proteins) actively engage
apoptosis. The mitochondrial death pathway to apoptosis is a major channel of physiological cell death
and a promising therapeutic target for human diseases [22]. Chalcone analogues exert cytotoxic activity
through different molecular mechanisms, of which apoptosis induction is a major mechanism
underlying cytotoxicity in various cancer-cell lines [5-7]. Notably, quinazoline has been recognized as
a privileged structure for the development of anti-cancer agents [23]. In the present study, we therefore
substituted either the A-ring or B-ring of chalcone with a 4-oxoquinazolin-2-yl group to generate a new
set of chalcone analogues 3a−s and 6a−s (Schemes 1 and 2), and evaluated their cytotoxic activity in
cultured human cancer-cell lines.
< Figure 1 >
2. Results and discussion
2.1. Synthesis
Scheme 1 outlines the synthetic pathway to derive chalcone analogues (3a−s) in which the B-ring
was substituted with a 4-oxoquinazolin-2-yl group. In step 1, reacting methyl 2-aminobenzoate with
triethyl orthoacetate and ammonium acetate under reflux for 30 h gave 2-methylquinazolin-4(3H)-one
(intermediate 1). In step 2, this product was oxidized with selenium dioxide to yield the
2-formyl-quinazoline-4(3H)-one (intermediate 2), as previously described [24]. It is interesting that use
of 1,4-dioxane as a solvent instead of ethanol (as reported in the literature [24]) shortened the reaction
time from 12−15 h to 2 h, and attenuated the demethylation side reaction that otherwise occurs under
prolonged periods of heating. Finally, condensation of this intermediate with various acetophenones
under alkaline conditions generated 19 chalcone analogues (compounds 3a−s) with
quinazolin-4(3H)-one as the B-ring.
< Scheme 1 >
Scheme 2 outlines the synthetic pathway to derive chalcone analogues (6a-s) in which the A-ring
was substituted with quinazolin-4(3H)-one. In step 1, reaction of methyl 2-aminobenzoate with triethyl
orthopropionate and ammonium acetate generated 2-ethylquinazolin-4(3H)-one (intermediate 4). In
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step 2, this product was oxidized by selenium dioxide to generate the 2-acetylquinazolin-4(3H)-one
(intermediate 5). Finally, condensation of intermediate 5 with different aromatic aldehydes or
heteroaromatic aldehydes, under alkaline conditions yielded 19 chalcone analogues (6a−s) with
quinazolin-4(3H)-one as the A-ring.
< Scheme 2 >
The final reactions producing compounds 3a−s and 6a−s were monitored by thin-layer
chromatograph (TLC) or high performance liquid chromatography (HPLC); only one spot or peak
1
corresponding to the product was observed. Moreover, H NMR spectral analysis of 3a−s and 6a−s
indicated that each proton on carbon atoms of the C=C double bond existed as a single doublet, with a
coupling constant of 15.6 or 16.2 Hz. All synthetic compounds (3a−s and 6a−s) contained a C=C
double bond in the three-carbon chain that can exist as a Z-isomer and/or E-isomer. The chemical
analyses of 3a−s and 6a−s, however, suggest that they exist as geometrical E-isomers.
2.2. Cytotoxic activity
The cytotoxic activity of compounds 3a−s and 6a−s towards human colorectal HCT-116 and
breast cancer MCF-7 cells was evaluated by MTS cell proliferation assay. Inhibition of cell
proliferation was determined 72 h after exposure to 20 µM of each compound. The compounds that
induced ≥50% inhibition compared to vehicle were considered “active”. The effects of these active
compounds were further analyzed across a range of concentrations to determine the IC50 values for
50% inhibition of cellular proliferation (Tables 1, 2 and 3). 5-Fluorouracil (5-FU) was used as a
positive control.
Compounds 3a−s have a 4-oxoquinazolin-2-yl group to serve as a B-ring, and a phenyl or
substituted phenyl group as an A-ring. Sixteen (3a−h, 3j−q) of nineteen final compounds exhibited a
cytotoxic effect on cultured HCT-116 and MCF-7 cells (IC50 < 20 µM); seven compounds (3b, 3e, 3f,
3j, 3l, 3n and 3o) were the most potent (IC50 < 5 µM). Among seven active compounds six contained
electron-donating group(s) (methoxy, hydroxyl and methyl groups) on the benzene ring, and only one
compound (3o) had a weak electron-withdrawing group (R1 = 4'-Br) on the benzene ring. Furthermore,
compounds with a strong electron-withdrawing group, namely, 3r (R1 = 4'-F) or 3s (R1 = 4'-CF3), were
inactive (IC50 > 20 µM). These results suggest that electron-donating groups at the benzene ring in
chalcone analogues are more favourable for cytotoxic activity than electron-withdrawing groups. When
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comparing the cytotoxic activity of closely related compounds in HCT-116 and MCF-7 cells, we found
that 3b (R1 = 2'-OCH3) was more potent than 3c (R1 = 3'-OCH3) and 3d (R1 = 4'-OCH3); 3e (R1 =
2',4'-diOCH3), 3f (R1 = 2',5'-diOCH3) and 3g (R1 = 2',4',6'-triOCH3) were more potent than 3h (R1 =
3',4',5'-triOCH3). These results imply that the presence of a methoxy group at the 2-position of the
benzene ring is critical for cytotoxic activity. As an example, compound 3f (R1 = 2',5'-diOCH3) had
relatively low IC50 values of 3.56 and 4.08 µM in HCT-116 and MCF-7 cells, respectively. In pyrazolic
chalcone analogues synthesized by Hawash et al. [25], three 2,5-dimethoxyphenyl-containing
analogues were very active against cancer cells tested (Huh-7, MCF-7 and HCT-116), which is similar
to our results in this study.
< Table 1 >
We also synthesized chalcone analogues 6a−s, in which the 4-oxoquinazolin-2-yl group serves as
the A-ring, and a benzene ring or a heterocycle serves as the B-ring. Only six of the nineteen
compounds with a quinazolin-4(3H)-one ring as the A-ring (6c, 6g, and 6k−n) exhibited weak
cytotoxicity in HCT-116 cells, and two of these (6c, 6g) also exhibited weak cytotoxicity in MCF-7
cells (IC50, 13.20−19.77 µM). All these active compounds contained a substituted benzene ring as the
B-ring. Compounds containing a heterocycle (such as thiophene, furan or pyridine) as the B-ring were
inactive in the cancer-cell lines tested. The active compounds were more active in HCT-116 than
MCF-7 cells, as determined by MTS assay, but were all less potent than the 3a−s series in terms of
their IC50. It had been disclosed in a review article that heteroaryl moieties either as the A-ring or
B-ring appear to enhance the cytotoxic activity of chalcones against many types of cancer cells [6].
Nevertheless, our results clearly indicate that the 4-oxoquinazolin-2-yl group as a B-ring substitute is
critical for cytotoxic activity.
< Table 2 >
Furthermore, the cytotoxicity of selective potent compounds 3b, 3e, 3f, 3j, 3l, 3n and 3o (IC50 < 5
µM in both HCT-116 and MCF-7 cells) were tested in other human cancer cells including A549 (lung
cancer), HeLa (cervical carcinoma), HT-29 (colorectal cancer) and MD-MBA-231 (triple-negative
breast cancer) as well as normal cells, MCF-10A (human breast epithelial cells) (Table 3). These
compounds exhibited obvious cytotoxicity in the tested cancer cells with IC50 values ranging from 6.18
to 19.49 µM, with the exception of compound 3o in HT29 cells (IC50 >20 µM). All these compounds
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were cytotoxic to MCF-10A cells, while the IC50 of 3f for MCF-10A (9.81 µM) is more than doubled
of that for MCF-7. As in HCT-116 and MCF-7 cells, compound 3f (R1 = 2',5'-diCH3O) also had
relatively low IC50 values in A549, HeLa, HT29 and MD-MBA-231 cells. Quinazoline derivatives
modified at C6 position (e.g., raltitrexed, trimetrexate) are major chemotherapeutic agents for
colorectal cancer [12,15], while there are no quinazoline derivatives modified at C2 position in clinical
use. Furthermore, 3f exhibited decent cytotoxicity against the triple-negative breast cancer, which lacks
target therapy options [26]. Thus, it was taken forward for further investigation into the mechanisms
underlying cytotoxicity in cancer cells.
< Table 3 >
2.3. Cell cycle profiling of HCT-116 cells following compound 3f exposure
MTS assay indicated that compound 3f had the most cytotoxic effect on HCT-116 cells compared
to all other chalcone analogues (IC50 = 3.56 µM). We next used flow cytometry to analyze the effects
of HCT-116 cell exposure to compound 3f on the cell-cycle profile (Fig. 2). We treated HCT-116 cells
with various concentrations of compound 3f (0.5×, 1.0× and 2.0× IC50), 5-FU (IC50), or Taxol (0.2 µM)
for 24 h and then analyzed cellular DNA content (Fig. 2A, B). 5-FU is an inhibitor of thymidylate
synthase (TS), which blocks DNA synthesis and arrests the cell cycle in S phase. Taxol promotes
microtubule assembly and protects the microtubule polymer from disassembly, thus arresting the cell
cycle in G2/M phase. We observed a significant, dose-dependent increase in the proportion of sub-G1
HCT-116 cells in response to 3f exposure, and no marked effect on the composition of other cell-cycle
phases (Fig. 2). Based on these data, 3f may elicit the fragmentation or degradation of nuclear DNA to
form hypodiploid cells with < 2N DNA content, and induce cell apoptosis.
< Figure 2 >
2.4. Detection of apoptosis induced by 3f by Annexin V-FITC/PI dual staining assay
We next performed an Annexin V-FITC/propidium iodide (PI) dual staining assay to confirm
whether compound 3f can induce apoptosis. Phosphatidylserine (PS) locates on the cytosolic (inner)
side of the cell membrane, and translocates to the extracellular (outer) surface during early apoptosis.
Annexin V has a high affinity for PS, and thus when fluorescently labelled with fluorescein
isothiocyanate (FITC), can be used to identify early-stage apoptotic cells. Propidium iodide (PI) is a
fluorescent intercalating agent that cannot cross the membrane of live cells. The membranes of cells in
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the latter stages of apoptosis and dead cells are permeable to PI, and their nuclei stain red. We exposed
HCT-116 cells or MCF-10A cells to 3f (1× or 2× IC50) for 24 h or staurosporine (STS; 1 µM) for 12 h,
and then stained the cells with Annexin V-FITC and PI (Fig. 3). STS is an ATP-competitive kinase
inhibitor that induces apoptosis [27,28] and was used as a positive control. Annexin V-FITC-positive
HCT-116 cells or MCF-10A cells were detected following both 3f and STS treatment, in a
dose-dependent manner (Fig. 3). We found that the percentage of Annexin V-FITC-positive cells
induced by 3f at the dose of 2× IC50 in HCT-116 cells was equivalent to that induced by STS (Fig. 3A
and 3B), whereas the percentage of apoptotic cells induced by the same dose of 3f in MCF-10A cells
was only 54% of that induced by STS (Fig. 3C and 3D). These data demonstrate that 3f induces
apoptosis in both colorectal cancer cells HCT-116 and immortalized mammary epithelial cells
MCF-10A, indicating that 3f has a higher potency inducing apoptosis in cancer cells than immortalized
normal cells.
< Figure 3 >
2.5. Detection of apoptosis in HCT-116 cells induced by compound 3f, using AO–EB double staining
assay
We performed an additional assay, based on acridine orange-ethidium bromide (AO–EB) double
staining to confirm that 3f induces apoptosis in HCT-116 cells. AO is a nucleic acid fluorescent cationic
dye that stains live and dead cells green, and EB stains cells with lost membrane integrity orange [29].
HCT-116 cells were treated with compound 3f (1× or 2× IC50) for 24 h or STS for 12 h, stained with
AO–EB and then visualized by fluorescence microscopy (Fig. 4). Few cells treated with DMSO alone
(negative control) underwent apoptosis, whereas the majority of cells treated with STS (positive control)
became apoptotic. Cells treated with 3f also underwent apoptosis, and the percentage of orange
fluorescent-stained cells increased dose-dependently. These data support that 3f induces apoptosis in
HCT-116 cells.
< Figure 4 >
2.6. Mitochondrial involvement in 3f-induced apoptosis of HCT-116 cells, determined by MitoSOX™
Red assay and mitochondrial membrane potential detection
Mitochondria stress or damage initiates apoptosis by increasing the permeability of the outer
mitochondrial membrane. This effect results in the release of pro-apoptotic factors into the cytosol and
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an increase in cytosolic levels of reactive oxygen species (ROS), such as superoxide anions and
hydrogen peroxide and reactive nitrogen species (RNS) [30]. We investigated whether 3f-induced
apoptosis was due to increased cellular ROS levels by using the fluoroprobe MitoSOXTM Red, which
selectively detects superoxide in the mitochondria of live cells. MitoSOXTM Red localizes within
mitochondria, is oxidized specifically by superoxide, binds mitochondrial DNA and then emits a red
fluorescence [30,31]. We treated HCT-116 cells with compound 3f (1× or 2× IC50) for 24 h, or STS (1
µM) for 12 h, and then stained with MitoSOX™ Red. We found that a red fluorescence was emitted in
response to both STS and 3f exposure, which increased with the increasing dose of 3f (Fig. 5A).
The depolarization of mitochondrial transmembrane potential (∆ψm) could be induced by the
opening of mitochondrial permeability transition pore when apoptosis happens [32]. The
metachromatic fluorochrome JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine
iodide) has been widely used for the detection of mitochondrial transmembrane potential (∆ψm)
depolarization [33]. In unperturbed condition, JC-1 is more concentrated in the mitochondrial matrix
(driven there by the ∆ψm), where it forms red-emitting aggregates. Apoptosis induced the loss of red
fluorescence and the gain of green-emitting monomers in cytoplasmic due to the disruption of
mitochondrial transmembrane potential (∆ψm) [34]. We treated HCT-116 cells with compound 3f (2×
IC50) for 24 h, or STS (1 µM) for 12 h, and then stained with JC-1. The JC-1 staining demonstrated that
3f, in line with STS treatment, resulted in red fluorescence loss while green fluorescence accumulation,
indicating that they dissipated the mitochondrial transmembrane potential (∆ψm) (Fig. 5B). These data
demonstrate that compound 3f induces apoptosis via the mitochondrial death pathway, elevating
mitochondrial superoxide generation and depolarizing mitochondrial transmembrane potential (∆ψm).
< Figure 5 >
2.7. Effects of compound 3f on caspase cleavage in HCT-116 cells
Apoptosis is governed by a cascade of caspase protein cleavage, which amplifies apoptotic
signalling [35]. Apoptotic caspases are divided into two categories: initiator caspases (2, 8, 9 and 10)
and executioner caspases (caspase 3, 6 and 7). Initiator caspases are activated by dimerization,
followed by auto-proteolytic cleavage. Once activated, initiator caspases cleave and activate
executioner caspases, leading to degradation of cellular components for apoptosis [35,36].
Poly(ADP-ribose) polymerase 1 (PARP1) is a response factor involved in DNA repair. During the early
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stages of apoptosis, PARP1 is cleaved by executioner caspases. The cleaved PARP1 (89 kDa) is a
hallmark of early apoptosis [37].
We exposed HCT-116 cells to compound 3f (2× IC50) for 4, 8 and 12 h and then analyzed the
expression of apoptosis-related proteins by Western blotting. After 12 h incubation we observed
marked accumulation of cleaved PARP1, caspase 3, caspase 7 and caspase 9 (Fig. 6). Cleaved caspase
9 was detected after only 8 h exposure, which is consistent with its initiator-caspase function. Cleavage
of these proteins in response to 3f exposure support that compound 3f initiates and executes apoptosis,
by inducing the mitochondrial death pathway.
< Figure 6 >
3. Conclusions
Using a bioisosteric replacement approach, we designed and synthesized two series of chalcone
analogues, known as 3a−s and 6a−s, whereby a 4-oxoquinazolin-2-yl group replaced the B-ring or
A-ring, respectively. Compounds within the 3a−s series were more cytotoxic than 6a−s in HCT-116
and MCF-7 cells, and among the 3a−s series 3f exhibited significant cytotoxic effect with an IC50 of
3.56 and 4.08 µM in HCT-116 and MCF-7 cells, respectively. Moreover, 3f had the potential of killing
triple-negative breast cancer cells, and 3f was less cytotoxic towards normal MCF-10A cells. We thus
chose compound 3f for further analysis to determine the mechanisms underlying 3f-induced
cytotoxicity. HCT-116 cells treated with compound 3f accumulated in the sub-G1 phase of the cell
cycle, indicating that these cells underwent apoptosis. This effect occurred in a dose-dependent manner
with increasing doses of compound 3f, but little effect of 3f was found on the overall cell cycle profile.
We then confirmed that compound 3f induced apoptosis by Annexin V-FITC/PI dual staining, AO-EB
staining, MitoSOX™ Red, and JC-1 staining assays. Finally, Western blot analysis indicated that
treatment of HCT-116 cells with 3f caused an increase of cleaved PARP1 and cleaved caspases 3, 7,
and 9, suggesting that this compound might induce the mitochondrial death pathway. However, its
molecular mechanism how this compound targets and induces apoptosis warrant further investigation.
Nevertheless, these data open avenues for the development of new anti-cancer compounds based on
chalcone analogues with a 4-oxoquinazolin-2-yl group.
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4. Experimental procedures
4.1. Chemistry
The melting points of all compounds were determined using XT5B microscopic melting point
apparatus and are presented uncorrected. 1H and 13C NMR spectra were recorded on a Varian
VNMRS-600 spectrometer at 600 MHz or 150 MHz using tetramethylsilane (TMS) as an internal
standard (Abbreviations: Ph = phenyl, Quin = quinazolin-4(3H)-one). High-resolution mass spectra
(electrospray ionization) (HRMS (ESI)) were recorded on a Thermo Scientific LTQ Orbitrap
Discovery (Bremen, Germany) mass spectrometer. 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 with a G1314B VWD (variable-wavelength detector), and the
profiles are presented in the supplementary materials. The purity of target compounds used in the
biological studies was in general ≥ 95%. All the chemical reagents were commercially available and
used without further purification, unless noted.
4.1.1. Preparation of 2-methylquinazolin-4(3H)-one (intermediate 1)
A mixture of methyl 2-aminobenzoate (6.04 g, 40 mmol), ammonium acetate (7.7 g, 0.1 mol) and
triethyl orthoacetate (25 mL, 0.28 mol) was heated under reflux for 30 h. The solution cooled to room
temperature, and the separated solid was collected by filtration and air-dried. The crude product was
purified by re-crystallization from ethanol to give intermediate 1 as a white solid (4.86 g, 76%), mp
226−229 °C (Lit. [38] mp 230−232°C). 1H NMR (600 MHz, DMSO-d6)
δ: 2.35 (s, 3H, CH3), 7.44 (t, J
= 7.8 Hz, 1H, Quin 6-H), 7.56 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.76 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H),
8.07 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 12.18 (br s, 1H, NH). HRMS (ESI) m/z: calcd for C9H9N2O
([M+H]+): 161.0715; found: 161.0708.
4.1.2. Preparation of 2-formylquinazolin-4(3H)-one (intermediate 2)
A mixture of 2-methylquinazolin-4(3H)-one (intermediate 1) (0.48 g, 3 mmol) and selenium
dioxide (0.66 g, 6 mmol) in 1,4-dioxane (50 mL) was heated under reflux for 2 h. After cooling to room
temperature, the mixture was filtered to remove a small amount of precipitate, and the filtrate was
adjusted with 5% aqueous sodium bicarbonate to pH 7. The separated solid was removed by filtration,
and the filtrate was extracted four times with dichloromethane (15 mL, each). The combined extracts
were washed with brine, and then dried over anhydrous sodium sulfate overnight. The solvent was
11

ACCEPTED MANUSCRIPT
removed by rotary evaporation, and the residue was re-crystallized from methanol to produce
1
intermediate 2 as a light yellow solid (0.35 g, 67%), mp 135−138°C (Lit. [24] mp 138−140°C). H
NMR (600 MHz, DMSO-d6) δ: 7.69 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.89 (dd, J = 7.8, 1.2 Hz, 1H,
Quin 8-H), 7.94 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.21 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 9.58 (s,
1H, CHO), 12.66 (br s, 1H, NH). HRMS (ESI) m/z: calcd for C9H7N2O2 ([M+H]+): 175.0508; found:
175.0502.
4.1.3. General preparation procedure for compounds 3a−s
An aqueous solution of 30% potassium hydroxide (1 mL) was added to a stirred solution of the
appropriate acetophenone (1.1 mmol) in methanol (10 mL). After stirring at room temperature for 20
min, 2-formylquinazoline-4(3H)-one (intermediate 2; 0.17 g, 1 mmol) was added and stirring was
continued for 24 h. The solution was adjusted to pH 6−7 with 6 mol/L hydrochloric acid, and the
precipitate was collected by filtration and air-dried. The crude product was purified by
re-crystallization from methanol to give compounds 3a−s.
4.1.3.1. (E)-2-(3-Oxo-3-phenylprop-1-enyl)quinazolin-4(3H)-one (3a)
Yield: 41%, white solid, mp 231−233 °C. HPLC retention time (rt) = 4.873 min (100%). 1H NMR
(600 MHz, DMSO-d6) δ: 7.34 (d, J = 16.2 Hz, 1H, =CHCO), 7.58 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.64 (t,
J = 7.8 Hz, 2H, Ph 3'-H, 5'-H), 7.74 (t, J = 7.8 Hz, 1H, Ph 4'-H), 7.78 (d, J = 7.8 Hz, 1H, Quin 8-H),
7.87 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.15 (d, J = 7.8 Hz, 2H, Ph 2'-H, 6'-H), 8.17 (dd, J = 7.8, 1.2
Hz, 1H, Quin 5-H), 8.39 (d, J = 16.2 Hz, 1H, =CH), 12.65 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6)
δ: 122.19, 126.38, 128.04, 128.29, 129.17 (2C), 129.48 (2C), 130.70, 134.36, 135.16, 136.26, 137.09,
148.87, 150.35, 161.95, 189.10. HRMS (ESI) m/z: calcd for C17H13N2O2 ([M+H]+): 277.0977; found:
277.0972.
4.1.3.2. (E)-2-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3b)
1
Yield: 31%, yellow solid, mp 225−227 °C. HPLC rt = 5.370 min (97%). H NMR (600 MHz,
DMSO-d6) δ: 3.91 (s, 1H, OCH3), 7.10 (t, J = 7.8 Hz, 1H, Ph 5'-H), 7.20 (d, J = 15.6 Hz, 1H, =CHCO),
7.25 (d, J = 7.8 Hz, 1H, Ph 3'-H), 7.56 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.58 (d, J = 7.8 Hz, 1H, Ph 6'-H ),
7.62 (td, J = 7.8, 1.2 Hz, 1H, Ph 4'-H), 7.74 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.85 (t, J = 7.8 Hz, 1H, Quin
7-H), 7.98 (d, J = 15.6 Hz, 1H, =CH), 8.14 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.58 (s, 1H, NH). 13C NMR
(150 MHz, DMSO-d6) δ: 56.44, 112.96, 121.15, 122.14, 126.33, 127.81, 128.13, 128.22, 130.31,
12

ACCEPTED MANUSCRIPT
134.49, 134.50, 135.06, 135.25, 148.86, 150.36, 158.65, 161.92, 191.77. HRMS (ESI) m/z: calcd for
C18H15N2O3 ([M+H]+): 307.1083; found: 307.1080.
4.1.3.3. (E)-2-(3-(3-Methoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3c)
1
Yield: 30%, white solid, mp 236−238 °C. HPLC rt = 6.512 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 3.87 (s, 3H, OCH3), 7.32 (d, J = 7.8 Hz, 1H, Ph 4'-H), 7.33 (d, J = 15.6 Hz, 1H, =CHCO),
7.56 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.58 (t, J = 7.8 Hz, 1H, Ph 5'-H), 7.61 (s, 1H, Ph 2'-H), 7.76 (d, J =
7.8 Hz, 1H, Quin 8-H), 7.78 (d, J = 7.8 Hz, 1H, Ph 6'-H), 7.87 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.17 (d, J
= 7.8 Hz, 1H, Quin 5-H), 8.36 (d, J = 15.6 Hz, 1H, =CH), 12.63 (s, 1H, NH). 13C NMR (150 MHz,
DMSO-d6) δ: 55.90, 113.66, 117.78, 120.34, 121.75, 122.18, 126.38, 128.07, 128.30, 130.66, 135.15,
136.38, 138.47, 148.86, 150.35, 160.11, 161.94, 188.81. HRMS (ESI) m/z: calcd for C18H15N2O3
([M+H]+): 307.1083; found: 307.1076.
4.1.3.4. (E)-2-(3-(4-Methoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3d)
1
Yield: 35%, yellow solid, mp 239−241 °C. HPLC rt = 6.361 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 3.90 (s, 3H, OCH3), 7.14 (d, J = 9.0 Hz, 2H, Ph 3'-H, 5'-H), 7.30 (d, J = 15.6 Hz, 1H,
=CHCO), 7.58 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.87 (td, J = 7.8, 1.2
Hz, 1H, Quin 7-H), 8.15 (d, J = 9.0 Hz, 2H, Ph 2'-H, 6'-H), 8.17 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H),
8.39 (d, J = 15.6 Hz, 1H, =CH), 12.62 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 55.70, 114.34
(2C), 121.70, 125.92, 127.52, 127.80, 129.62, 130.36, 131.22 (2C), 134.69, 135.10, 148.45, 150.06,
161.51, 163.84, 186.64. HRMS (ESI) m/z: calcd for C18H15N2O3 ([M+H]+): 307.1083; found: 307.1074.
4.1.3.5. (E)-2-(3-(2,4-Dimethoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3e)
1
Yield: 18%, yellow solid, mp 185−187 °C. HPLC rt = 5.082 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 3.88 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 6.69 (dd, J = 8.4, 2.4 Hz, 1H, Ph 5'-H), 6.73 (d,
J = 2.4 Hz, 1H, Ph 3'-H), 7.22 (d, J = 15.6 Hz, 1H, =CHCO), 7.56 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.69
(d, J = 8.4 Hz, 1H, Ph 6'-H), 7.74 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.85 (td, J = 7.8, 1.2 Hz, 1H, Quin
7-H), 8.14 (d, J = 15.6 Hz, 1H, =CH), 8.14 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 12.58 (s, 1H, NH). 13C
NMR (150 MHz, DMSO-d6) δ: 56.16, 56.57, 99.08, 106.84, 120.82, 122.10, 126.31, 127.65, 128.19,
132.88, 133.28, 135.02, 135.82, 148.95, 150.58, 161.29, 161.98, 165.20, 188.73. HRMS (ESI) m/z:
calcd for C19H17N2O4 ([M+H]+): 337.1188; found: 337.1184.
13

ACCEPTED MANUSCRIPT
4.1.3.6. (E)-2-(3-(2,5-Dimethoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3f)
1
Yield: 41%, yellow solid, mp 185−187 °C. HPLC rt = 5.247 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 3.77 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.11 (m, 1H, Ph 6'-H), 7.19 (m, 2H, Ph 3'-H,
4'-H), 7.21 (d, J = 15.6 Hz, 1H, =CHCO), 7.56 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.74 (d, J = 7.8 Hz,
1H, Quin 8-H), 7.85 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 7.99 (d, J = 15.6 Hz, 1H, =CH), 8.14 (dd, J =
7.8, 1.2 Hz, 1H, Quin 5-H), 12.58 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 56.05, 56.94, 114.41,
114.55, 120.21, 122.15, 126.33, 127.82, 128.23, 128.48, 134.59, 135.07, 135.17, 148.86, 150.34,
152.92, 153.53, 161.91, 191.37. HRMS (ESI) m/z: calcd for C19H17N2O4 ([M+H]+): 337.1188; found:
337.1184.
4.1.3.7. (E)-2-(3-Oxo-3-(2,4,6-trimethoxyphenyl)prop-1-enyl)quinazolin-4(3H)-one (3g)
1
Yield: 50%, yellow solid, mp 212−214 °C. HPLC rt = 3.779 min (99%). H NMR (600 MHz,
DMSO-d6) δ: 3.76 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 6.35 (s, 2H, Ph 3'-H, 5'-H), 6.99 (d, J = 16.2
Hz, 1H, =CHCO), 7.48 (d, J = 16.2 Hz, 1H, =CH), 7.55 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.71 (d, J
= 7.8 Hz, 1H, Quin 8-H), 7.84 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.12 (dd, J = 7.8, 1.2 Hz, 1H, Quin
5-H), 12.49 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 56.02, 56.39 (2C), 91.55 (2C), 110.60,
122.09, 126.32, 127.84, 128.17, 135.06, 135.52, 136.74, 148.80, 150.33, 158.91 (2C), 161.83, 163.09,
192.80. HRMS (ESI) m/z: calcd for C20H19N2O5 ([M+H]+): 367.1294; found: 367.1292.
4.1.3.8. (E)-2-(3-Oxo-3-(3,4,5-trimethoxyphenyl)prop-1-enyl)quinazolin-4(3H)-one (3h)
1
Yield: 53%, orange solid, mp 223−225 °C. HPLC rt = 6.052 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 3.80 (s, 3H, OCH3), 3.92 (s, 6H, 2OCH3), 7.33 (d, J = 15.6 Hz, 1H, =CHCO), 7.44 (s, 2H,
Ph 2'-H, 6'-H), 7.59 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.78 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.88 (td, J
= 7.8, 1.2 Hz, 1H, Quin 7-H), 8.17 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 8.35 (d, J = 15.6 Hz, 1H, =CH),
12.60 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 56.28 (2C), 60.27, 106.53 (2C), 121.66, 125.96,
127.62, 127.73, 130.08, 131.91, 134.71, 135.79, 142.73, 148.26, 149.99, 153.01 (2C), 161.54, 187.28.
HRMS (ESI) m/z: calcd for C20H19N2O5 ([M+H]+): 367.1294; found: 367.1290.
4.1.3.9. (E)-2-(3-(4-Aminophenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3i)
Yield: 14%, orange red solid, mp 253−255 °C. HPLC rt = 2.418 min (95%). 1H NMR (600 MHz,
DMSO-d6) δ: 6.37 (s, 2H, NH2), 6.66 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.24 (d, J = 15.6 Hz, 1H,
=CHCO), 7.57 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.76 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.86 (t, J = 7.8 Hz,
14

ACCEPTED MANUSCRIPT
1H, Quin 7-H), 7.91 (d, J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 8.16 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.36 (d, J =
15.6 Hz, 1H, =CH), 12.58 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 113.32 (2C), 122.07, 124.96,
126.35, 127.76, 128.17, 131.45, 131.95 (2C), 134.00, 135.09, 149.01, 150.80, 155.08, 162.03, 185.07.
HRMS (ESI) m/z: calcd for C17H14N3O2 ([M+H]+): 292.1086; found: 292.1083.
4.1.3.10. (E)-2-(3-(4-Hydroxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3j)
1
Yield: 31%, yellow solid, mp 280−282 °C. HPLC rt = 3.427 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 6.95 (d, J = 7.8 Hz, 2H, Ph 3'-H, 5'-H), 7.29 (d, J = 15.6 Hz, 1H, =CHCO), 7.58 (t, J =
7.2 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.2 Hz, 1H, Quin 8-H), 7.87 (t, J = 7.2 Hz, 1H, Quin 7-H), 8.06 (d,
J = 7.8 Hz, 2H, Ph 2'-H, 6'-H), 8.16 (d, J = 7.2 Hz, 1H, Quin 5-H), 8.38 (d, J = 15.6 Hz, 1H, =CH),
10.61 (s, 1H, OH), 12.61 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6, CDCl3) δ: 116.09 (2C), 122.09,
126.33, 127.83, 128.15, 128.75, 130.95, 131.89 (2C), 135.04, 135.13, 148.89, 150.53, 162.03, 163.36,
186.76. HRMS (ESI) m/z: calcd for C17H13N2O3 ([M+H]+): 293.0926; found: 293.0922.
4.1.3.11. (E)-2-(3-(4-Hydroxy-3-methoxyphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3k)
1
Yield: 47%, yellow solid, mp 244−246 °C. HPLC rt = 1.257 min (100%). H NMR (600 MHz,
DMSO-d6) δ: 3.90 (s, 3H, OCH3), 6.98 (d, J = 8.4 Hz, 1H, Ph 5'-H), 7.30 (d, J = 15.6 Hz, 1H, =CHCO),
7.57 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.61 (d, J = 1.8 Hz, 1H, Ph 2'-H), 7.76 (d, J = 7.8, 1H, Quin 8-H),
7.79 (dd, J = 8.4, 1.8 Hz, 1H, Ph 6'-H), 7.86 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.16 (dd, J = 7.8, 1.2
Hz, 1H, Quin 5-H), 8.38 (d, J = 15.6 Hz, 1H, =CH), 10.30 (s, 1H, OH), 12.60 (s, 1H, NH). 13C NMR
(150 MHz, DMSO-d6) δ: 55.72, 111.54, 115.15, 121.68, 124.40, 125.93, 127.47, 127.79, 128.65,
130.36, 134.67, 134.76, 148.05, 148.49, 150.12, 152.80, 161.55, 186.23. HRMS (ESI) m/z: calcd for
C18H15N2O4 ([M+H]+): 323.1032; found: 323.1031.
4.1.3.12. (E)-2-(3-(2-Methylphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3l)
Yield: 48%, light yellow solid, mp 198−200 °C. HPLC rt = 7.872 min (96%). 1H NMR (600 MHz,
DMSO-d6) δ: 2.44 (s, 3H, CH3), 7.16 (d, J = 15.6 Hz, 1H, =CHCO), 7.39 (d, J = 7.8 Hz, 1H, Ph 3'-H),
7.41 (t, J = 7.8 Hz, 1H, 5'-H), 7.52 (t, J = 7.8 Hz, 1H, Ph 4'-H), 7.57 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.75
(d, J = 7.8 Hz, 1H, Quin 8-H), 7.78 (d, J = 7.8 Hz, 1H, Ph 6'-H), 7.86 (t, J = 7.8 Hz, 1H, Quin 7-H),
7.95 (d, J = 15.6 Hz, 1H, =CH), 8.15 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.59 (s, 1H, NH). 13C NMR (150
MHz, DMSO-d6) δ: 20.77, 122.14, 126.37, 127.99, 128.25, 129.56, 132.04, 132.13, 132.22, 134.12,
135.14, 136.69, 137.64, 137.90, 148.81, 150.25, 161.88, 193.93. HRMS (ESI) m/z: calcd for
15

ACCEPTED MANUSCRIPT
C18H15N2O2 ([M+H]+): 291.1134; found: 291.1128.
4.1.3.13. (E)-2-(3-(4-Methylphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3m)
1
Yield: 62%, white solid, mp 246−248 °C. HPLC rt = 9.081 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 2.43 (s, 3H, CH3), 7.32 (d, J = 15.6 Hz, 1H, =CHCO), 7.44 (d, J = 7.8 Hz, 2H, Ph 3'-H,
5'-H), 7.58 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.87 (td, J = 7.8, 1.2 Hz,
1H, Quin 7-H), 8.06 (d, J = 7.8 Hz, 2H, Ph 2'-H, 6'-H), 8.16 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.39 (d, J =
15.6 Hz, 1H, =CH), 12.64 (s, 1H, NH). 13C NMR (150 MHz, CDCl3, CF3CO2D) δ: 21.82, 119.12,
121.69, 126.60, 127.98, 129.67 (2C), 130.13 (2C), 130.40, 131.22, 132.74, 138.18, 138.40, 147.66,
152.33, 161.13, 187.55. HRMS (ESI) m/z: calcd for C18H15N2O2 ([M+H]+): 291.1134; found: 291.1129.
4.1.3.14. (E)-2-(3-(2,4-Dimethylphenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3n)
1
Yield: 45%, light yellow solid, mp 200−202 °C. HPLC rt = 18.080 min (95%). H NMR (600
MHz, DMSO-d6) δ: 2.37 (s, 3H, CH3), 2.44 (s, 3H, CH3), 7.17 (d, J = 15.6 Hz, 1H, =CHCO), 7.20 (s,
1H, Ph 3'-H), 7.22 (d, J = 7.8 Hz, 1H, Ph 5'-H), 7.57 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.75 (d, J =
7.8 Hz, 1H, Quin 8-H), 7.76 (d, J = 7.8 Hz, 1H, Ph 6'-H), 7.86 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.01
(d, J = 15.6 Hz, 1H, =CH), 8.15 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 12.59 (s, 1H, NH). 13C NMR (150
MHz, DMSO-d6) δ: 21.11, 21.43, 122.13, 126.35, 126.91, 127.92, 128.23, 130.26, 132.90, 134.00,
134.68, 135.10, 136.10, 138.60, 142.59, 148.85, 150.34, 161.90, 192.77. HRMS (ESI) m/z: calcd for
C19H17N2O2 ([M+H]+): 305.1290; found: 305.1287.
4.1.3.15. (E)-2-(3-(4-Bromophenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3o)
1
Yield: 58%, white solid, mp 232−234 °C. HPLC rt = 13.329 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 7.34 (d, J = 15.6 Hz, 1H, =CHCO), 7.59 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.8
Hz, 1H, Quin 8-H), 7.85 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.87 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.07 (d,
J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 8.16 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.34 (d, J = 15.6 Hz, 1H, =CH),
12.63 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 122.19, 126.38, 128.09, 128.30, 128.63, 130.29,
131.11 (2C), 132.56 (2C), 135.16, 136.06, 136.58, 148.84, 150.26, 161.91, 188.21. HRMS (ESI) m/z:
calcd for C17H12BrN2O2 ([M+H]+): 355.0082, 357.0062; found: 355.0080, 357.0059.
4.1.3.16. (E)-2-(3-(4-Chlorophenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3p)
1
Yield: 28%, white solid, mp 231−233 °C. HPLC rt = 11.392 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.33 (d, J = 15.6 Hz, 1H, =CHCO), 7.59 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.70 (d, J = 8.4
16

ACCEPTED MANUSCRIPT
Hz, 2H, Ph 3'-H, 5'-H), 7.77 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.87 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.15 (d,
J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 8.16 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.36 (d, J = 15.6 Hz, 1H, =CH),
12.63 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 122.19, 126.38, 128.09, 128.29, 129.61 (2C),
130.33, 131.04 (2C), 135.16, 135.74, 136.56, 139.35, 148.84, 150.26, 161.91, 188.00. HRMS (ESI) m/z:
calcd for C17H12ClN2O2 ([M+H]+): 311.0587, 313.0558; found: 311.0585, 313.0565.
4.1.3.17. (E)-2-(3-(3,4-Dichlorophenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3q)
1
Yield: 35%, white solid, mp 229−231 °C. HPLC rt = 18.943 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.33 (d, J = 15.6 Hz, 1H, =CHCO), 7.59 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.78 (d, J = 7.8
Hz, 1H, Quin 8-H), 7.87 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 7.90 (d, J = 8.4 Hz, 1H, Ph 5'-H), 8.08 (dd,
J = 8.4, 1.8 Hz, 1H, Ph 6'-H), 8.16 (d, J = 7.8 Hz, 1H, Quin 5-H), 8.33 (d, J = 1.8 Hz, 1H, Ph 2'-H),
8.33 (d, J = 15.6 Hz, 1H, =CH), 12.61 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 122.17, 125.21,
126.40, 127.89, 128.32, 129.12, 130.98, 131.85, 132.55, 134.96, 135.23, 137.00, 137.21, 148.79,
150.20, 161.92, 187.20. HRMS (ESI) m/z: calcd for C17H11Cl2N2O2 ([M+H]+): 345.0198, 347.0168;
found: 345.0195, 347.0164.
4.1.3.18. (E)-2-(3-(4-Fluorophenyl)-3-oxoprop-1-enyl)quinazolin-4(3H)-one (3r)
1
Yield: 22%, white solid, mp 252−254 °C. HPLC rt = 5.717 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.33 (d, J = 15.6 Hz, 1H, =CHCO), 7.47 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.59 (t, J =
7.8 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.87 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.16 (d,
J = 7.8 Hz, 1H, Quin 5-H), 8.24 (dd, J = 8.4, 5.4 Hz, 2H, Ph 2'-H, 6'-H), 8.38 (d, J = 15.6 Hz, 1H,
=CH), 12.63 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 116.60 (d, J = 21.9 Hz, 2C), 122.17,
126.40, 128.10, 128.28, 130.56, 132.27 (d, J = 9.75 Hz, 2C), 133.84 (d, J = 2.1 Hz), 135.20, 136.33,
148.84, 150.34, 161.97, 165.90 (d, J = 251.4 Hz), 187.68. HRMS (ESI) m/z: calcd for C17H12FN2O2
([M+H]+): 295.0883; found: 295.0877.
4.1.3.19. (E)-2-(3-Oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)quinazolin-4(3H)-one (3s)
1
Yield: 41%, white solid, mp 234−236 °C. HPLC rt = 9.929 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 7.37 (d, J = 15.6 Hz, 1H, =CHCO), 7.59 (t, J = 7.8 Hz, 1H, Quin 6-H), 7.77 (d, J = 7.8
Hz, 1H, Quin 8-H), 7.87 (t, J = 7.8 Hz, 1H, Quin 7-H), 8.0 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 8.16 (d, J
= 7.8 Hz, 1H, Quin 5-H), 8.31 (d, J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 8.37 (d, J = 15.6 Hz, 1H, =CH), 12.66
(s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 122.22, 124.18 (q, J = 271.95 Hz), 126.40, 126.43 (q,
17

ACCEPTED MANUSCRIPT
J = 3.9 Hz, 2C), 128.16, 128.32, 129.95 (2C), 130.40, 133.40 (q, J = 31.95 Hz), 135.20, 137.05, 140.25,
148.82, 150.18, 161.90, 188.69. HRMS (ESI) m/z: calcd for C18H12F3N2O2 ([M+H]+): 345.0851; found:
345.0849.
4.1.4. Preparation of 2-ethylquinazolin-4(3H)-one (intermediate 4)
A mixture of methyl 2-aminobenzoate (6.04 g, 40 mmol), ammonium acetate (7.7 g, 100 mmol)
and triethyl orthopropionate (25 mL) was heated under reflux for 30 h. After cooling to room
temperature, the separated solid was collected by filtration and air-dried, which was re-crystallized
from ethanol to give intermediate 4 as a white solid (5.01 g, 72%), mp 228−229 °C (Lit. [39] mp
229−231 °C). 1H NMR (600 MHz, DMSO-d6) δ: 1.25 (t, J = 7.8 Hz, 3H, CH2CH3), 2.63 (q, J = 7.8 Hz,
2H, CH2CH3), 7.46 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.60 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.77 (td, J
= 7.8, 1.2 Hz, 1H, Quin 7-H), 8.09 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H), 12.16 (s, 1H, NH). HRMS (ESI)
m/z: calcd for C10H11N2O ([M+H]+): 175.0871; found: 175.0865.
4.1.5. Preparation of 2-acetylquinazolin-4(3H)-one (intermediate 5)
A mixture of 2-ethylquinazolin-4(3H)-one (intermediate 4) (0.52 g, 3 mmol) and selenium dioxide
(0.66 g, 6 mmol) in 1,4-dioxane (50 mL) was heated under reflux for 1 h. After cooling to room
temperature, a small amount of black solid was removed by filtration, and the filtrate was adjusted to
pH 7−8 with a 5% aqueous solution of sodium bicarbonate. The separated solid was collected by
filtration, and the filtrate was extracted four times with dichloromethane (15 mL, each). The combined
extracts were washed with brine and dried over anhydrous sodium sulfate overnight. The solvent was
removed by rotary evaporation, and the crude product was re-crystallized from methanol to produce
1
intermediate 5 as an orange-brown solid (0.34 g, 61%), mp 174−176 °C. H NMR (600 MHz,
DMSO-d6) δ: 2.64 (s, 3H, CH3CO), 7.66 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.85 (dd, J = 7.8, 1.2 Hz,
1H, Quin 8-H), 7.91 (td, J = 7.8, 1.2 Hz, 1H, Quin 7-H), 8.19 (dd, J = 7.8, 1.2 Hz, 1H, Quin 5-H),
12.25 (s, 1H, NH). HRMS (ESI) m/z: calcd for C10H9N2O2 ([M+H]+): 189.0664; found: 189.0658.
4.1.6. General preparation procedure for compounds 6a−s
An aqueous solution of 30% potassium hydroxide (1 mL) was added to a stirred solution of
2-acetylquinazolin-4(3H)-one (5) (0.17 g, 1 mmol) in methanol (10 mL). After 20 min stirring at room
temperature, the appropriate aromatic aldehyde or heteroaromatic aldehyde (1.1 mmol) was added and
stirring was continued for 24 h. The solution was adjusted to pH 6−7 with 6 mol/L hydrochloric acid.
18

ACCEPTED MANUSCRIPT
The precipitate was collected by filtration and air-dried, which was re-crystallized from methanol to
give compounds 6a−s.
4.1.6.1. (E)-2-(3-Phenylacryloyl)quinazolin-4(3H)-one (6a)
1
Yield: 48%, brown solid, mp 171−173 °C. HPLC rt = 10.675 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.52 (m, 3H, Ph 3'-H, 4'-H, 5'-H), 7.69 (m, 1H, Quin 6-H), 7.87 (m, 2H, Ph 2'-H, 6'-H),
7.94 (d, J = 16.2 Hz, 1H, =CHCO), 7.95 (m, 2H, Quin 7-H, 8-H), 8.12 (d, J = 16.2 Hz, 1H, =CH), 8.22
(d, J = 7.8 Hz, 1H, Quin 5-H), 12.42 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 120.15, 123.66,
126.61, 129.09, 129.39, 129.49 (2C), 129.62 (2C), 131.83, 134.62, 135.24, 146.07, 147.65, 148.74,
161.32, 183.64. HRMS (ESI) m/z: calcd for C17H13N2O2 ([M+H]+): 277.0977; found: 277.0977.
4.1.6.2. (E)-2-(3-(2-Methoxyphenyl)acryloyl)quinazolin-4(3H)-one (6b)
1
Yield: 39%, brown solid, mp 175−177 °C. HPLC rt = 13.352 min (94%). H NMR (600 MHz,
DMSO-d6) δ: 3.95 (s, 1H, OCH3), 7.08 (t, J = 7.8 Hz, 1H, Ph 5'-H), 7.17 (d, J = 7.8 Hz, 1H, Ph 3'-H),
7.52 (t, J = 7.8 Hz, 1H, Ph 4'-H), 7.68 (m, 1H, Quin 6-H), 7.85 (d, J = 7.8 Hz, 1H, Ph 6'-H), 7.94 (m,
2H, Quin 7-H, 8-H), 8.17 (s, 2H, CH=CH), 8.22 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.37 (s, 1H, NH). 13C
NMR (150 MHz, DMSO-d6) δ: 56.30, 112.47, 120.12, 121.37, 122.95, 123.63, 126.60, 129.11, 129.35,
129.59, 133.59, 135.21, 140.85, 147.67, 148.77, 159.20, 161.32, 183.73. HRMS (ESI) m/z: calcd for
C18H15N2O3 ([M+H]+): 307.1083; found: 307.1082.
4.1.6.3. (E)-2-(3-(3-Methoxyphenyl)acryloyl)quinazolin-4(3H)-one (6c)
1
Yield: 47%, brown solid, mp 169−171 °C. HPLC rt = 11.509 min (98%). H NMR (600 MHz,
DMSO-d6) δ: 3.85 (s, 3H, OCH3), 7.10 (dd, J = 7.8, 1.2 Hz, 1H, Ph 4'-H), 7.41 (t, J = 2.4 Hz, Ph 2'-H),
7.44 (d, J = 7.8 Hz, 1H, Ph 6'-H), 7.45 (d, J = 7.8 Hz, 1H, 5'-H), 7.69 (td, J = 7.8, 1.2 Hz, 1H, Quin
6-H), 7.91 (d, J = 16.2 Hz, 1H, =CHCO), 7.94 (d, J = 7.8 Hz, 1H, Quin 8-H), 7.95 (d, J = 7.8 Hz, 1H,
Quin 7-H), 8.10 (d, J = 16.2 Hz, 1H, =CH), 8.22 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.42 (s, 1H, NH). 13C
NMR (150 MHz, DMSO-d6) δ: 55.72, 114.46, 117.67, 120.42, 121.70, 123.64, 126.58, 129.08, 129.33,
130.59, 135.14, 136.00, 146.03, 147.62, 148.66, 160.10, 161.28, 183.62. HRMS (ESI) m/z: calcd for
C18H15N2O3 ([M+H]+): 307.1083; found: 307.1080.
4.1.6.4. (E)-2-(3-(4-Methoxyphenyl)acryloyl)quinazolin-4(3H)-one (6d)
1
Yield: 43%, yellow solid, mp 189−191 °C. HPLC rt = 2.030 min (100%). H NMR (600 MHz,
DMSO-d6) δ: 3.85 (s, 3H, OCH3), 7.07 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.68 (m, 1H, Quin 6-H), 7.84
19

ACCEPTED MANUSCRIPT
(d, J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 7.91 (d, J = 16.2 Hz, 1H, =CHCO), 7.93 (m, 2H, Quin 7-H, 8-H),
7.98 (d, J = 16.2 Hz, 1H, =CH), 8.22 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.35 (s, 1H, NH). 13C NMR (150
MHz, DMSO-d6) δ: 55.93, 115.16 (2C), 117.51, 123.60, 126.60, 127.30, 129.05, 129.27, 131.60 (2C),
135.21, 146.27, 147.72, 148.91, 161.32, 162.49, 183.40. HRMS (ESI) m/z: calcd for C18H15N2O3
([M+H]+): 307.1083; found: 307.1080.
4.1.6.5. (E)-2-(3-(2,4-Dimethoxyphenyl)acryloyl)quinazolin-4(3H)-one (6e)
1
Yield: 46%, yellow solid, mp 180−182 °C. HPLC rt = 12.074 min (97%). H NMR (600 MHz,
DMSO-d6) δ: 3.87 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.67 (dd, J = 8.4, 2.4 Hz, 1H, Ph 5'-H), 6.69 (d,
J = 2.4 Hz, 1H, Ph 3'-H), 7.67 (m, 1H, Quin 6-H), 7.81 (d, J = 8.4 Hz, 1H, Ph 6'-H), 7.93 (m, 2H, Quin
7-H, 8-H), 8.04 (d, J = 16.2 Hz, 1H, =CHCO), 8.12 (d, J = 16.2 Hz, 1H, =CH), 8.21 (d, J = 7.8 Hz, 1H,
Quin 5-H), 12.28 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 56.09, 56.42, 98.88, 107.20, 116.08,
117.19, 123.56, 126.58, 129.06, 129.20, 131.50, 135.17, 141.28, 147.74, 148.98, 161.07, 161.31,
164.29, 183.46. HRMS (ESI) m/z: calcd for C19H17N2O4 ([M+H]+): 337.1188; found: 337.1187.
4.1.6.6. (E)-2-(3-(3,4-Dimethoxyphenyl)acryloyl)quinazolin-4(3H)-one (6f)
1
Yield 51%, yellow solid, mp 184−186 °C. HPLC rt = 3.731 min (98%). H NMR (600 MHz,
DMSO-d6) δ: 3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.09 (d, J = 9.0 Hz, 1H, Ph 5'-H), 7.46 (m, 2H,
Ph 2'-H, 6'-H), 7.68 (m, 1H, Quin 6-H), 7.90 (d, J = 16.2 Hz, 1H, =CHCO), 7.94 (m, 2H, Quin 7-H,
8-H), 7.97 (d, J = 16.2 Hz, 1H, =CH), 8.21 (d, J = 7.8 Hz, 1H, Quin 5-H), 12.36 (s, 1H, NH). 13C NMR
(150 MHz, DMSO-d6) δ: 56.13 (2C), 111.69, 112.22, 117.74, 123.58, 124.52, 126.59, 127.47, 129.06,
129.25, 135.19, 146.92, 147.72, 148.97, 149.50, 152.43, 161.32, 183.51. HRMS (ESI) m/z: calcd for
C19H17N2O4 ([M+H]+): 337.1188; found: 337.1186.
4.1.6.7. (E)-2-(3-(3,5-Dimethoxyphenyl)acryloyl)quinazolin-4(3H)-one (6g)
1
Yield: 51%, brown solid, mp 181−183 °C. HPLC rt = 12.351 min (98%). H NMR (600 MHz,
DMSO-d6) δ: 3.83 (s, 6H, 2OCH3), 6.66 (t, J = 1.8 Hz, 1H, Ph 4'-H), 7.02 (d, J = 1.8 Hz, 2H, Ph 2'-H,
6'-H), 7.68 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.86 (d, J = 16.2 Hz, 1H, =CHCO), 7.93 (td, J = 7.8, 1.2
Hz, 1H, Quin 7-H), 7.96 (d, J = 7.8 Hz, 1H, Quin 8-H), 8.07 (d, J = 16.2 Hz, 1H, =CH), 8.22 (d, J = 7.8
Hz, 1H, Quin 5-H), 12.42 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 55.93 (2C), 103.96, 107.28
(2C), 120.86, 123.65, 126.59, 129.15, 129.38, 135.20, 136.57, 146.23, 147.64, 148.75, 161.26 (2C),
161.32, 183.82. HRMS (ESI) m/z: calcd for C19H17N2O4 ([M+H]+): 337.1188; found: 337.1186.
20

ACCEPTED MANUSCRIPT
4.1.6.8. (E)-2-(3-(3,4,5-Trimethoxyphenyl)acryloyl)quinazolin-4(3H)-one (6h)
1
Yield: 33%, yellow solid, mp 189−191 °C. HPLC rt = 9.931 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 3.74 (s, 3H, OCH3), 3.89 (s, 6H, 2OCH3), 7.21 (s, 2H, Ph 2'-H, 6'-H), 7.68 (t, J = 7.8 Hz,
1H, Quin 6-H), 7.89 (d, J = 15.6 Hz, 1H, =CHCO), 7.95 (m, 2H, Quin 7-H, 8-H), 8.01 (d, J = 15.6 Hz,
1H, =CH), 8.22 (d, J = 7.8, 1H, Quin 5-H), 12.39 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ:
56.59 (2C), 60.64, 107.23 (2C), 119.57, 123.61, 126.60, 129.10, 129.31, 130.16, 135.19, 140.92,
146.85, 147.67, 148.91, 153.62 (2C), 161.31, 183.79. HRMS (ESI) m/z: calcd for C20H19N2O5
([M+H]+): 367.1294; found: 367.1291.
4.1.6.9. (E)-2-(3-(4-Methylphenyl)acryloyl)quinazolin-4(3H)-one (6i)
1
Yield: 39%, orange solid, mp 186−188 °C. HPLC rt = 17.735 min (96%). H NMR (600 MHz,
DMSO-d6) δ: 2.38 (s, 3H, CH3), 7.33 (d, J = 7.8 Hz, 2H, Ph 3'-H, 5'-H), 7.68 (m, 1H, Quin 6-H), 7.76
(d, J = 7.8 Hz, 2H, Ph 2'-H, 6'-H), 7.91 (d, J = 15.6 Hz, 1H, =CHCO), 7.94 (m, 2H, Quin 7-H, 8-H),
8.07 (d, J = 15.6 Hz, 1H, =CH), 8.22 (d, J = 7.8, 1H, Quin 5-H), 12.39 (s, 1H, NH). 13C NMR (150
MHz, DMSO-d6) δ: 21.61, 118.95, 123.60, 126.60, 129.07, 129.36, 129.56 (2C), 130.25 (2C), 131.89,
135.24, 142.18, 146.20, 147.64, 148.74, 161.35, 183.52. HRMS (ESI) m/z: calcd for C18H15N2O2
([M+H]+): 291.1134; found: 291.1132.
4.1.6.10. (E)-2-(3-(4-Bromophenyl)acryloyl)quinazolin-4(3H)-one (6j)
Yield: 68%, light yellow solid, mp 202−204 °C. HPLC rt = 1.704 min (95%). 1H NMR (600 MHz,
DMSO-d6) δ: 7.69 (m, 1H, Quin 6-H), 7.71 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.83 (d, J = 8.4 Hz, 2H,
Ph 2'-H, 6'-H), 7.91 (d, J = 16.2 Hz, 1H, =CHCO), 7.94 (m, 2H, Quin 7-H, 8-H), 8.12 (d, J = 16.2 Hz,
1H, =CH), 8.22 (d, J = 7.8, 1H, Quin 5-H), 12.43 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ:
120.90, 123.67, 125.25, 126.62, 129.09, 129.44, 131.36 (2C), 132.61 (2C), 133.90, 135.26, 144.67,
147.63, 148.69, 161.32, 183.61. HRMS (ESI) m/z: calcd for C17H12BrN2O2 ([M+H]+): 335.0082,
357.0062; found: 355.0076, 357.0060.
4.1.6.11. (E)-2-(3-(4-Chlorophenyl)acryloyl)quinazolin-4(3H)-one (6k)
Yield: 46%, light yellow solid, mp 198-200 °C. HPLC rt = 2.539 min (98%). 1H NMR (600 MHz,
DMSO-d6) δ: 7.57 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.69 (m, 1H, Quin 6-H), 7.91 (d, J = 8.4 Hz, 2H,
Ph 2'-H, 6'-H), 7.92 (m, 3H, Quin 7-H, 8-H; =CHCO), 8.11 (d, J = 16.2 Hz, 1H, =CH), 8.22 (d, J = 7.8,
1H, Quin 5-H), 12.43 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 120.87, 123.68, 126.63, 129.09,
21

ACCEPTED MANUSCRIPT
129.43, 129.67 (2C), 131.19 (2C), 133.59, 135.25, 136.29, 144.56, 147.63, 148.69, 161.31, 183.60.
HRMS (ESI) m/z: calcd for C17H12ClN2O2 ([M+H]+): 311.0587, 313.0558; found: 311.0587, 313.0569.
4.1.6.12. (E)-2-(3-(4-Fluorophenyl)acryloyl)quinazolin-4(3H)-one (6l)
1
Yield: 31%, pink solid, mp 189−191 °C. HPLC rt = 13.651 min (98%). H NMR (600 MHz,
DMSO-d6) δ: 7.35 (t, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 7.68 (m, 1H, Quin 6-H), 7.94 (d, J = 16.2 Hz, 1H,
=CHCO), 7.95 (m, 5H, Ph 2'-H, 6'-H; Quin 7-H, 8-H; =CHCO), 8.06 (d, J = 16.2 Hz, 1H, =CH), 8.22
(d, J = 7.8 Hz, 1H, Quin 5-H), 12.42 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 116.68 (d, J =
21.75 Hz, 2C), 120.0, 123.64, 126.61, 129.06, 129.39, 131.31 (d, J = 3.0 Hz), 131.96 (d, J = 8.7 Hz,
2C), 135.23, 144.85, 147.63, 148.69, 161.32, 163.42 (d, J = 248.85 Hz), 183.57. HRMS (ESI) m/z:
calcd for C17H12FN2O2 ([M+H]+): 295.0883; found: 295.0882.
4.1.6.13. (E)-2-(3-(4-Cyanophenyl)acryloyl)quinazolin-4(3H)-one (6m)
1
Yield: 61%, pink solid, mp 200−202 °C. HPLC rt = 1.288 min (100%). H NMR (600 MHz,
DMSO-d6) δ: 7.69 (m, 1H, Quin 6-H), 7.95 (m, 5H, Quin 7-H, 8-H; Ph 2'-H, 6'-H; =CHCO), 8.06 (d, J
= 7.8 Hz, 2H, Ph 3'-H, 5'-H), 8.21 (m, 2H, =CH, Quin 5-H), 12.48 (s, 1H, NH). 13C NMR (150 MHz,
DMSO-d6, CF3COOD) δ: 113.31, 121.14, 121.63, 127.11, 128.83, 129.15, 129.61 (2C), 130.73, 133.05
(2C), 136.51, 138.85, 145.51, 145.95, 147.49, 163.88, 182.46. HRMS (ESI) m/z: calcd for C18H12N3O2
([M+H]+): 302.0930; found: 302.0926.
4.1.6.14. (E)-2-(3-(4-Nitrophenyl)acryloyl)quinazolin-4(3H)-one (6n)
Yield: 69%, light yellow solid, mp 206−208 °C. HPLC rt = 1.353 min (99%). 1H NMR (600 MHz,
DMSO-d6) δ: 7.70 (m, 1H, Quin 6-H), 7.95 (m, 2H, Quin 7-H, 8-H), 8.01 (d, J = 16.2 Hz, 1H, =CHCO),
8.15 (d, J = 8.4 Hz, 2H, Ph 2'-H, 6'-H), 8.23 (d, J = 8.4, 1H, Quin 5-H), 8.25 (d, J = 16.2 Hz, 1H, =CH),
8.32 (d, J = 8.4 Hz, 2H, Ph 3'-H, 5'-H), 12.50 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 123.72,
124.09, 124.60 (2C), 126.66, 129.12, 129.56, 130.48 (2C), 135.30, 140.93, 142.94, 147.57, 148.58,
148.89, 161.32, 183.59. HRMS (ESI) m/z: calcd for C17H12N3O4 ([M+H]+): 322.0828; found: 322.0826.
4.1.6.15. (E)-2-(3-(Furan-2-yl)acryloyl)quinazolin-4(3H)-one (6o)
1
Yield: 65%, yellow solid, mp 187−189 °C. HPLC rt = 5.546 min (97%). H NMR (600 MHz,
DMSO-d6) δ: 6.75 (dd, J = 3.6, 1.8 Hz, 1H, Furan-2-yl 4'-H), 7.20 (d, J = 3.6 Hz, 1H, Furan-2-yl 3'-H),
7.68 (td, J = 7.8, 1.8 Hz, 1H, Quin 6-H), 7.73 (d, J = 15.6 Hz, 1H, =CHCO), 7.87 (d, J = 15.6 Hz, 1H,
=CH), 7.93 (m, 2H, Quin 7-H, 8-H), 8.01 (d, J = 1.2 Hz, 1H, Furan-2-yl 5'-H), 8.21 (d, J = 7.8, 1H,
22

ACCEPTED MANUSCRIPT
Quin 5-H), 12.36 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 109.98, 113.95, 116.72, 119.63,
123.62, 126.58, 129.09, 129.34, 131.89, 135.20, 147.66, 148.63, 151.41, 161.27, 183.01. HRMS (ESI)
m/z: calcd for C15H11N2O3 ([M+H]+): 267.0770; found: 267.0764.
4.1.6.16. (E)-2-(3-(Thiophen-2-yl)acryloyl)quinazolin-4(3H)-one (6p)
1
Yield: 37%, brown solid, mp 174−176 °C. HPLC rt = 11.772 min (94%). H NMR (600 MHz,
DMSO-d6) δ: 7.25 (td, J = 4.8, 1.2 Hz, 1H, Thiophen-2-yl 4'-H), 7.67 (td, J = 7.2, 1.8 Hz, 1H, Quin
6-H), 7.76 (d, J = 4.8 Hz, 1H, Thiophen-2-yl 3'-H), 7.80 (d, J = 15.6 Hz, 1H, =CHCO), 7.88 (d, J = 4.8
Hz, 1H, Thiophen-2-yl 5'-H), 7.93 (m, 2H, Quin 7-H, 8-H), 8.12 (d, J = 15.6 Hz, 1H, =CH), 8.21 (d, J
= 8.4, 1H, Quin 5-H), 12.37 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 118.29, 123.63, 126.60,
129.11, 129.35, 129.57, 132.08, 135.13, 135.21, 138.81, 140.03, 147.62, 148.67, 161.28, 182.97.
HRMS (ESI) m/z: calcd for C15H11N2O2S ([M+H]+): 283.0541; found: 283.0537.
4.1.6.17. (E)-2-(3-(Pyridin-2-yl)acryloyl)quinazolin-4(3H)-one (6q)
1
Yield: 28%, orange solid, mp 190−192 °C. HPLC rt = 3.559 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.50 (m, 1H, Pyridin-2-yl 5'-H), 7.69 (td, J = 7.8, 1.2 Hz, 1H, Quin 6-H), 7.85 (d, J = 7.8
Hz, 1H, Pyridin-2-yl 3'-H), 7.91 (d, J = 15.6 Hz, 1H, =CHCO), 7.93-7.97 (m, 3H, Quin 7-H, 8-H;
Pyridin-2-yl 4'-H), 8.22 (d, J = 7.8, 1H, Quin 5-H), 8.51 (d, J = 15.6 Hz, 1H, =CH), 8.75 (d, J = 4.8 Hz,
1H, Pyridin-2-yl 6'-H), 12.44 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 123.34, 123.71, 125.83,
126.62, 126.83, 129.15, 129.50, 135.28, 137.88, 144.40, 147.65, 148.64, 150.75, 152.55, 161.30,
183.94. HRMS (ESI) m/z: calcd for C16H12N3O2 ([M+H]+): 278.0930; found: 278.0925.
4.1.6.18. (E)-2-(3-(Pyridin-3-yl)acryloyl)quinazolin-4(3H)-one (6r)
1
Yield: 48%, brown solid, mp 197−200 °C. HPLC rt = 3.408 min (95%). H NMR (600 MHz,
DMSO-d6) δ: 7.54 (t, J = 6.0 Hz, 1H, Pyridin-3-yl 5'-H), 7.69 (m, 1H, Quin 6-H), 7.96 (m, 3H, Quin
7-H, 8-H; =CHCO), 8.21 (d, J = 16.2 Hz, 1H, =CH), 8.22 (d, J = 7.8, 1H, Quin 5-H), 8.34 (d, J = 7.8
Hz, 1H, Pyridin-3-yl 6'-H), 8.67 (d, J = 4.2 Hz, 1H, Pyridin-3-yl 4'-H), 9.01 (s, 1H, Pyridin-3-yl 2'-H),
12.47 (s, 1H, NH). 13C NMR (150 MHz, DMSO-d6) δ: 122.10, 123.68, 124.62, 126.63, 129.10, 129.49,
130.52, 135.27, 135.58, 142.62, 147.60, 148.60, 151.06, 152.05, 162.75, 183.58. HRMS (ESI) m/z:
calcd for C16H12N3O2 ([M+H]+): 278.0930; found: 278.0927.
4.1.6.19. (E)-2-(3-(Pyridin-4-yl)acryloyl)quinazolin-4(3H)-one (6s)
1
Yield: 19%, brown solid, mp 218−220 °C. HPLC rt = 3.462 min (94%). H NMR (600 MHz,
23

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DMSO-d6) δ: 7.70 (m, 1H, Quin 6-H), 7.81 (d, J = 6.0 Hz, 2H, Pyridin-4-yl 2'-H, 6'-H), 7.88 (d, J =
16.2 Hz, 1H, =CHCO), 7.95 (m, 2H, Quin 7-H, 8-H), 8.23 (d, J = 7.8, 1H, Quin 5-H), 8.28 (d, J = 16.2
Hz, 1H, =CH), 8.72 (d, J = 6.0 Hz, 2H, Pyridin-4-yl 3'-H, 5'-H), 12.51 (s, 1H, NH). 13C NMR (150
MHz, DMSO-d6) δ: 123.77, 126.05 (2C), 126.53, 128.88, 129.34, 129.54, 134.84, 138.88, 142.52 (2C),
147.36, 147.90, 151.37, 161.11, 183.17. HRMS (ESI) m/z: calcd for C16H12N3O2 ([M+H]+): 278.0930;
found: 278.0927.
4.2. Biological assays
4.2.1. Cell lines and culture conditions
HCT-116 and MCF-7 cells were cultured in Dulbecco modified Eagle medium (DMEM, Hyclone)
supplemented with 10% fetal bovine serum (FBS, Hyclone) and standard antibiotics (Hyclone), at
37 °C in a humidified, 5% CO2 incubator.
4.2.2. MTS cell proliferation assay
MTS cell proliferation assay was performed as previously described [40]. Cell viability was
assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega). The data
were obtained from triplicate wells in three independent experiments.
4.2.3. Cell cycle profile analysis of HCT-116 cells
HCT-116 cells were treated with compound 3f (0.5×, 1× and 2× IC50), 5-FU (IC50), or taxol (0.2
µM) for 24 h. The cells were harvested with trypsin, fixed in 75% ice-cold ethanol, and stained with
propidium iodide (PI) at room temperature for 30 min. The stained cells were analyzed for DNA
content using a BD Accuri C6 flow cytometer (BD Biosciences, USA). The cell cycle distribution was
analyzed using FlowJo software.
4.2.4. Annexin V-FITC/PI dual staining assay
The Annexin V-FITC/PI dual staining assay was used to determine the percentage of apoptotic
cells. Cells (3 × 106/mL per well) were plated in 6-well culture plates and allowed to grow for 24 h. The
cells were treated with compound 3f (1× or 2× IC50) for 24 h, and then harvested with trypsin. The
harvested cells were washed twice with ice-cold PBS, and incubated in the dark at room temperature in
100 mL 1× binding buffer containing 1 µL Annexin V-FITC and 12.5 mL PI. After 15 min incubation,
cells were analyzed for percentage undergoing apoptosis using a BD Accuri C6 flow cytometer (BD
Biosciences, USA).
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4.2.5. Acridine orange–ethidium bromide (AO–EB) fluorescence staining assay
HCT-116 cells (3 × 106/mL per well) were plated in 6-well culture plates and exposed to different
concentrations of compound 3f (1× or 2× IC50) for 24 h or staurosporine (STS; 1 µM) for 12 h. The
cells were washed twice with ice-cold PBS, and then stained with 1 mL of dye mixture containing 1
µg/mL AO and 1 µg/mL EB in PBS and incubated at room temperature for 5 min in the dark. The
stained cells were observed under a Nikon fluorescence microscope (TE2000-U, Nikon Inc., Japan) at
10× magnification, using excitation wavelength 488 nm and emission wavelength 550 nm.
4.2.6. MitoSOX™ Red staining assay
Mitochondrial superoxide production was determined using MitoSOX™ Red mitochondrial
superoxide indicator. HCT-116 cells (3 × 106/mL per well) were plated in 6-well culture plates and
allowed to grow for 24 h. Cells were treated with different concentrations of compound 3f (1× or 2×
IC50) for 24 h or STS (1 µM) for 12 h. After treatment, cells were washed twice with ice-cold PBS and
incubated with MitoSOX™ Red mitochondrial superoxide indicator (5 µM) in PBS for 10 min at 37 °C
in the dark. The stained cells were observed under a Nikon fluorescence microscope (TE2000-U, Nikon
Inc., Japan) at 10× magnification, and an excitation wavelength of 488 nm and an emission wavelength
of 550 nm.
4.2.7. Mitochondrial membrane potential monitored by JC-1 staining
HCT-116 cells were seeded in 6 well plate and treated with compound 3f (2× IC50) for 24 h, or
STS (1 µM) for 12 h. They were then washed with PBS, added fresh medium (1 mL) and JC-1
(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide, Beyotime, C2006)
molecular probe (1 mL). After 20 min incubation at 37 °C, rinsed twice with PBS. Visualization of
JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) were done using filter
(emission 488 and 550 nm, respectively) by 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.
4.2.8. Western blotting
Cultured HCT-116 cells were treated with compound 3f for 4, 8, or 12 h. After treatment, cells
were harvested and total proteins were extracted using sample buffer (62.5 mM Tris-HCl (pH 6.8), 2%
SDS, 0.05% bromophenol blue, 20% Glycerin, 5% β-mercaptoethanol). The extracted protein samples
25

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were separated on a 4−18% gradient SDS-polyacrylamide gel and transferred onto polyvinylidene
difluoride membranes. The membranes were blocked in 0.05% PBST containing 5% skim milk for 30
min at room temperature, and incubated with primary antibody overnight at 4 °C. Antibodies against
PARP1 (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. After washing with PBST, the membranes were incubated with
HRP-conjugated Goat Anti-Mouse IgG (115-035-116) or Donkey Anti-Rabbit (711-035-152)
secondary antibodies (Jackson ImmunoResearch) at room temperature for 1 h. The immunoblots were
visualized with ECL reagent and imaged using ImageQuantTM LAS 4000 software (GE Healthcare Life
Sciences).
4.2.9. Statistical analysis method
Student’s t-test for means, chi-squared tests, Mann-Whitney U test and Kruskall-Wallis analysis of
variance by ranks were considered significant for p values < 0.05.
Acknowledgments
This work was supported by the 973 project (Project No. 2013CB911002), National Natural
Science Foundation of China (Project No. 31530016 and 31761133012), the Shenzhen Science and
Technology Innovation Commission (Project No. JCYJ20170412113009742) and Capacity Building
for Sci-Tech Innovation−Fundamental Scientific Research Funds (Grant No. 025185305000/208). The
authors would like to thank Dr. Jessica Tamanini (Shenzhen University Health Science Centre and
ETediting) for proof reading the manuscript prior to submission.
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