Novel hybrids derived from aspirin and chalcones potently suppress colorectal cancer in vitro and in vivo

Article abstract of DOI:10.1039/C8MD00284C

Colorectal cancer (CRC) remains the fourth leading cause of cancer deaths around the world despite the availability of many approved small molecules for treatment. The issues lie in the potency, selectivity and targeting of these compounds. Therefore, new

Full text of DOI:10.1039/C8MD00284C

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DOI: 10.1039/C8MD00284C
Novel hybrids derived from aspirin and chalcones potently suppress
colorectal cancer in vitro and in vivo
Shan Lu, a, * Obinna N Obianom, b and Yong Ai b, **
aCollege of Pharmacy, Hubei University of Chinese Medicine, Hubei 430065, PR
China
bDepartment of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, MD 21201, United States
* Corresponding author. College of Pharmacy, Hubei University of Chinese Medicine,
Hubei 430065, PR China. Eꢀmail address: lushan9805@163.com (S. Lu).
** Corresponding author. Department of Pharmaceutical Sciences, University of
Maryland School of Pharmacy, Baltimore, MD 21201, United States. Eꢀmail address:
aiyong0508@126.com (Y. Ai).
1

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Abstract
Colorectal cancer (CRC) remains the fourth leading cause of cancer deaths in the
world despite the availability of many approved small molecules for treatments. The
issues lie in the potency, selectivity and targeting of these compounds. Therefore, new
strategies and targets are needed to optimize and develop novel treatments for CRC.
Here, a group of novel hybrids derived from aspirin and chalcones were designed and
synthesized based on recent reports of their individual benefits to CRC targeting and
selectivity. The most active compound 7h inhibited proliferation of CRC cell lines
with better potency compared to 5ꢀFluorouracil, a current therapy for CRC.
Importantly, 7h had 8ꢀfold less inhibitory activity against nonꢀcancer CCD841 cells.
In addition, 7h inhibited CRC growth via the inhibition of the cell cycle in G1 phase.
Furthermore, 7h induced apoptosis by activating caspase 3 and PARP cleavage as
well as increasing ROS in CRC cells. Finally, 7h significantly retarded the CRC cells
growth in a mouse xenograft model. These findings suggest that 7h may have a
potential to treat CRC.
2

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Introduction
Colorectal cancer (CRC) originates from the inner walls of the colon and the
rectum. It is the third most commonly diagnosed malignancy and the fourth leading
cause of cancerꢀrelated death worldwide.1,2 Common treatments for CRC include
5ꢀFluorouracil (5ꢀFU), capecitabine, Irinotecan, and Oxaliplatin. The metastasis of
CRC has fueled the discovery of many novel targets in recent years, and a number of
groups have developed newer drugs to specifically alter these new targets. For
example, the development of vascular endothelial growth factor (VEGF) and
epidermal growth factor receptor (EGFR) inhibitors.3 However, in many cases, there
has been reports of mild to grave side effects encountered with these new drugs due to
their unselectivity towards cancer cells.4ꢀ8 For this reason, novel therapeutics for CRC
with reduced side effects is still urgently needed.
Acetylsalicylic acid, commonly known as Aspirin (ASA, 1, Fig.1), is a widely
used nonꢀsteroidal antiꢀinflammatory drug (NSAID) that has been shown to be
effective for the prevention and remission of cancers, especially in CRC.9ꢀ14 To
improve its relatively low anticancer potency, many novel synthetic ASA derivatives
have been developed including HSꢀreleasing ASA,15ꢀ17 HNOꢀ and NOꢀreleasing
ASA,18ꢀ22
dual nitric oxideꢀ and hydrogen sulfideꢀreleasing ASA,23,24
phosphoꢀASA,25 6ꢀgingerolꢀbased ASA prodrug,26 resveratrolꢀbased ASA prodrug,27
SeꢀASA,28 and Asplatin.29,30 These ASA derivatives are more cytotoxic and
chemoꢀpreventive against CRC cells than ASA, hence, the advancement of new ASA
derivatives with more favorable and safer profiles continues to be a welcome
advancement.
Natural products and their derivatives provide a diverse source of biologically
active lead compounds for drug discovery and development.31ꢀ34 For example,
chalcones (2, Fig. 1) are naturally occurring compounds, which bear an
α,βꢀunsaturated ketone moiety and can act as Michael acceptors. Chalcones exert
anticancer activity through a number of mechanisms including inhibition of
thioredoxin reductases (TrxRs),35ꢀ38 nuclear factor κB (NFꢀκB),39ꢀ47 tubulin
polymerization,48ꢀ60 as well as inhibition of receptor tyrosine kinases such as EGFR61
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and vascular endothelial growth factor receptor 2 (VEGFRꢀ2).62 More so, chalcones
are recognized as a privileged scaffold for the incorporation of different molecules or
pharmacophores using linkers such as amide, diol, esters or the use of a triazoles via
click chemistry.63ꢀ78 These hybrids typically retain or enhance the biological potency
and selectivity of the parent chalcones.
The aforementioned investigations led us to hypothesize that a combination of
ASA and chalcones can be an attractive strategy to selectively treat CRC. Accordingly,
we synthesized a novel class of hybrids derived from aspirin and chalcones (7a-p,
Fig.1), and evaluated their bioactivity in vitro and in vivo against CRC cells.
Fig. 1. Chemical structures of ASA, chalcones and hybrids 7a-p.
Results and discussion
Chemistry
The target compounds 7a-p were synthesized as depicted in Scheme 1. Initially,
aspirin 1 was converted to the acid chloride 3 upon treatment with thionyl chloride.
Then the corresponding chalcones 6a-p were prepared by the Wittig Reaction starting
from aldehydes 4a-f in moderate to high yields. Next, reaction of acid chloride 3 with
6a-l in dry CH2Cl2, in the presence of the nonꢀnucleophilic base trimethylamine
(TEA), furnished the respective target compounds 7a-p in good yields. The structures
of all hybrids were fully characterized by their 1H and 13C NMR spectroscopic data as
well as by mass spectral analysis.
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Scheme 1. Synthesis of hybrids 7a-p. Reagents and conditions: (a) SOCl2, anhydrous
CH2Cl2, reflux, 2h; (b) i) PPh3, anhydrous THF, rt, 16 h; ii) KOH, MeOH, rt, 12h; (c)
aldehydes, anhydrous toluene, 60 ºC , 8h; (d) TEA, anhydrous CH2Cl2, 12h.
Biological evaluation
Assessment of in vitro anti-proliferative activity
The antiꢀproliferative activity of compounds 7a-p against HCTꢀ8 and DLDꢀ1 cells
was initially evaluated using MTT assays with 5ꢀFU as a positive control. As shown
in Table 1, compounds 7c, 7f, 7h, and 7i at 10 ꢁM displayed potent antiꢀproliferative
activity (>50%) against both HCTꢀ8 and DLDꢀ1 cells, and this was more potent than
that of 5ꢀFU. It was obvious that R1 and R2 substituents on the benzene ring of the
hybrids may be crucial for their anticancer activity. In general, the compounds with a
Cl or NO2 on the left benzene ring (7c and 7f) showed more potent inhibitory activity
than those bearing an Me, OMe, Br, or CN group (7a, 7b, 7d, or 7e). Moreover, the
compounds with an OMe or F at the 5ꢀposition of the beta benzene ring (7h and 7i)
generally showed more potent inhibitory activity than those bearing an 3ꢀOMe,
4ꢀOMe, 5ꢀCl, 5ꢀBr, or 5ꢀMe group (7l, 7g, 7j, 7k, or 7m).
The active compounds 7h and 7i were further assayed for their antiꢀproliferative
activity against two CRC cell lines (Table 1). The IC50 values of 7h were 2.4 and 2.7
ꢁM against HCTꢀ8 and DLDꢀ1 cells, respectively, which is superior to that of 5FU,
individual drugs 6h (IC50 = 19.7, 14.7 ꢁM) and ASA (IC50 > 100 ꢁM), and even the
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combination of 6h and ASA (IC50 = 6.3, 6.9 ꢁM, physical mixture, mole ratio, 1:1).
Similarly, compound 7i displays more potent anticancer activity than its individual
moieties 6i (IC50 = 25.6, 13.2 ꢁM) and ASA (IC50 > 100 ꢁM), and the combination of
6i and ASA (IC50 = 9.3, 8.0 ꢁM). These results confirm that the antitumor activity of
7h and 7i may be most likely attributed to the synergic effects derived from their
chalcone and ASA moieties. Given the improved potency of 6h+ASA and 6i+ASA, it
is probable that 7h and 7i may be acting as prodrugs and this will be tested in future
studies. In nonꢀtumorigenic cells, CCD841, 7h had 8ꢀfold less inhibitory activity (IC50
= 19.4 ꢁM) than against the cancer cells tested. Hence, 7h preferably inhibited the
proliferation of CRC cells and was selected for further investigation.
HCT-8
DLD-1
100
75
50
25
0
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 5-FU
Fig. 2. Inhibition of CRC cell proliferation. Cells were treated with, or without, the
indicated compounds at 10 ꢁM for 72 h, and the cell proliferation was measured. The
inhibition (%) of each compound was determined. Data are presented as the mean (%)
± SD of each compound from three independent experiments.
Table 1. The antiꢀproliferative activity of compounds 7h and 7i.
IC50 (ꢁM) a
Compounds
HCTꢀ8
2.4 ± 0.1
2.7 ± 0.5
DLDꢀ1
2.7 ± 0.2
3.5 ± 0.4
CCD841
19.4 ± 0.9
ND
7h
7i
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19.7 ± 2.5
14.7 ± 1.8
6.9 ± 0.9
13.2 ± 1.4
8.0 ± 0.6
>100
ND
6h
6h+ASA
6i
6.3 ± 0.9
25.6 ± 3.2
9.3 ± 1.0
>100
ND
ND
6i+ASA
ASA
ND
ND
5ꢀFU
28.9 ± 2.7
33.0 ± 2.1
35.7 ± 3.2
aData are expressed as mean IC50 (µM) ± SD of each compound from three
independent experiments. IC50 is the drug concentration inhibiting 50% of the cell
proliferation. ND, not determined.
Effect of 7h on cell cycle arrest in CRC cells
Since the anticancer agents can prevent the proliferation of cancer cells by
blockade of the cell cycle at a specific checkpoints, we performed cell cycle analysis
after treatment of HCTꢀ8 cells with compound 7h. In short, HCTꢀ8 cells were treated
with varying concentrations (0, 2.5, 5, and 10 ꢁM) of 7h for 24 h and then stained
with propidium iodide (PI), followed by flow cytometry analysis. Figure 3 shows that
treatment with 7h induced HCTꢀ8 cell cycle arrest at G0/G1 in a concentration
dependent manner – 48.08% (2.5 ꢁM), 51.03% (5 ꢁM), and 56.46% (10 ꢁM), higher
than that of the vehicleꢀtreated control. As a result, the fraction of CRC cells at the G2
phase was significantly lower in 7hꢀtreated than vehicleꢀtreated control. This suggests
that the antiꢀcancer mechanisms of 7h may include its ability to induce cycle arrest in
the CRC cells.
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Fig. 3. Effects of 7h on cell cycle distribution in HCTꢀ8 cells. Cells were treated with
the indicated concentrations of 7h for 24 h and stained with PI, followed by flow
cytometry analysis.
Effect of 7h on apoptosis in CRC cells
To determine whether the inhibitory effects of 7h on CRC cell proliferation are
accompanied by enhanced apoptosis, we next carried out Annexin VꢀFITC and PI
staining on 7h treated cells. HCTꢀ8 cells were incubated with vehicle, or 7h (2.5, 5,
and 10 ꢁM) for 24 h. Then flow cytometry analysis was used to determine the
apoptotic status of the cells. We observed that treatment with 7h induced apoptosis in
HCTꢀ8 cells in a concentration dependent manner (Fig. 4). This suggests that 7h
treatment significantly increases apoptosis in cancer cells as a result of its antiꢀcancer
effects.
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Fig. 4. Effects of 7h on the induction of apoptosis. HCTꢀ8 cells were treated with the
indicated concentrations of 7h or vehicle (control) for 24 h. The cells were stained
with Annexin VꢀFITC and propidium iodide (PI), followed by flow cytometry
analysis.
To understand the mechanism of 7hꢀinduced apoptosis, we further examined the
changes of the intracellular proteins related to apoptosis such as caspaseꢀ3, and
poly(ADPꢀribose)polymerase (PARP) in HCTꢀ8 cells treated with 7h (0, 2.5, 5, and
10 ꢁM). As shown in Figure 5, 7h significantly induced the cleavage of both caspase
3 and PARP in a doseꢀdependent manner in the cells. Overall, our results confirm that
compound 7h inhibits the growth of CRC cells by inducing apoptosis in addition to its
effects on cell cycle.
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Fig. 5. Effect of 7h on the expression of apoptotic markers. The relative protein
expression levels of PARP and Caspase 3 were measured following 7h treatment in
HCTꢀ8 cells. Cells were treated with increasing concentrations of 7h or control (0.1%
DMSO) for 24 h and immunoblotting of the whole cell lysate was carried out to
determine the protein levels.
Compound 7h increased ROS levels in CRC cells
Previous studies have demonstrated that the anticancer effect of chalcones is
associated with increase in ROS levels.35, 79, 80 To investigate whether induced
cytotoxic effect of 7h is related to ROS production, we measured ROS levels in
7hꢀtreated CRC cells by flow cytometry analysis. Again, we treated HCTꢀ8 with
different concentrations of 7h, and measured the ROS levels. As shown in Figure 6, a
dramatic increase (from 3.67 to 53.64) in the levels of ROS generation in HCTꢀ8 cells
was observed after treatment with 7h. These results indicate that the induction of cell
death by 7h may be due to its effect on ROS production.
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Fig. 6. Effect of compound 7h on ROS levels in CRC cells. Flow cytometry analysis
of HCTꢀ8 cells treated with the indicated concentrations of 7h for 24 h and stained
with the fluorogenic dye 2',7'ꢀdichlorofluorescin diacetate (DCFHꢀDA).
In vivo anticancer activity of 7h
To evaluate the in vivo anticancer activity of 7h, BALB/c nude mice were
inoculated subcutaneously with HCTꢀ8 cells. After the establishment of solid tumor,
the mice were randomly treated intraperitoneally with 7h (60 mg/kg) or the vehicle
consisting of Tween 80/PBS (5:95) daily for 32 consecutive days, respectively.
Treatment with 60 mg/kg of 7h significantly reduced the volume of implanted colon
tumors (P < 0.001 vs. the vehicleꢀtreated control, Figs. 7A and 7B). More so, the
tumor weights in the mice treated with 7h at 60 mg/kg were 42% (w/w) less than that
in the vehicleꢀtreated controls (1.95 ± 0.42 g vs 1.13 ± 0.07 g, P < 0.001, Fig. 7C)
while the body weights of the mice were not significantly changed by treatment with
7h (Fig. 7D). Together, our data clearly demonstrated that 7h inhibited the growth of
implanted tumors in vivo with little offꢀtarget cell toxicity.
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Fig. 7. Anticancer effects of 7h in mice inoculated with HCTꢀ8 cells. (A) Images of
the tumors. The effect of intraperitoneal 7h (60 mg/kg) on tumor volumes (B), tumor
weight (C) and body weight (D) in a HCTꢀ8 xenograft model. Data are expressed as
the mean ± SD from each group of mice (n = 6 per group). ( ∗∗∗) P < 0.001 vs the
vehicleꢀtreated.
Conclusion
In summary, we have designed and synthesized a novel group of hybrids derived
from aspirin and chalcones 7a-p. We found that compound 7h had potent and
selective antiꢀproliferative activity against CRC cells in vitro. In addition, treatment
with 7h induced CRC cell cycle arrest at G1 phase and apoptosis in vitro, increased
the relative levels of cleaved caspase 3 and PARP as well as the ROS in HCTꢀ8 cells.
Finally, treatment with 7h significantly inhibited the growth of implanted CRC cancer
in mice. These findings may provide a proof of principle that 7h may be potential
chemotherapeutic agents for the intervention of CRC.
Experimental protocols
Chemical analysis
All chemicals were obtained from commercial suppliers and used without further
purification unless noted specifically. Analytical thin layer chromatography was
1
13
visualized by ultraviolet light at 254 nM. H and C NMR spectra were recorded on
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Bruker Avance 400, and tetramethylsilane (TMS) was used as a reference. Data for 1H
are reported as follows: chemical shift (ppm), and multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet). Data for 13C NMR are reported as ppm.
High resolution mass spectrometry (HRMS) spectra were recorded on an Agilent
Technologies LC/MSD TOF instrument. Aspirin 2 and αꢀbromoacetophenones 4a-f
were commercially available.
Synthesis of intermediate 3
Intermediate 3 were prepared according to the method described previously81 wtih
minor modification. To a solution of aspirin (180 mg, 1 mmol) in anhydrous CH2Cl2
(2 mL), SOCl2 (1 mL) was added, and the mixture was refluxed under nitrogen for 2h.
The reaction solution was evaporated in vacuo to give corresponding crude
intermediate 3, which was used in the next without further purification.
Synthesis of intermediates 5a-f
Intermediates 5a-f were prepared according to the method described previously
82ꢀ85
wtih minor modification. Triphenyl phosphine (PPh3, 1 mmol) was dissolved in
anhydrous THF (2 mL). To this mixture was added αꢀbromoacetophenones 4a-f (1
mmol). The mixture was stirred at room temperature for 16h. The resulting white
precipitate was collected by filtration, washed with n-hexane (3 × 20 mL). The white
solid was dissolved in MeOH (5 mL). To this solution was added KOH (10 mmol)
dissolved in H2O (5 mL). The mixture was allowed to stir at room temperature (rt) for
12h. The MeOH was removed under reduced pressure and the crude reaction mixture
extracted with DCM (3 × 20 mL). The combined organic layers were washed with
water (3 × 5 mL) and dried over anhydrous sodium sulfate. The solvent was
evaporated in vacuo to give corresponding crude intermediate 5a-f, which was used in
the next without further purification.
Synthesis of 6a-p
Chalcones 6a-p were prepared using a known synthetic method procedure.85
A
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mixture of a corresponding aldehyde (1.0 mmol) with intermediates 5a-f (1.0 mmol)
in anhydrous toluene (2.0 mL) was stirred at 60 ºC under nitrogen for 8 h. The solvent
was evaporated in vacuo to give corresponding crude intermediate 6a-p, which were
directly used in the next without further purification. (data in Supplementary
Materials)
General procedures for the preparation of 7a-p
A mixture of 6a-p (1 mmol) and TEA (2 mmol) in anhydrous CH2Cl2 (5 mL) was
stirred at 0 °C. Intermediate 3 (1 mmol) in anhydrous CH2Cl2 (2 mL) was sequentially
added dropwise, and the mixture was stirred at room temperature for 12 h. The
reaction solution was quenched by adding water and extracted with CH2Cl2. The
organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered,
and evaporated under reduced pressure. The resulting crude product was purified by
flash chromatography on silica gel column to provide the compounds 7a-p (53ꢀ88%),
respectively.
(E)ꢀ2ꢀ(3ꢀoxoꢀ3ꢀ(pꢀtolyl)propꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7a). The title
compound was prepared according to the general procedure, as described above in 66%
1
yield. HꢀNMR (400 MHz, CDCl3) δ 8.47 (d, J = 6.8 Hz, 1H), 8.09 (d, J = 15.6 Hz,
1H), 8.01ꢀ7.99 (m, 3H), 7.88 (t, J = 7.2, 7.6 Hz, 1H), 7.75ꢀ7.52 (m, 4H), 7.45ꢀ7.36 (m,
13
4H), 2.57 (s, 3H), 2.48 (m, 3H); CꢀNMR (100 MHz, CDCl3) δ 189.9, 169.7, 162.6,
151.4, 149.5, 143.6, 137.7, 135.3, 134.9, 132.2, 131.3, 129.3, 128.7, 128.5, 127.9,
126.6, 126.4, 124.6, 124.2, 123.4, 122.0, 21.7, 21.0; HRMS (ESI): calcd for C25H21O5
[M + H]+ 401.1389, found 401.1395.
(E)ꢀ2ꢀ(3ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7b).
The title compound was prepared according to the general procedure, as described
1
above in 77% yield. HꢀNMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.0 Hz, 1H),
7.94ꢀ7.90 (m, 3H), 7.81 (d, J = 6.8 Hz, 1H), 7.69 (t, J = 8.0, 7.2 Hz, 1H), 7.57 (d, J =
16.0 Hz, 1H, COCH=CH), 7.50ꢀ7.41 (m, 2H), 7.35 (6.8, 8.0 Hz, 1H), 7.28ꢀ7.23 (m,
2H), 6.88 (d, J = 8.0 Hz, 2H), 3.83 (s, 3H, ArꢀOCH3), 2.31 (s, 3H, CH3CO); 13CꢀNMR
(100 MHz, CDCl3) δ 188.6, 169.7, 163.4, 162.7, 151.4, 149.4, 137.2, 135.0, 132.2,
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131.2, 130.9, 130.7, 128.5, 128.0, 126.7, 126.4, 124.4, 124.3, 123.4, 122.1, 113.8,
55.4, 21.0; HRMS (ESI): calcd for C25H21O6 [M + H]+ 417.1338, found 417.1344.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7c). The
title compound was prepared according to the general procedure, as described above
1
in 80% yield. HꢀNMR (400 MHz, CDCl3) δ 8.60 (d, J = 7.6 Hz, 1H), 8.20 (d, J =
15.6 Hz, 1H), 8.15ꢀ8.11 (m, 3H), 8.05 (t, J = 7.6, 8.0 Hz, 1H), 7.85ꢀ7.65 (m, 6H),
7.61ꢀ7.56 (m, 2H), 2.63 (s, 3H); 13CꢀNMR (100 MHz, CDCl3) δ 189.4, 169.6, 162.6,
151.4, 149.5, 139.1, 138.8, 136.1, 135.1, 132.2, 131.6, 130.0, 128.8, 128.7, 127.7,
126.7, 126.4, 124.3, 124.2, 123.4, 121.9, 21.0; HRMS (ESI): calcd for C24H18ClO5 [M
+ H]+ 421.0843, found 421.0849.
(E)ꢀ2ꢀ(3ꢀ(4ꢀbromophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7d). The
title compound was prepared according to the general procedure, as described above
1
in 60% yield. HꢀNMR (400 MHz, CDCl3) δ 8.43 (d, J = 7.6 Hz, 1H), 8.03 (d, J =
16.0 Hz, 1H, COCH=CH), 7.95 (d, J = 7.6 Hz, 1H), 7.90ꢀ7.88 (m, 3H), 7.68ꢀ7.50 (m,
6H), 7.42 (t, J = 8.0 Hz, 2H), 2.46 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ
189.7, 169.7, 162.6, 151.4, 149.5, 138.9, 136.5, 135.1, 132.2, 131.8, 131.6, 130.1,
128.7, 127.8, 127.6, 126.8, 126.4, 126.3, 124.3, 124.2, 123.4, 121.9, 21.0; HRMS
(ESI): calcd for C24H18BrO5 [M + H]+ 465.0338, found 465.0345.
(E)ꢀ2ꢀ(3ꢀ(4ꢀcyanophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7e). The
title compound was prepared according to the general procedure, as described above
1
in 53% yield. HꢀNMR (400 MHz, CDCl3) δ 8.23 (d, J = 7.6 Hz, 1H), 7.88ꢀ7.73 (m,
5H), 7.60 (d, J = 7.6 Hz, 2H), 7.52 (t, J = 8.0 Hz, 1H), 7.44ꢀ7.36 (m, 3H), 7.26 (d, J =
7.6 Hz, 2H), 2.29 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 190.0, 169.6,
162.5, 151.5, 149.6, 141.1,149.2, 135.3, 132.3, 132.1, 132.0, 129.4, 128.9, 128.8,
127.3, 126.9, 126.4, 124.4, 124.2, 123.5, 121.8, 118.0, 115.6, 21.0; HRMS (ESI):
calcd for C25H18NO5 [M + H]+ 412.1185, found 412.1198.
(E)ꢀ2ꢀ(3ꢀ(4ꢀnitrophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate (7f). The
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title compound was prepared according to the general procedure, as described above
in 67% yield. 1HꢀNMR (400 MHz, DMSOꢀd6) δ 8.27ꢀ8.20 (m, 6H), 7.92 (d, J = 15.6
Hz, 1H, COCH=CH), 7.83 (t, J = 6.8, 8.0 Hz, 1H), 7.73 (d, J = 15.6 Hz, 1H,
COCH=CH), 7.61 (t, J = 6.8, 8.0 Hz, 1H), 7.53 (t, J = 8.0, 6.8 Hz, 1H), 7.46 (t, J =
7.6, 7.2 Hz, 1H), 7.40ꢀ7.35 (m, 2H), 2.22 (s, 3H, CH3CO); 13CꢀNMR (100 MHz,
DMSOꢀd6) δ 189.0, 169.6, 162.8, 151.2, 150.2, 149.9, 142.2, 138.3, 136.0, 132.8,
132.2, 130.2, 128.9, 127.4, 127.3, 127.1, 124.9, 124.6, 124.2, 123.9, 121.9, 21.1;
HRMS (ESI): calcd for C24H18NO7 [M + H]+ 432.1083, found 432.1094.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀ5ꢀmethoxyphenyl 2ꢀacetoxybenzoate
(7g). The title compound was prepared according to the general procedure, as
described above in 88% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.38 (d, J = 8.0 Hz, 1H),
7.96 (m, 5H), 7.56ꢀ7.35 (m, 5H), 7.02 (d, J = 8.0 Hz, 1H), 6.90 (s, 1H), 3.98 (s, 3H,
ArꢀOCH3), 2.42 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 189.5, 169.7, 162.4,
151.5, 150.9, 138.9, 136.4, 135.2, 132.2, 129.9, 128.7, 126.4, 124.3, 121.8, 121.6,
120.1, 113.3, 108.6, 55.8, 21.0; HRMS (ESI): calcd for C25H20ClO6 [M + H]+
451.0948, found 451.0955.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀ4ꢀmethoxyphenyl 2ꢀacetoxybenzoate
(7h). The title compound was prepared according to the general procedure, as
described above in 78% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.51 (d, J = 8.0 Hz, 1H),
8.06ꢀ8.04 (m, 3H), 7.96 (t, J = 8.0 Hz, 1H), 7.70ꢀ7.66 (m, 2H), 7.59ꢀ7.57 (m, 2H),
7.50ꢀ7.48 (m, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 4.13 (s, 3H,
ArꢀOCH3), 2.55 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 189.5, 169.7, 163.0,
157.6, 151.4, 143.1, 139.1, 138.9, 136.0, 135.0, 132.1, 130.0, 128.8, 128.3, 126.4,
124.4, 124.3, 124.2, 122.0, 117.2, 112.9, 55.8, 21.0; HRMS (ESI): calcd for
C25H20ClO6 [M + H]+ 451.0948, found 451.0953.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀ4ꢀfluorophenyl
2ꢀacetoxybenzoate
(7i). The title compound was prepared according to the general procedure, as
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described above in 59% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.14 (d, J = 7.2 Hz, 1H),
7.72ꢀ7.67 (m, 3H), 7.60 (t, J = 7.2, 8.0 Hz, 1H), 7.37ꢀ7.29 (m, 3H), 7.23 (d, J = 8.0 Hz,
2H), 7.13ꢀ7.07 (m, 3H), 2.19 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 188.9,
169.6, 162.6, 160.4 (d, J = 245 Hz), 151.4, 145.4, 139.3, 137.4, 135.8, 135.2, 132.1,
130.4, 130.0, 129.3, 128.9, 126.4, 125.0, 124.9, 124.3, 121.7, 118.3 (d, J = 24.4 Hz),
114.4 (d, J = 24.4 Hz), 21.0; HRMS (ESI): calcd for C24H17ClFO5 [M + H]+ 439.0749,
found 439.0757.
(E)ꢀ4ꢀchloroꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate
(7j). The title compound was prepared according to the general procedure, as
described above in 70% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.39 (d, J = 8.0 Hz, 1H),
7.99ꢀ7.84 (m, 5H), 7.63ꢀ7.55 (m, 3H), 7.50 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 8.0, 7.6 Hz,
2H), 2.44 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 188.8, 169.6, 162.4, 151.5,
147.9, 139.4, 137.1, 135.8, 135.3, 132.2, 132.1, 131.2, 130.0, 129.3, 128.9, 128.0,
126.5, 125.0, 124.8, 124.3, 121.6, 21.0; HRMS (ESI): calcd for C24H17Cl2O5 [M + H]+
455.0453, found 455.0459.
(E)ꢀ4ꢀbromoꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate
(7k). The title compound was prepared according to the general procedure, as
described above in 83% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.13 (d, J = 8.0 Hz, 1H),
7.79 (s, 1H), 7.73ꢀ7.66 (m, 3H), 7.60 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H),
7.37ꢀ7.29 (m, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.04 (d, J = 8.0
Hz, 1H), 2.18 (s, 3H); 13CꢀNMR (100 MHz, CDCl3) δ 188.7, 169.6, 162.3, 151.5,
148.4, 139.4, 137.0, 135.8, 135.3, 134.2, 132.1, 131.0, 130.0, 129.7, 128.9, 126.5,
125.1, 125.0, 124.3, 121.6, 119.9, 21.0; HRMS (ESI): calcd for C24H17BrClO5 [M +
H]+ 498.9948, found 498.9959.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀ6ꢀmethoxyphenyl 2ꢀacetoxybenzoate
(7l). The title compound was prepared according to the general procedure, as
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described above in 65% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.26 (d, J = 7.2 Hz, 1H),
7.85ꢀ7.78 (m, 3H), 7.66 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 15.6 Hz, 1H, COCH=CH),
7.38 (t, J = 8.0, 7.2 Hz, 1H), 7.31ꢀ7.19 (m, 5H), 7.01 (d, J = 8.0 Hz, 1H), 3.78 (s, 3H,
ArꢀOCH3), 2.25 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 189.3, 169.5, 161.9,
151.8, 151.4, 139.1, 139.0, 138.8, 136.1, 134.8, 132.4, 130.0, 128.8, 127.0, 126.3,
124.4, 124.3, 122.0, 119.8, 114.1, 56.1, 21.0; HRMS (ESI): calcd for C25H20ClO6 [M
+ H]+ 451.0948, found 451.0953.
(E)ꢀ2ꢀ(3ꢀ(4ꢀchlorophenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)ꢀ4ꢀmethylphenyl 2ꢀacetoxybenzoate
(7m). The title compound was prepared according to the general procedure, as
described above in 75% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.36 (d, J = 7.6 Hz, 1H),
7.95ꢀ7.90 (m, 3H), 7.81 (t, J = 6.8, 8.0 Hz, 1H), 7.68 (s, 1H), 7.57ꢀ7.50 (m, 2H),
7.44ꢀ7.37 (m, 3H), 7.33 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 2.53 (s, 3H,
ArꢀCH3), 2.39 (s, 3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 189.5, 169.7, 162.8,
151.4, 147.4, 139.0, 136.5, 136.1, 135.0, 132.4, 132.2, 130.0, 129.0, 128.8, 127.2,
126.4, 124.3, 123.9, 123.1, 122.0, 21.0; HRMS (ESI): calcd for C25H20ClO5 [M + H]+
435.0999, found 435.1006.
(E)ꢀ4ꢀmethoxyꢀ2ꢀ(3ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl
2ꢀacetoxybenzoate (7n). The title compound was prepared according to the general
procedure, as described above in 79% yield. 1HꢀNMR (400 MHz, CDCl3) δ 7.90 (d, J
= 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 16.0 Hz, 1H, COCH=CH), 7.30 (t,
J = 7.6, 8.0 Hz, 1H), 7.13 (d, J = 16.0 Hz, 1H, COCH=CH), 7.03 (t, J = 7.6, 8.0 Hz,
1H), 6.89 (s, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.63 (dd, J = 8.0
Hz, 1H), 6.49 (d, J = 8.0 Hz, 2H), 3.49 (s, 3H, ArꢀCH3), 3.45 (s, 3H, ArꢀCH3), 1.92 (s,
3H, CH3CO); 13CꢀNMR (100 MHz, CDCl3) δ 188.7, 169.7, 163.4, 163.1, 157.6, 151.3,
143.0, 137.3, 134.9, 132.2, 130.9, 130.6, 128.7, 126.4, 124.6, 124.2, 124.1, 122.1,
116.8, 113.8, 112.7, 55.7, 55.4, 21.0; HRMS (ESI): calcd for C26H23O7 [M + H]+
447.1444, found 447.1453.
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(E)ꢀ4ꢀbromoꢀ2ꢀ(3ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate
(7o). The title compound was prepared according to the general procedure, as
described above in 85% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.0 Hz, 1H),
7.92ꢀ7.90 (m, 3H), 7.78 (d, J = 15.6 Hz, 1H, COCH=CH), 7.65 (t, J = 7.6, 8.0 Hz,
1H), 7.53ꢀ7.49 (m, 2H), 7.38 (t, J = 6.8, 8.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.11 (d,
J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 3.80 (s, 3H, ArꢀCH3), 2.27 (s, 3H, CH3CO);
13CꢀNMR (100 MHz, CDCl3) δ 187.9, 169.6, 163.6, 162.4, 151.4, 148.3, 135.5, 135.2,
133.8, 132.2, 130.9, 130.8, 130.4, 130.1, 126.4, 125.3, 125.1, 124.3, 121.7, 119.8,
113.8, 55.5, 21.0; HRMS (ESI): calcd for C25H20BrO6 [M + H]+ 495.0443, found
495.0449.
(E)ꢀ4ꢀchloroꢀ2ꢀ(3ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenꢀ1ꢀyl)phenyl 2ꢀacetoxybenzoate
(7p). The title compound was prepared according to the general procedure, as
described above in 74% yield. 1HꢀNMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.0 Hz, 1H),
7.92 (d, J = 8.0 Hz, 2H), 7.81ꢀ7.75 (m, 2H), 7.67 (t, J = 6.8, 8.0 Hz, 1H), 7.52 (d, J =
15.6, 1H, COCH=CH), 7.41ꢀ7.40 (m, 2H), 7.19 (t, J = 6.4, 8.0 Hz, 2H), 6.87 (d, J =
8.0 Hz, 2H), 3.83 (s, 3H, ArꢀOCH3), 2.27 (s, 3H, CH3CO); 13CꢀNMR (100 MHz,
CDCl3) δ 183.3, 164.9, 158.8, 157.7, 146.7, 143.0, 130.9, 130.4, 127.4, 127.4, 126.2,
126.1, 125.7, 124.9, 123.1, 121.7, 120.6, 120.0, 119.5, 116.9, 109.1, 50.7, 16.2;
HRMS (ESI): calcd for C25H20ClO6 [M + H]+ 451.0948, found 451.0956.
Biological assays
MTT assay
HCTꢀ8, DLDꢀ1, and CCD841 cell lines were purchased from American Tissue
Culture Collection (ATCC, Rockville, MD, USA). HCTꢀ8, DLDꢀ1 and CCD841 cells
were seeded in 96ꢀwell plates, then treated with vehicle alone or tested compounds for
72 h. Then 20 ꢁL of MTT (5 mg/mL, in PBS) was added to each well and further
incubated for another 4 h. The MTT formazan formed by viable cells was dissolved in
DMSO (150 ꢁL), and absorbance was measured using a microplate reader (570 nm).
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Cell cycle analysis
HCTꢀ8 cells were treated with 7h at concentrations of 0, 2.5, 5 and 10 ꢁM for 24 h,
respectively. The cells were harvested, fixed with 70% ethanol for 2 h, and incubated
with PI/RNase staining buffer for 15 min at room temperature. The DNA content in
the different groups of cells was assessed by flow cytometry.
Apoptosis Analysis
HCTꢀ8 cells were incubated in sixꢀwell plates (1 × 105/well) and treated with
DMSO (1%), and compound 7h at concentrations of 2.5, 5 and 10 ꢁM for 24 h,
respectively. The cells were collected, washed with PBS, and stained with
FITCꢀAnnexinꢀV and PI. Apoptosis was determined by flow cytometry.
Western Blotting
The assay was performed according to the method described previously wtih
minor modification.86 HCTꢀ8 cells were incubated in sixꢀwell plates (1 ×106/well)
overnight and treated with vehicle DMSO (0.1%, v/v) alone or 7h for 24 h. The cells
were harvested and lysed at 4 °C for 30 min in a lysis buffer. The cell lysates were
centrifuged at 15000 rpm for 15 min at 4 °C, and the supernatants were collected. The
protein concentration in the cell lysates was determined using the bicinchonininc acid
protein assay kit. Protein samples were separated by SDSꢀpolyacrylamide gel
electrophoresis (7.5% gel, 20 ꢁg per lane) and then transferred to polyvinylidene
difluoride (PVDF) membranes. The membranes were blocked with skim milk (5%) in
Trisꢀbuffered saline containing 0.05% Tween 20 and sequentially incubated with
primary antibodies [antiꢀcaspase 3, anticleaved caspase 3, antiꢀPARP, anticleaved
PARP, antiꢀGAPDH (Cell Signaling, Boston, MA)] and followed by enhanced
chemiluminescence detection.
Determination of ROS generation
The assay was performed according to the method described previously wtih
minor modification.87 HCTꢀ8 cells treated with different concentrations of 7h (0, 2.5,
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5, 10 ꢁM) for 24 h. After being loaded with 10 ꢁM DCFHꢀDA at 37 C for 20 min and
washed three times with PBS buffer to remove excess dye. The fluorescence intensity,
used as a fluorescent indicator of intracellular ROS, was recorded in a plate reader
and measured with the flow cytometer with an excitation/emission (Ex/Em) frequency
of 488/530 nm.
In vivo tumor growth inhibition experiment
The assay was performed according to the method described previously wtih
minor modification.88 Individual male BALB/c nude mice (5ꢀ6 weeks, 18ꢀ20 g,
Experimental Animal Research Center, Chinese Academy of Military Medical
Sciences) were inoculated subcutaneously with HCTꢀ8 cells (1 × 107/mouse,
American Tissue Culture Collection, ATCC, Rockville, MD, USA). After the solid
tumors were established and allowed to reach 180ꢀ200 mm3, the mice were
randomized and treated intraperitoneally with 7h (60 mg/kg) or the same volume of
vehicle consisting of Tween 80/PBS (5:95) daily for 32 consecutive days. The tumor
growth was recorded every 4 days from the measurement of length and width using a
vernier caliper and calculated as tumor volumes (TV) with the following formula: TV
(mm3) = width2 (length/2). The tumor growth inhibition rate (weight per unit weight,
w/w, %) was calculated as described previously.88 The experimental protocols were
evaluated and approved by the Ethics Committee of the Hubei University of Chinese
Medicine. All animal experiments were complied with the International Guiding
Principles for Animals Research (A CIOMS Ethical Code for Animal Experimentation,
WHO Chronicle, 39 (2): 51ꢀ56, 1985).
Conflicts of Interest
There are no conflicts to declare.
Acknowledgments
This work was supported by grants from Qing Miao Plan of Hubei University of
Chinese Medicine (2016ZZX013).
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Table of contents entry
Novel hybrids derived from aspirin and chalcones potently suppress
colorectal cancer in vitro and in vivo
Shan Lu, a, * Obinna N Obianom, b and Yong Ai b, **
A novel group of hybrids derived from aspirin and chalcones were designed and
synthesized. The most active compound 7h had potent and selective anti-proliferative
activity against CRC cells in vitro. 7h induced CRC cell cycle arrest at G1 phase and
apoptosis in vitro, increased the relative levels of cleaved caspase 3 and PARP as well
as the ROS in HCT-8 cells. 7h significantly inhibited the growth of implanted CRC
cancer in mice.