Synthesis and biological evaluations of monocarbonyl curcumin inspired pyrazole analogues as potential anti-colon cancer agent

Article abstract of DOI:10.2147/DDDT.S244865

Purpose: The monocarbonyl analogs of curcumin (MCACs) have been widely studied for their promising antitumor activity. Pyrazole is a five-membered aromatic heterocyclic system with various bioactivities incorporated frequently in drugs. However, few of MCACs inspired pyrazole analogues were investigated. To search for more potent cytotoxic agents based on MCACs, a series of new 1,5-diaryl/heteroaryl-1,4-pentadien-3-ones inspired pyrazole moiety was synthesized and evaluated on their anti-colon cancer activities. Methods: Fifteen new compounds were synthesized and characterized by spectral datum, and then they were tested preliminarily by MTT assay for their cytotoxic activities against a panel of four human cancer cell lines, namely, gastric (SGC-7901), liver (HepG2), lung (A549), and colon (SW620) cancer cells. Compound 7h exhibited excellent selectivity and outstanding anti-proliferation activity against SW620 cells among these 15 compounds. Further, the mechanisms were investigated by transwell migration and invasion assay, clonogenic assay, cell apoptosis analysis, cell cycle analysis, Western blot analysis. Results: The IC₅₀ value of 7h against SW620 cells was 12 nM, being more potent than curcumin (IC₅₀ = 9.36 μM), adriamysin (IC₅₀ = 3.28 μM) and oxaliplatin (IC₅₀ = 13.33 μM). Further assays showed that 7h inhibited SW620 cell migration, invasion and colony formation obviously, which was due to its ability to induce cell cycle arrest in the G2/M and S phases and apoptosis. Western blot assay revealed that 7h decreased the protein expression of ATM gene, which may primarily contribute to its anticancer activity against SW620 cells. Conclusion: A new MCACs 7h was synthesized and found to exhibit excellent anti-proliferation activity against SW620 cells. Further studies indicated that 7h exerted its anticancer activity against SW620 cells probably via decreasing the ATM protein expression. The present study suggested that 7h was a promising candidate as an anti-colon cancer drug for future development.

Full text of DOI:10.2147/DDDT.S244865

Drug Design, Development and Therapy
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Open Access Full Text Article
O R I G I N A L R E S E A R C H
Synthesis and Biological Evaluations of
Monocarbonyl Curcumin Inspired Pyrazole
Analogues as Potential Anti-Colon Cancer Agent
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Zhenli Min1,2,
*
Purpose: The monocarbonyl analogs of curcumin (MCACs) have been widely studied for
Yue Zhu1,3,
*
their promising antitumor activity. Pyrazole is a five-membered aromatic heterocyclic system
with various bioactivities incorporated frequently in drugs. However, few of MCACs
inspired pyrazole analogues were investigated. To search for more potent cytotoxic agents
based on MCACs, a series of new 1,5-diaryl/heteroaryl-1,4-pentadien-3-ones inspired pyr-
azole moiety was synthesized and evaluated on their anti-colon cancer activities.
Methods: Fifteen new compounds were synthesized and characterized by spectral datum,
and then they were tested preliminarily by MTT assay for their cytotoxic activities against
a panel of four human cancer cell lines, namely, gastric (SGC-7901), liver (HepG2), lung
(A549), and colon (SW620) cancer cells. Compound 7h exhibited excellent selectivity and
outstanding anti-proliferation activity against SW620 cells among these 15 compounds.
Further, the mechanisms were investigated by transwell migration and invasion assay,
clonogenic assay, cell apoptosis analysis, cell cycle analysis, Western blot analysis.
Results: The IC50 value of 7h against SW620 cells was 12 nM, being more potent than
curcumin (IC50 = 9.36 μM), adriamysin (IC50 = 3.28 μM) and oxaliplatin (IC50 = 13.33 μM).
Further assays showed that 7h inhibited SW620 cell migration, invasion and colony forma-
tion obviously, which was due to its ability to induce cell cycle arrest in the G2/M and
S phases and apoptosis. Western blot assay revealed that 7h decreased the protein expression
of ATM gene, which may primarily contribute to its anticancer activity against SW620 cells.
Conclusion: A new MCACs 7h was synthesized and found to exhibit excellent anti-
proliferation activity against SW620 cells. Further studies indicated that 7h exerted its
anticancer activity against SW620 cells probably via decreasing the ATM protein expression.
The present study suggested that 7h was a promising candidate as an anti-colon cancer drug
for future development.
Xing Hong1
Zhijun Yu1,2
Min Ye1,2
Qiong Yuan1,2
Xiamin Hu4
1Hubei Province Key Laboratory of
Occupational Hazard Identification and
Control, Wuhan University of Science
and Technology, Wuhan 430081, People’s
Republic of China; 2New Medicine
Innovation and Development Institute,
College of Medicine, Wuhan University of
Science and Technology, Wuhan 430081,
People’s Republic of China; 3Stem Cell
Lab, Puren Hospital Affiliated to Wuhan
University of Science and Technology,
Wuhan, Hubei 430081, People’s Republic
of China; 4College of Pharmacy, Shanghai
University of Medicine & Health Sciences,
Shanghai, People’s Republic of China
*These authors contributed equally to
this work
Keywords: 1, 5-diheteroarylpenta-1, 4-dien-3-one, colon cancer, cell proliferation, cell
apoptosis, ATM gene
Introduction
Colon cancer is the third most common cancer and the second leading cause of
cancer death worldwide.1 Although target therapies and immunotherapy have
achieved progresses in recent years, their applications and efficacies are still far
from satisfactory for an advanced-stage colon cancer.2 Chemotherapy is still con-
sidered the most effective for colon cancer.3 However, cytotoxic drugs, such as
cisplatin and paclitaxel, can lead to side effects and chemoresistance, imposing
Correspondence: Xiamin Hu; Qiong Yuan
Tel +862165883592; +86 27 68893640
Email huxm@sumhs.edu.cn;
yuanqiong@wust.edu.cn
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you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For
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http://doi.org/10.2147/DDDT.S244865

Min et al
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a financial burden on patients and impairing their quality rings. These MCACs display multiple biological activities,
of life. Therefore, development of a novel potent anti- such as antitumor,16–21 anti-inflammatory,22–24 antioxidant25
colon cancer agent with a low toxicity is urgently required. and neuroprotection.25 Meanwhile, most of the MCACs
Natural products play an important role in a process of
drug discovery, and many drugs used clinically are of natural
origins. Curcumin is a primary bioactive compound isolated
show better stabilities and activities than curcumin does in
both in vivo and in vitro model.
Among these derivatives, various aryl or heteroaryl rings
from the turmeric, a dietary spice made from the rhizome of were incorporated into the 1, 5-position of MCACs to
Curcuma longa (Figure 1). It possesses versatile biological explore bioactivities, including pyrazine,17 chromone,19
activities. However, curcumin has as yet achieved a limited indole,20 imidazole,21,26 quinoline,27 quinazoline,28 and
success clinically although it had been studied in a number of piperidone29 moieties, nevertheless, few of which were
clinical trials,4 and even there have been some controversies related to pyrazolyl group. Pyrazole, as a five-membered
about its potential as a pharmaceutical agent recently.5–8 The aromatic heterocyclic system, has attracted considerable
nature of instability as well as pharmacokinetic deficiencies attentions in development of pharmacological molecules,
of curcumin resulted from an unstable β-diketone moiety are and many marketed drugs (Figure 1) bearing this moiety
one of the reasons for the fails and controversies. In spite of display a variety of biological activities, such as anti-tumor
these shortcomings, curcumin has still aroused interests of (ruxolitinib, crizotinib), anti-inflammatory (Celecoxib), and
many scientists to overcome them as it is safe and a dietary antiobesity (Rimonabant).30,31 Therefore, we envisioned that
spice in some countries. The problem can be addressed in incorporating substituted pyrazole structure to replace the
part by a chemical structural modification of curcumin above-mentioned aryl or heteroaryl rings of MCACs may be
besides a pharmaceutical way.9,10 Indeed, great attempts beneficial to search for new anticancer drugs. In an effort to
have already been made by researchers to chemical modifi- discover chemical entities active against colon cancer, this
cations, and a large number of curcumin analogues have been background motivated us to introduce pyrazole moiety to
synthesized.11–15 In this process, a major chemical class, one terminal of MCACs and investigate their bioactivities
namely the MCACs, evolves that is characterized by 1, (Figure 1). Presented here was a study on the synthesis and
5-diaryl/heteroaryl penta-1, 4-dien-3-one and incorporating anti-cancer evaluations of a series of new MCACs which
a range of alternative substituent groups into the terminal aryl inspired a pyrazole moiety.
O
O
O
N
N
OCH3
OH
H3CO
HO
OCH3
OH
1 Curcumin
2
CH3
N
O
O
S
O
H2N
N
OCH3
OCH3
N
N
N
N
N
HN
HN
OCH3
CF3
5 Celecoxib
4 Ruxolitinib
3
NH2
N
Cl
O
F
R'
O
N
N
NH
N
R
Cl
7 target compounds
6 Crizotinib
HN
Figure 1 Chemical structures of Curcumin, MCACs, drugs and target compounds containing pyrazolyl ring.
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solution was neutralized by saturated NaHCO3 solution and
kept stirring for 30 min. The solid precipitated, filtered. The
residue was recrystallized from ethanol to generate 10 as
a light yellow solid, m.p: 145.0–146.5°C, yield: 38.2%.
Materials and Methods
Chemistry
All reagents and solvents were obtained from commer-
cially available sources and were used without further
purification. Reaction progress was monitored using ana-
lytical thin-layer chromatography (TLC) on precoated
silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-
Dao, China) plates and spots were detected under UV light
(254 nm). Melting points were determined on a WRS-2B
digital melting point apparatus and uncorrected. IR spectra
were recorded using a Nicolet 380 Fourier-transform infra-
red (FTIR) spectrophotometer (Thermo, USA) from KBr
General Procedure for Synthesis of 11a–11c
To a solution of 2-methoxyphenol, 3, 4-methylenedioxyphe-
nol or ethanol (27 mmol, 1.0 equiv) in dry DMF (20 mL) was
added KOH solid (1.73g, 29.7 mmol, 1.1 equiv) while stir-
ring. The mixture was stirred at 40°C for 1 h, to which 10
(5.96g, 27 mmol, 1.0equiv) was added. The solution was
stirred at 110°C for 5–6 h, and then cooled to ambient tem-
perature, poured into ice-water. The mixture was stirred for 15
min, followed by extraction with ethyl acetate (3×20 mL).
The combined organic layers were dried over Na2SO4 and
concentrated and purified by silica gel column chromatogra-
phy (hexanes/EtOAc = (10–6): 1) to give 11a–11c.
1
pellets. H NMR and 13C NMR spectra were recorded on
a Bruker AVANCE III-600 NMR spectrometer (Bruker
Biospin Co., Switzerland) with tetramethylsilane (TMS)
as the internal standard and CDCl3 as solvent and known
chemical shifts of residual proton signals of deuterated
solvents (1H NMR δ: 7.26 for CDCl3 and δ: 3.33 for H2
O) or carbon signals of deuterated solvents (13C NMR δ:
77.16 for CDCl3) as internal standard. MS (ESI) measure-
ment was conducted on an Agilent 1100 LC-MS spectro-
meter (Agilent, Palo Alto, USA).
5-(2-methoxyphenoxy)-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (11a)
1
A yellow solid, m.p: 73.4–74.5°C, 68.1% yield. H NMR
(600 MHz, CDCl3) δ: 9.84 (s, 1H, CHO), δ 7.65 (d, J = 7.8
Hz, 2H,H-2′, 6′), 7.35 (t, J = 7.9 Hz, 2H, H-3′, 5′), 7.24 (t,
J =7.4 Hz, 1H, H-4′), 7.10 (d, J = 8.3 Hz, 1H, H-6″), 7.05
(t, J = 7.4 Hz, 1H, H-4″), 7.02 (t, J = 7.8 Hz, 1H, H-5″),
6.95 (d, J = 8.2 Hz, 1H, H-3″), 3.83 (s, 3H, OCH3), 2.53
(s, 3H, CH3).
The synthetic route of target molecules 7a–7o was
depicted in Scheme 1. All the target compounds and
some intermediates were synthesized according to the
following procedures.
5-(benzo[d][1,3] dioxol-5-yloxy)-3-methyl-1-phenyl-
1H-pyrazole-4-carbaldehyde (11b)
A white solid, m.p:106.6–107.4°C, 48.6% yield. H-NMR
Synthesis of 1-phenyl-3-methyl-
5-pyrazolone 932
1
Tungstophosphoric acid (1% mol) was dissolved in 40 mL
water, and then phenylhydrazine (3 mmol) was added
under stirring. To the mixture was slowly added ethyl
acetoacetate (3 mmol) over a period of 20 min at room
temperature. The mixture was further heated under reflux
for 3 h. After completion of the reactions, the mixture was
cooled to room temperature and solid was filtered off and
washed with H2O (40 mL). The crude products were dried
and purified by recrystallization from ethanol to give 9 as
a yellow powder, m.p: 125.5–127.1°C, yield: 78%.
(600 MHz, CDCl3) δ: 9.58 (s, 1H, CHO), 7.63 (d, J = 7.7
Hz, 2H, H-2′, 6′), 7.43 (t, J = 7.9 Hz, 2H, H-3′, 5′), 7.34 (t,
J = 7.4 Hz, 1H, H-4′), 6.69 (d, J = 8.5 Hz, 1H, H-5″), 6.59
(s, 1H, H-2″), 6.46 (d, J = 5.9 Hz, 1H, H-6″), 5.96 (s, 2H,
OCH2O), 2.53 (s, 3H, CH3).
5-ethoxy-3-methyl-1-phenyl-1H-pyrazole
-4-carbaldehyde (11c)
A light yellow oil, 40.1% yield. 1H-NMR (600 MHz,
CDCl3) δ: 9.53 (s, 1H, CHO), 7.68 (d, J = 8.3 Hz, 2H,
H-2′, 6′), 7.43 (t, J = 7.7 Hz, 2H, H-3′, 5′), 7.33 (t, J = 7.4
Hz, 1H, H-4′), 4.02 (q, J = 7.0 Hz, 2H, OCH2), 2.53 (s,
3H, CH3), 1.31 (t, J = 7.1 Hz, 3H, CH3).
Synthesis of 5-chloro-3-methyl-1-phenyl-
1H-pyrazole-4-carbaldehyde 10
To a solution of 0.52 g compound9 (3 mmol, 1.0 equiv) in dry
DMF (2 mL) was added dropwise POCl3 (0.42 mL, 4.5 mmol,
General Procedure for Synthesis of Various
1.5 equiv) slowly while cooling in an ice-water bath. The (E)-4-phenylbut-3-en-2-ones13a–13f
mixture was stirred at 80°C for 2 h, and then cooled to room To a stirred solution of various benzaldehydes, 12 (3.6
temperature, followed by pouring into ice-water (50 mL). The mmol) in acetone (2 mL) was added 0.7 mL of 10%
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R1
Cl
NHNH2
O
N
N
iii
N
N
ii
i
CHO
N
N
CHO
11a-11c
9
8
10
11a R1=2-methoxyphenoxy,
11b R1= C2H5O,
11c R1=3,4-methylenedioxyphenoxy
O
O
R1
R2
R3
CHO
R2
R3
R2
R3
v
iv
N
N
R4
12
R4
R4
13a-13f
7a-7m
O
13a R2=H;
R3=H;
R4=H
R4=H
R2
R3
vi
13b R2=OCH3; R3=OH;
N
N
13a-13f
13c R2=OCH3; R3=OCH3; R4=OCH3
13d R2=OCH3; R3=OCH3; R4=H
13e R2=Br;
13f R2=Br;
R3=OH;
R3=OH;
R4=Br
R4=Hr
R4
7n-7o
7g-7i: R1=
7a: R1 =Cl ;
7b: R1 =Cl ;
7c: R1 = Cl;
7d: R1 =Cl;
7e: R1 =Cl;
7f: R1 =Cl;
R2=H;
R3=H;
R4=H
R2=OCH3; R3=OH; R4=H
7g: R2=OCH3; R3=OCH3; R4=H
7h: R2=OCH3; R3=OCH3; R4=OCH3
7i: R2=OCH3; R3=OH; R4=H
H3CO
R2=OCH3; R3=OCH3; R4=OCH3
R2=H;
R2=Br;
R2=Br;
R3=OCH3; R4=H
O
R3=OH;
R4=Br
R3=OH; R4=H
7j-7l: R1=
7j: R2=OCH3; R3=OCH3; R4=H
7k: R2=OCH3; R3=OCH3; R4=OCH3
7l: R2=OCH3; R3=OH; R4=H
7m: R1 =OC2H5; R2=OCH3; R3=OH; R4=H
7n: R2=OCH3; R3=OCH3; R4=OCH3
O
O
7o: R2=OCH3; R3=OH;
R4=H
O
Scheme 1 Synthesis of target molecules 7a-7o; Reagents and conditions: (i) CH3COCH2COOC2H5, tungstophosphoric acid, H2O, 100°C, 3h, 78%; (ii) POCl3, DMF, 80°C,
3h, 42%; (iii) C2H5OH or substituted phenol, KOH, 100°C; (iv) CH3COCH3, 10%NaOH, rt, 2–4h, 47%; (v) 10 or 11a-11c, 10%NaOH, C2H5OH, rt, 12h, 20%; (vi) 1-methyl-
1H-pyrazole-4-carbaldehyde, 10%NaOH, C2H5OH, rt, 12h, 17%.
aqueous NaOH solution at room temperature. The reaction
General Procedure for Synthesis of
was allowed to stir for 3–5 h till it was completed. The
Target Compounds (7a–7o)
reaction mixture was poured into ice-water (30mL) and
To a stirred solution of 13a–13f(2.0 mmol) and 11a–11c (2.2
neutralized by 5% aqueous HCl while keeping stirring for
mmol) in ethanol (10 mL) was added 0.9 mL of 10%
20 min. Then it was extracted with ethyl acetate
aqueous NaOH solution at room temperature. The reaction
(3×15 mL). The combined organic layers were dried over
was allowed to stir for 12–18 h till it was completed. The
Na2SO4 and concentrated and purified by silica gel column
reaction mixture was poured into ice-water (50mL) and
neutralized by 5% aqueous HCl while keeping stirring for
chromatography (hexanes/EtOAc = (8–5): 1) to give
13a–13f.
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20 min. Then it was extracted with ethyl acetate (20×3 mL). 140.34, 137.61, 132.10, 130.27, 129.13, 128.66, 125.40,
The combined organic layers were dried over Na2SO4, con- 124.96, 123.98, 114.17, 105.57, 60.98, 56.19, 14.33. ESI-
centrated and purified by silica gel column chromatography MS m/z: 439.14[M+H]+, calcd for C24H23ClN2O4: 438.13.
[hexanes/EtOAc = (7–3): 1] to give products, which were
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-(3, 4-dimethoxyphenyl) penta-1, 4-dien-3-one
(7d)
recrystallized from ethanol to afford 7a–7o.
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-phenylpenta-1, 4-dien-3-one (7a)
A
yellow solid, m.p: 137.9–139.8°C, 50.1%
yield.1H-NMR (600 MHz, CDCl3) δ: 7.68 (d, J = 16.0
Hz, 2H, vinyl H), 7.54 (d, J = 7.5 Hz, 2H, H-2′, 6′), 7.49 (t,
J = 7.8 Hz, 2H, H-3′, 5′), 7.43 (t, J = 7.3 Hz, 1H, H-4′),
7.20 (dd, J = 8.2, 1.6 Hz, 1H, H-6″), 7.14 (d, J = 1.4 Hz,
1H, H-2″), 7.05 (d, J = 16.1 Hz, 1H, vinyl H), 6.90 (d, J =
15.9 Hz, 1H, vinyl H), 6.88 (d, J = 8.3 Hz, 1H, H-5″), 3.94
(s, 3H, OCH3), 3.92 (s, 3H, OCH3), 2.51 (s, 3H, CH3).
13C NMR (150 MHz, CDCl3) δ: 188.45, 151.35, 149.97,
149.22, 143.19, 137.64, 131.73, 129.12, 128.62, 127.76,
124.96, 124.22, 124.02, 123.16, 114.21, 111.06, 109.87,
55.98, 55.93, 14.33. IR (KBr, cm−1): 1657.6, 1591.1. ESI-
MS m/z: 409.13[M+H]+, calcd for C23H21ClN2O3: 408.12.
A yellow solid, m.p:123.8–124.5°C, 16.1% yield. 1H NMR
(600 MHz, CDCl3) δ: 7.74 (d, J = 15.9 Hz, 1H, vinyl H),
7.69 (d, J = 16.1 Hz, 1H, vinyl H), 7.63 (td, J = 5.4, 1.8
Hz, 2H, H-2′, 6′), 7.55 (dt, J = 7.3, 0.8 Hz, 2H, H-3′, 5′),
7.50 (td, J = 6.7, 1.6 Hz, 2H, H-2″, 6″), 7.45 (dt, J = 7.3,
1.3 Hz, 1H, H-4′), 7.43–7.41 (m, 3H, H-3″, 4″, 5″), 7.06
(d, J = 15.9 Hz, 1H, vinyl H), 7.05 (d, J = 16.1 Hz, 1H,
vinyl H), 2.52 (s, 3H, CH3). 13C NMR (600 MHz, CDCl3)
δ: 188.62, 150.01, 143.09, 137.62, 134.81, 132.15, 130.44,
129.13, 128.92, 128.66, 128.38, 125.69, 124.98, 124.32,
114.14, 14.34. IR (KBr, cm−1): 1651, 1621. ESI-MS m/z:
349.11[M+H]+, calcd for C21H17ClN2O: 348.10.
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-(3,5-dibromo-4-hydroxyphenyl)penta-
1,4-dien-3-one (7e)
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-(3-methoxy-4-methylphenyl) penta-1, 4-dien-
3-one (7b)
A yellow solid, m.p: 198.2–200.9°C, 56.5% yield. 1H-NMR
(600 MHz, CDCl3) δ: 7.72 (s, 2H, H-2″, 6″), 7.69 (d, J =
16.1 Hz, 1H, vinyl H), 7.55–7.52 (m, 3H, vinyl H, H-2′, 6′),
7.51–7.48 (m, 2H, H-3′, 5′), 7.44 (tt, J = 7.3, 1.3 Hz, 1H,
H-4′), 6.99 (d, J = 16.1 Hz, 1H, vinyl H), 6.91 (d, J = 15.8
Hz, 1H, vinyl H), 6.15 (s, 1H,Ar-OH), 2.50 (s, 3H, CH3).
13C-NMR (150 MHz, CDCl3) δ: 187.86, 150.92, 150.09,
139.69, 137.57, 132.57, 131.85, 130.01, 129.15, 128.82,
128.71, 125.74, 124.98, 124.17, 114.07, 110.45, 14.33. IR
(KBr, cm−1): 3068, 1643, 1574. ESI-MS m/z: 534.94[M
+H]+, calcd for C22H17Br2ClN2O2: 534.93.
1
A yellow solid, m.p:144.9–146.1°C, 11.5% yield. H NMR
(600 MHz, CDCl3) δ: 7.68 (d, J = 16.1 Hz, 1H, vinyl H),
7.67 (d, J = 15.8 Hz, 1H, vinyl H), 7.55 (d, J = 8.6 Hz, 2H,
H-2′, 6′), 7.50 (t, J =7.8Hz, 2H, H-3′, 5′), 7.44 (t, J=7.4 Hz,
1H, H-4′), 7.19 (d, J= 8.2, 1.7Hz, 1H, H-6″), 7.12 (d, J=
1.7Hz, 1H, H-2″), 7.06 (d, J = 16.1 Hz, 1H, vinyl H), 6.95
(d, J = 8.2 Hz,1H, H-5″), 6.89 (d, J = 15.8 Hz, 1H, vinyl H),
5.97 (s, 1H, Ar-OH), 3.96 (s, 3H, OCH3), 2.51 (s, 3H, CH3).
13C NMR (150 MHz, CDCl3) δ: 188.53, 149.98, 148.30,
146.86, 143.43, 137.65, 131.71, 129.12, 128.63, 127.35,
124.98, 124.27, 123.73, 123.45, 114.87, 114.22, 109.83,
55.99, 14.30. IR (KBr, cm−1): 3108, 1639. ESI-MS m/z:
395.12[M+H]+, calcd for C22H19ClN2O3: 394.10.
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-Pyrazol-
4-yl)-5-(3-bromo-4-hydroxyphenyl) penta-1, 4-dien-
3-one (7f)
(1E, 4E)-1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-(3, 4, 5-trimethoxyphenyl) penta-1, 4-dien-
1
A yellow solid, m.p:187.2–188.4°C, 50.5%yield. H NMR
3-one (7c)
(600 MHz, CDCl3) δ: 7.75 (d, J = 1.9 Hz, 1H, H-1″), 7.68 (d,
1
A yellow solid, m.p:87.5–88.7°C, 19.6% yield. H-NMR J = 16.1 Hz, 1H, vinyl H), 7.61 (d, J = 15.8 Hz, 1H, vinyl H),
(600 MHz, CDCl3) δ: 7.69 (d, J = 16.1 Hz, 1H, vinyl H), 7.55 (d, J = 7.9 Hz, 2H, H-2′, 6′), 7.50 (td, J = 8.2, 1.9 Hz, 2H,
7.64 (d, J = 15.8 Hz, 1H, vinyl H), 7.54 (d, J = 7.7 Hz, 2H, H-3′, 5′), 7.49 (d, J = 1.8 Hz, 1H, H-6″), 7.43 (t, J = 7.3 Hz,
H-2′, 6′), 7.49 (t, J = 7.8 Hz, 2H, H-3′, 5′), 7.43 (t, J = 7.3 Hz, 1H, H-4′), 7.05 (d, J = 8.4 Hz, 1H, H-5″), 7.01 (d, J = 16.1
1H, H-4′), 7.06 (d, J = 16.1 Hz, 1H, vinyl H), 6.91 (d, J = Hz, 1H, vinyl H), 6.91 (d, J = 15.8 Hz, 1H, vinyl H), 5.85 (s,
15.8 Hz, 1H, vinyl H), 6.84 (s, 2H, H-2″, 6″), 3.90 (s, 6H, 1H, Ar-OH), 2.50 (s, 3H, CH3).13C NMR (150 MHz, CDCl3)
OCH3), 3.88 (s, 3H, OCH3), 2.51 (s, 3H, CH3). 13C-NMR δ: 188.30, 154.22, 150.03, 141.30, 137.60, 132.20, 132.07,
(150 MHz, CDCl3) δ: 188.35, 153.44, 150.01, 143.16, 129.56, 129.13, 129.07, 128.71, 128.68, 124.99, 124.63,
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124.54, 124.34, 116.53, 114.17, 114.13, 110.88, 14.33. IR (d, J = 16.0 Hz, 1H, vinyl H), 7.42 (d, J = 15.9 Hz, 1H, vinyl
(KBr, cm−1): 3066, 1616, 1596. ESI-MS m/z: 443.01[M H), 7.35 (t, J = 7.9 Hz, 2H, H-3′, 5′), 7.25 (d, J = 5.6 Hz, 1H,
+H]+, calcd for C21H16BrClN2O2: 442.99.
H-4′), 7.06 (d, J = 8.2 Hz, 1H, H-4‴), 7.03 (t, J = 6.6 Hz, 2H,
H-5‴, 6‴), 6.96 (d, J = 7.9Hz, 1H, H-6″), 6.91 (d, J = 8.2 Hz,
1H, H-6″), 6.78 (t, J = 8.0 Hz, 2H, H-3‴), 6.74 (d, J = 16.0
Hz, 1H, vinyl H), 6.70 (s, 1H, H-2″), 6.67 (d, J = 15.9 Hz, 1H,
vinyl H), 5.91 (s, 1H, Ar-OH), 3.92 (s, 3H, OCH3), 3.90 (s,
3H, OCH3), 2.49 (s, 3H, CH3).13C NMR (150 MHz, CDCl3)
δ: 188.81, 149.55, 148.98, 148.31, 148.04, 146.78, 144.84,
142.69, 137.47, 131.34, 129.00, 127.51, 127.31, 124.70,
124.16, 123.29, 123.10, 122.38, 121.11, 115.84,
114.75,112.97, 109.68, 105.38, 56.21, 55.94, 14.28. IR
(KBr, cm−1): 3435, 1642, 1564. ESI-MS m/z: 483.17 [M
+H]+, calcd for C29H26N2O5: 482.16.
(1E, 4E)-1-(5-(2-methoxyphenoxy)-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-5-(3, 4-dimethoxyphenyl)
penta-1,4-dien-3-one (7g)
A yellow solid, m.p:159.7–160.2°C, 10.5% yield.1H NMR
(600 MHz, CDCl3) δ: 7.65 (d, J = 7.8 Hz, 2H, H-2′, 6′), 7.47
(d, J = 16.0 Hz, 1H, vinyl H), 7.44 (d,J = 15.9 Hz, 1H, vinyl
H), 7.35 (t, J = 7.9 Hz, 2H, H-3′, 5′), 7.24 (t, J =7.4 Hz, 1H,
H-4′),7.10 (dd, J =8.3, 1.9 Hz, 1H, H-4‴), 7.04 (d, J = 1.7 Hz,
1H, H-6″), 7.02 (td, J = 8.4, 1.3 Hz, 1H, H-5‴), 6.95 (dd, J =
8.2, 1.2 Hz, 1H, H-6‴), 6.86 (d, J = 8.3 Hz, 1H, H-2″), 6.78
(dd, J = 8.3, 1.3 Hz, 1H, H-5″), 6.75 (d, J = 15.9 Hz, 1H,
vinyl H), 6.70–6.68 (m, 2H, H-3‴, vinyl H), 3.91 (s, 6H,
OCH3), 3.90 (s, 3H, OCH3),2.49 (s, 3H, CH3). 13C NMR
(150 MHz, CDCl3) δ: 188.74, 151.18, 149.55, 149.20,
148.97, 148.29, 144.84, 142.48, 137.48, 131.34, 129.00,
127.92, 127.30, 124.69, 124.47, 123.29, 122.80, 122.35,
121.11, 115.83, 112.94, 111.06, 109.86, 105.40, 56.20,
55.96, 55.91, 14.30. IR (KBr, cm−1): 1621, 1591. ESI-MS
m/z: 497.20 [M+H]+, calcd for C30H28N2O5: 496.20.
(1E, 4E)-1-(5-(benzo[d][1,3]dioxol-6-yloxy)-
3-methyl-1-phenyl-1H-pyrazol-4-yl)-
5-(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (7j)
1
A yellow solid, m.p:151.4–153.6°C, 12.8% yield. H-NMR
(600 MHz, CDCl3) δ: 7.60 (dd, J = 8.6, 1.1 Hz, 2H, H-2′, 6′),
7.49 (d, J = 16.0 Hz, 1H, vinyl H), 7.43 (d, J = 15.9 Hz, 1H,
vinyl H), 7.37 (td, J = 7.4, 1.6 Hz, 2H, H-3′, 5′), 7.27 (tt, J =
7.4, 1.0 Hz, 1H, H-4′), 7.11 (dd, J = 8.3, 1.9 Hz, 1H, H-6″),
7.06 (d, J = 1.9 Hz, 1H, H-2″), 6.86 (d, J = 8.3 Hz, 1H, H-5″),
6.74 (d, J = 15.2 Hz, 1H, vinyl H), 6.71 (d, J = 15.9 Hz, 1H,
vinyl H), 6.65 (d, J = 8.5 Hz, 1H, H-5‴), 6.51 (d, J = 2.6 Hz,
1H, H-2‴), 6.35 (dd, J = 8.5, 2.6 Hz, 1H, H-6‴), 5.89 (s, 2H,
OCH2O), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 2.49 (s,
3H, CH3). 13C-NMR (150 MHz, CDCl3) δ: 188.63, 151.27,
150.80, 149.73, 149.22, 148.74, 147.60, 143.86, 142.69,
137.33, 131.06, 129.14, 127.81, 127.46, 124.58, 123.36,
122.90, 122.33, 111.05, 109.86, 108.28, 107.16, 105.79,
101.74, 98.20, 55.97, 55.92, 14.15. IR (KBr, cm−1): 1668,
1616, 1506. ESI-MS m/z: 511.17 [M+H]+, calcd for
C30H26N2O6: 510.16.
(1E, 4E)-1-(5-(2-methoxyphenoxy)-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-
5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (7h)
A yellow solid, m.p:144.2–144.3°C, 10.9% yield.1H-NMR
(600 MHz, CDCl3) δ: 7.65 (d, J = 7.7 Hz, 2H, H-2′, 6′),
7.48 (d, J = 15.9 Hz, 1H, vinyl H), 7.40 (d, J = 15.9 Hz,
1H, vinyl H), 7.35 (t, J = 7.9 Hz, 2H, H-3′, 5′), 7.25 (t,
J =4.3 Hz, 1H, H-4′), 7.02 (td, J = 8.3, 1.4 Hz, 1H, H-4‴),
6.94 (dd, J = 8.2, 1.2 Hz, 1H, H-6‴), 6.78 (dd, J = 8.5, 1.4
Hz, 1H, H-5‴),6.76 (d, J = 15.9 Hz, 1H, vinyl H), 6.75 (s,
2H, H-2″, 6″), 6.72 (d, J = 15.9 Hz, 1H, vinyl H), 6.68 (dd,
J = 8.1, 1.4 Hz, 1H, H-3‴), 3.90 (s, 3H, OCH3), 3.89 (s,
6H, OCH3),3.87 (s, 3H, OCH3), 2.49 (s, 3H, CH3).
13C-NMR (150 MHz, CDCl3) δ: 188.63, 153.40, 149.58,
148.93, 148.32, 144.83, 142.47, 140.17, 137.43, 131.69,
130.43, 129.01, 127.36, 125.85, 124.67, 123.06, 122.37,
121.12, 115.78, 112.91, 105.46, 105.41, 60.98, 56.20,
56.17, 14.31. IR (KBr, cm−1): 1667, 1619. ESI-MS m/z:
527.22 [M+H]+, calcd for C31H30N2O6: 526.21.
(1E, 4E)-1-(5-(benzo[d][1,3]dioxol-6-yloxy)-
3-methyl-1-phenyl-1H-pyrazol-4-yl)-
5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (7k)
1
A yellow solid, m.p:157.7–158.2°C, 16.8% yield. H-NMR
(600 MHz, CDCl3), δ: 7.59 (dd, J = 8.7, 0.66 Hz, 2H, H-2′,
6′), 7.51 (d, J = 16.0 Hz, 1H, vinyl H), 7.39–7.36 (m, 3H,
vinyl H, H-3′, 5′), 7.27 (t, J = 7.4 Hz, 1H, H-4′), 6.75 (s, 3H,
H-2″, 6″, 2‴), 6.73 (d, J = 16.0 Hz, 1H, vinyl H), 6.64 (d, J =
15.9 Hz, 1H, vinyl H), 6.51 (d, J = 2.6 Hz, 1H, H-5‴), 6.35
(dd, J = 8.5, 2.6 Hz, 1H, H-6‴), 5.88 (s, 2H, OCH2O), 3.88 (s,
6H, OCH3), 3.87 (s, 3H, OCH3), 2.49 (s, 3H, CH3).
(1E, 4E)-1-(5-(2-methoxyphenoxy)-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-5-(4-hydroxy-
3-methoxyphenyl)-penta-1,4-dien-3-one (7i)
A yellow solid, m.p:116.4–118.1°C, 11.4% yield. H-NMR 13C-NMR (150 MHz, CDCl3), δ: 188.53, 153.41, 150.78,
1
(600 MHz, CDCl3) δ: 7.66 (d, J = 7.7 Hz, 2H, H-2′, 6′), 7.46 149.75, 148.74, 147.65, 143.86, 142.65, 140.24, 137.28,
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131.41, 130.32, 129.15, 127.50, 125.94, 123.11, 122.32, (d, J = 15.8 Hz, 1H, vinyl H), 6.89 (d, J = 15.9 Hz, 1H,
108.26, 107.15, 105.77, 105.45, 101.76, 98.17, 60.97,
56.18, 14.18. IR (KBr, cm−1): 1667, 1622. ESI-MS m/z:
541.19 [M+H]+, calcd for C31H28N2O7: 540.19.
vinyl H), 6.83 (d, J = 16.1 Hz, vinyl H), 6.81 (s, 2H, ArH),
3.91 (s, 3H, NCH3), 3.89 (s, 6H, OCH3), 3.87 (s, 3H,
OCH3). 13C-NMR (150 MHz, CDCl3), δ: 188.50, 153.42,
142.79, 140.21, 138.81, 133.77, 130.93, 130.33, 125.08,
123.21, 118.95, 105.44, 60.97, 56.15, 39.21. IR
(KBr, cm−1): 1669, 1621. ESI-MS m/z: 329.15 [M+H]+,
calcd for C18H20N2O4: 328.14.
(1E, 4E)-1-(5-(benzo[d][1,3]dioxol-6-yloxy)-
3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-(4-hydroxy-
3-methoxyphenyl)penta-1,4-dien-3-one (7l)
A
yellow solid, m.p:152.2–153.9°C, 10.7% yield.
1H-NMR (600 MHz, CDCl3), δ: 7.60 (d, J = 7.9 Hz, 2H,
H-2′, 6′), 7.49 (d, J = 16.0 Hz, 1H, vinyl H), 7.41 (d, J =
16.0 Hz, 1H, vinyl H), 7.38 (t, J = 8.0 Hz, 2H, H-3′, 5′),
7.28 (t, J = 7.4 Hz, 1H, H-4′), 7.08 (dd, J = 8.1, 1.6 Hz,
1H, H-6″), 7.04 (s, 1H, H-2″), 6.92 (d, J = 8.2 Hz, 1H,
H-5″), 6.74 (d, J = 16.0 Hz, 1H, vinyl H), 6.69 (d, J = 15.9
Hz, 1H, vinyl H), 6.66 (d, J = 8.5 Hz, 1H, H-5‴), 6.51 (d,
J = 2.5 Hz, 1H, H-2‴), 6.35 (dd, J = 8.5, 2.6 Hz, 1H,
H-6‴), 5.90 (s, 2H,OCH2O), 5.88 (s, 1H, Ar-OH), 3.93 (s,
3H, OCH3), 2.50 (s, 3H, CH3). 13C NMR (150 MHz,
CDCl3), δ: 188.72, 150.77, 149.76, 148.74, 148.15,
147.58, 146.81, 143.87, 142.94, 137.31, 131.05, 129.15,
127.48, 127.37, 124.28, 123.34, 123.26, 122.34, 114.77,
109.64, 108.29, 107.13, 105.78, 101.75, 98.20, 55.95,
14.14. IR (KBr, cm−1): 3334, 1647, 1592. ESI-MS m/z:
597.17 [M+H]+, calcd for C29H24N2O6: 496.16.
(1E, 4E)-1-(4-hydroxy-3-methoxyphenyl)-5-(1-methyl-
1H-pyrazol-4-yl) penta-1, 4-dien-3-one (7o)
A yellow solid, m.p:153.3–153.5°C, 14.1% yield. 1H NMR
(600 MHz, CDCl3), δ 7.77: (s, 1H,Pyrazole-H), 7.63 (d,
J = 15.8 Hz, 1H, vinyl H), 7.61 (d, J = 15.9 Hz, 1H, vinyl
H), 7.59 (s, 1H,Pyrazole-H), 7.15 (dd, J = 8.2, 1.7 Hz, 1H,
H-6′), 7.09 (d, J =1.7 Hz, 1H, H-2′), 6.93 (d, J = 8.2 Hz,
1H, H-5′), 6.85 (d, J = 15.8 Hz, 1H, vinyl H), 6.82 (d, J =
15.8 Hz, 1H, vinyl H), 5.91 (s, 1H, Ar-OH), 3.94 (s, 3H,
NCH3), 3.92 (s, 3H, OCH3). 13C NMR (150 MHz,
CDCl3), δ: 188.67, 148.19, 146.84, 143.07, 138.80,
133.40, 130.83, 127.38, 123.42, 123.34, 119.02, 114.84,
109.69, 55.94, 39.19. IR (KBr, cm−1): 1667, 1617. ESI-
MS m/z: 285.12 [M+H]+, calcd for C16H16N2O3: 284.11.
Biology
Cell Culture and Reagents
(1E, 4E)-1-(5-ethoxy-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-5-(4-hydroxy-3-methoxyphenyl) penta-1,4-dien-
3-one (7m)
The human cancer cell lines SGC-7901, HepG2, A549,
SW620 and human normal colon epithelial cells HCoEpiC
were purchased from the Cell Collection Center of Wuhan
University and maintained in Gibco RPMI medium 1640
(Gibco Company, Grand Island, NY, USA) supplemented
with 10% fetal bovine serum (Gibco Company, Grand
Island, NY, USA), 1% penicillin and streptomycin in a 5%
CO2 atmosphere at 37°C. Curcumin was purchased from
Sigma Company (C1386, Sigma-Aldrich Chemical
Company, Louis, Missouri, USA). Compound 7h and cur-
cumin were, respectively, dissolved in DMSO at 20 mM,
which were then diluted to different concentrations in the
prepared medium. Primary antibodies against ATM (2873,
Cell Signaling Technology, Trask Lane Danvers, MA,
USA), PARP (9542, Cell Signaling Technology, Trask
Lane Danvers, MA, USA), cyclin B1 (sc-7393, Santa
Cruz Biotechnology, Delaware Ave Santa Cruz, CA,
USA), cyclin D1 (sc-6283, Santa Cruz Biotechnology,
Delaware Ave Santa Cruz, CA, USA), CC3 (9662, Cell
Signaling Technology, Trask Lane Danvers, MA, USA),
A yellow solid, m.p:157.3–157.7°C, 12.6% yield. 1H NMR
(600 MHz, CDCl3) δ: 7.69 (d, J = 8.3 Hz, 2H, H-2′, 6′),
7.64 (d, J = 15.8 Hz, 2H, vinyl H), 7.45 (t, J = 7.7 Hz, 2H,
H-3′, 5′), 7.32 (t, J = 7.4 Hz, 1H, H-4′), 7.17 (d, J = 8.2
Hz, 1H, H-6″), 7.11 (s, 1H, H-2″), 6.94 (d, J = 7.8 Hz, 1H,
H-5″), 6.92 (d, J = 15.5 Hz, 1H, vinyl H), 6.86 (d, J = 15.5
Hz, 1H, vinyl H), 5.88 (s, 1H, Ar-OH), 4.04 (q, J = 7.0 Hz,
2H, OCH2), 3.95 (s, 3H, OCH3), 2.45 (s, 3H, CH3), 1.30
(t, J = 6.9 Hz, 3H, CH3).13C NMR (150 MHz, CDCl3) δ:
188.71, 153.09, 149.07, 148.11, 146.81, 142.79, 137.95,
132.64, 129.13, 127.50, 127.19, 124.16, 123.25, 122.41,
122.39, 114.81, 109.74, 104.75, 71.27, 55.97, 15.34,
14.41. IR (KBr, cm−1): 1619, 1593. ESI-MS m/z: 405.18
[M+H]+, calcd for C24H24N2O4: 404.17.
(1E, 4E)-1-(3, 4, 5-trimethoxyphenyl)-5-(1-methyl-
1H-pyrazol-4-yl) penta-1, 4-dien-3-one (7n)
A yellow solid, m.p:111.2–111.7°C, 12.8% yield. 1H NMR
(600 MHz, CDCl3), δ: 7.78 (s, 1H,Pyrazole-H), 7.62 (d,
J = 15.8 Hz, 1H, vinyl H), 7.60 (s, 1H,Pyrazole-H), 7.59 Bcl-2 (ab196495, Abcam Inc., Cambridge, MA, USA),
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plus all secondary antibodies, were obtained from Thermo supplemented with 10% FBS was placed over the lower
(Thermo Fisher Scientific, Waltham, MA, USA).
chamber and then 7h at different concentrations were
added. After incubation for 24 h, the upper layer of cells
was removed by cotton wool. The cells on the lower sur-
face were fixed in methanol and stained with 0.1% crystal
violet, and then imaged using an IX71 inverted micro-
scope (Olympus, Tokyo, Japan). The number of migrating
cells in a total of five randomly selected fields was
counted. All experiments were performed in triplicate.
Cell Viability Assay
A MTT assay was performed to assess cell viability accord-
ing to the instructions of the manufacturer (Sigma-Aldrich
Chemical Company, Louis, Missouri, USA). Briefly, 1×104
SW620 cells were plated onto 96-well plates. After cultured
overnight, the cells were treated with different concentrations
of 7h and curcumin (0, 0.5, 1.25, 2.5, 5, 10 and 20 μM) for
24 h, 48 h and 72 h. At the end of each treatment, 20 μL of Clonogenic Assay
MTT (5 mg/mL) was added. After incubation for 4 h, the In order to examine the survival of cells treated with 7h,
medium was carefully discarded and dimethyl sulfoxide (100 SW620 cells were plated (1×106 per well) in a 6-well plate
μL) was added. Absorbance was recorded at a wavelength of and incubated overnight. After exposure to different con-
490 nm by a Universal Microplate Reader (Bio-Tek centrations of 7h for 48 h, the viable cells were counted
Instruments, Winooski, USA), using a blank well as control. and then seeded into 6-well plate in a density of 5,000
Cell viability and IC50 values of compounds for the four cells per well. The cells were then incubated for 14 days at
cancer cell lines were determined by comparison with con- 37°C in a humidified 5% CO2 atmosphere. All the colonies
trol. Each experiment was repeated at least 3 independent were stained with 2% crystal violet and cells were counted
times.
manually.
Transfection
Cell Apoptosis Analysis
SW620 cells were seeded into 6-well plates and trans- The apoptotic cells were detected with an Annexin
fected with Atm CRISPR Activation Plasmid (sc-400,192- V-FITC/PI apoptosis detection kit (Biouniqure, Beijing,
ACT, Santa Cruz Biotechnology, Delaware Ave Santa China) according to the manufacturers’ instructions.
Cruz, California, USA) and lipofectamine 2000 Briefly, 1×106 SW620 cells were incubated in 6-well
(Invitrogen Inc., Carlsbad, CA, USA) by following the plate overnight and treated with various concentrations of
manufacture’s instruction. After the indicated periods of 7h and curcumin for 48 h. Cells were harvested by cen-
incubation, the cells were subjected to further analysis.
trifugation, washed with PBS, then resuspended in 500 μL
of binding buffer with 5 μL propidium iodide (PI) and kept
in the dark for 5 min at room temperature. Next, 5 μL
FITC-conjugated anti-Annexin V antibody was added,
which was kept in the dark for 10 min at room tempera-
ture. Apoptosis was analyzed by a flow cytometer (Becton
Dickinson, Mountain View, CA, USA).
Transwell Migration and Invasion Assay
For transwell migration assays, 1×106 cells were plated
into the top chamber of a transwell (Corning, Acton, MA,
USA) with a porous membrane (8.0-μm pore size). Then,
the cells were plated in medium with minimal serum
(0.5% FBS), and medium supplemented with additional
serum (10% FBS) was used to produce a chemoattractant Cell Cycle Analysis
effect in the lower chamber. Compound 7h at different Exponentially growing SW620 cells were seeded in
concentrations was added. The cells were incubated for a 6-well plate and incubated overnight. The cells were
24 h at 37°C and cells that did not migrate through the treated, respectively, with curcumin and 7h and then cul-
pores were removed using a cotton swab. Cells on the tured for 48 h. At the end of treatment period, cells were
lower surface of the membrane were stained with crystal collected and fixed with ice-cold 75% (v/v) ethanol and
violet (Sigma-Aldrich Chemical Company, Louis, kept at 4°C overnight. The cells were collected and
Missouri, USA). The migrated cells were manually quan- washed with ice-cold PBS. Then, the cell pellets were re-
tified. The inhibition rates of migrated cells were calcu- suspended at 1×106 cells/mL in PBS and incubated with
lated using the untreated group as 100%.
0.1 mg/mL RNase I and 50 mg/mL Propidium iodide (PI)
As for in vitro cellular invasion analysis, briefly, 1×106 at 37°C for 30 min. DNA contents were determined with
cells in serum-free medium were seeded into the upper a flow cytometer (Becton Dickinson, Mountain View,
chamber of an insert coated with Matrigel. Medium CA, USA).
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Min et al
Figure 2 The inhibitions of cell viability for the synthesized compounds (7a–7o) on human cancer cell lines SGC-7901 (A), HepG2 (B), A549 (C) and SW620 (D). The cells
were treated with 7a-7o (20 μM) for 72 h, and then the cell viability was determined by MTTassay, curcumin as positive control. Results were presented as Mean S.D (n=3)
and analyzed by GraphPad Prism 6.0 followed by the Student t-test. *P < 0.05, **p< 0.01 or ***p < 0.001 vs curcumin.
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Western Blot Analysis
The expressions of several proteins were detected by
Western blot analysis. The SW620 cells were treated,
respectively, with 7h, Atm CRISPR Activation Plasmid,
7h+ Atm CRISPR Activation Plasmid. The harvested
SW620 cells were washed by PBS and lysed with cell
lysis buffer (Cell Signaling, Danvers, MA, USA). The
protein concentrations were tested by a BCA Protein
Assay kit (Thermo Fisher Scientific, Waltham, MA,
USA). Equal amounts of protein samples were prepared
and fractionated by electrophoresis in a sodium dodecyl
sulfonate-polyacrylamide gel and transferred onto
a polyvinylidene fluoride membrane. Skimmed milk
powder was added, and the samples were incubated for
2 h. Next, appropriate primary antibodies were added,
and the sample was finally incubated at 4°C overnight.
The membranes were incubated for 1 h with secondary
antibodies at room temperature and then washed three
times with tris-buffered saline and Tween 20. Protein
bands were subsequently detected by electrogenerated
chemiluminescence assay.
Statistical Analysis
All data analyses were conducted using IBM SPSS,
Version 19.0 (IBM Corp., Armonk, NY, USA). Statistical
comparisons were performed using the Student t-test.
P-values < 0.05 were considered statistically significant.
Results were expressed as means standard deviations.
Results and Discussion
Synthesis of MCACs
The condensation of phenylhydrazine with ethyl acetoace-
tate afforded 1-phenyl-3-methyl-5-pyrazolone 9, which
was followed by the Vilsmeier reaction to produce
5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
10. Compound 10 was treated with ethanol or substituted
phenols in the presence of KOH to give substituted
1H-pyrazole-4-carbaldehydes 11a–11c. Various substituted
benzaldehydes conducted the Claisen-Schmid condensa-
tion with acetone in the presence of 10%NaOH to furnish
α, β-unsaturated ketones 13a–13f. Using the same NaOH-
catalysed Claisen-Schmidt condensations of 13a–13f with
10, 11a–11c or 1-methyl-1H-pyrazole-4-carbaldehyde, the
target compounds 7a–7o were finally obtained. All the
target compounds 7a–7o were well characterized by spec-
troscopic techniques such as IR, NMR and ESI-MS, which
were in full accordance with the depicted structures. The
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Figure 3 Compound 7h inhibited clone formation (A and B), migration (C and D) and invasion (E and F) on SW620 cells. SW620 cells were treated with 7h at the
concentration of 1.25 μM, 2.5 μM and 5 μM for 48h respectively, the cells treated with DMSO as control and with curcumin (5 μM) as positive control. Results were
presented as Mean SD (n = 3) and analyzed by GraphPad Prism 6.0 followed by the Student t-test. *P < 0.05 or **P < 0.01 or ***P < 0.001 vs control, ++P < 0.01 or +++P <
0.001 vs curcumin.
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Figure 4 The effect of 7h on apoptosis in SW620 cells. SW620 cells were treated with 7h (1.25, 2.5 or 5 μM) for 48 h, and the cell apoptotic rate was detected with FCM
assay (A and B), the cells with DMSO as control and with curcumin (5 μM) as positive control. (C) The effects of 7h on the protein expression of PARP, cleaved-caspase 3
and Bcl-2 were tested by Western blot. Data were presented as Mean SD (n = 3) and analyzed by GraphPad Prism 6.0 followed by the Student t-test. *P < 0.05, **P < 0.01
or ***P < 0.001 vs control, +P < 0.05, ++P < 0.01 or +++P < 0.001 vs curcumin.
proton NMR of 7a–7o exhibited two pair of doublets in HepG2, A549 and SW620 cells were treated with these
the aromatic region with J values between 15.5 and 16.1
Hz for the alkene protons present in the penta-1, 4-dien-
3-one skeleton, providing evidence of (E)-stereochemistry.
Before used for biological assays, all synthesized com-
pounds 7a–7o were determined their purity (all >97%)
by HPLC.
synthesized compounds (Figure 2). Among them, com-
pounds 7a, 7h and 7i exhibited excellent inhibitory activities,
especially in SW620 cells with the ratio of cell viability
<50%. Further, the IC50 values of compounds 7a, 7h, 7i
were determined at the time of 24h, 48h and 72h, respec-
tively, and curcumin, adriamycin and oxaliplatin were used
as positive control. The results are shown in Table 1.
Compound 7h exhibited more potent inhibitory activity
against SW620 cells with IC50 value of 0.012 μM than the
three positive controls. Furthermore, in order to evaluate its
cytotoxicity to normal cells, 7h was chosen to test its inhibi-
tory effects on the proliferation of human normal colon
epithelial cells HCoEpiC at the time of 72 h, and the IC50
value was more than 100μM, implying that 7h exhibited
selective cytotoxicity to cancer cells. The preliminary study
Effect of the Synthesized Compounds on
Cell Viability
The inhibitory effects of the synthesized compounds on the
proliferation of human cancer cell lines GC-7901, HepG2,
A549 and SW620 were assessed by MTT assay at
a concentration of 20μM respectively, and curcumin was
used as positive control. The results indicated that most of
the synthesized compounds decreased cell viability signifi-
cantly as compared to curcumin, especially after 72 h when on structure–activity relationship (SAR), taking that in
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Figure 5 The effect of 7h on cell cycle. (A and B) SW620 cells were treated with 7h (1.25, 2.5 or 5 μM) for 48 h, and then the cell cycle distribution was detected by PI
staining, the cells with DMSO as control and with curcumin (5 μM) as positive control. (C) Western blotting was used to detect the expression of cyclin B1 and cyclin D1
protein. Data were presented as Mean SD (n = 3) and analyzed by GraphPad Prism 6.0 followed by the Student t-test. **P < 0.01 or ***P < 0.001 vs control, +P < 0.05 or ++
+P < 0.001 vs curcumin.
SW620 cells as an example, indicated that on the terminal metastatic site where they proliferate.33 Thus, an ability
to inhibit the migration and invasion of cancer cells is
very important for an anticancer compound. As 7h exhib-
ited excellent inhibitory activities against the prolifera-
tion of SW620 cancer cells, further, its effects on
inhibitions of the migration and colony formation of
SW620 cells were evaluated at the various concentrations
of 1.25μM, 2.5μM and 5μM after the cells were treated
with 7h for 48 h. The cells treated with DMSO and
curcumin (5 μM) were used as blank and positive control
correspondingly. The results indicated that 7h decreased
the number of migration and invasion in a dose-
dependent manner (Figure 3A–D), and there was
a significant reduction in the percentages of cell migra-
tion and invasion especially at the concentration of 5μM
as compared with both control groups. As shown in
containing pyrazolyl ring of these MCACs ethoxy or phe-
noxy groups resulted in better activity than chlorine atom.
Excepted for 7g, 2-methoxyphenoxy group was introduced
into 7h, 7i to replace the chlorine atom, which showed the
most potent activity. On another terminal of these MCACs,
trimethoxyphenoxy (3, 4, 5-OCH3) group together with both
3-methoxy and 4-hydroxyl groups might contribute to good
activities.
Effect of 7h on Migration and Colony
Formation in SW620 Cells
The migration and invasion of cancer cells due to their
promoted penetration into the lymphatic system and
blood vessels can lead to metastasis dissemination, and
then cancer cells undergo extravasation into a newly Figure 3E and F, the clonogenic quantity of
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Figure 6 Compound 7h down-regulated ATM protein expression in SW620 cells. (A) SW620 cells were treated with curcumin (5 μM) and 7h (5 μM) for 48 h, and then
ATM protein expression was detected, the cells were treated with DMSO as control group; (B) SW620 cells were treated with ATM plasmid (Atm CRISPR Activation) for
24 h and then combined with or without 7h for 48 h, and the cells with negative plasmid as control. Data were presented as Mean SD (n = 3) and analyzed by GraphPad
Prism 6.0 followed by the Student t-test. **P < 0.01 or ***P < 0.001 vs control, ++P < 0.01 vs compound 7h, ###P < 0.001 vs ATM, ==P < 0.01 vs curcumin.
SW620 cells treated with 7h (5μM) decreased nearly by cell cycle was determined, the cells treated with DMSO as
thirteen-fold as compared to the positive control. These control and curcumin (5μM) as positive control. As shown in
results indicated that 7h could significantly inhibit cell Figure 5A and B, compound 7h at the concentration of 5μM
proliferation.
increased the percentages of G2/M phase cells from 10% to
21% and that of S phase cells from 43% to 66%, while
decreased the percentages of G0/G1 phase cells from 46% to
11%. Compared to the control and the positive control group,
compound 7h significantly increased the percentages of G2/M
phase cells at the concentration of 5μM. The results indicated
that 7h inhibited SW620 cell proliferation by inducing cell
cycle arrest at G2/M and S phase. Meanwhile, the expressions
of cyclin B1 and cyclin D1 were also determined by Western
blot assay, and the results (Figure 5C) showed that 7h increased
cyclin B1 protein expression and decreased cyclin D1 protein
expression at the concentration of 5μM in SW620 cells.
Effect of 7h on SW620 Cell Apoptosis
In order to evaluate the effect of 7h on cell apoptosis, SW620
cells were treated with 7h at the concentrations of 1.25μM,
2.5μM and 5μM for 48 h, respectively, and then the apoptosis
was analyzed by flow cytometry, using the cells treated with
DMSO and curcumin (5 μM) as blank and positive control
correspondingly. The results (Figure 4A and B) showed that
the treatment with 7h increased the apoptotic rate from
18.64% of vehicle control to 19.69% (1.25 μM), 47.4%
(2.5 μM) and 87.3% (5 μM) in a concentration-dependent
manner. Compared to curcumin group, 7h significantly
increased the cell apoptotic rate at the concentration of
2.5μM and 5μM. On the other hand, Western blotting result
(Figure 4C) showed that 7h markedly increased the protein
expression of cleaved-caspase 3 and PARP and decreased
Bcl-2 expression at the concentration of 5μM in SW620 cells
as compared with curcumin (5μM) group, which suggested
that 7h promoted SW620 cell apoptosis.
Compound 7h Regulated ATM
Expression on SW620 Cells
The ATM gene is a DNA repair gene and is located on the
chromosome llq22-q23. Studies have shown that the ATM
gene may become a new potential target for the treatment
of colon cancer,34 and then we hypothesized that the
inhibitory effect of 7h on the proliferations of SW620
cells might be related to ATM pathway. Therefore,
SW620 cells were treated with 7h (5μM) and curcumin
Effect of 7h on SW620 Cell Cycle
SW620 cells were treated with 7h at the concentrations of (5μM) for 48 h, and then ATM protein expressions were
1.25μM, 2.5μM and 5μM for 48h, respectively, and then the determined, the cells treated with DMSO as control. The
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Min et al
Figure 7 The effects of 7h on apoptosis, cell cycle, migration and invasion were partly dependent on ATM pathway in SW620 cells. SW620 cells were transfected with ATM
plasmid (Atm CRISPR Activation) for 24 h and then combined with or without 7h for 48 h, and the cells were treated with negative plasmid as control. The clone formation
(A and B) and cell migration and invasion (C–F) were detected; The apoptotic rate and apoptotic relative protein expression (G–I) were detected by FCM and Western
blot assay; The cell cycle arrest, cell cycle arrest-related protein expression (J–L) were also detected. Results were presented as Mean SD (n = 3) and analyzed by
GraphPad Prism 6.0 followed by the Student t-test. *P < 0.05, **P < 0.01 or ***P < 0.001 vs control, +P < 0.05, ++P < 0.01 or +++P < 0.001 vs compound 7h, ##P < 0.01 or
###P < 0.001 vs ATM.
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Western blotting results revealed that 7h markedly formation inhibitions, possibly due to its ability to
decrease the ATM expression in SW620 cells.
decreased ATM protein expression as compared to both
control and curcumin (5μM) groups (Figure 6A). Further,
in order to confirm whether 7h could really down-
regulated ATM signal, SW620 cells were transfected
with the ATM activated plasmids that over-expressed
ATM protein for 24 h. The cells were treated with 7h for
48 h, using the cells treated with negative plasmids as
control. The result showed that the ATM activated plas-
mids reversed the down-regulation of ATM expression
induced by 7h (Figure 6B), which also proved that 7h
decreases the ATM protein expression in SW620 cells.
Conclusion
A serial of monocarbonyl analogs of curcumin containing
pyrazole moiety was designed and synthesized whose struc-
tures were confirmed by spectroscopic techniques. Their
inhibitory activities against four human cancer lines, SGC-
7901, HepG2, A549 and SW620, were evaluated. Of these
15 newly synthesized compounds, (1E,4E)-1-(5-(2-methox-
yphenoxy)-3- methyl-1-phenyl-1H-pyrazol-4-yl)-5-(3, 4,
5-trimethoxyphenyl) penta-1, 4-dien-3-one 7h exhibited
excellent selectivity and outstanding anti-proliferation activ-
ity against SW620 cells with a IC50 value of 12 nM, which
was more potent than curcumin (IC50 = 9.36 μM), adriamysin
(IC50 = 3.28 μM) and oxaliplatin (IC50 = 13.33 μM).
In order to investigate the mechanisms that 7h exerted
its inhibitory effect on SW620 cells proliferation, further
assays were performed. The results showed that 7h inhib-
ited cell migration, invasion and colony formation of
SW620 colon cancer cells obviously, which was due to
its ability to induce cell cycle arrest in the G2/M and
S phases and apoptosis. Ultimately, 7h decreased the
expression of ATM protein, which may primarily contri-
bute to its anticancer activity against SW620 cells. All the
data indicated that 7h could be identified and developed as
a novel potential anti-colon cancer agent in the future.
Effects of 7h on Cell Apoptosis, Cycle,
Migration and Invasion in SW620 Cells
Were Partly via ATM Pathway
As 7h decreased the ATM protein expression, in order to
investigate whether its effects on SW620 cells migration,
invasion, cell cycle and apoptosis were via ATM pathway,
the related assays were performed, and SW620 cells were
transfected with or without ATM activated plasmids were
used for comparison. The cells were treated with 7h
(5μM), using the cells with negative plasmids as control.
The result demonstrated that the transfection with ATM
activated plasmids could reverse the up-regulation of the
apoptotic rate induced by 7h (Figure 7G and H). Similarly,
the Western blotting result indicated that the expressions
of cleaved-caspase 3, PARP and Bcl-2 induced by 7h in
SW620 cells were all decreased by 7h combined with
ATM activated plasmid (Figure 7I). Meanwhile, the treat-
ment with 7h+ATM significantly restored cell cycles that
were obviously different from the ones induced only by 7h
(Figure 7J and K). Correspondingly, 7h+ATM clearly
reduced the up-regulation of cyclin B1 protein expression
while enhanced the protein expression of cyclin D1 as
compared to treatment only with 7h (Figure 7L). Finally,
the present study also demonstrated that 7h+ATM signifi-
cantly enhanced the decline of the migration and invasion
number induced only by 7h (Figure 7C–F). Although 7h
decreased the amount of clone formation, the amount was
drastically increased from 1.33 1.01 to 8.22 2.22 after
treatment with 7h+ATM for 48 h(Figure 7A and B). In
brief, the treatment combined 7h with ATM protein could
almost reverse all the effects resulted only from 7h, which
suggested that 7h exerted its activities, such as induction
of cell apoptosis, cycle arrest, migration and clone
Abbreviations
MCACs, monocarbonyl analogs of curcumin; MTT,
methyl thiazolyl tetrazolium; IC50, 50% inhibitory concen-
tration; ATM, ataxia-telangiectasia mutated; PARP, poly
ADP-ribose polymerase; CRISPR, clustered regularly
interspaced short palindromic repeats; FBS, fetal calf
serum.
Acknowledgment
The financial supports by the National Natural Science
Foundation of China (No. 81770377) and the Natural
Science Foundation of Hubei Province (No.
2017CFB448) is gratefully acknowledged.
Disclosure
The authors report no conflicts of interest in this work.
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