Design and biological evaluation of novel hybrids of 1, 5-diarylpyrazole and Chrysin for selective COX-2 inhibition

Article abstract of DOI:10.1016/j.bmc.2018.07.022

The overexpress of COX-2 was clearly associated with carcinogenesis and COX-2 as a possible target has long been exploited for cancer therapy. In this work, we described the design and synthesis of a series of diarylpyrazole derivatives integrating with c

Full text of DOI:10.1016/j.bmc.2018.07.022

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and biological evaluation of novel hybrids of 1, 5-diarylpyrazole and
Chrysin for selective COX-2 inhibition
Shen-Zhen Rena,1, Zhong-Chang Wanga,⁎,1, Xiao-Hua Zhua, Dan Zhua, Zhang Lia, Fa-Qian Shena,
Yong-Tao Duanc, Han Caoa, Jing Zhaob,⁎, Hai-Liang Zhua,⁎
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, PR China
State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210023, PR China
Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Zhengzhou Children's Hospital, Zhengzhou 450018, PR China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pyrazole
Chysin
COX-2 inhibitor
Antiproliferative
Docking
The overexpress of COX-2 was clearly associated with carcinogenesis and COX-2 as a possible target has long
been exploited for cancer therapy. In this work, we described the design and synthesis of a series of dia-
rylpyrazole derivatives integrating with chrysin. Among them, compound e9 exhibited the most potent in-
hibitory activity against COX-2 and antiproliferative activity against Hela cells with IC50 value of 1.12 μM.
Further investigation revealed that e9 could induce apoptosis of Hela cells by mitochondrial depolarization and
block the G1 phase of cell cycle in a dose-dependent manner. Besides, molecular docking simulation results was
further confirmed that e9 could bind well with COX-2. In summary, compound e9 may be promising candidates
for cancer therapy.
1. Introduction
invasion.10,11 In the past few decades, a large number of COX-2 in-
hibitors, named coxibs, have been discovered, such as Celecoxib, Val-
Inflammation is a complex process that reflects the changes in cellar
and molecular components of host and is associated with various dis-
ease such as arthritis, diabetic, asthma and cancer.1,2 Traditional non-
steroidal anti-inflammatory drugs (tNSAIDs) are used widely for the
treatment of pain and inflammatory disorders.3 In addition, numerous
in vitro and in vivo studies have demonstrated that these tNSAIDs are
able to prevent the proliferation of tumor by increasing apoptosis and
altering the microenvironment of tumor.4 The therapeutic effects of
tNSAIDs are related to the inhibition effect of cyclooxygenase (COX),
which is a rate-limiting enzymes in the biosynthesis of prostaglandins
(PGs) and exists three isoforms: COX-1, COX-2 and COX-3.5,6 However,
long-term therapy with tNSAIDs usually causes some side-effects such
as gastrointestinal ulcer, bleeding, and hepatotoxicity, which are
mainly due to the inhibition of COX-1.7,8 Therefore, the development of
selective COX-2 inhibitors seems to be a potential approach to reduce
the side-effects of tNSAIDs.
decoxib, Parecoxib, and Rofecoxib (Fig. 1). Multiple lines evidences
have demonstrated that these inhibitors can reduce side-effects of
tNSAIDs and exhibit efficacy in many cancer studies such as anti-
proliferation, apoptosis, prevention of metastasis and invasion.12 Un-
fortunately, numerous clinical studies have demonstrated that the use
of COX-2 inhibitors is associated with an increased risk of cardiovas-
cular and myocardial infarction.8,13 Due to increased risk of cardio-
vascular events, some coxibs have been withdrawn from market, such
as Rofecoxib and Valdecoxib.14,15
In general, the most famous Coxibs can be characterized by five-
membered heterocyclic cores and 1, 5-diarylsubstitution, such as
Celecoxib, Valdecoxib, Parecoxib, and Rofecoxib (Fig. 1).12,16–18 Based
on this knowledge, we have extracted from these Coxibs and employed
1, 5-diarylpyrazole as the scaffold of compounds. Recently, our group
have identified a series novel 1, 5-diarylpyrazole derivatives as selec-
tive COX-2 inhibitors and evaluated their anticancer activities in vitro
and vivo.19–22 Inspired by the obtained interesting results of our pre-
vious studies, we became interest in exploring more powerful and high-
efficiency COX-2 inhibitors as anticancer candidates. Thus, we hy-
bridized 1, 5-diarylpyrazole scaffold with Chrysin moiety and designed
COX-2 is inducible and overexpression in several solid malig-
nancies, such as breast, prostate and colorectal cancer.9 Many reports
have revealed that the overexpression of COX-2 is associated with the
occurrence of carcinogens, tumor proliferation, metastasis and
⁎
Corresponding authors.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
1
https://doi.org/10.1016/j.bmc.2018.07.022
Received 25 April 2018; Received in revised form 28 June 2018; Accepted 12 July 2018
0968-0896/©2018PublishedbyElsevierLtd.
Pleasecitethisarticleas:Zhu,H.-L.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.07.022

S.-Z. Ren et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 1. Structures of reference compounds and design strategy of the target compounds.
a series of hybrids of diarylpyrazole and Chrysin as COX-2 inhibitors for
anticancer candidates. Chrysin, 5, 7-dihydroxyflavone (Compound 5,
Fig. 1), was chosen due to it was an important pharmacophore and
possess all kind of biological properties such as antivirus, antibacterial,
anti-inflammatory, antioxidant and anticancer.23,24 At present, many
novel chrysin derivatives have been synthesized and the structure-ac-
tivity relationship studies indicated that modification in C-7 position of
chrysin can improve its pharmaceutical activity and pharmacoki-
netics.25,26
On the basis of this finding, we designed 30 novel hybrids of 1, 5-
diarylpyrazole and Chrysin to explore their COX-2 inhibition and an-
ticancer activities. The bioassay results revealed that many of the target
compounds exhibited more potent inhibitory activities in the enzymatic
and cellular assays compared with the reported diarylpyrazole deriva-
tives (Tables 2 and 3).19–22 Among them, compound e9 displayed the
most potency against COX-2 (IC50 = 2.23 μM) and cancer cell line pa-
nels (IC50 = 1.12 μM for Hela cells) without toxicity to normal cells
(IC50 = 193 μM for 293T cells). Docking simulations were also per-
formed to explore the binding modes of these compounds at the active
site of COX-2.
The synthesis of the chrysin-pyrazol derivatives was described in
Scheme 2. 5,7-dihydroxy-2-phenyl-4H-chromen-4-one was reacted with
ethyl 2-bromoacetate in the presence of potassium carbonate in acetone
to produce an intermediate, which then hydrolyzed by potassium hy-
droxide and several drops water in methanol to give the last inter-
mediate (d). Furthermore, 2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yl)oxy)acetic acid (d) was reacted with corresponding compound c in
the presence EDC·HCl, DMAP and triethylamine through esterification
condensation reaction to obtain the corresponding target compounds
e1–e30. These synthesized compounds were characterized by 1H NMR
spectroscopy, 1C NMR spectroscopy, melting point, ESI-MS and ele-
mental analysis, in accordance with their depicted structures.
2.2. In vitro COX-2 inhibitory activities
The ability of the hybrid compounds of diarylpurazole and Chrysin
to inhibit COX-1 and COX-2 were evaluated using COX-1/COX-2 ELSIA
kit. Celecoxib as standard compound was selected for comparison. The
results were summarized in Table 2, which suggested that majority of
the tested compounds exhibited excellent COX-2 inhibitory activity
with IC50 values varying from 0.23 to 8.58 μM, but showed relative
weaker activities to COX-1. Among them, compound e9 displayed the
most significant COX-2 inhibitory activity with IC50 value 0.23 μM, si-
milar to the reference COX-2 selective inhibitor Celecoxib
(IC50 = 0.36 μM).
2. Results and discussion
2.1. Chemistry
A series of novel diarylpyrazole derivatives integrating with chrysin
were synthesized in this study and their structures were outlined in
Table 1. As shown in Scheme 1, compounds c1–c30 were synthesized
by a three-step procedure. First, the substituted acetophenone were
reacted with dimethyl oxalate in the presence of excess sodium meth-
oxide as catalyst in methanol to produce the intermediate (a1–a15).
And then intermediate (b1–b30) were obtained according to the reac-
tion of diverse chalcones with substituted phenylhydrazines in refluxing
ethanol. Finally, these methyl ester (b1–b30) were reduced by lithium
aluminium hydride to be the corresponding alcohols (c1–c30).
Then, the structure-activity relationship (SAR) studies were per-
formed to determine how the substituents affect the COX-2 inhibitory
activity. It was clear that electon-donating groups substituted on the
para-position of A ring exhibited better COX-2 inhibitory activity than
electon-withdrawing
groups,
comparing
compound
e2
(IC50 = 0.93 μM), e8 (IC50 = 0.54 μM) and e22 (IC50 = 1.66 μM).
Similarly, when the para-position of B ring was substituted by electon-
donating group, the COX-2 inhibitory activity was also better than
electon-withdrawing groups. Moreover, comparing the COX-2 in-
hibitory activity of compound e8 (IC50 = 0.54 μM) and e11
2

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Table 2
In vitro COX-1/COX-2 inhibitory activity of the designed compounds.
Table 1
Structures of compounds e1–e30.
Compounds
IC50
SD (μM)a
Selectivity Indexbb (SI)
COX-1
COX-2
e1
e2
e3
e4
e5
e6
e7
e8
32.93
45.04
41.24
40.93
39.12
32.16
27.62
41.34
47.48
46.45
25.52
> 50
30.16
43.20
> 50
> 50
44.80
49.64
36.85
43.20
39.45
42.05
44.82
33.76
> 50
27.89
33.12
32.93
29.74
41.08
46.89
3:29
1.57
1.55
2.11
2.12
1.23
2.08
1.98
2.73
1.83
1.07
2.20
0.93
0.42
1.44
1.36
1.19
1.77
0.54
0.23
0.35
1.21
2.54
2.84
3.53
1.41
2.12
4.43
4.80
0.422
2.12
1.08
1.66
1.39
5.94
6.83
5.02
8.58
4.33
5.02
4.79
0.36
0.34
0.14
0.09
0.43
0.17
0.27
0.17
0.04
0.02
0.03
0.58
0.42
0.44
0.12
0.25
0.55
1.04
0.18
13.13
48.44
96.03
28.42
28.73
27.02
15.59
75.87
206.45
131.55
21.00
> 20
10.62
12.23
> 35
> 23
10.11
10.33
87.26
20.38
36.54
25.33
32.11
5.68
e9
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
e29
e30
Celecoxib
Compounds
R1
R2
R3
2.57
2.12
e1
H
H
H
e2
e3
e4
e5
e6
e7
e8
e9
CH3
CH3
CH3
H
H
H
OCH3
OCH3
OCH3
H
F
F
H
Cl
Cl
H
H
H
Br
Br
H
H
H
CH3
CH3
CH3
H
H
H
OCH3
H
H
F
H
H
CH3
Cl
H
CH3
Cl
H
CH3
Cl
H
H
CH3
H
H
CH3
H
CH3
Cl
H
CH3
H
CH3
H
H
1.21
1.95
1.88
2.14
1.84
2.37
1.93
2.08
0.38
0.14
0.05
0.33
0.57
1.08
2.02
2.37
1.79
0.87
0.62
0.47
0.05
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
e28
e30
> 7
5.56
3.86
7.61
5.92
8.58
130.25
1.99
2.01
2.29
2.74
3.02
2.19
H
Cl
Cl
Cl
H
H
H
a
The concentration of test compound required to produce 50% inhibition of
COX-1/COX-2 is the mean of four determinations.
b
In vitro COX-2 selectivity index (COX-2 IC50/COX-1 IC50).
NO2
NO2
H
Cl
Cl
H
NO2
Cl
Cl
Cl
H
2.4. Compound e9 caused cell apoptosis in Hela cells
CH3
Cl
H
CH3
H
Cl
CF3
CF3
H
To determine whether compound e9 has any apoptotic effects in
Hela cells, we applied Annexin V/PI double staining assay to quantify
the apoptotic percentage in control and treatment groups. Hela cells
were treated with varying concentration (0, 1.25, 2.5, 5 and 10 μM) of
compound e9 for 24 h. As shown in Fig. 2A and B, no apoptotic or
necrotic cells were observed in control group, while the percentage of
apoptotic cells was significantly elevated to 48.30% after treated with
10 μM compound e9 for 24 h. In addition, after treating Hela cells with
5 μM compound e9 for increased time (0, 12, 24, 36 and 48 h), the
percentage of apoptotic cells was significantly increasing from 3.81% to
80.24% (Fig. 2C and D). In conclusion, compound e9 could induce Hela
cells apoptosis in a dose and time-dependent manner.
H
CF3
(IC50 = 1.22 μM), we found that the substitution on the meta-position
of A ring was more efficient than other position. The above analysis
suggested that electon-donating groups substituted on the para-position
of A ring and B ring play a crucial role in the inhibitory activity.
2.3. In vitro antiproliferative activities
According to the COX-2 inhibitory activities, all target compounds
were evaluated by using traditional MTT method for in vitro anti-
proliferative activities against four cancer cell lines (A549: human lungs
cell line; HeLa: human cervix cell line; MCF-7: human breast cancer cell
line; B16-F10: murine melanoma cell line) and one human normal cell
line (293T: human kidney epithelial cell line). Celecoxib and Chrysin
were selected as the positive controls. The results were summarized in
Table 3. Most of the tested compounds exhibited excellent growth in-
hibitory effects especially for Hela cells. Among them, compound e9
could inhibit Hela cells with IC50 value of 1.12 μM, superior to the
positive controls Celecoxib (IC50 = 15.69 μM) and Chrysin
(IC50 = 25.05 μM). Interestingly, we found that the antiproliferative
activities correlated well with their COX-2 inhibitory activities, sug-
gested that electon-donating groups substituted on the para-position of
phenyl ring were necessary to enhance their anti-tumor activities.
2.5. Mitochondrial membrane potential analysis
Mitochondrial plays a crucial role in regulating cellular functions,
and the depolarization of mitochondrial member is a prelude in the
propagation of apoptosis. The change of mitochondrial member po-
tential (MMP, ΔΨm) could be monitored by fluorescent dye JC-1. Red
fluorescence represents the J-aggregates formation in normal polarized
mitochondrial, whereas green fluorescence represents the J-monomers
formation in depolarized mitochondrial. As shown in Fig. 3, the treat-
ment of Hela cells with gradient concentrations (0, 1.25, 2.5, 5 and
10 μM) of compound e9 could induced the loss of MMP in dose-de-
pendent manner. Our results suggested that compound e9 could induce
mitochondrial dysfunction, and then induce Hela cells apoptosis.
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Table 3
Antiproliferative activities of selected compounds against four cancer cell lines and cytotoxicity towards non-cancer cells.
Compounds
IC50 (μM)
SDa
MCF-7
F10
A549
Hela
293T
e1
e2
e3
e4
e5
e6
e7
e8
3.47
5.43
1.02
0.77
0.34
1.53
1.61
0.62
1.86
0.87
0.55
1.57
2.59
3.51
17.63
2.85
4.33
4.95
1.76
10.4
12.42
6.92
6.24
65.85
10.85
15.55
20.77
7.86
0.23
0.52
0.82
3.23
4.49
2.87
4.69
> 100
11.62
32.12
10.55
6.41
12.66
13.64
12.04
4.62
0.88
0.53
1.83
0.54
2.41
4.78
2.87
12.36
1.95
1.31
4.06
2.72
1.12
0.37
0.48
1.02
173.25
315.32
197.03
222.21
315.97
398.43
161.95
256.64
193.94
290.21
317.02
187.23
265.34
197.33
226.56
299.22
187.52
161.42
271.81
315.87
278.43
192.11
216.22
348.44
233.27
273.36
268.08
162.06
211.83
271.05
319.36
224.86
4.21
3.76
2.07
2.88
1.95
3.58
1.69
2.11
2.85
1.99
3.06
1.66
2.07
3.68
3.05
2.78
2.07
2.63
2.19
3.18
1.99
3.68
3.62
2.95
3.04
1.95
2.98
3.08
2.96
2.47
3.05
2.48
3.235
7.86
10.79
3.255
23.43
7.1
3.23
> 100
5.12
3.82
17.8
9.02
46.97
> 100
32.91
21.98
3.94
1.69
1.04
0.32
1.19
0.77
0.57
1.62
2.07
0.97
1.29
0.37
0.33
0.72
0.55
0.62
0.85
1.22
1.74
0.63
e9
0.45
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
e29
e30
Chrysin
Celecoxib
0.83
0.49
0.95
0.57
2.07
5.65
1.47
0.92
1.52
1.01
13.62
> 100
> 100
2.81
1.65
2.67
1.06
3.03
1.87
0.99
1.68
2.06
1.03
0.66
40.16
> 100
3.68
4.71
8.17
0.77
0.52
0.82
0.66
2.56
0.43
2.69
1.72
0.42
1.07
0.52
2.09
1.79
2.06
1.03
3.04
1.12
0.93
1.31
1.03
1.87
2.11
0.37
1.07
0.64
0.37
1.32
1.87
0.92
1.75
3.85
5.73
25.51
> 100
6.67
24.67
2.04
18.36
30.93
6.28
6.06
4.72
6.61
4.32
2.41
4.34
3.58
8.48
2.96
> 100
32.12
15.62
2.71
21.73
6.44
7.61
6.08
9.85
7.44
0.62
2.01
0.41
2.04
1.95
0.53
0.89
0.89
1.02
0.93
0.32
2.31
2.41
0.66
0.95
1.17
0.92
0.76
1.22
1.63
0.87
22.65
17.02
5.88
> 100
5.52
> 100
3.62
8.02
5.07
6.68
47.8
16.08
22.1628
15.8256
12.56
3.29
22.16
12.08
11.02
6.21
0.57
0.55
0.86
0.88
0.66
1.86
8.01
25.66
26.78
6.53
17.45
19.15
4.33
25.05
15.69
1.06
0.79
1.05
0.97
1.49
a
Antiproliferation activity and cytotoxicity of the synthesized were measured using the MTT assay. Data shown are the mean
experiments (n = 3).
SD of three independent
incubated with increased concentration of compound e9 mostly accu-
mulate in G1 phase with the percentage increased from 55.51% to
76.02%, along with concomitant losses in G2/M phase. These results
suggested that compound e9 could induce cell-cycle arrest in G1 phase
with a dose-dependent manner.
Scheme 1. Reagents and conditions: (i) Sodium methoxide, dimethyl oxalate,
MeOH, refluxing, 6 h; (ii) phenylhydrazine hydrochloride, MeOH, refluxing,
6 h; (iii) lithium aluminium hydride, THF, 0 °C, 10 min, 45 °C, 2 h.
2.7. Molecular docking
Molecular docking studies were performed to investigate the
binding models of Celecoxib and compound e9 in COX-2. Celecoxib and
compound e9 were fitted into the activity pocket of COX-2 (PDB code:
3PGH) using Discovery Studio 3.5 (Accelrys, Co. Ltd). Then, the binding
models were predicted and detailed protein-molecular interactions
were exhibited in Fig. 5. As displayed in Fig. 5A and B, Celecoxib binds
to COX-2 with binding free energy of −36.56 kcal/mol via five hy-
drogen bonds with Trp 387, Thr 212, Asn 382 and Tyr 148. Compared
to Celecoxib, compound e9 was well embedded into the activity pocket
of COX-2 with binding free energy of −83.77 kcal/mol via two hy-
drogen bonds, one Pi-Sigma bong and four Pi-Pi bonds (Fig. 5C). The
oxygen atom of the chrysin ring of compound e9 formed two hydrogen
bonds with the amino hydrogen of Arg 222 (angle NH…O = 121.00°,
distance = 2.21 Å) and the animo hydrogen of His 207 (angle NH…
O = 102.46°, distance = 2.98 Å). In addition, van der Waals was also
crucial to the binding of compound e9 with COX-2. The 3D models
shown in Fig. 5D more clearly suggested that compound e9 was nicely
inserted into the activity pocket of COX-2. These docking results cor-
related well with their COX-2 inhibitory activities and demonstrated
that compound e9 may be a potential inhibitor of COX-2.
Scheme 2. Reagents and conditions: (i) Acetone, K2CO3, ethyl 2-bromoacetate,
70 °C, 6 h; Then, MeOH, KOH, refluxing, 2 h; (ii) EDC·HCl, DMAP, triethyla-
mine, CH2Cl2, rt, 8 h.
2.6. Compound e9 induces cell-cycle arrest in HeLa cells
To further study whether the antiproliferation of compound e9 was
due to the cell cycle arrest, a flow cytometry analysis was performed.
Hela cells were treated with gradient concentration (0, 1.25, 2.5, 5 and
10 μM) of compound e9 for 24 h and stained with propidium iodide
(PI). The results exhibited in Fig. 4 clearly shown that the population of
cells in G1 phase dramatically increased with increased concentration
of compound e9. Compared with the control group, Hela cells
4

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Fig. 2. Compound e9 induced apoptosis in Hela cells. (A, B) Effect of treatment of e9 (0, 1.25, 2.5, 5 and 10 μM) for 24 h on the apoptosis of Hela cells was
determined; (C, D) Effect of treatment of e9 (5 μM) for 12, 24, 36 or 48 h on the apoptosis of Hela cells was determined; The early apoptotic cells are located in the
lower right quadrant (Annexin V–FITC+/PI− cells), and the late apoptotic cells are in the upper right quadrant (Annexin V–FITC+/PI+ cells). Data are shown as
mean
SD of three independent experiments; *p < 0.05, **p < 0.01.
3. Conclusion
Chemical Reagent Co. Ltd. (Nanjing, China) and used in this study
without further purification. All the 1H NMR spectra 13C NMR spectra
were recorded on Bruker DPX400 or Bruker DPX600 model spectro-
meter in DMSO‑d6, and chemical shifts (δ) are reported as parts per
million (ppm). Melting points were determined on a X4 MP apparatus
(Jingsong Corp, Shanghai, China). Thin layer chromatography (TLC)
was performed on the glass backed silica gel sheets (Silica Gel 60
GF254) and visualized in UV light (254 nm and 365 nm). Column
chromatography was performed using silica gel (200–300 mesh) and
eluting with ethyl acetate and petroleum ether (bp. 30–60 °C).
Elemental analyses were performed on a CHN-O-Rapid instrument and
were within 0.4% of the theoretical values.
In order to discovery novel COX-2 inhibitors, we designed and
synthesized a series of novel diarylpyrazole derivatives integrating with
chrysin. The biology activities of these compounds were tested in var-
ious experiments. The results suggested that most of the synthesized
compounds exhibited potent inhibitory activities against COX-2 with
satisfying selectivity. Among them, compound e9 displayed the most
potent COX-2 inhibitory activities and antiproliferative activities
against Hela cells with IC50 value of 1.12 μM. Moreover, compound e9
could induce Hela cells apoptosis by mitochondrial depolarization in a
dose-dependent manner. The further studies found that compound e9
could arrest cell cycle arrest in G1 phase. In addition, molecular
docking was performed to learn the interaction between compound e9
and COX-2. The results shown that compound e9 could bind well with
COX-2, which might contribute to its COX-2 inhibitory activities and
antiproliferative activities. In summary, our studies of these well de-
signed compounds may be great helpful for the development of novel
antitumor candidates in the future.
4.2. General procedure for the synthesis of compounds a1–a15
Corresponding substituted acetophenone (8 mmol) was added to the
suspension of sodium methoxide (16 mmol) in methanol (40 mL). Then,
dimethyl oxalate (16 mmol) was added, and the mixture was allowed to
proceed under reflux for 6 h. After cooling to room temperature, the
reaction mixture was poured into ice water (100 mL), acidified with
dilute hydrochloric acid (1 M) to pH 3–4. The precipitate was isolated
4. Experimental section
by filtration and washed with ethanol and petroleum ether (VEtOH
VPE = 2:1). The product was dried under vacuum to obtain target
compound a (a1–a15).
/
4.1. Materials and measurements
All chemicals (reagent grade) used were purchased from Nanjing
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Fig. 3. Mitochondrial membrane potential were reduced by compound e9. Hela cells treated with 0, 1.25, 2.5, 5 and 10 μM e9 for 12 h were collected and analyzed.
CCCP was used as a positive control.
4.3. General procedure for the synthesis of compounds b1–b30
the crude product was purified by recrystallization from ethanol and
petroleum ether (VEtOH/VPE = 1:2) to obtain compound b (b1–b30).
A solution of compound a (1 mmol) and substituted phenylhy-
drazine hydrochloride (2 mmol) in methanol (20 mL) was heated to
reflux for 6 h under vigorous stirring. After cooling to room tempera-
ture, the reaction mixture was partitioned between ethyl acetate
(100 mL) and water (100 mL × 2). The organic layer was dried over
anhydrous sodium sulfate, and concentrated under vacuum. Further,
4.4. General procedure for the synthesis of compounds c1–c30
To
a solution of compound b (3 mmol) in anhydrous tetra-
hydrofuran (20 mL), 0.17 g of lithium aluminium hydride (4.5 mmol)
was added slowly. The mixture was stirred at 0 °C for 10 min, and then
Fig. 4. Effect of compound e9 on cell-cycle progression of HeLa cells was determined by flow cytometry analysis. Hela cells treated with 0, 1.25, 2.5, 5 and 10 μM e9
for 24 h were collected and analyzed. The percentage of cells in G1 graph are showed in the dot plots. (G1 phase, green; S phase, yellow and G2/M phase, blue)
*p < 0.05, **p < 0.01.
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Fig. 5. Binding modes of Celecoxib and compound e9 with COX-2 (PDB code: 3PGH). (A, B) The interaction between Celecoxib and key amino acid residues in the
activity pocket of COX-2; (B) The interaction between compound e9 and key amino acid residues in the activity pocket of COX-2. The hydrogen bond (green) is
displayed as dotted arrows.
heated to 45 °C for another 2 h. The reaction mixture was extracted with
ethyl acetate (100 mL) and water (100 mL × 3). The organic layer was
dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was further purified by silica gel column
chromatography (VAcOEt/VPE = 2:1) to obtain compound c (c1–c30).
washed with water and brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography (VEtOAc/VPE = 1:1) to give the
compound e (e1–e30).
4.6.1. (1, 5-Diphenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl)oxy)acetate (e1)
4.5. General procedure for the synthesis of 2-((5-hydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl)oxy)acetic acid (d)
Light yellow powder, yield 62%; mp: 189.6–191.8 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.09 (d, J = 7.4 Hz, 2H),
7.66–7.60 (m, 1H), 7.58 (t, J = 7.5 Hz, 3H), 7.43–7.35 (m, 3H),
7.35–7.28 (m, 3H), 7.27–7.22 (m, 2H), 7.21–7.16 (m, 2H), 7.06 (s, 1H),
6.89 (d, J = 2.3 Hz, 1H), 6.65 (s, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.09 (s,
2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.47, 164.04, 161.70,
157.66, 147.97, 144.12, 139.92, 132.65, 131.00, 130.11, 129.60,
129.56, 129.06, 128.96, 128.81, 128.36, 126.93, 125.69, 108.09,
To
a
solution of 5,7-dihydroxy-2-phenyl-4H-chromen-4-one
(2 mmol) in acetone (20 mL), potassium carbonate (2 mmol) and ethyl
2-bromoacetate (1 mmol) were added, and then the mixture was heated
at 70 °C for 6 h. The reaction mixture was cooled to room temperature
and the precipitate was isolated by filtration. The crude product was
dissolved in 20 mL methanol, potassium hydroxide (5 mmol) and sev-
eral drops water (1 mL) were added in sequence. The mixture was re-
fluxing for another 2 h under vigorous stirring. When cooled to room
temperature, the reaction mixture was poured into ice water and
acidified to reach pH 3–4. The precipitate was filtered off and washed
with water (10 mL). The resulted product dried under vacuum to obtain
the target compound d.
105.88, 105.81, 99.07, 94.15, 65.47, 60.68. MS(ESI): 545.21 [M+H]+
.
Anal. Calcd for C33H24N2O6: C, 72.79; H, 4.44; N, 5.14%; Found: C,
72.73; H, 4.52; N, 5.02.
4.6.2. (1-Phenyl-5-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-4-oxo-
2-phenyl-4H-chromen-7-yl)oxy)acetate (e2)
Light yellow powder, yield 55%; mp: 172.5–173.1 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.09 (d, J = 7.6 Hz, 2H),
7.66–7.56 (m, 3H), 7.43–7.35 (m, 3H), 7.24 (d, J = 7.2 Hz, 2H), 7.10
(d, J = 8.0 Hz, 2H), 7.08–7.02 (m, 3H), 6.90 (d, J = 2.1 Hz, 1H), 6.58
(s, 1H), 6.48 (d, J = 2.1 Hz, 1H), 5.27 (s, 2H), 5.09 (s, 2H), 2.27 (s, 3H).
13C NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.47, 164.05, 164.04,
161.70, 157.66, 147.92, 144.17, 140.01, 138.46, 132.65, 131.00,
129.63, 129.59, 129.55, 128.65, 128.30, 127.21, 126.92, 125.67,
107.75, 105.88, 105.80, 99.07, 94.14, 65.47, 60.71, 21.21. MS(ESI):
559.08 [M+H]+. Anal. Calcd for C34H26N2O6: C, 73.11; H, 4.69; N,
5.02%; Found: C, 72.97; H, 4.54; N, 5.04.
4.6. General procedure for the synthesis of compounds e1–e30
A solution of 2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)
acetic acid (d) (0.31 g, 1 mmol), 1-(3-Dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC·HCl) (0.29 g, 1.5 mmol), 4-(di-
methylamino)pyridine (DMAP) (0.18 g, 1.5 mmol), triethylamine
(0.60 mL, 4 mmol) and corresponding compound c (1.5 mmol) in di-
chloromethane (20 mL) was stirred at room temperature for 8 h. Then,
the reaction mixture was partitioned between dilute hydrochloric acid
(30 mL, 1 M) and dichloromethane (50 mL × 3). The organic layer was
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4.6.3. (1, 5-Diptolyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-4-oxo-2-phenyl-
4H-chromen-7-yl)oxy)acetate (e3)
C
34H25ClN2O6: C, 68.86; H, 4.25; N, 4.72%; Found: C, 68.82; H, 4.81;
N, 4.79.
Light yellow powder, yield 63%; mp: 153.2–154.5 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.09 (d, J = 7.6 Hz, 2H),
7.66–7.55 (m, 3H), 7.19 (d, J = 8.2 Hz, 2H), 7.13–7.08 (m, 4H),
7.08–7.03 (m, 3H), 6.89 (d, J = 2.1 Hz, 1H), 6.56 (s, 1H), 6.48 (d,
J = 2.1 Hz, 1H), 5.26 (s, 2H), 5.08 (s, 2H), 2.31 (s, 3H), 2.27 (s, 3H).
13C NMR (150 MHz, DMSO‑d6) δ: 184.16, 166.46, 165.11, 164.21,
160.89, 159.21, 157.08, 146.55, 143.57, 135.38, 133.56, 132.32,
131.08, 130.34, 127.15, 126.28, 126.11, 125.37, 122.73, 112.43,
105.88, 105.21, 104.02, 99.58, 94.31, 65.37, 58.26, 52.79, 22.06. MS
(ESI): 573.62 [M+H]+. Anal. Calcd for C35H28N2O6: C, 73.41; H, 4.93;
N, 4.89%; Found: C, 73.40; H, 4.87; N, 4.83.
4.6.8. (5-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e8)
Light yellow powder, yield 69%; mp: 143.6–144.2 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.12–8.06 (m, 2H), 7.66–7.61
(m, 1H), 7.58 (m, 2H), 7.42–7.35 (m, 3H), 7.27–7.22 (m, 2H),
7.11–7.05 (m, 3H), 6.89 (d, J = 2.3 Hz, 1H), 6.88–6.83 (m, 2H), 6.55
(s, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.27 (s, 2H), 5.08 (s, 2H), 3.73 (s, 3H).
13C NMR (150 MHz, DMSO‑d6) δ: 182.59, 168.46, 164.05, 161.67,
159.71, 157.67, 147.83, 144.00, 140.03, 132.67, 131.00, 130.15,
129.62, 129.54, 128.26, 126.94, 125.67, 122.33, 114.49, 107.47,
105.89, 105.81, 99.08, 94.15, 65.46, 60.72, 55.61. MS(ESI): 575.17 [M
+H]+. Anal. Calcd for C34H26N2O7: C, 71.07; H, 4.56; N, 4.88%;
Found: C, 70.93; H, 4.49; N, 4.91.
4.6.4. (1-(4-Chlorophenyl)-5-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e4)
Light yellow powder, yield 45%; mp: 172.6–173.3 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.11–8.06 (m, 2H), 7.65–7.61
(m, 1H), 7.58 (m, 2H), 7.49–7.44 (m, 2H), 7.28–7.23 (m, 2H), 7.13 (d,
J = 7.6, 2H), 7.09–7.04 (m, 3H), 6.89 (d, J = 2.3 Hz, 1H), 6.59 (s, 1H),
6.47 (d, J = 2.3 Hz, 1H), 5.27 (s, 2H), 5.08 (s, 2H), 2.28 (s, 3H). 13C
NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.45, 164.05, 161.68, 157.67,
148.32, 144.29, 138.77, 138.68, 132.67, 131.00, 129.76, 129.57,
128.73, 127.21, 126.94, 108.06, 105.89, 99.07, 94.17, 65.45, 60.61,
21.24. MS(ESI): 593.21 [M+H]+. Anal. Calcd for C34H25ClN2O6: C,
68.86; H, 4.25; N, 4.72%; Found: C, 68.75; H, 4.28; N, 4.72.
4.6.9. (5-(4-Methoxyphenyl)-1-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hy-
droxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e9)
Yellow powder, yield 51%; mp: 163.4–164.6 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.83 (s, 1H), 8.09 (d, J = 7.5 Hz, 2H), 7.63 (t, J = 7.2 Hz,
1H), 7.58 (t, J = 7.6 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 7.13–7.06 (m,
5H), 6.89 (d, J = 2.1 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H),
6.48 (d, J = 2.2 Hz, 1H), 5.26 (s, 2H), 5.08 (s, 2H), 3.73 (s, 3H), 2.32 (s,
3H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.56, 168.46, 164.03, 164.01,
161.69, 159.67, 157.65, 147.60, 143.90, 137.68, 137.67, 132.62,
130.98, 130.11, 129.95, 129.58, 126.90, 125.49, 122.43, 114.47,
107.31, 105.86, 105.80, 99.06, 94.11, 65.47, 60.76, 55.59, 21.05. MS
(ESI): 589.21 [M+H]+. Anal. Calcd for C35H28N2O7: C, 71.42; H, 4.79;
N, 4.76%; Found: C, 71.43; H, 4.72; N, 4.83.
4.6.5. (1-Phenyl-5-(m-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl)oxy)acetate (e5)
Yellow powder, yield 71%; mp: 163.6–164.1 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.83 (s, 1H), 8.07 (d, J = 7.5 Hz, 2H), 7.59 (m, 3H),
7.41–7.36 (m, 3H), 7.24 (d, J = 7.0 Hz, 2H), 7.18–7.12 (m, 2H), 7.05
(d, J = 23.0 Hz, 2H), 6.88 (m, J = 7.6, 4.9 Hz, 2H), 6.62 (s, 1H), 6.47
(d, J = 2.1 Hz, 1H), 5.29 (s, 2H), 5.08 (s, 2H), 2.22 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ: 182.56, 168.47, 164.02, 161.70, 157.64,
147.94, 144.22, 139.97, 138.36, 132.63, 130.98, 130.04, 129.58,
129.51, 129.37, 128.85, 128.30, 126.91, 125.92, 125.63, 108.00,
105.86, 105.79, 99.04, 94.15, 65.47, 60.69, 21.34. MS(ESI): 559.03 [M
+H]+. Anal. Calcd for C34H26N2O6: C, 73.11; H, 4.69; N, 5.02%;
Found: C, 73.17; H, 4.79; N, 5.13.
4.6.10. (1-(4-Chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazol-3-yl)
methyl 2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e10)
Light yellow powder, yield 77%; mp: 143.5–144.3 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.10 (d, J = 7.6 Hz, 2H), 7.64 (t,
J = 7.3 Hz, 1H), 7.59 (t, J = 7.6 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.26
(d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 7.07 (s, 1H), 6.89 (d,
J = 8.6 Hz, 3H), 6.56 (s, 1H), 6.48 (d, J = 2.2 Hz, 1H), 5.27 (s, 2H),
5.08 (s, 2H), 3.74 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ: 184.43,
169.62, 165.26, 163.74, 157.17, 148.92, 144.83, 138.52, 136.53,
131.78, 131.03, 129.88, 129.77, 129.31, 128.93, 125.95, 125.63,
106.19, 105.87, 105.31, 99.33, 94.26, 67.46, 59.19, 20.22. MS(ESI):
609.33 [M+H]+. Anal. Calcd for C34H25ClN2O7: C, 67.05; H, 4.24; N,
4.60%; Found: C, 66.99; H, 4.23; N, 4.78.
4.6.6. (5-(M-tolyl)-1-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-4-oxo-2-
phenyl-4H-chromen-7-yl)oxy)acetate (e6)
Yellow powder, yield 55%; mp: 155.3–156.1 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.83 (s, 1H), 8.08 (d, J = 7.6 Hz, 2H), 7.60 (m, 3H), 7.14
(m,7H), 7.05 (s, 1H), 6.87 (d, J = 11.6 Hz, 2H), 6.59 (s, 1H), 6.47 (s,
1H), 5.27 (s, 2H), 5.08 (s, 2H), 2.31 (s, 3H), 2.23 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ: 187.12, 165.43, 165.01, 164.11, 161.83,
158.35, 157.36, 144.75, 142.77, 135.02, 133.48, 131.32, 131.11,
130.08, 128.32, 126.16, 126.01, 126.47, 124.63, 113.53, 106.83,
105.31, 104.77, 99.53, 94.01, 67.41, 57.25, 53.19, 24.53. MS(ESI):
573.52 [M+H]+. Anal. Calcd for C35H28N2O6: C, 73.41; H, 4.93; N,
4.89%; Found: C, 73.37; H, 4.84; N, 4.86.
4.6.11. (5-(3-Methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydro-
xy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e11)
Yellow powder, yield 59%; mp: 187.6–188.2 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.82 (s, 1H), 8.03 (d, J = 7.3 Hz, 2H), 7.61–7.50 (m, 3H),
7.44–7.34 (m, 3H), 7.28–7.17 (m, 3H), 6.99 (s, 1H), 6.91–6.79 (m, 2H),
6.75–6.65 (m, 3H), 6.45 (s, 1H), 5.30 (s, 2H), 5.06 (s, 2H), 3.61 (s, 3H).
13C NMR (150 MHz, DMSO‑d6) δ: 182.56, 168.46, 164.02, 161.70,
159.50, 157.64, 147.90, 143.97, 139.97, 132.62, 131.27, 130.98,
130.19, 129.57, 129.55, 128.42, 126.90, 125.79, 121.05, 114.56,
114.30, 108.08, 105.86, 105.80, 99.04, 94.13, 65.47, 60.68, 55.40. MS
(ESI): 575.22 [M+H]+. Anal. Calcd for C34H26N2O7: C, 71.07; H, 4.56;
N, 4.88%; Found: C, 71.13; H, 4.59; N, 4.66.
4.6.7. (1-(4-Chlorophenyl)-5-(m-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e7)
Light yellow powder, yield 52%; mp: 157.2–157.9 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.84 (s, 1H), 8.09 (d, J = 7.5 Hz, 2H), 7.63 (t,
J = 7.3 Hz, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 7.26
(d, J = 8.7 Hz, 2H), 7.19 (m, 2H), 7.12 (s, 1H), 7.06 (s, 1H), 6.93–6.85
(m, 2H), 6.63 (s, 1H), 6.48 (d, J = 2.2 Hz, 1H), 5.28 (s, 2H), 5.09 (s,
2H), 2.25 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.46,
164.03, 161.70, 157.65, 148.34, 144.35, 138.74, 138.55, 132.65,
132.58, 130.99, 129.82, 129.60, 129.53, 129.44, 128.99, 127.13,
126.92, 126.01, 125.64, 108.36, 105.87, 105.80, 99.03, 94.17, 65.46,
4.6.12. (5-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e12)
Light yellow powder, yield 66%; mp: 190.1–190.6 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.14–8.07 (m, 2H), 7.67–7.61
(m, 1H), 7.61–7.54 (m, 2H), 7.44–7.36 (m, 3H), 7.26–7.20 (m, 4H),
7.20–7.11 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 2.3 Hz, 1H), 6.65 (s, 1H),
6.48 (d, J = 2.3 Hz, 1H), 5.28 (s, 2H), 5.09 (s, 2H). 13C NMR (150 MHz,
DMSO‑d6) δ: 182.59, 168.45, 164.06, 161.67, 157.68, 147.95, 143.13,
60.60, 21.36. MS(ESI): 593.21 [M+H]+
.
Anal. Calcd for
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139.74, 132.69, 131.11, 129.63, 128.45, 126.95, 125.72, 116.18,
116.03, 108.13, 105.90, 99.08, 94.17, 65.46, 60.64. MS(ESI): 563.44
[M+H]+. Anal. Calcd for C33H23FN2O6: C, 70.46; H, 4.12; N, 4.98%;
Found: C, 70.44; H, 4.09; N, 5.07.
(s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 184.52, 169.73, 165.26,
160.54, 156.28, 146.33, 145.23, 138.55, 134.73, 131.01, 129.42,
129.05, 127.54, 124.01, 116.66, 116.31, 107.53, 104.63, 99.50, 94.21,
66.43, 60.32. MS(ESI): 579.37 [M+H]+
.
Anal. Calcd for
33H23ClN2O6: C, 68.46; H, 4.00; N, 4.84%; Found: C, 68.47; H, 4.01;
N, 4.94.
C
4.6.13. (5-(4-Fluorophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl) methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e13)
Light yellow powder, yield 73%; mp: 189.6–191.4 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.11–8.05 (m, 2H), 7.65–7.60
(m, 1H), 7.57 (m, 2H), 7.24–7.17 (m, 4H), 7.18–7.09 (m, 4H), 7.05 (s,
1H), 6.88 (d, J = 2.3 Hz, 1H), 6.62 (s, 1H), 6.46 (d, J = 2.3 Hz, 1H),
5.27 (s, 2H), 5.07 (s, 2H), 2.31 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
δ: 182.58, 168.46, 164.04, 163.22, 161.68, 161.59, 157.66, 147.73,
143.03, 137.91, 137.38, 132.66, 131.02, 130.04, 129.60, 126.93,
126.68, 125.54, 116.16, 116.01, 107.96, 105.88, 105.80, 99.07, 94.15,
65.47, 60.66, 21.05. MS(ESI): 577.28 [M+H]+. Anal. Calcd for
4.6.18. (5-(3-Chlorophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl) methyl-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e18)
Light yellow powder, yield 55%; mp: 159.5–161.5 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.07 (d, J = 7.1 Hz, 2H),
7.63–7.55 (m, 3H), 7.35–7.28 (m, 3H), 7.18 (m, 3H), 7.11 (d,
J = 8.3 Hz, 2H), 7.04 (s, 1H), 6.88 (d, J = 2.3 Hz, 1H), 6.62 (s, 1H),
6.47 (d, J = 2.3 Hz, 1H), 5.28 (s, 2H), 5.08 (s, 2H), 2.31 (s, 3H). 13C
NMR (150 MHz, DMSO‑d6) δ: 182.59, 168.46, 164.06, 161.67, 157.67,
147.72, 144.01, 137.82, 137.54, 132.68, 131.01, 129.97, 129.62,
129.05, 128.78, 126.95, 125.51, 107.89, 105.90, 105.80, 99.08, 94.16,
65.46, 60.69, 21.05. MS(ESI): 593.46 [M+H]+. Anal. Calcd for
C
34H25FN2O6: C, 70.83; H, 4.37; N, 4.86%; Found: C, 70.73; H, 4.32; N,
4.79.
C
34H25ClN2O6: C, 68.86; H, 4.25; N, 4.72%; Found: C, 69.03; H, 4.24;
4.6.14. (5-(3-Fluorophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e14)
N, 4.86.
Yellow powder, yield 74%; mp: 142.5–143.2 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.82 (s, 1H), 8.06 (d, J = 6.3 Hz, 2H), 7.59 (dd, J = 24.8,
6.0 Hz, 3H), 7.44–7.32 (m, 4H), 7.26 (d, J = 5.3 Hz, 2H), 7.17 (s, 1H),
7.06–6.95 (m, 3H), 6.87 (s, 1H), 6.73 (s, 1H), 6.46 (s, 1H), 5.30 (s, 2H),
5.08 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.56, 168.45, 164.02,
163.09, 161.70, 161.47, 157.65, 148.05, 142.79, 139.69, 132.64,
132.21, 131.13, 130.98, 129.62, 128.62, 126.90, 125.81, 125.01,
115.91, 115.77, 115.71, 115.56, 108.52, 105.83, 99.04, 94.15, 65.46,
60.59. MS(ESI): 563.25 [M+H]+. Anal. Calcd for C33H23FN2O6: C,
70.46; H, 4.12; N, 4.98%; Found: C, 70.39; H, 4.13; N, 5.01.
4.6.19. (5-(3-Chlorophenyl)-1-(4-chlorophenyl)-1H-pyrazol-3-yl)methyl
2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e19)
Light yellow powder, yield 49%; mp: 192.6–193.1 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.11–8.07 (m, 2H), 7.65–7.61
(m, 1H), 7.60–7.56 (m, 2H), 7.50–7.44 (m, 2H), 7.36–7.34 (m, 2H),
7.30–7.25 (m, 2H), 7.23–7.17 (m, 2H), 7.06 (s, 1H), 6.89 (d,
J = 2.3 Hz, 1H), 6.65 (s, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.29 (s, 2H),
5.08 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.60, 168.46, 164.06,
161.69, 157.68, 148.37, 144.26, 138.70, 132.69, 132.67, 131.01,
129.86, 129.68, 129.63, 129.59, 129.20, 129.17, 128.90, 127.39,
127.24, 126.96, 108.41, 105.91, 105.81, 99.07, 94.19, 65.46, 60.59.
MS(ESI): 613.07 [M+H]+. Anal. Calcd for C33H22Cl2N2O6: C, 64.61; H,
3.61; N, 4.57%; Found: C, 64.77; H, 3.65; N, 4.51.
4.6.15. (5-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e15)
Light yellow powder, yield 68%; mp: 177.6–179.3 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (d, J = 3.3 Hz, 1H), 8.09 (d, J = 7.5 Hz,
2H), 7.63 (t, J = 7.3 Hz, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.44–7.36 (m,
5H), 7.27–7.23 (m, 2H), 7.21–7.14 (m, 2H), 7.06 (s, 1H), 6.89 (d,
J = 2.1 Hz, 1H), 6.66 (d, J = 20.7 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H),
5.29 (s, 2H), 5.08 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.59,
168.45, 164.06, 161.67, 157.67, 148.08, 142.89, 139.68, 133.77,
132.68, 130.99, 130.56, 129.62, 129.14, 128.81, 128.55, 126.94,
125.74, 108.30, 105.89, 99.08, 94.17, 65.46, 60.60. MS(ESI): 579.09
[M+H]+. Anal. Calcd for C33H23ClN2O6: C, 68.46; H, 4.00; N, 4.84%;
Found: C, 68.47; H, 4.03; N, 4.92.
4.6.20. (5-(4-Bromophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e20)
Light yellow powder, yield 63%; mp: 188.6–189.3 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (d, J = 3.5 Hz, 1H), 8.12–8.05 (m, 2H),
7.63 (t, J = 7.3 Hz, 1H), 7.58 (t, J = 7.5 Hz, 2H), 7.43–7.36 (m, 3H),
7.35–7.29 (m, 2H), 7.24 (m, 2H), 7.20–7.10 (m, 2H), 7.06 (d,
J = 1.3 Hz, 1H), 6.66 (d, J = 22.1 Hz, 1H), 6.48 (t, J = 2.4 Hz, 1H),
5.29 (s, 2H), 5.08 (d, J = 2.4 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:
182.57, 168.48, 164.04, 161.67, 157.66, 147.97, 144.13, 142.95,
139.89, 132.68, 132.05, 130.96, 130.78, 130.07, 129.70, 129.62,
129.57, 129.06, 128.79, 128.39, 126.92, 125.70, 108.26, 108.06,
4.6.16. (5-(4-Chlorophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e16)
Light yellow powder, yield 72%; mp: 142.7–143.4 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.08 (d, J = 7.4 Hz, 2H), 7.60
(m, 3H), 7.37 (d, J = 8.3 Hz, 2H), 7.22–7.10 (m, 7H), 7.05 (s, 1H), 6.88
(s, 1H), 6.63 (d, J = 25.0 Hz, 1H), 6.47 (s, 1H), 5.28 (s, 2H), 5.08 (s,
2H), 2.32 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.59, 168.46,
164.05, 161.68, 157.67, 147.85, 142.80, 138.03, 137.32, 133.71,
132.68, 131.00, 130.54, 130.10, 129.62, 129.13, 126.94, 125.56,
108.12, 107.15, 105.89, 105.81, 99.08, 94.15, 65.47, 60.62, 57.69,
21.07. MS(ESI): 593.05 [M+H]+. Anal. Calcd for C34H25ClN2O6: C,
68.86; H, 4.25; N, 4.72%; Found: C, 68.82; H, 4.33; N, 4.72.
105.85, 105.79, 99.07, 94.13, 65.45, 60.68. MS(ESI): 623.17 [M+H]+
.
Anal. Calcd for C33H23BrN2O6: C, 63.57; H, 3.72; N, 4.49%; Found: C,
63.62; H, 3.89; N, 4.41.
4.6.21. (5-(4-Bromophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e21)
Yellow powder, yield 62%; mp: 142.6–143.7 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.82 (s, 1H), 8.09 (d, J = 7.4 Hz, 2H), 7.63 (t, J = 7.3 Hz,
1H), 7.58 (t, J = 7.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.21 (d,
J = 8.2 Hz, 2H), 7.12 (m, 4H), 7.06 (s, 1H), 6.89 (d, J = 2.3 Hz, 1H),
6.66 (s, 1H), 6.47 (d, J = 2.3 Hz, 1H), 5.27 (s, 2H), 5.08 (s, 2H), 2.32 (s,
3H). 13C NMR (150 MHz, DMSO‑d6) δ: 188.78, 169.32, 165.22, 162.50,
158.07, 149.55, 144.38, 140.21, 138.50, 134.78, 133.11, 129.53,
129.44, 128.81, 127.71, 126.08, 109.25, 106.81, 99.26, 94.51, 66.01,
4.6.17. (5-(3-Chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl-((5-
hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e17)
Light yellow powder, yield 66%; mp: 166.8–167.2 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (d, J = 2.3 Hz, 1H), 8.11–8.06 (m, 2H),
7.65–7.61 (m, 1H), 7.58 (m, 2H), 7.44–7.37 (m, 3H), 7.33–7.30 (m,
2H), 7.27–7.22 (m, 2H), 7.20–7.12 (m, 2H), 7.06 (d, J = 3.4 Hz, 1H),
6.89 (m, 1H), 6.70 (s, 1H), 6.48 (t, J = 2.6 Hz, 1H), 5.29 (s, 2H), 5.09
61.47, 20.33. MS(ESI): 637.52 [M+H]+
34H25BrN2O6: C, 64.06; H, 3.95; N, 4.39%; Found: C, 63.97; H, 3.88;
N, 4.36.
.
Anal. Calcd for
C
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4.6.22. (5-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e22)
128.79, 128.45, 127.46, 126.91, 125.61, 108.39, 105.87, 99.03, 94.17,
65.46, 60.59, 21.07. MS(ESI): 627.31 [M+H]+. Anal. Calcd for
Light yellow powder, yield 75%; mp: 138.9–139.1 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.13 (d, J = 8.8 Hz, 2H), 8.07 (d,
J = 7.4 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H),
7.46–7.41 (m, 5H), 7.28 (dd, J = 7.5, 1.9 Hz, 2H), 7.05 (s, 1H), 6.89 (d,
J = 2.2 Hz, 1H), 6.84 (s, 1H), 6.48 (d, J = 2.2 Hz, 1H), 5.31 (s, 2H),
5.09 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.54, 168.46, 164.01,
163.99, 161.67, 157.64, 148.42, 147.36, 142.01, 139.48, 136.26,
132.62, 130.94, 129.86, 129.57, 128.87, 126.86, 125.83, 124.19,
109.39, 105.84, 105.80, 99.08, 94.10, 65.46, 60.49. MS(ESI): 590.05
[M+H]+. Anal. Calcd for C33H23N3O8: C, 67.23; H, 3.93; N, 7.13%;
Found: C, 67.13; H, 4.02; N, 7.11.
C34H24Cl2N2O6: C, 65.08; H, 3.86; N, 4.46%; Found: C, 64.98; H, 3.92;
N, 4.42.
4.6.27. (1-(4-Chlorophenyl)-5-(3,4-dichlorophenyl)-1H-pyrazol-3-yl)
methyl 2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e27)
Light yellow powder, yield 49%; mp: 177.6–178.2 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.20–7.99 (m, 2H), 7.67–7.61
(m, 1H), 7.61–7.55 (m, 3H), 7.50 (td, J = 8.4, 3.0 Hz, 2H), 7.38–7.33
(m, 1H), 7.33–7.27 (m, 2H), 7.12–7.05 (m, 2H), 6.89 (t, J = 2.2 Hz,
1H), 6.77 (d, J = 19.0 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 5.29 (s, 2H),
5.08 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 185.33, 169.12, 165.05,
160.28, 158.57, 146.36, 144.77, 139.93, 138.36, 133.03, 130.25,
130.11, 129.78, 129.44, 129.02, 128.71, 128.22, 126.07, 125.88,
125.63, 107.31, 105.96, 105.32, 99.27, 97.88, 65.42, 62.62, 20.56. MS
(ESI): 647.20 [M+H]+. Anal. Calcd for C33H21Cl3N2O6: C, 61.18; H,
3.27; N, 4.32%; Found: C, 61.02; H, 3.24; N, 4.29.
4.6.23. (5-(4-Nitrophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e23)
Yellow powder, yield 60%; mp: 154.2–155.7 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.82 (s, 1H), 8.14 (d, J = 8.6 Hz, 2H), 8.08 (d,
J = 7.5 Hz, 2H), 7.62 (t, J = 7.1 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.43
(d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H),
7.06 (s, 1H), 6.92–6.86 (m, 1H), 6.82 (s, 1H), 6.48 (d, J = 1.8 Hz, 1H),
5.30 (s, 2H), 5.09 (s, 2H), 2.33 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
δ: 182.50, 168.46, 163.98, 163.95, 161.68, 157.60, 148.27, 148.08,
141.81, 139.41, 135.10, 132.59, 131.43, 130.91, 130.64, 129.81,
129.53, 128.85, 126.84, 125.91, 123.60, 123.23, 108.92, 105.79,
105.77, 99.03, 94.10, 65.47, 60.54. MS(ESI): 604.23 [M+H]+. Anal.
Calcd for C34H25N3O8: C, 67.66; H, 4.18; N, 6.96%; Found: C, 67.51; H,
4.04; N, 7.07.
4.6.28. (1-Phenyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)methyl
2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e28)
Light yellow powder, yield 62%; mp: 163.5–164.7 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.09 (d, J = 7.1 Hz, 2H),
7.72–7.51 (m, 5H), 7.47–7.32 (m, 5H), 7.28–7.22 (m, 2H), 7.06 (s, 1H),
6.90 (d, J = 2.3 Hz, 1H), 6.78 (s, 1H), 6.48 (d, J = 2.2 Hz, 1H), 5.30 (s,
2H), 5.09 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.53, 168.45,
164.01, 161.70, 157.63, 148.25, 142.60, 139.60, 134.02, 132.59,
130.95, 129.75, 129.55, 129.50, 128.70, 126.87, 125.95, 125.80,
108.92, 105.81, 99.05, 94.09, 65.46, 60.56. MS(ESI): 613.06 [M+H]+
.
4.6.24. (5-(3-Nitrophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydroxy-
4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e24)
Anal. Calcd for C34H23F3N2O6: C, 66.67; H, 3.78; N, 4.57%; Found: C,
66.76; H, 3.72; N, 4.67.
Light yellow powder, yield 71%; mp: 182.1–183.3 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.03 (d, J = 7.3 Hz, 2H),
7.61–7.50 (m, 3H), 7.44–7.34 (m, 3H), 7.28–7.17 (m, 3H), 6.99 (s, 1H),
6.91–6.79 (m, 2H), 6.75–6.65 (m, 3H), 6.45 (s, 1H), 5.30 (s, 2H), 5.06
(s, 2H), 3.61 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ: 185.23, 167.42,
163.22, 160.24, 158.34, 146.25, 142.37, 138.09, 133.32, 130.22,
129.52, 128.03, 127.25, 125.66, 117.98, 115.43, 109.35, 107.44,
99.33, 94.52, 66.37, 60.25. MS(ESI): 590.11 [M+H]+. Anal. Calcd for
4.6.29. (1-(P-tolyl)-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)methyl
2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e29)
Light yellow powder, yield 59%; mp: 165.2–166.4 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.82 (s, 1H), 8.09 (d, J = 7.2 Hz, 2H),
7.69–7.55 (m, 5H), 7.39 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H),
7.17–7.12 (m, 2H), 7.06 (s, 1H), 6.90 (d, J = 2.3 Hz, 1H), 6.76 (s, 1H),
6.48 (d, J = 2.3 Hz, 1H), 5.29 (s, 2H), 5.08 (s, 2H), 2.32 (s, 3H). 13C
NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.46, 164.04, 161.68, 157.67,
148.03, 142.50, 138.23, 137.22, 134.11, 132.66, 130.99, 130.19,
129.54, 129.12, 128.90, 126.92, 125.96, 125.63, 125.33, 123.53,
108.74, 105.84, 99.09, 94.14, 65.47, 60.56, 21.06. MS(ESI): 627.39 [M
+H]+. Anal. Calcd for C35H25F3N2O6: C, 67.09; H, 4.02; N, 4.47%;
Found: C, 67.16; H, 3.97; N, 4.51.
C
33H23N3O8: C, 67.23; H, 3.93; N, 7.13%; Found: C, 67.32; H, 4.02; N,
7.02.
4.6.25. (5-(3,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-3-yl)methyl 2-((5-hydro-
xy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e25)
Yellow powder, yield 81%; mp: 125.6–127.4 °C; 1H NMR (600 MHz,
DMSO‑d6) δ: 12.83 (s, 1H), 8.08 (d, J = 7.2 Hz, 2H), 7.65–7.60 (m, 1H),
7.60–7.55 (m, 2H), 7.45–7.37 (m, 4H), 7.32 (t, J = 7.9 Hz, 1H), 7.27
(m, 3H), 7.10 (d, J = 7.8 Hz, 1H), 7.05 (d, J = 3.8 Hz, 1H), 6.89 (d,
J = 2.3 Hz, 1H), 6.70 (m, 1H), 6.47 (d, J = 2.3 Hz, 1H), 5.29 (s, 2H),
5.09 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 182.58, 168.45, 164.03,
161.68, 157.66, 148.07, 142.57, 139.63, 133.69, 132.67, 132.03,
130.99, 129.68, 128.87, 128.65, 128.46, 127.49, 126.93, 125.82,
4.6.30. (1-Phenyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)methyl
2-((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl)oxy)acetate (e30)
Light yellow powder, yield 64%; mp: 182.9–183.7 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.83 (s, 1H), 8.08 (d, J = 7.1 Hz, 2H),
7.71–7.53 (m, 5H), 7.50–7.41 (m, 5H), 7.28 (dd, J = 7.7, 2.0 Hz, 2H),
7.05 (s, 1H), 6.89 (d, J = 2.3 Hz, 1H), 6.83 (s, 1H), 6.47 (d, J = 2.3 Hz,
1H), 5.31 (s, 2H), 5.09 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:
182.52, 168.45, 163.98, 163.97, 161.71, 157.61, 148.17, 142.52,
139.56, 132.73, 132.58, 130.96, 130.19, 129.90, 129.69, 129.63,
129.53, 129.48, 128.72, 126.85, 125.93, 125.82, 125.56, 125.52,
125.49, 125.23, 125.20, 125.18, 125.09, 123.29, 108.64, 107.68,
105.81, 105.77, 98.98, 94.13, 65.47, 60.58, 57.68. MS(ESI): 613.28 [M
+H]+. Anal. Calcd for C34H23F3N2O6: C, 66.67; H, 3.78; N, 4.57%;
Found: C, 66.63; H, 3.72; N, 4.66.
108.54, 105.89, 99.04, 94.19, 65.46, 60.57. MS(ESI): 613.55 [M+H]+
.
Anal. Calcd for C33H22Cl2N2O6: C, 64.61; H, 3.61; N, 4.57%; Found: C,
64.51; H, 3.65; N, 4.49.
4.6.26. (5-(3,4-Dichlorophenyl)-1-(p-tolyl)-1H-pyrazol-3-yl) methyl 2-
((5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl) oxy) acetate (e26)
Light yellow powder, yield 62%; mp: 144.5–145.2 °C; 1H NMR
(600 MHz, DMSO‑d6) δ: 12.84–12.82 (m, 1H), 8.09 (m, 2H), 7.65–7.61
(m, 1H), 7.60–7.55 (m, 2H), 7.41–7.36 (m, 1H), 7.32–7.29 (m, 1H),
7.25–7.20 (m, 2H), 7.17–7.12 (m, 2H), 7.10–7.04 (m, 2H), 6.89 (t,
J = 2.3 Hz, 1H), 6.76–6.65 (m, 1H), 6.47 (d, J = 2.3 Hz, 1H), 5.28 (s,
2H), 5.08 (s, 2H), 2.33 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ:
182.56, 168.44, 164.03, 161.68, 157.64, 147.84, 142.47, 138.10,
137.26, 133.69, 132.65, 132.13, 130.98, 130.84, 130.07, 129.59,
4.7. Cell culture
A549 (human lungs cell line), HeLa (human cervix cell line), MCF-7
(human breast cancer cell line), B16-F10 (murine melanoma cell line)
and 293T (human kidney epithelial cell line) were preserved in the
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State Key Laboratory of Pharmaceutical Biotechnology of Nanjing
University. Cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum (FBS, BI), 100 U/mL
penicillin and 100 mg/mL streptomycin (Hyclone), and incubated at
37 °C in a humidified atmosphere containing 5% CO2.
4.12. Cell cycle assay
Hela cells were seeded in six-well plates (2 × 105 cells/well) and
incubation at 37 °C for 12 h. Then, the cells were treated with com-
pound e9 at final concentrations of 0, 1.25, 2.5, 5 and 10 μM for 24 h.
After incubation, the cells were harvested by trypsinization and washed
twice with ice-cold PBS. Subsequently, the cells were fixed in ice-cold
70% ethanol for a least 24 h. After being centrifuged, the cells treated
with RNase A (0.1 mg/mL) at 37 °C for 30 min, and stained with PI
(5 μg/mL) for 30 min. Cell-cycle distribution was analyzed by a flow
cytometer (Becton Dickinson, USA). Statistical analysis was done using
Flowjo 7.6.1 software.
4.8. In vitro COX inhibition assay
The ability of the synthesized compounds to inhibit COX-1 and
COX-2 was determined by COX-1/COX-2 ELISA Kit (catalog number
701170 and 701180, Cayman Chemical). COX-1 or COX-2 enzyme was
pre-incubated with test compounds at 0 μM, 1 μM, 10 μM and 100 μM in
the supplied reaction buffer (0.1 M Tris–HCl, pH 8.0, 5 mM EDTA,
2 mM phenol and 1 μM heme) for 10 min at 37 °C. The reactions were
initiated by adding arachidonic acid to a final concentration of 100 μM
and incubated 2 min at 37 °C for. Afterwards, 1 M HCl was added to the
reaction mixtures to stop the reaction, followed by one tenth the vo-
lume of saturated stannous chloride (50 mg/mL). The reaction mixtures
were incubated for 5 min at room temperature, and the amount of
PGF2α produced from PGH2 by reduction with stannous chloride was
measured using enzyme immunoassay at 450 nm.
4.13. Molecular docking
Molecular docking of compound e9 into the three dimensional X-ray
structure of Matrix COX-2 (PDB code: 3PGH) was carried out using the
Discovery Studio (version 3.5) as implemented through the graphical
user interface DS-CDOCKER protocol. The crystal structure was ob-
tained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/
home/home.do). For protein preparation, the hydrogen atoms were
added, and the water and impurities were removed. Then, compound
e9 was inserted into he binding pocket of COX-2 based on the binding
mode. Types of interactions of the docked protein with ligand were
analyzed at the end of molecular docking.
4.9. Anti-proliferative assay
The antiproliferation activities of the synthesized compounds
against Hela, A549, MCF-7, F10 and 293T cell lines were evaluated
using a standard MTT-based colorimetric assay with some modification.
Briefly, the cells were seeded at a density of 5 × 104 cells/well into 96-
well plates and then allowed to adhere for 12 h at 37 °C, 5% CO2 at-
mosphere. Then the cells were treated with the synthesized compounds
at final concentrations of 0, 0.1, 1, 10 and 100 μM with Celecoxib as
positive reference. After 48 h incubation, cell viability was assessed by
the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reduction assay carried out strictly according to the
manufacturer’s instructions (Sigma). The absorbance (OD 570 nm) was
read on an ELISA reader (ELx800, BioTek, USA) with reference wave-
length of 630 nm. The IC50 was calculated according to the dose-de-
Acknowledgements
The work was financed by Public Science and Technology Research
Fund Project of Ocean (No. 201505023).
A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmc.2018.07.022.
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