Discovery of novel sulfonamide-containing aminophosphonate derivatives as selective COX-2 inhibitors and anti-tumor candidates

Article abstract of DOI:10.1016/j.bioorg.2020.104390

As an essential enzyme with a variety of physiological functions, Cyclooxygenase-2 (COX-2) is also closely related to carcinoma due to the observed overexpression. In this work, a novel series of sulfonamide-containing aminophosphonate derivatives (A1-A25

Full text of DOI:10.1016/j.bioorg.2020.104390

Bioorganic Chemistry 105 (2020) 104390
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel sulfonamide-containing aminophosphonate derivatives
as selective COX-2 inhibitors and anti-tumor candidates
,
,
,*
Bo Zhanga, Xiu-Ting Hua, Jin Gua, Yu-Shun Yanga *, Yong-Tao Duanb *, Hai-Liang Zhua
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, PR China
b Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases, Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou University,
Zhengzhou 450018, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
As an essential enzyme with a variety of physiological functions, Cyclooxygenase-2 (COX-2) is also closely
related to carcinoma due to the observed overexpression. In this work, a novel series of sulfonamide-containing
aminophosphonate derivatives (A1-A25) were developed as selective COX-2 inhibitors and anti-cancer candi-
dates. The top hit compound A23 presented applicative COX-2 inhibitory activity (IC50 = 0.28 µM) and anti-
proliferative capability against several cancer cell lines (IC50 = 2.34–16.43 µM for HeLa, MCF-7, HCT116 and
HepG2 cells). Among them, A23 has the most significant inhibitory effect on HCT116 cells, which were com-
parable with that of the positive controls respectively (eg: IC50 = 8.73 µM for HCT116). The binding pattern of
A23 was inferred by the molecular docking simulation. Moreover, A23 could induce the apoptosis via a
mitochondrion-dependent mode and cause the arrest of the cell-cycle in G1 stage. A further investigation in the
checkpoints of apoptosis indicated that the node Bcl-2 might connect the selective COX-2 inhibition and the anti-
tumor activity. Therefore, this work brought new information for developing COX-2 inhibitors in anti-tumor
therapies in future.
Cyclooxygenase-2
Anti-tumor
Sulfonamide moiety
Aminophosphonate derivatives
Apoptosis induction
Mitochondrion-dependent mode
1. Introduction
steroidal anti-inflammatory drugs (NSAIDs) [9], there have been a
plenty sample size to check their performances in the anti-proliferation,
Prostaglandin (PGs), a vital series of aliphatic acids, play significant
role in the physiological and pathological processes including platelet
aggregation, cardiovascular balance, inflammation and tumor progres-
sion. Cyclooxygenase (COX), transforming arachidonic acid into PGs, is
the rate-limiting enzyme of the biosynthesis of PGs [1–3]. There exist at
least two isoenzymes of COX. One is COX-1, the structural protein found
in blood vessels, stomach and kidney, thus functioning in the vasocon-
striction, gastric mucosal injury and renal regulation, respectively [4].
The other is COX-2, the inducible enzyme anchored in the nuclear
membrane, showing an obvious overexpression during the pathological
events [5]. It has been known for a long time that the expression of COX-
2 is up-regulated significantly after the occurrence of inflammation [6].
Recently, the interest of the investigators on COX-2 has been drawn to its
overexpression in a variety of tumors such as breast cancer and
pancreatic cancers [7]. The level of COX-1 indicated no obvious varia-
tion in these cancer models [8]. Thus selectively inhibiting COX-2 seems
a favorable strategy for designing therapeutic methods for carcinomas.
Since a large number of COX-2 inhibitors have been discovered as non-
apoptosis induction and metastasis prevention [10]. Therefore, seeking
the connection between COX-2 inhibition and anti-tumor activity has
become an interesting research topic [11].
Glancing at the numbers of references each year in Web of Science
during the period of 2000–2020 (Fig. 1), we could conclude that COX-2
was a hotspot in this field and approximate 30% of the reports were
associated with cancers. Many factors or nodes in the pathways, such as
PTEN [12], PI3K/Akt/mTOR/S6K1 [13], MAPK [14], NF-κB [15], TNF-
α
[16], CDK [17] and Bcl-2 [18], were involved to draw the mechanism
of COX-2 inhibitors for anti-tumor applications. However, till now, no
clear map was depicted due to several problems including the target-
missing caused by nonspecific structure, the complex inflammation-
tumor interactions, and the sophisticated environmental factors. As a
solution strategy, we attempted to explore the potential mechanism with
selective inhibitors, key checkpoints and simplified indicators.
In this work, we designed the novel compounds on the structural
basis of known COX-2 inhibitors such as Celecoxib [19] and Valdecoxib
[20] (shown in Fig. 2). The five-membered nitrogen heterocyclic core
* Corresponding authors.
E-mail addresses: ys_yang@nju.edu.cn (Y.-S. Yang), duanyongtao860409@163.com (Y.-T. Duan), zhuhl@nju.edu.cn (H.-L. Zhu).
https://doi.org/10.1016/j.bioorg.2020.104390
Received 23 July 2020; Received in revised form 4 October 2020; Accepted 15 October 2020
Available online 20 October 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
and sulfonamide moiety were both retained to maintain the basic COX-2
inhibition and selectivity according to the reports [21,22]. The other
fragment we introduced, the aminophosphonate group, was recently
investigated in enhancing both the anti-tumor and anti-inflammation
effects [23–28]. The designed compounds were preliminarily checked
by virtual screening of ADMET and molecular docking simulation. The
prepared series was then evaluated according to their performance on
COX-2 and several cancer cells lines. After confirming the
mitochondrion-dependent apoptosis induction and cell-cycle arrest,
further investigation in the checkpoints was conducted to infer the
connection between the selective COX-2 inhibition and the anti-tumor
activity by this series.
significantly better selectivity (Selectivity Index: 172.32 vs. 93.25). A
glance at the substitutes led to some hints about the Structure-Activity
Relationship (SAR). For COX-1 inhibition, the IC50 values of all this se-
ries were in a narrow range (25.63–51.62 μM, almost within 2-fold).
Cheerfully, in this aspect, these series showed less potency than Cele-
coxib, which conversely contributed to the selectivity towards COX-2.
With close COX-1 inhibition, better performance against COX-2 meant
better selectivity as well. In the case of electron-withdrawing groups at
R1 (-F, -Cl, -CF3), R2 substitutes seemed to prefer a smaller size, with the
corresponding examples (“>” meant “better than”): A1 > A2, A3 > A4,
A7 > A8, A9 > A10, A11 > A12, A16 > A17. The only exception was A5
and A6 with the othro-fluoro group, being strong electron-withdrawing
and close to the pyrazole core. On the contrary, in the case of electron-
donating groups at R1 (-Br(para), -Me, -OMe), R2 substitutes seemed to
prefer a bulky size, with the corresponding examples: A15 > A14, A20
> A19, A22 > A21, A25 > A24. Fortunately, we involved methyl at R2,
which brought fluctuation to cause more favorable (A18, A23) or un-
favorable (A13) results. Although the difficult in synthesis increased at
this site, an aliphatic group and a corresponding discussion seemed an
interesting point in the near future.
2. Results and discussion
2.1. Chemistry
The chemical synthesis route to compounds 4a-4k are outlined in
Scheme 1. First, the substituted acetophenones were reacted with
dimethyl oxalate in sodium methoxide-methanol solution to produce the
intermediates 2a-2k. Then the intermediate 3a-3k were obtained
through the reaction of 2a-2k with 4-hydrazinylbenzenesulfonamide.
Finally, these pyrazole methyl ester 3a-3k was hydrolyzed by sodium
hydroxide into the corresponding acids 4a-4k. The synthesis of com-
pounds A1-A25 was described in Schemes 2. The corresponding acids
4a-4k was activated with EDC⋅HCl, DMAP and HOBt at 0 ◦C for 30 min,
then reacted with different aminophosphonate derivatives to produce
the target compounds A1-A25 through amide condensation reaction.
Aminophosphonate derivatives were synthesized according to the
literature [29]. The solvent used in the above steps was anhydrous
dichloromethane. The target compounds A1-A25 were purified by col-
umn chromatography and their structures were shown in Table 1.
Compounds A1-A25 were reported for the first time and characterized
by 1H NMR spectra, 1C NMR spectra, melting point and ESI-MS, in
accordance with their depicted structures.
2.3. Molecular docking simulation
To visualize the possible binding pattern of the hit compound A23
into COX-2, the molecular docking simulation was performed with the
Protein Crystal Structure of COX-2 bearing Celecoxib (PDB code: 3LN1).
The obtained maps were depicted in Fig. 2. As shown in Fig. 3A and the
detailed Fig. 3B, the general extension directions were similar between
A23 and Celecoxib, which might explain their close potency. However,
longer substitutes of A23 seemed to bring more potential interactions
with the key residues in COX-2, while at the same time avoid the off-
target effect which was usually caused by the small molecular size.
When we compared the possible interactions in detail with Fig. 3C and
D, we found that A23 included more interactions with more residues.
Although COX-2 seemed a typical case which agreed with the Lipinski’s
“Rule of Five” in hydrogen bonds, its interactions relied seriously on the
sulfonamide and electron-withdrawing trifluoromethyl. However, as we
discussed in the preliminary SAR after the COX-2 inhibition, in the case
of A23, when retaining the important sulfonamide, an electron-donating
group allowed the backbone to extend further. Afterwards the sub-
stitutes around the pyrazole core were redistributed, which led to the
key interactions with almost all the rings and chains of A23. Based on
the fact that the COX-2 inhibition ability of A23 and Celecoxib, we
supposed that a better selectivity and less off-target effect might resulted
in better potency on cancer cells.
2.2. COX-2 inhibitory activities and selectivity
The initial task of this work was the evaluation of the selective COX-2
inhibition of the synthesized compounds. By using the human COX-1/
COX-2 ELSIA kit, we obtained the required data including both the
inhibitory activities of COX-2 and COX-1. As shown in Table 2, all this
series indicated potent inhibition upon COX-2 and a majority of them
suggested obvious selectivity from COX-1. Among them, the most
attractive compound, A23, showed a comparable COX-2 inhibitory ac-
tivity with the positive control Celecoxib and A33 in another study, Zhu
et al. [30] (IC50 values: 0.28
μM vs. 0.24 μM and 0.17 μM) but a
Fig. 1. The numbers of references each year in Web of Science during the preriod of 2000–2020 for COX-2 only and COX-2 with cancers. (Searching keywords: COX-
2, Cancer; Searching date: 2020.07.15).
2

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
2.4. Anti-proliferation assay
could block cell mitosis at G1 phase. This result was different from a
commonly reported G2/M phase arrest, inferring that our series might
experience less off-target effect.
The anti-proliferative activities of all compounds against four tumor
cell lines (HeLa, human cervical cancer cells; MCF-7, human breast
cancer cells; HCT116, human colon cancer cells; HepG2, human liver
cancer cells) was determined by employing MTT method. Besides, the
cytotoxicity was checked by the epithelial cell line 293T. As shown in
Table 3, a majority of these series indicated potent anti-proliferative
activity against all the investigated cancer cells and low toxicity upon
the epithelial cells. Since the pathway from COX-2 down to tumor is not
clear, there might be complex factors all through the whole process.
Therefore, no rigid one-to-one correspondence could be generated be-
tween the COX-2 inhibition and the anti-tumor effect. But still, we found
that the top hits in COX-2 inhibitory assay (A3, A7, A9, A11, A23, A25)
also showed better potency against the growth of the cancer cells. As the
chosen hit above, A23 seemed potential for cancer treatment (IC50
Mitochondrial membrane potential decrease is the earliest change in
cell apoptosis. Herein the apoptosis-inducing effect of A23 on HCT116
cells were determined by JC-1 assay. HCT116 cells were treated with
compound A23 (0, 2.5, 5, 10, and 20 µM) for 24 h and stained with JC-1.
As shown in Fig. 6, along with the concentration increased from 2.5 µM
to 20 µM, the fluorescence of cells gradually changed from red to green.
The results indicated that A23 could induce the apoptosis of HCT116
cells in a dose-dependent manner.
2.6. Investigations on the apoptosis checkpoints
The above results attributed the anti-tumor effect to cell cycle arrest.
Herein we studied the apoptosis checkpoints to hint the connection
between the selective COX-2 inhibition and the anti-tumor activity. The
involved checkpoints included c-parp, Bcl-2, BAX, and Caspase 3, 4, 6, 7,
8, 9. The results in Fig. 7 indicated that the apoptosis event caused by
our series was correlated to almost all the checkpoints except for Cas-
pase 4 and 8. At least we could conclude at this step that the apoptosis
values: 9.71
12.51 M for HepG2). This result is much better than that of Celecoxib
(IC50 values: 25.72
μ
M for HeLa, 16.43
μ
M for MCF-7, 2.34
μ
M for HCT116,
μ
μM) and 7m (IC50 values: 8.07
μM) in in another
study, Wang et al. [31], being comparable with the anti-cancer drug
Cisplatin. Thus, A23 was further studied in the subsequent experiments
with the most affected HCT116 cells.
was through
a mitochondrion-dependent mode. Afterwards, we
attempted to track back the upstream node. As the key point of PI3K/
Akt/mTOR/S6K1 and MAPK pathways, Akt and ERK were checked
(Fig. S1 in Supporting Information). Surprisingly, both of the checked
points were affected when COX-2 was inhibited. This result indicated
that in the anti-cancer events, COX-2 might be in the upper position of
both the pathways, while the unbalanced transduction in these path-
ways converge at Bcl-2. Therefore, we could preliminarily infer that Bcl-
2 might be regulated by COX-2 with a following induction of apoptosis.
In the near future, we would seek the key nodes between Bcl-2 and COX-
2 with a more detailed interaction net.
2.5. Cell apoptosis, cell cycle arrest and mitochondrial membrane
potential.
The COX-2-related anti-tumor effect was reported to be associated
with the induction of cell apoptosis. As shown in Fig. 4, along with the
increase of A23 concentration (0, 5, 10 and 20 µM), the 48 h-incubated
cells also exhibited an increased percentage of apoptotic cells (9.22%,
20.5%, 23.4%, 42.5%). This result inferred that A23 could induce cell
apoptosis in a dose-dependent manner. Besides, the proportion of early
apoptosis cells was equal to that of late apoptosis ones.
Since cell cycle arrest was another reported phenomenon through
the COX-2-related anti-tumor cases, we also studied this factor by using
propidium iodide (PI) staining. With the same time and concentration
conditions, a cell cycle arrest at G1 phase was observed. As shown in
Fig. 5, with the increased concentration of A23, the percentage of cells
arrested in G1 phase exhibited a corresponding increase (58.77%,
67.24%, 72.44%, 79.96%). Therefore, in a dose-dependent manner, A23
3. Conclusion
In this work, a novel series of sulfonamide-containing amino-
phosphonate derivatives were developed as selective COX-2 (the medi-
ator of cell survival, proliferation and apoptosis) inhibitors and anti-
cancer candidates. Via evaluating both the COX-2 inhibition and selec-
tivity, we chose A23 as the top hit, which exhibited applicative COX-2
Fig. 2. The designing concept of the series in this work as selective COX-2 inhibitors.
3

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
Scheme 1. Reagents and conditions: (i) Dimethyl oxalate, Sodium methoxide, MeOH, reflux, 12 h, ice water, dilute hydrochloric acid; (ii) 4-hydrazinobenzenesul-
fonamide hydrochloride, MeOH, 70 ◦C, 4–6 h; (iii) NaOH, MeOH, 70 ◦C, 4 h.
Scheme 2. Reagents and conditions: (i), EDC⋅HCl, DMAP, HOBt, CH2Cl2, r.t, overnight.
Table 1
Table 2
COX-1/COX-2 inhibitory activity of compounds A1-A25 and Celecoxib.
Compounds
IC50 ± SD (
μ
M)a
Selectivity Indexb (SI)
COX-1
COX-2
Structures of compounds A1-A25.
A1
45.74 ± 1.25
39.65 ± 2.01
32.05 ± 0.92
45.26 ± 0.45
44.08 ± 1.08
41.25 ± 1.31
27.63 ± 1.82
45.36 ± 0.91
32.17 ± 0.38
38.76 ± 1.36
36.24 ± 0.85
42.18 ± 1.09
35.92 ± 0.23
35.86 ± 1.59
48.51 ± 2.14
38.24 ± 1.61
30.73 ± 0.78
47.14 ± 1.19
41.21 ± 0.54
46.43 ± 1.05
51.62 ± 0.71
25.63 ± 1.26
48.25 ± 1.93
35.19 ± 1.47
29.55 ± 0.39
22.38 ± 0.65
1.19 ± 0.18
1.21 ± 0.26
0.27 ± 0.02
3.94 ± 0.21
4.63 ± 0.52
3.17 ± 0.13
0.31 ± 0.08
6.32 ± 1.37
0.28 ± 0.03
0.45 ± 0.14
0.33 ± 0.11
2.19 ± 0.24
3.96 ± 0.62
2.37 ± 0.35
0.53 ± 0.04
2.74 ± 1.57
5.68 ± 0.82
0.95 ± 0.35
5.16 ± 0.26
1.28 ± 0.15
1.27 ± 0.29
0.45 ± 0.07
0.28 ± 0.05
0.54 ± 0.16
0.35 ± 0.03
0.24 ± 0.09
38.44
32.77
118.70
11.49
9.52
A2
A3
A4
Code
R1
R2
Code
R1
R2
A5
A6
13.01
89.13
7.18
A1
4-
Phenyl
A14
4-Bromo
Phenyl
A7
Fluoro
4-
A8
A2
4-
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
4-Bromo
4-
A9
114.89
86.13
109.82
19.26
9.07
Fluoro
3-
Bromophenyl
Phenyl
Bromophenyl
Phenyl
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
Celecoxib
A3
4-
Fluoro
3-
Trifluoromethyl
4-
A4
4-
4-
Fluoro
2-
Bromophenyl
Phenyl
Trifluoromethyl
4-Methyl
Bromophenyl
Methyl
15.13
91.53
13.96
5.41
A5
Fluoro
2-
A6
4-
4-Methyl
Phenyl
Fluoro
4-
Bromophenyl
Phenyl
49.62
7.99
A7
4-Methyl
4-
Chloro
4-
Bromophenyl
Phenyl
36.27
40.65
56.96
172.32
65.16
84.43
93.25
A8
4-
4-Methoxy
4-Methoxy
3-Methoxy
3-Methoxy
3-Methoxy
Chloro
3-
Bromophenyl
Phenyl
A9
4-
Chloro
3-
Bromophenyl
Methyl
A10
A11
A12
A13
4-
Chloro
2-
Bromophenyl
Phenyl
Phenyl
a
Chloro
2-
The concentration of test compound required to produce 50% inhibition of
COX-1/COX-2 is the mean of four determinations.
4-
4-
b
Chloro
4-
Bromophenyl
Methyl
Bromophenyl
In vitro COX-2 selectivity index (COX-2 IC50/COX-1 IC50).
Bromo
Ala513 and so on. The anti-proliferation effect was inferred to be led
through an apoptosis induction via a mitochondrion-dependent mode
with the cell cycle arrest at G1 stage. After investigating the checkpoints
of apoptosis, we preliminarily deduced that the node Bcl-2 might con-
nect the selective COX-2 inhibition and the anti-tumor activity.
Although more elaborate mechanism from COX-2 to Bcl-2 seems still
difficult for us to reveal with a single study, we will try our best to depict
inhibitory activity (IC50 = 0.28 ± 0.05 µM) and anti-proliferative
capability against several cancer cell lines. The selectivity of A23 to-
wards COX-2 (Selectivity Index 172.32) was much better than the con-
trol Celecoxib. Molecular docking simulation hinted that this kind of
potency and selectivity might be related to the binding interactions with
the key residues including Val102, Arg106, Val335, Tyr371, Arg499,
4

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
Fig. 3. The binding patterns of hit compound A23 and Celecoxib into the active site of COX-2 (PDB code: 3LN1). (A) 3D binding map showing the site; (B) Detailed
conformations in the binding site; (C) 2D binding pattern of A23 into COX-2; (D) 2D binding pattern of Celecoxib into COX-2.
the panorama of this map for developing COX-2 inhibitors in anti-tumor
therapies in future.
compounds 2a-2k (1 mmol) in anhydrous methanol (20 mL) was stirred
at reflux for 6 h. After confirming completion of the reaction, the reac-
tion mixture was poured into water. The solid compounds 3a-3k were
isolated by filtration.
4. Experimental section
4.1. Materials and measurements
4.4. General procedure for compounds 4a-4k
All chemical reagents and solvents purchased from Aladdin (China)
were of analytical grade. All the melting points were determined with an
X4 MP apparatus. NMR spectra were recorded in CDCl3 and DMSOd6 on
a Bruker DPX600 model spectrometer. A series of cell function experi-
ments were analyzed by flow cytometry (BD FACSCalibur Flow
Cytometry) and Fluorescence microscope (OLYMPUS IX71). The PCR
amplification was evaluated on a LightCycler ® 480 II System (Roche).
All cells were purchased from American type culture collection and
conserved at State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing. Cells were used in experiments after 3
times of passages.
Sodium hydroxide (4 mmol) and compounds 3a-3k (1 mmol) were
added in anhydrous methanol and heated at reflux for 6 h. Then the
reaction mixture was acidified with the hydrochloric acid solution (1
mol/L) and extracted with ethyl acetate (3 × 100 mL). The combined
organic layer was dried over anhydrous sodium sulfate and concentrated
in vacuo to obtain compound 4a-4k.
4.5. General procedure for compounds A1-A25
EDC (1.2 mmol), HOBT (1.2 mmol) and DMAP (0.5 mmol) were
added to a solution of 4a-4 k (1 mmol) in anhydrous dichloromethane.
◦
After stirring at 0 C for 30 min, a-aminophosphonate (2 mmol) was
4.2. General procedure for compounds 2a-2k
added to the mixture and stirred for 5 h at room temperature. The crude
product was purified by column chromatography with ethyl acetate and
petroleum ether (V: V = 1:1) to give the final product A1-A25.
Different substituted acetophenones (1 mmol) were added to anhy-
drous methanol, followed by dimethyl oxalate (2 mmol) and sodium
methoxide (2 mmol). The above mixture was stirred at reflux for 12 h.
After cooling to room temperature, the mixture solution was poured into
ice water (150 mL) and treated with dilute hydrochloric acid (1 mol/L)
to pH = 4. The precipitate was filtered, washed and dried in vacuo [32].
4.5.1. Diethyl((5-(4-fluorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxamido)(phenyl)methyl)phosphonate (A1)
Yellow solid, yield 71.2%, m.p.: 87.6–88.2 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.71 (dd, J = 9.8, 3.5 Hz, 1H), 7.91 (d, J = 8.6 Hz, 3H), 7.68
(d, J = 8.3 Hz, 1H), 7.60 (d, J = 8.6 Hz, 3H), 7.54 (s, 2H), 7.40–7.36 (m,
4H), 7.28 (t, J = 8.8 Hz, 2H), 7.17 (s, 1H), 5.70 (dd, J = 21.7, 9.8 Hz,
1H), 4.08 (d, J = 7.2 Hz, 2H), 3.98–3.94 (m, 1H), 3.85 (dd, J = 17.9, 7.9
Hz, 1H), 1.21 (t, J = 7.0 Hz, 3H), 1.10 (t, J = 7.0 Hz, 3H). ESI-MS: 587.15
4.3. General procedure for compounds 3a-3k
A mixture of 4-hydrazinylbenzenesulfonamide (2 mmol) and
5

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
4.5.3. Diethyl((5-(3-fluorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxamido)(phenyl)methyl)phosphonate (A3)
Table 3
Anti-proliferative activities of compounds A1-A25, Celecoxib and Cisplatin.
Light yellow solid, yield 64.1%, m.p.: 105.1–105.6 ◦C. 1H NMR (600
MHz, DMSO‑d6) δ 8.72 (dd, J = 9.8, 3.5 Hz, 1H), 7.90 (d, J = 8.6 Hz,
2H), 7.59 (dd, J = 16.3, 8.2 Hz, 4H), 7.53 (s, 2H), 7.45 (d, J = 6.2 Hz,
1H), 7.38 (t, J = 7.6 Hz, 2H), 7.33 (s, 1H), 7.23 (s, 3H), 7.09 (d, J = 7.8
Hz, 1H), 5.68 (dd, J = 21.7, 9.8 Hz, 1H), 4.06 (d, J = 7.2 Hz, 2H), 3.94 (s,
1H), 3.86–3.80 (m, 1H), 1.20 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H).
ESI-MS: 587.15 [M+H]+. Anal. Calcd for: C27H28FN4O6PS C, 55.29; H,
4.81; N, 9.55.
Code
IC50 ± SD (
μ
M)a
HeLa
MCF-7
HCT116
HepG2
293 T
A1
13.81 ±
1.57
10.12 ±
1.65
16.51 ±
1.28
10.53 ±
0.42
117.75 ±
1.34
A2
19.02 ±
1.36
23.04 ±
0.37
21.38 ±
0.26
16.34 ±
0.81
206.48 ±
1.01
A3
11.08 ±
0.04
5.32 ±
0.65
10.45 ±
1.03
8.73 ±
1.26
83.17 ±
3.12
A4
13.27 ±
0.96
18.07 ±
0.49
16.43 ±
0.52
17.28 ±
0.74
104.56 ±
1.51
4.5.4. Diethyl((4-bromophenyl)(5-(3-fluorophenyl)-1-(4-
A5
21.74 ±
0.78
15.76 ±
1.37
16.12 ±
0.39
21.17 ±
1.09
185.87 ±
0.63
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A4)
Yellow solid, yield 53.8%, m.p.: 90.1–90.7 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.81 (dd, J = 9.7, 3.7 Hz, 1H), 7.89 (d, J = 11.0 Hz, 2H),
7.60–7.57 (m, 3H), 7.54 (d, J = 7.0 Hz, 2H), 7.52 (s, 2H), 7.44 (d, J =
22.1 Hz, 2H), 7.29–7.20 (m, 3H), 7.09 (d, J = 7.8 Hz, 1H), 5.68 (dd, J =
22.0, 9.7 Hz, 1H), 4.10–4.04 (m, 2H), 3.99–3.95 (m, 1H), 3.91–3.86 (m,
1H), 1.20 (t, J = 7.0 Hz, 3H), 1.12 (t, J = 7.0 Hz, 3H). ESI-MS: 665.06
[M+H] +. Anal. Calcd for C27H27BrFN4O6PS: C, 48.73; H, 4.09; N, 8.42.
A6
28.58 ±
0.31
26.83 ±
0.18
20.46 ±
0.57
28.16 ±
0.95
125.49 ±
1.14
A7
7.25 ±
0.23
>100
18.04 ±
1.41
7.64 ±
0.82
106.02 ±
1.42
A8
20.19 ±
0.61
28.09 ±
1.38
>100
16.71 ±
1.04
>300
A9
18.38 ±
1.92
6.39 ±
0.15
11.93 ±
0.36
>100
157.36 ±
0.97
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
Celecoxib
Cisplatin
21.44 ±
0.85
12.47 ±
0.68
13.20 ±
1.03
15.78 ±
0.72
>300
4.5.5. Diethyl((5-(2-fluorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxamido) (phenyl)methyl)phosphonate(A5)
14.52 ±
1.34
16.94 ±
0.25
16.58 ±
0.43
13.96 ±
0.26
114.23 ±
1.81
Yellow solid, yield 75.4%, m.p.: 87.5–88.4 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.80 (dd, J = 9.8, 3.4 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.60
(d, J = 7.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 5H), 7.48 (d, J = 6.9 Hz, 3H),
7.39 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.8 Hz, 1H), 7.13 (s, 1H), 5.70 (dd, J
= 21.7, 9.8 Hz, 1H), 4.10–4.03 (m, 2H), 3.98–3.93 (m, 1H), 3.87–3.81
(m, 1H), 1.21 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ESI-MS:
587.15 [M+H] +. Anal. Calcd for C27H28FN4O6PS: C, 55.29; H, 4.81;
N, 9.55.
35.28 ±
0.19
20.91 ±
0.57
34.87 ±
1.26
32.44 ±
2.27
91.12 ±
1.42
9.54 ±
0.72
24.82 ±
0.91
>100
7.25 ±
0.49
218.94 ±
0.47
17.85 ±
0.58
21.98 ±
1.24
21.06 ±
0.89
29.24 ±
0.27
165.21 ±
2.18
27.62 ±
0.21
>100
7.42 ±
0.58
30.61 ±
1.08
95.77 ±
2.63
45.32 ±
1.63
18.35 ±
0.72
22.97 ±
0.13
>100
152.38 ±
0.45
25.05 ±
0.25
>100
32.36 ±
0.95
27.39 ±
1.05
171.59 ±
1.27
4.5.6. Diethyl((4-bromophenyl)(5-(2-fluorophenyl)-1-(4-
41.24 ±
1.61
>100
23.91 ±
0.52
28.67 ±
0.67
153.72 ±
1.94
sulfamoylphenyl)-1H-pyrazol-3-carboxamido)methyl)phosphonate(A6)
Yellow solid, yield 52.7%, m.p.: 93.2–94.6 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.88 (dd, J = 9.7, 3.5 Hz, 1H), 7.86 (d, J = 8.6 Hz, 2H),
7.61–7.53 (m, 7H), 7.50 (s, 3H), 7.32 (t, J = 7.9 Hz, 1H), 7.29–7.25 (m,
1H), 7.18 (s, 1H), 5.70 (dd, J = 22.0, 9.7 Hz, 1H), 4.10–4.03 (m, 2H),
4.00–3.95 (m, 1H), 3.90 (dd, J = 16.6, 9.4 Hz, 1H), 1.21 (t, J = 7.0 Hz,
3H), 1.12 (t, J = 7.0 Hz, 3H). ESI-MS: 665.06 [M+H] +. Anal. Calcd for
>100
27.04 ±
1.86
26.14 ±
0.27
>100
211.02 ±
2.05
22.56 ±
0.78
>100
40.35 ±
2.01
>100
>300
14.05 ±
1.25
20.61 ±
0.43
21.58 ±
0.81
18.45 ±
0.12
197.83 ±
1.18
24.88 ±
2.16
>100
8.74 ±
0.65
19.33 ±
0.55
143.37 ±
1.51
C27H27BrFN4O6PS: C, 48.73; H, 4.09; N, 8.42.
9.71 ±
0.47
16.43 ±
0.62
2.34 ±
0.27
12.51 ±
1.18
205.95 ±
2.36
4.5.7. Diethyl((5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxamido)(phenyl)methyl)phosphonate(A7)
12.39 ±
1.08
8.52 ±
0.94
13.16 ±
0.78
10.75 ±
0.53
171.54 ±
1.29
Yellow solid, yield 56.0%, m.p.: 86.1–86.9 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.72 (dd, J = 9.7, 3.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H),
7.68–7.59 (m, 4H), 7.58 (d, J = 7.8 Hz, 2H), 7.53 (s, 2H), 7.38 (t, J = 7.7
Hz, 2H), 7.33 (q, J = 7.4, 6.8 Hz, 2H), 7.25 (s, 1H), 7.21 (d, J = 7.9 Hz,
1H), 5.68 (dd, J = 21.7, 9.8 Hz, 1H), 4.09–4.02 (m, 2H), 3.97–3.92 (m,
1H), 3.83 (dd, J = 17.3, 8.6 Hz, 1H), 1.20 (t, J = 7.0 Hz, 3H), 1.09 (t, J =
7.0 Hz, 3H). ESI-MS: 603.12 [M+H] +. Anal. Calcd for C27H28ClN4O6PS:
C, 53.78; H, 4.68; N, 9.29.
>100
31.65 ±
0.39
23.13 ±
1.35
18.84 ±
1.05
139.26 ±
2.24
28.85 ±
1.27
83.49 ±
0.61
25.72 ±
0.26
29.25 ±
1.91
182.32 ±
1.08
10.06 ±
0.54
23.18 ±
0.83
8.73 ±
1.09
28.36 ±
0.43
89.27 ±
2.15
a
IC50: The concentration of compounds inhibits 50% of cell growth. Data are
shown as the mean ± SD of three independent experiments (n = 3).
[M+H] +. Anal. Calcd for C27H28FN4O6PS: C, 55.29; H, 4.81; N, 9.55.
4.5.8. Diethyl((4-bromophenyl)(5-(4-chlorophenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A8)
White solid, yield 60.8%, m.p.: 102.3–104.2 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.83 (dd, J = 9.7, 3.7 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.60
(d, J = 11.9 Hz, 4H), 7.56 (d, J = 7.5 Hz, 2H), 7.53 (s, 2H), 7.50 (d, J =
8.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 5.70 (dd, J = 22.0,
9.7 Hz, 1H), 4.11–4.04 (m, 2H), 4.01–3.95 (m, 1H), 3.93–3.87 (m, 1H),
1.21 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.0 Hz, 3H). ESI-MS: 681.03 [M+H]
+. Anal. Calcd for C27H27BrClN4O6PS: C, 47.56; H, 3.99; N, 8.22.
4.5.2. Diethyl((4-bromophenyl)(5-(4-fluorophenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate (A2)
1
◦
White solid, yield 59.6%, m.p.: 92.5–93.7 C. H NMR (600 MHz,
DMSO‑d6) δ 8.81 (dd, J = 9.6, 3.5 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H),
7.62–7.55 (m, 6H), 7.54 (s, 2H), 7.38 (dd, J = 8.5, 5.3 Hz, 2H), 7.28 (t, J
= 8.8 Hz, 2H), 7.17 (s, 1H), 5.71 (dd, J = 22.0, 9.7 Hz, 1H), 4.11–4.04
(m, 2H), 3.98 (s, 1H), 3.91 (dd, J = 16.7, 9.2 Hz, 1H), 1.21 (t, J = 7.1 Hz,
3H), 1.13 (t, J = 7.0 Hz, 3H). ESI-MS: 665.06 [M+H] +. Anal. Calcd for
C
27H27BrFN4O6PS: C, 48.73; H, 4.09; N, 8.42.
4.5.9. Diethyl((5-(3-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-
carboxamido)(phenyl)methyl)phosphonate(A9)
White solid, yield 80.2%, m.p.: 125.4–126.5 ◦C. 1H NMR (600 MHz,
6

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
Fig. 4. Hit compound A23 induced apoptosis in HCT116 cells. (A) The result of HCT116 cells treated with different concentrations of A23 (0, 5, 10, and 20 µM) for
48 h; (B) Apoptotic ratios of HCT116 cells. Data are shown as mean ± SD of three independent experiments; **p < 0.01, ***p < 0.001. Statistical analyses performed
with a two-tailed Student’s t-test with unequal variance.
Fig. 5. (A) The variations in cell cycle of HCT116 cells with treatment of hit compound A23. (B) The percentage of cells in the G1 phase. Data are shown as mean ±
SD of three independent experiments; *p < 0.05, **p < 0.01. Statistical analyses performed with a two-tailed Student’s t-test with unequal variance.
Fig. 6. Effect of compound A23 on mitochondrial membrane potential of HCT116 cells. Scale bar: 100 μm.
7

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
Fig. 7. Effects of compound A23 after treatment for 48h on HCT116 cells detected by qRT-PCR and Western blotting. Statistical analyses performed with a two-tailed
Student’s t-test with unequal variance.
DMSO‑d6) δ 8.71 (dd, J = 9.8, 3.6 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H),
7.62–7.54 (m, 4H), 7.54–7.46 (m, 4H), 7.38 (t, J = 7.7 Hz, 2H), 7.33 (d,
J = 8.5 Hz, 3H), 7.20 (s, 1H), 5.68 (dd, J = 21.7, 9.8 Hz, 1H), 4.10–4.01
(m, 2H), 3.97–3.91 (m, 1H), 3.87–3.80 (m, 1H), 1.20 (t, J = 7.0 Hz, 3H),
1.09 (t, J = 7.0 Hz, 3H). ESI-MS: 603.12 [M+H] +. Anal. Calcd for
4.5.13. Diethyl(1-(5-(4-bromophenyl)-1-(4-sulfamoylphenyl)-1H-
pyrazole-3-carboxamido)ethyl)phosphonate(A13)
Yellow solid, yield 6.2%, m.p.: 143.2–143.7 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.38 (d, J = 9.3 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.67 (d, J
= 8.3 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.55 (s, 2H), 7.31 (d, J = 8.3 Hz,
2H), 7.19 (s, 1H), 4.58 (dd, J = 15.6, 7.9 Hz, 1H), 4.10 (dd, J = 16.0, 9.1
Hz, 4H), 1.43 (dd, J = 11.9, 4.7 Hz, 3H), 1.29–1.26 (m, 6H). ESI-MS:
585.05 [M+H] +. Anal. Calcd for C22H26BrN4O6PS: C, 45.14; H, 4.48;
N, 9.57.
C
27H28ClN4O6PS: C, 53.78; H, 4.68; N, 9.29.
4.5.10. Diethyl((4-bromophenyl)(5-(3-chlorophenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A10)
White solid, yield 51.9%, m.p.: 112.7–113.9 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.95–8.73 (m, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.69–7.61 (m,
4H), 7.60–7.49 (m, 6H), 7.35 (t, J = 7.8 Hz, 1H), 7.30–7.12 (m, 2H),
5.71 (dd, J = 21.9, 9.6 Hz, 1H), 4.14–4.04 (m, 2H), 4.03–3.95 (m, 1H),
3.90 (q, J = 9.3, 8.8 Hz, 1H), 1.21 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.0 Hz,
3H). ESI-MS: 681.03 [M+H] +. Anal. Calcd for C27H27BrClN4O6PS: C,
47.56; H, 3.99; N, 8.22.
4.5.14. Diethyl((5-(4-bromophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-
3-carboxamido)(phenyl)methyl)phosphonate(A14)
Light yellow solid, yield 58.3%, m.p.: 102.4–103.6 ◦C. 1H NMR (600
MHz, DMSO‑d6) δ 8.74–8.68 (m, 1H), 7.89 (d, J = 8.4 Hz, 2H),
7.69–7.54 (m, 6H), 7.51 (s, 2H), 7.35 (dt, J = 33.6, 7.3 Hz, 3H), 7.26 (d,
J = 8.3 Hz, 2H), 7.20 (s, 1H), 5.70–5.64 (m, 1H), 4.09–4.02 (m, 2H),
3.96–3.91 (m, 1H), 3.86–3.80 (m, 1H), 1.20 (t, J = 7.0 Hz, 3H), 1.09 (t, J
+
4.5.11. Diethyl((5-(2-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-
3-carboxamio)(phenyl)methyl)phosphonate(A11)
=
7.0 Hz, 3H). ESI-MS: 647.07 [M+H]
. Anal. Calcd for
C
27H28BrN4O6PS: C, 50.09; H, 4.36; N, 8.65.
Yellow solid, yield 78.4%, m.p.: 88.5–90.3 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.82 (dd, J = 9.8, 3.3 Hz, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.59
(dd, J = 13.7, 7.0 Hz, 3H), 7.54–7.46 (m, 7H), 7.39 (t, J = 7.5 Hz, 2H),
7.33 (t, J = 7.2 Hz, 1H), 7.13 (s, 1H), 5.70 (dd, J = 21.7, 9.8 Hz, 1H),
4.10–4.04 (m, 2H), 3.98–3.93 (m, 1H), 3.87–3.82 (m, 1H), 1.21 (t, J =
7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ESI-MS: 603.12 [M+H]+. Anal.
Calcd for C27H28ClN4O6PS: C, 53.78; H, 4.68; N, 9.29.
4.5.15. Diethyl((4-bromophenyl)(5-(4-bromophenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A15)
Yellow solid, yield 63.5%, m.p.: 95.3–96.9 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.81 (dd, J = 9.7, 3.7 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.63
(d, J = 8.5 Hz, 2H), 7.61–7.56 (m, 4H), 7.56–7.53 (m, 2H), 7.51 (s, 2H),
7.26 (d, J = 8.5 Hz, 2H), 7.20 (s, 1H), 5.68 (dd, J = 22.0, 9.7 Hz, 1H),
4.10–4.03 (m, 2H), 3.96 (d, J = 7.4 Hz, 1H), 3.89 (dd, J = 16.6, 9.5 Hz,
1H), 1.20 (t, J = 7.0 Hz, 3H), 1.12 (t, J = 7.0 Hz, 3H). ESI-MS: 724.98
[M+H] +. Anal. Calcd for C27H27Br2N4O6PS: C, 44.65; H, 3.75; N, 7.71.
4.5.12. Diethyl((4-bromophenyl)(5-(2-chlorophenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A12)
Yellow solid, yield 67.1%, m.p.: 91.8–93.4 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.89 (dd, J = 9.7, 3.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.83
(d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.3 Hz, 1H), 7.61–7.54 (m, 4H), 7.54 (d,
J = 1.5 Hz, 1H), 7.52–7.38 (m, 5H), 7.12 (s, 1H), 5.70 (dd, J = 21.9, 9.7
Hz, 1H), 4.08 (dd, J = 12.4, 7.9 Hz, 2H), 3.98 (dd, J = 8.8, 5.9 Hz, 1H),
3.90 (dd, J = 18.1, 8.0 Hz, 1H), 1.21 (t, J = 7.0 Hz, 3H), 1.12 (t, J = 7.0
Hz, 3H). ESI-MS: 681.03 [M+H] +. Anal. Calcd for C27H27BrClN4O6PS:
C, 47.56; H,3.99; N, 8.22.
4.5.16. Diethyl(phenyl(1-(4-sulfamoylphenyl)-5-(4-(trifluoromethyl)
phenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A16)
Yellow solid, yield 65.7%, m.p.: 76.9–77.4 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.76 (dd, J = 9.8, 3.5 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.80
(d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.59–7.56 (m, 2H), 7.54 (d,
J = 8.3 Hz, 4H), 7.39 (t, J = 7.7 Hz, 3H), 7.30 (s, 1H), 5.68 (dd, J = 21.7,
9.8 Hz, 1H), 4.06 (q, J = 8.8, 8.4 Hz, 2H), 3.97–3.92 (m, 1H), 3.84 (dd, J
= 16.5, 9.4 Hz, 1H), 1.20 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H).
ESI-MS: 637.14 [M+H] +. Anal. Calcd for C28H28F3N4O6PS: C, 52.83; H,
4.43; N, 8.80.
8

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
4.5.17. Diethyl ((4-bromophenyl)(1-(4-sulfamoylphenyl)-5-(4-
(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate
(A17)
(m, 4H), 7.31 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.98 (dd, J = 8.3, 2.0 Hz,
1H), 6.89 (d, J = 2.3 Hz, 1H), 6.81 (d, J = 7.9 Hz, 1H), 4.66–4.40 (m,
1H), 4.11–4.01 (m, 4H), 3.69 (s, 3H), 1.37 (dd, J = 16.6, 7.4 Hz, 3H),
1.24 (dt, J = 11.8, 7.0 Hz, 6H). ESI-MS: 537.15 [M+H] +. Anal. Calcd for
C23H29N4O7PS: C, 51.49; H, 5.45; N, 10.44.
Yellow solid, yield 82.6%, m.p.: 109.5–110.2 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.87 (dd, J = 9.7, 3.6 Hz, 1H), 7.95–7.90 (m, 2H), 7.80 (d, J
= 8.3 Hz, 2H), 7.68–7.58 (m, 4H), 7.55 (d, J = 11.3 Hz, 6H), 7.30 (s,
1H), 5.71 (dd, J = 22.0, 9.7 Hz, 1H), 4.12–4.04 (m, 2H), 4.03–4.00 (m,
4.5.24. Diethyl((5-(3-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-
pyrazole-3-carboxamido)(phenyl)methyl)phosphonate(A24)
1H), 3.91 (dd, J = 9.5, 6.3 Hz, 1H), 1.21 (t, J = 7.1 Hz, 3H), 1.13 (t, J =
+
7.2 Hz, 3H). ESI-MS: 715.05 [M+H]
.
Anal. Calcd for
Yellow solid, yield 59.0%, m.p.: 130.1–130.8 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.69 (dd, J = 9.8, 3.5 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H),
7.64–7.55 (m, 4H), 7.53 (s, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.32 (dt, J =
15.9, 7.6 Hz, 2H), 7.19 (s, 1H), 6.98 (dd, J = 8.3, 2.0 Hz, 1H), 6.89 (s,
1H), 6.81 (d, J = 7.7 Hz, 1H), 5.68 (dd, J = 21.7, 9.8 Hz, 1H), 4.06 (q, J
= 8.7, 8.3 Hz, 2H), 3.98–3.91 (m, 1H), 3.84 (q, J = 8.7 Hz, 1H), 3.69 (s,
3H), 1.20 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ESI-MS: 599.17
[M+H] +. Anal. Calcd for C28H31N4O7PS: C, 56.18; H, 5.22; N, 9.36.
C
28H27BrF3N4O6PS: C, 47.00; H, 3.80; N, 7.83.
4.5.18. Diethyl(1-(1-(4-sulfamoylphenyl)-5-(p-tolyl)-1H-pyrazole-3-
carboxamido)ethyl)phosphonate(A18)
Yellow solid, yield 8.4%, m.p.: 135.8–136.3 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.19 (d, J = 9.7 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.55 (d, J
= 8.6 Hz, 2H), 7.50 (s, 2H), 7.21 (q, J = 8.2 Hz, 4H), 7.07 (s, 1H), 4.33
(dd, J = 10.0, 4.8 Hz, 1H), 4.04 (dd, J = 16.1, 8.9 Hz, 4H), 2.32 (s, 3H),
1.84–1.75 (m, 2H), 1.22 (dt, J = 19.1, 7.0 Hz, 6H), 0.91 (t, J = 7.3 Hz,
3H). ESI-MS: 521.15 [M+H] +. Anal. Calcd for C23H29N4O6PS: C, 53.07;
H, 5.62; N, 10.76.
4.5.25. Diethyl((4-bromophenyl)(5-(3-methoxyphenyl)-1-(4-
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A25)
Yellow solid, yield 62.5%, m.p.: 78.3–79.2 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.79 (dd, J = 9.5, 3.2 Hz, 1H), 7.90 (d, J = 8.5 Hz, 2H),
7.62–7.51 (m, 8H), 7.21 (q, J = 8.0 Hz, 4H), 7.12 (s, 1H), 5.70 (dd, J =
21.9, 9.7 Hz, 1H), 4.12–4.04 (m, 2H), 4.02–3.95 (m, 1H), 3.90 (q, J =
9.7, 9.0 Hz, 1H), 2.32 (s, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.0 Hz,
3H). ESI-MS: 677.08 [M+H] +. Anal. Calcd for C28H30BrN4O7PS: C,
49.64; H, 4.46; N, 8.27.
4.5.19. Diethyl(phenyl(1-(4-sulfamoylphenyl)-5-(p-tolyl)-1H-pyrazole-3-
carboxamido)methyl)phosphonate(A19)
White solid, yield 70.3%, m.p.: 121.4–122.6 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.67 (dd, J = 9.8, 3.6 Hz, 1H), 7.88 (d, J = 8.6 Hz, 2H), 7.57
(d, J = 8.6 Hz, 4H), 7.51 (s, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.33 (d, J = 6.5
Hz, 1H), 7.25–7.15 (m, 4H), 7.11 (s, 1H), 5.67 (dd, J = 21.7, 9.8 Hz, 1H),
4.09–4.02 (m, 2H), 3.97–3.92 (m, 1H), 3.83 (dd, J = 17.3, 8.6 Hz, 1H),
2.31 (s, 3H), 1.21 (s, 3H), 1.08 (s, 3H). ESI-MS: 583.17 [M+H] +. Anal.
Calcd for C28H31N4O6PS: C, 57.72; H, 5.36; N, 9.62.
4.6. Cell culture
HeLa (Human epithelial cervical cancer cell line), MCF-7 (human
breast cancer cell line), HCT116 (human colon cancer cell line), HepG2
(human hepatoma cell line), and 293T (human embryonic kidney cell
line) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% fetal bovine serum (FBS, BI), 100 U/mL peni-
cillin and 100 mg/mL streptomycin, and maintained in a humidified
incubator with 5% CO2 at 37 ◦C。
4.5.20. Diethyl((4-bromophenyl)(1-(4-sulfamoylphenyl)-5-(p-tolyl)-1H-
pyrazole-3-carboxamido)methyl)phosphonate(A20)
White solid, yield 77.4%, m.p.: 118.3–120.7 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.79 (dd, J = 9.5, 3.2 Hz, 1H), 7.90 (d, J = 8.5 Hz, 2H),
7.62–7.51 (m, 8H), 7.21 (q, J = 8.0 Hz, 4H), 7.12 (s, 1H), 5.70 (dd, J =
21.9, 9.7 Hz, 1H), 4.12–4.04 (m, 2H), 4.02–3.95 (m, 1H), 3.90 (q, J =
9.7, 9.0 Hz, 1H), 2.32 (s, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.0 Hz,
3H). ESI-MS: 661.08 [M+H] +. Anal. Calcd for C28H30BrN4O6PS: C,
50.84; H, 4.57; N, 8.47.
4.7. COX inhibition assay
The inhibitory ability of the synthesized compounds on COX-1 and
COX-2 was determined by COX-1/COX-2 ELISA Kit. COX-1 or COX-2
enzyme was pre-incubated with test compounds at various concentra-
tions in the supplied buffer (0.1 M Tris–HCl, pH 8.0, 5 mM EDTA, 2 mM
4.5.21. Diethyl((5-(4-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-
pyrazole-3-carboxamido)(phenyl)methyl)phosphonate(A21)
Yellow solid, yield 55.2%, m.p.: 98.5–99.6 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.65 (dd, J = 9.8, 3.6 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H),
7.62–7.55 (m, 4H), 7.52 (s, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.7
Hz, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 6.97 (d, J = 8.8 Hz, 2H),
5.67 (dd, J = 21.7, 9.8 Hz, 1H), 4.09–4.03 (m, 2H), 3.97–3.92 (m, 1H),
3.83 (dd, J = 10.2, 1.5 Hz, 1H), 3.77 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H),
1.09 (t, J = 7.0 Hz, 3H). ESI-MS: 599.17[M+H] +. Anal. Calcd for
phenol and 1 μ
M heme) for 10 min at 37 ◦C. 10 µL arachidonic acid (100
µM) was added to initiate the reactions, which incubated 2 min at 37 ◦C.
Then the reaction was stopped by adding 50 µL of 1 M HCl, following by
one-tenth the volume of saturated stannous chloride (50 mg/mL). The
reaction mixture was incubated at room temperature for 5 min. Ac-
cording to the detection method of the kit, the wavelength of each well
at 450 nm was measured by enzyme immunoassay [30].
C
28H31N4O7PS: C, 56.18; H, 5.22; N, 9.36.
4.5.22. Diethyl((4-bromophenyl)(5-(4-methoxyphenyl)-1-(4-
4.8. Anti-proliferation assay
sulfamoylphenyl)-1H-pyrazole-3-carboxamido)methyl)phosphonate(A22)
Yellow solid, yield 79.8%, m.p.: 85.9–87.2 ◦C. 1H NMR (600 MHz,
DMSO‑d6) δ 8.75 (dd, J = 9.7, 3.7 Hz, 1H), 7.89 (d, J = 8.7 Hz, 2H),
7.65–7.56 (m, 4H), 7.56–7.51 (m, 4H), 7.23 (d, J = 8.8 Hz, 2H), 7.07 (s,
1H), 6.97 (d, J = 8.8 Hz, 2H), 5.69 (dd, J = 22.0, 9.7 Hz, 1H), 4.10–4.03
(m, 2H), 4.01–3.97 (m, 1H), 3.90 (dd, J = 9.5, 7.7 Hz, 1H), 3.77 (s, 3H),
1.20 (t, J = 7.0 Hz, 3H), 1.12 (t, J = 7.0 Hz, 3H). ESI-MS: 677.08 [M+H]
+. Anal. Calcd for C28H30BrN4O7PS: C, 49.64; H, 4.46; N, 8.27.
The anti-proliferative activity of the synthesized products against
HeLa, MCF-7, HCT116, HepG2 and 293T cell lines was assessed using a
modified standard (MTT) – based colorimetric assay. The cells grown to
log phase were plated at a density of 5000 cells per well on a 96-well
plate and treated with gradient concentrations (0.01, 0.1, 1, 10 and
100 μM) of tested compounds for 48 h. After that, MTT (10 mg/ mL)
(Sigma, USA) added and incubated for 4 h at 37 ◦C. 150 µL DMSO was
added in each well and shaken for 5 min. The absorbance (OD 570 nm)
was measured at a wavelength of 630 nm on an ELISA reader (ELx800,
BioTek, USA). IC50 values of compounds were calculated by comparison
with DMSO treated control wells. Each drug concentration was repeated
in three wells. Each assay was carried out three times for each cell line.
4.5.23. Diethyl(1-(5-(3-methoxyphenyl)-1-(4-sulfamoylphenyl)-1H-
pyrazole-3-carboxamido)ethyl)phosphonate(A23)
Yellow solid, yield 5.1%, m.p.: 108.6–110.3 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 8.30 (d, J = 9.4 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.60–7.49
9

B. Zhang et al.
Bioorganic Chemistry 105 (2020) 104390
4.9. Cell apoptosis assay
Scientific, Rockford, IL, USA). The protein levels were normalized by
probing the same blots with a GAPDH antibody [34]. The antibodies
were purchased from the following sources: anti-c-Caspase 6: (Cell
Signaling Technology Inc., USA) , anti-c-Caspase 7: (Cell Signaling
Technology Inc., USA), anti-c-Caspase 9: (Cell Signaling Technology
Inc., USA), anti-AKT1(Cell Signaling Technology Inc., USA), anti-p-
ERK1/2 (Cell Signaling Technology Inc., USA), anti-c-parp: (Cell
Signaling Technology Inc., USA), and anti-GAPDH (Cell Signaling
Technology Inc., USA). ImageJ software was used to quantify the protein
levels.
HCT116 cells were plated six-well plates at 106/well, and treated
with compound A23 at final concentrations of 0, 5, 10, and 20 μM for 48
h. The cells were collected by trypsin, washed with PBS and resuspend
into 500 μL of binding buffer. Then Annexin-V/FITC (5 μL) and PI (5 μL)
were added to the above buffer and incubated for 15 min at RT (25 ◦C)
away from light. The cell apoptosis was analyzed with FACS Calibur
flow cytometer (Becton Dickinson, San Jose, CA, USA). Statistical
analysis was performed with the Flowjo 7.6.1 software (Emerald Biotech
Co. Ltd., Hangzhou, China).
Declaration of Competing Interest
4.10. Cell cycle assay
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
HCT116 cells were treated with compound A23 at indicated con-
centrations (0, 5, 10, and 20 μM) for 48 h, washed twice with ice-cold
PBS and fixed with 70% ethanol at 4 ◦C overnight. The cells were
centrifuged at 4000 rpm and treated with RNase A (0.1 mg/mL). After
Acknowledgements
incubation at 37 ◦C for 30 min, 400
μL propidium iodide (PI) was mixed
We would like to express our gratitude to our colleagues, Chen-Wen
Shao and Kang-Min Zhou, for their help in the experiments.
with the cells. Analysis was performed with FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA, USA).
Appendix A. Supplementary material
4.11. Mitochondrial membrane potential evaluation
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104390.
Mitochondrial depolarization in HCT116 cells were measured by JC-
1 dye (CBIC2(3), Beyotime, Haimen, China). HCT116 cells grown in six-
well plates were treated with different concentrations (0, 2.5, 5, 10, and
20 µM) of compound A23 for 24 h. Then the culture medium was
removed and HCT116 cells were stained with JC-1 staining solution (5
mg/mL) for 20 min. After being washed twice with PBS, the stained cells
were observed fluorescence microscope (OLYMPUS IX71) [31].
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