Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2019.03.008

In this paper, 41 hybrid compounds containing diaryl-1,5-diazole and morpholine structures acting as dual COX-2/5-LOX inhibitors have been designed, synthesized and biologically evaluated. Most of them showed potent antiproliferative activities and COX-2/5-LOX inhibitory in vitro. Among them, compound A33 displayed the most potency against cancer cell lines (IC₅₀ = 6.43–10.97 μM for F10, HeLa, A549 and MCF-7 cells), lower toxicity to non-cancer cells than celecoxib (A33: IC₅₀ = 194.01 μM vs. celecoxib: IC₅₀ = 97.87 μM for 293T cells), and excellent inhibitory activities on COX-2 (IC₅₀ = 0.17 μM) and 5-LOX (IC₅₀ = 0.68 μM). Meanwhile, the molecular modeling study was performed to position compound A33 into COX-2 and 5-LOX active sites to determine the probable binding models. Mechanistic studies demonstrated that compound A33 could block cell cycle in G2 phase and subsequently induced apoptosis of F10 cells. Furthermore, compound A33 could significantly inhibit tumor growth in F10-xenograft mouse model, and pharmacokinetic study of compound A33 indicated that it showed better stability in vivo. In general, compound A33 could be a promising candidate for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.03.008

Accepted Manuscript
Design, synthesis and evaluation of novel diaryl-1,5-diazoles derivatives bearing
morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents
Zhang Li, Zhong-Chang Wang, Xin Li, Muhammed Abbas, Song-Yu Wu, Shen-Zhen
Ren, Qi-Xing Liu, Yi Liu, Peng-Wen Chen, Yong-Tao Duan, Peng-Cheng Lv, Hai-
Liang Zhu
PII:
S0223-5234(19)30219-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.008
EJMECH 11178
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 November 2018
Revised Date: 4 March 2019
Accepted Date: 4 March 2019
Please cite this article as: Z. Li, Z.-C. Wang, X. Li, M. Abbas, S.-Y. Wu, S.-Z. Ren, Q.-X. Liu, Y. Liu, P.-
W. Chen, Y.-T. Duan, P.-C. Lv, H.-L. Zhu, Design, synthesis and evaluation of novel diaryl-1,5-diazoles
derivatives bearing morpholine as potent dual COX-2/5-LOX inhibitors and antitumor agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.03.008.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Design, synthesis and evaluation of novel diaryl-1,5-diazoles
derivatives
bearing
morpholine
as
potent
dual
COX-2/5-LOX inhibitors and antitumor agents
a,
†
a,†,
a
a
Zhang Li , Zhong-Chang Wang *, Xin Li , Muhammed Abbas, Song-Yu Wu ,
a
a
a
b
c
Shen-Zhen Ren , Qi-Xing Liu , Yi Liu , Peng-Wen Chen , Yong-Tao Duan ,
a,
a,
Peng-Cheng Lv *, Hai-Liang Zhu *
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
2
10023, PR China
b
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing
10023, PR China
2
c
Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases,
Zhengzhou Children's Hospital, Zhengzhou 450018, PR China
A series of novel dual COX-2/5-LOX inhibitors have been synthesized and
evaluated for their anti-cancer activity as potential COX-2/5-LOX inhibitors.
Compound A33 was the most active.

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
Design, synthesis and evaluation of novel diaryl-1,5-diazoles
derivatives
bearing
morpholine
as
potent
dual
COX-2/5-LOX inhibitors and antitumor agents
a,
†
a,†,
a
a
Zhang Li , Zhong-Chang Wang *, Xin Li , Muhammed Abbas, Song-Yu Wu ,
a
a
a
b
c
Shen-Zhen Ren , Qi-Xing Liu , Yi Liu , Peng-Wen Chen , Yong-Tao Duan ,
a,
a,
Peng-Cheng Lv *, Hai-Liang Zhu *
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
1
1
1
1
1
1
1
1
1
1
2
2
2
210023, PR China
b
School of Chemistry and Chemical Engineering, Nanjing University, Nanjing
210023, PR China
c
Henan Provincial Key Laboratory of Children’s Genetics and Metabolic Diseases,
Zhengzhou Children's Hospital, Zhengzhou 450018, PR China
Declarations of interest: none
*Corresponding
author.
Tel.
&
fax:
+86-025-89682572;
zhuhl@nju.edu.cn
e-mail:
(ZHL),
wangzhongchang2006@163.com
pengcheng.lui@gmail.com (LPC)
(WZC),
†These two authors equally contributed to this paper.
1

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
Abstract:
In this paper, 41 hybrid compounds containing diaryl-1,5-diazole and morpholine
structures acting as dual COX-2/5-LOX inhibitors have been designed, synthesized
and biologically evaluated. Most of them showed potent antiproliferative activities
and COX-2/5-LOX inhibitory in vitro. Among them, compound A33 displayed the
most potency against cancer cell lines (IC = 6.43-10.97 µM for F10, HeLa, A549
5
0
and MCF-7 cells), lower toxicity to non-cancer cells than celecoxib (A33: IC =
5
0
194.01 µM vs. celecoxib: IC = 97.87 µM for 293T cells), and excellent inhibitory
5
0
activities on COX-2 (IC = 0.17 µM) and 5-LOX (IC = 0.68 µM). Meanwhile, the
5
0
50
molecular modeling study was performed to position compound A33 into COX-2 and
5-LOX active sites to determine the probable binding models. Mechanistic studies
demonstrated that compound A33 could block cell cycle in G2 phase and
subsequently induced apoptosis of F10 cells. Furthermore, compound A33 could
significantly inhibit tumor growth in F10-xenograft mouse model, and
pharmacokinetic study of compound A33 indicated that it showed better stability in
vivo. In general, compound A33 could be a promising candidate for cancer therapy.
Keywords: diaryl-1,5-diazoles, morpholine, cyclooxygenase-2, 5-lipoxygenase,
anticancer
2

ACCEPTED MANUSCRIPT
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1. Introduction:
Inflammatory response exhibited pleiotropic effects in the development of cancer.
On one hand, inflammation contributes to carcinogenesis, tumor growth, invasion,
metastatic spread, and malignant transformation. On the other hand, it also stimulates
immune effector mechanisms that might limit tumor growth [1]. However, numerous
studies have proved that chronic inflammation is generally detrimental and can
increase the risk of cancer[2-3]. And tumor micro-environments contain various
inflammatory mediators, such as cytokines, chemokines and growth factors, which
accelerate extravasations of tumor cells through the stroma and promote the
development of carcinogenesis by participating in complex signaling processes[4].
Thus, targeting these factors associated with inflammation in cancer cells can offer
rational treatment strategies for cancer.
The potent inflammation mediators are derivatives of arachidonic acid (AA) a
poly-unsaturated fatty acid produced from membrane phospholipids. AA metabolism
includes two principal pathways, one is the cyclooxygenase (COX) pathway, which
converts AA to prostaglandins (PGs) and thromboxanes (TXs); the other is
lipoxygenase (LOX) pathway, which produces a collection of leukotrienes (LTs) and
hydroxyeicosatetranoic acids (HETEs)[5-6]. It is well known that COX exists in two
isoforms COX-1 and COX-2, among which COX-1 is a constructive enzyme found in
most normal tissues as a “house-keeper” one, while COX-2 is an inducible enzyme
found in macrophages, fibroblasts and leukocytes, and is up-regulated in a variety of
tumor types[7-9]. 5-LOX, a human non-heme enzyme, is a vital member of LOXs and
3

ACCEPTED MANUSCRIPT
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
responsible for the production of leukotriene B (LTB ) and 5-HETE, which could
4
4
enhance cell proliferation, inhibit apoptosis, and promote cancer development[10-11].
The over-expression and activities of COX-2 and 5-LOX in tumor cells are closely
related to cell cycle, blocking apoptosis and stimulating angiogenesis. Lots of results
indicated that elevated levels of COX-2 and 5-LOX would result in elevation of
downstream prostaglandin E (PGE ) and LTB levels respectively[12]. PGE , a major
2
2
4
2
inflammation mediator with meaningful biological functions, could increase the
motility and metastatic potential of tumor cells, promote tumor angiogenesis, induce
local immunosuppression and suppress apoptosis[13-14]. Meanwhile, COX-2 itself
could promote tumor cells survival by lowing the levels of unesterified AA, and
COX-2 peroxidase has also been shown to convert many procarcinogens into ultimate
carcinogens, thereby promoting the deterioration of tumors[15-16]. Given the
previous research, COX-2 and 5-LOX with strikingly similar biological functions are
frequent co-expression. Inhibition of the COX pathway would switch AA metabolism
to the LOX pathway and vice versa, resulting in elevated levels of leukotrienes or
prostaglandins, which in turn cause fatal side effects[17]. Drugs acting on individual
molecular targets would produce unfavorable activity, toxicity, and drug resistance,
whereas drugs acting on multiple targets synchronously are less prone to drug
resistance, reduce side effects, and better exert drug activity, produce better
therapeutic effects[18]. It is believed that compounds which could inhibit
COX-2/5-LOX concurrently would shut off the production of mediators of
inflammation from the AA pathway. Thus dual COX-2/5-LOX inhibitors established a
4

ACCEPTED MANUSCRIPT
8
9
9
9
9
9
9
9
9
9
9
9
0
1
2
3
4
5
6
7
8
9
worthy rational approach to obtain effective and safer anti-tumor agents.
Diaryl five-membered heteroatom system is a characteristic structure in numerous
selective COX-2 inhibitors displaying higher COX-2 selectivity and better safety
profile, such as Celecoxib, Rofecoxib, and SC558[19-21]. Meanwhile, structure
activity relationship (SAR) studies of COX-2 indicated the significance of
aminosulfonyl (SO NH ) pharmacophore for COX-2 selectivity[22-23]. Furthermore,
2
2
some antioxidant 5-LOX inhibitors such as Phenidone and BW-755C are comprised
of pyrazole that the core pharmacophore which also appear to inhibit the COX
isoforms[24-25]. The pharmacological effects of morpholine derivatives are of great
significance in different biological fields. Emerging evidence demonstrated that
morpholine and its analogs have shown favorable anti-inflammation, anti-cancer,
anti-oxidant and anti-microbial activities[26], as well as reduced gastrointestinal and
cardiovascular side effects[27-28]. For example, the cis-2,3-disubstituted morpholine
aprepitant 4 is used to treat nausea and vomiting caused by chemotherapy[29].
Moroxydine, a common morpholine-containing drug, is mainly used to treat viral
influenza. The introduction of morpholine derivatives is expected to inhibit
COX-2/5-LOX simultaneously, enhance physiological activities and reduce toxic side
effects[30-31].
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
Over the past few years, we had reported a series of diarylpyrazole derivatives as
selective COX-2 or dual COX-2/5-LOX inhibitors and successfully obtained some
potent anticancer candidates[32-36]. Based on the above findings, a series of
compounds incorporating diaryl-1,5-diazoles and morpholine pharmacophores were
5

ACCEPTED MANUSCRIPT
1
1
1
1
1
11
12
13
14
15
designed and synthesized to explore more potent and safer dual COX-2/5-LOX
inhibitors for anticancer candidates (Figure 1). Hereupon, the synthesis, in vitro and
in vivo biological evaluation has been reported. Meanwhile, docking studies were
performed to understand the possible models of the most potent compound A33 into
both COX-2/5-LOX active sites in order to explain their anti-cancer activities.
1
1
16
17
1
1
1
1
1
1
1
18
19
20
21
22
23
24
Fig 1. Structure of Celecoxib, Phenidone and Moroxydine and targeted compounds as dual
COX-2/5-LOX inhibitors
2. Result and discussion
2.1 Chemistry
The synthetic route of starting materials 4a-4k has been described in the previous
study (Scheme 1)[36]. Catalyzed by excess sodium methoxide, the substituted
acetophenone reacted with dimethyl oxalate to form diverse chalcones 2a-2k, then
6

ACCEPTED MANUSCRIPT
1
1
1
25
26
27
2a-2k were reacted with 4-hydrazinylbenzenesulfonamide to obtain the intermediate
3a-3k. Finally, 3a-3k were hydrolyzed by potassium hydroxide into corresponding
pyrazolesulfonamide carboxylic acid 4a-4k. The solvent used in the above three steps
was methanol.
1
1
28
29
a
Scheme 1
H NO S
H NO S
2 2
2
2
O
O
N N
N
N
i
R1
ii
iii
R1
R1
R1
O
OH
O
R2
2a-2k
O
R2
R2
R2
O
O
1
a-1k
3a-3k
4
a-4k
1
1
30
31
a
Reagents and conditions: (i) dimethyl oxalate, methanol, reflux
6
h; (ii)
1
32
4-hydrazinylbenzenesulfonamide, methanol, reflux, 6 h; (iii) KOH, methanol, reflux, 2 h.
1
33
The synthesis of target compounds A1-A41 was outlined in Scheme 2 and 3. The
starting materials 4a-4k were activated by EDC·HCl, HOBt and DMAP at 0 � for
half an hour, followed by esterification condensation reaction with different
morpholine derivatives to form the corresponding target compound A1-A41. The
purified compounds were prepared by subsequent column chromatography and
crystallization. The structures of the target compounds A1-A41 were shown in Table
1
1
1
1
1
1
1
1
1
34
35
36
37
38
39
40
41
42
1
1 and 2. All targeted compounds were first reported and characterized by H NMR
1
spectroscopy, C NMR spectroscopy, melting point, ESI-MS and elemental analysis,
in accordance with their depicted structures.
a
Scheme 2
1
1
43
44
a
Reagents and conditions: (i) EDC·HCl, HOBt, DMAP, dichloromethane, 0 � , Revitalite, 0.5 h,
7

ACCEPTED MANUSCRIPT
1
1
1
45
46
47
RT, over night.
a
Scheme 3
1
1
48
49
a
Reagents and conditions: (i) EDC·HCl, HOBt, DMAP, dichloromethane, 0 � , Revitalite, 0.5 h,
RT, over night.
1
1
50
51
Table 1． Structure of target compounds A1-A22
1
52
1
2
Compds
A1
R
R
X
S
H
H
F
F
A2
O
S
A3
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
A4
H
O
S
A5
Cl
Cl
A6
O
S
A7
CH
CH
3
CH
CH
F
2
2
O
O
A8
3
O
S
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
F
O
O
S
CH
CH
Br
Br
H
3
3
S
O
S
CH
O
O
3
H
CH
O
S
3
CH
CH
O
O
3
H
3
H
H
H
H
H
O
S
3
CF
CF
H
O
S
3
H
O
1
1
1
53
54
55
8

ACCEPTED MANUSCRIPT
1
1
56
57
Table 2． Structure of target compounds A23-A41
1
58
1
Compds
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
R
Y
NH
O
n
3
3
2
3
2
2
2
2
3
2
3
3
2
3
3
2
3
3
2
H
H
H
Cl
Cl
Cl
F
NH
O
O
NH
O
F
NH
O
F
CF
CF
CF
O
3
NH
O
3
3
CH
CH
CH
O
3
O
3
O
3
NH
O
NH
NH
O
CH
CH
CH
CH
CH
CH
CH
CH
O
O
O
O
3
3
3
3
2
2
2
2
NH
O
1
1
1
59
60
61
Furthermore, the crystal structure of A8 (CCDC: 1845660) was determined by
X-ray diffraction analysis. The crystal data showed in Figure 2 and Table 3. The data
is provided free of charge by The Cambridge Crystallographic Data Center.
9

ACCEPTED MANUSCRIPT
1
1
62
63
Fig 2. Crystal structure diagram of compound A8.
1
64
Table 3. Crystal data for compound A8
Compounds
CCDC number
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
A8
1845660
C H N O S
2
2
24
4
5
456.51
Monoclinic
P 2 /c
1
7.8754(7)
16.3675(13)
16.9128(14)
93.7768(16)
2175.3(3)
4
b (Å)
c (Å)
β (°)
3
V (Å )
Z
-
3
D (g cm )
1.394
c
-
1
µ (mm )
F (000)
0.191
960.0
θ (°)
2.489-27.523
4270/3444
0.0378, 0.0930
0.0501, 0.0982
1.052
Reflections collected/unique
R , wR [I >2σ(I)]
1
2
R , wR [all data]
1
2
2
Goodness-of-fit on F
1
1
65
66
1
0

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
2.2. MTT assay
All synthesized compounds A1-A41 were evaluated for in vitro antiproliferative
activity on five cell lines (F10: murine melanoma, Hela: human cervical cancer, A549:
human lung cancer, MCF-7: human breast cancer, 293T: human renal epithelium.) by
using MTT assay. Celecoxib and an intermediate product 4c (Figure 3) served as
positive control. As shown in Table 4, most target compounds could effectively
inhibit the proliferation of the four test cancer cell lines compared with Celecoxib and
exhibited lower cytotoxic effects according to the safety trial on 293T cell lines.
Among them, compounds A19 and A33 showed superior growth inhibitory effects to
that of Celecoxib (IC : 8.16-10.21 µM of A19, 6.43-10.97 µM of A33 vs.
5
0
11.04-15.68 µM of Celecoxib), and it was worth noting that both compounds have
1
CF group substitution in the para-position of phenyl ring (R ). Meanwhile, the
3
intermediate 4c has an IC50 value ranging from 23.79 to 26.51 µM, it was apparent
that the introduction of morpholine derivatives significantly enhances the
anti-proliferative activity. Compounds A1 and A3 showed equivalent
anti-proliferative activity to celecoxib, but were much less toxic to non-cancer cell
line than celecoxib, which also indicated that the introduction of morpholine
derivatives also plays a positive role in reducing the toxicity of normal cells(e.g. F10:
10.35 µM of A1 vs. 13.27 µM of Celecoxib; 293T:176.15 µM of A1 vs. 97.87 µM of
Celecoxib). Interestingly, it was found that these hybrid compounds (A1, A3, A19,
A33) were linked by the amide linkage. Therefore, it can be assumed that the
hydrophobicity of hybrid compounds also contributes to the improvement of
1
1

ACCEPTED MANUSCRIPT
1
89
antiproliferative activity.
1
90
1
1
1
91
92
93
Fig. 3. Structure of 4
Table 4. Antiproliferative activities of target compounds, celecoxib, and 4c against different
cancer cell lines, and cytotoxicity towards non-cancer cells.
a,b
Cell lines (IC , µM ± SD）
5
0
Compds
A1
F10
Hela
A549
MCF-7
293T
10.35±0.78 13.23±0.87 14.49±0.59 14.39±1.57 176.15±3.68
30.14±0.36 51.27±1.27 25.71±0.16 18.94±0.16 179.76±1.02
13.94±1.09 14.78±0.57 15.43±0.55 12.54±0.46 191.35±1.17
14.63±0.84 17.92±1.59 24.57±1.24 15.79±1.31 113.59±2.19
A2
A3
A4
A5
＞100
＞100
＞100
＞100
＞100
＞100
39.79±0.21 231.63±3.15
＞100 ＞300
16.51±0.83 168.71±0.59
A6
A7
32.26±0.48 21.57±0.83 ＞100
A8
18.64±0.32 27.15±0.91 41.71±2.17 15.01±1.24 115.54±0.82
26.40±0.93 29.17±1.23 18.19±0.91 18.19±0.50 173.71±1.57
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
20.47±1.51
21.37±1.22
＞100
＞100
27.47±0.68 20.01±2.19 205.91±0.81
22.79±1.13 17.89±1.38 189.67±0.56
15.49±1.28 13.57±1.33 19.91±0.92 18.13±0.12 105.79±0.83
12.14±0.25 15.95±0.81 17.65±1.28 14.56±0.37 197.47±2.57
＞100
12.98±0.11
29.17±1.42
13.17±0.59 24.10±0.17 17.19±0.54 19.71±0.94 210.71±0.65
20.81±1.50 21.57±1.36 19.59±0.76 16.03±1.23 206.76±0.56
8.16±0.19
21.79±1.08 27.65±0.55 25.19±1.03 19.98±0.31 198.79±0.48
＞100
16.17±0.38
21.82±0.54 27.42±0.71 20.47±0.93 18.12±1.27 156.15±2.68
22.68±1.89 22.42±0.85 17.69±0.71 17.19±0.51 95.47±0.48
30.79±1.09 21.79±1.07 ＞100
26.79±1.22 21.37±0.43 ＞100
22.62±1.05 19.97±0.17 17.91±0.69 160.40±0.97
20.3±0.47 15.98±1.33 17.95±1.84 177.38±0.98
＞100
＞100
25.98±0.91 134.09±3.15
9.94±0.25 10.21±1.52 9.25±1.06
135.51±1.29
＞100
＞100
30.79±0.37 18.87±0.62 163.54±0.90
26.12±0.35 16.49±0.27 184.48±3.87
39.17±0.95 151.35±1.37
22.14±2.49 188.71±0.98
21.98±1.95 25.37±0.14 22.98±0.48 25.79±0.48 121.96±0.59
＞100
18.13±1.28
19.97±1.26 19.48±0.92 22.97±0.79 111.86±1.51
＞100 ＞100 19.31±0.85 145.81±1.24
1
2

ACCEPTED MANUSCRIPT
A30
A31
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
＞100
＞100
＞100
＞100
＞100
＞100
15.79±1.36 112.25±2.39
＞100 ＞300
21.50±0.81 24.56±0.62 27.79±1.28 16.69±1.05 147.78±0.98
6.43±0.14 8.08±0.14 10.97±0.57 7.65±0.82 194.01±0.67
14.40±069 29.79±0.77 15.17±0.04 14.71±0.88 179.47±1.18
19.47±0.93 20.41±0.15 17.59±0.42 15.01±1.27 143.79±0.62
13.71±0.34 26.71±1.27 18.19±0.36 19.79±0.69 169.40±0.17
12.98±0.03
24.17±1.34 20.81±1.57 16.51±0.53 25.79±0.92 155.09±2.19
＞100 ＞100 ＞100 15.38±0.67 178.71±0.55
12.88±0.22 19.19±0.98 17.83±0.08 15.01±0.33 137.91±2.28
20.17±1.71 29.78±0.11 21.47±1.12 18.79±0.98 115.52±1.79
23.79±0.78 26.51±0.81 22.46±1.21 24.81±1.01 189.57±0.78
13.27±1.09 15.68±0.89 11.04±1.94 12.57±1.51 97.87±0.48
＞100
19.17±0.81 16.31±0.48 199.86±1.46
4c
Celecoxib
a
1
1
94
95
IC : Concentration inhibits 50% of cell growth.
50
b
Data are shown as the mean ± SD of three independent experiments (n=3)
1
1
1
1
2
2
2
2
2
2
2
2
2
2
96
97
98
99
00
01
02
03
04
05
06
07
08
09
2.3. In vitro COX-2 and 5-LOX inhibitory activities
Both COX-2 and 5-LOX inhibitory activities of target compounds were evaluated
in vitro, compared with Celecoxib and Zileuton as the corresponding positive
controls. As summarized in Table 5.1, most of the target compounds could effectively
suppress COX-2 and 5-LOX activities with IC50 values varying from 0.17-7.64 µM
and 0.68-3.41 µM respectively (Celecoxib, IC = 0.25 µM; Zileuton, IC = 0.83
5
0
50
µM).
Wherein, hybrid compounds substituted with electron withdrawing groups on the
1
2
benzene ring (R , R ) exhibited better COX-2 inhibitory activity than those substituted
2
1
with electron donating groups. For example, when R was hydrogen, different R
contributed variously to COX-2 inhibitory activity and showed the order as CF > F >
3
Cl > Br > H > CH > CH O > CH CH O (e.g. A19, IC = 0.19 µM vs. A7, IC = 0.97
3
3
3
2
50
50
µM). Consistently, it was found that the presence of carbon chains could slightly
increase the ability of hybrid compounds to inhibit COX-2, comparing compound
1
3

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
10
11
12
13
14
15
16
17
18
19
20
A19 (IC = 0.19 µM) and compound A33 (IC = 0.17 µM). For the 5-LOX inhibitory
5
0
50
activity, the IC50 values of the two groups of hybrid compounds containing carbon
chains (A23-A41) and without carbon chains (A1-A22) were 0.68-1.01 µM and
0.71-3.41 µM, respectively. Therefore, the increase in hydrophobicity might play a
positive role in the inhibition of 5-LOX to some extent. In addition, it was noteworthy
that the 5-lox inhibitory activity of the hybrid compounds linking with ester linkage
(e.g. A39 and A41) was slightly better than those linking with amide linkage(e.g. A38
and A40).
Furthermore, based on the results of COX-2 and 5-LOX inhibition and
antiproliferative test, compounds A3, A19, A33, and A34 were selected to test the
selectivity between COX-1 and COX-2. As shown in Table 5.2, these compounds
inhibited favorable COX-1/COX-2 selectivity.
2
2
21
22
Table 5.1． In vitro COX-2 and 5-LOX inhibitory activities
a
Compds
IC ± SD (µM)
50
COX-2
5-LOX
A1
A2
0.19±0.09
0.23±0.13
0.22±0.07
0.24±0.02
0.23±0.05
0.35±0.09
0.97±0.18
2.99±0.21
0.21±0.05
0.23±0.07
0.93±0.23
0.89±0.14
1.28±0.12
0.31±0.17
1.95±0.28
2.25±0.21
1.21±0.26
1.32±0.13
2.01±0.15
0.87±0.08
1.33±0.29
2.31±0.34
1.79±0.41
0.97±0.21
0.95±0.22
1.27±0.12
0.89±0.17
1.24±0.12
1.57±0.31
1.21±0.21
0.98±0.08
1.19±0.23
0.96±0.09
1.01±0.18
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
1
4

ACCEPTED MANUSCRIPT
A18
A19
2.01±0.25
0.19±0.02
0.39±0.04
0.67±0.15
0.69±0.21
0.42±0.19
0.56±0.27
0.64±0.16
5.47±0.25
0.25±0.09
0.31±0.07
0.21±0.07
0.16±0.02
3.57±0.76
0.19±0.04
0.17±0.07
0.21±0.08
0.87±0.09
1.05±0.14
0.71±0.28
0.85±0.29
7.64±1.16
0.91±0.11
1.11±0.15
0.25±0.03
--
0.93±0.16
0.71±0.13
2.59±0.35
3.41±0.19
1.19±0.17
0.93±0.24
0.82±0.19
0.96±0.19
0.85±0.21
0.87±0.19
0.89±0.11
0.84±0.15
1.01±0.23
0.75±0.08
0.81±0.08
0.68±0.17
0.75±0.12
0.91±0.15
0.87±0.23
0.98±0.06
0.86±0.31
0.72±0.09
0.84±0.23
0.79±0.09
--
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
Celecoxib
Zileuton
0.83±0.11
a
2
2
2
23
24
25
Values are means of four determinations
Table 5.2. In vitro COX-1 and COX-2 inhibitory activities of selected compounds
a
Compds
IC ± SD (µM)
50
b
Selectivity Index(SI)
COX-1
COX-2
A3
A19
A33
29.35±0.88
30.79±1.02
32.06±2.79
28.52±2.23
24.47±0.35
0.21±0.07
0.19±0.02
0.17±0.07
0.21±0.08
0.25±0.03
139.76
162.05
188.58
135.80
97.88
A34
Celecoxib
a
2
2
2
2
2
2
26
27
28
29
30
31
The concentration of test compound required to produce 50% inhibition of COX-1/COX-2 is the
mean of four determinations.
b
selectivity index (COX-1 IC /COX-2 IC )
5
0
50
1
5

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
2.4. Molecular modeling (docking) studies selectivity index
In order to examine the binding mode and interaction, docking study between
compound A33 which had balanced COX-2/5-LOX inhibitory activities and the
COX-2 (PDB: 3LN1) and 5-LOX (PDB: 3V99) enzymes were performed (Figure 4).
The high-resolution structural information about the COX-2 active site had been
reported in detail in the previous research[19]. As illustrated in Figure 4A, compound
A33 was well inserted into the active pocket of the COX-2 by three hydrogen bonds
and the docking score up to 56.34. The fluoro atom formed a hydrogen bond with
Arg-106 (angle NH...F = 110.33°, distance = 2.65
sulfonamide group formed a hydrogen bond with Tyr-371 (angle OH...O = 133.36°,
distance = 1.97 ) and the secondary amine of the amide in linkage offered hydrogen
bond to the oxygen atom of Ser-339(angle NH...O = 118.93°, distance = 2.11 ).
Å), one oxygen atom of the
Å
Å
Furthermore, Van der Waals, carbon-hydrogen bonds, π-sigma bonds, and other
weaker interactions also increased the binding affinity of the compound with COX-2.
As illustrated in Figure 4B, the 3D model shows that the bulge of compound A33 was
inserted into the dent of COX-2 completely.
Similarly, based on the previous study on the crystal structure of human 5-LOX
enzyme[37]. The docking study was continued to explore the binding of compound
A33 to 5-LOX. As shown in Figure 4C, compound A33 was well embedded into the
active site of 5-LOX by six hydrogen bonds, Van Der Waals, Carbon-hydrogen bond,
Alkyl, and other weak interaction. Fluorine atom of trifluoromethyl formed an
H-bond with Glu 172 (angle NH...F = 128.18°, distance = 2.80 Å). Two oxygen atoms
1
6

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
54
55
56
57
58
59
60
61
62
63
64
of sulfonamide formed two H-bonds with Lys 173 (angle NH...O = 144.82°, distance
= 2.66
Moreover, the secondary amine in linkage formed an H-bond with Ala 672 (angle
O...NH = 151.98°, distance = 2.04 ) and the carbonyl in linkage formed two
H-bonds with Gln 363 (angle NH...O = 116.11°, distance = 2.07 ) and His 367
Å) and Asn 554 (angle NH...O = 153.33°, distance = 2.21 Å) respectively.
Å
Å
(angle NH...O = 96.82°, distance = 2.82 Å). These interactions play important role in
improving the stability of the complex and increasing the inhibitory activity of 5-LOX.
The model of 3D (Figure 4D) showed that compound A33 was inserted into the
activity pocket of 5-LOX nicely. In general, these docking studies showed that the
hybrids of morpholine derivatives and pyrazole sulfonamide which acted as
COX-2/5-LOX dual inhibitors might be promising potent agents.
2
65
17

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
66
67
68
69
70
71
Fig.4. Binding mode of compound A33 with the COX-2 (PDB code: 3LN1) and 5-LOX (PDB
code: 3V99). A) 2D diagram of the interaction between compound A33 and COX-2; B) 3D
models of compound A33 binding with the active site, only interacting residues are displayed. The
hydrogen bond (green) is displayed as dotted arrows. C) 2D diagram of the interaction between
compound A33 and 5-LOX (PDB code: 3V99); D) 3D models of compound A33 in the 5-LOX
pocket. Green lines represent hydrogen bonds.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
2.5. Compound A33 induced tumour cell apoptosis
Previous studies had shown that selective COX-2 inhibitors could both inhibit the
cell cycle and induce apoptosis of tumor cells, such as Celecoxib[38],
Nimesulide[39]. Therefore, according to the antiproliferative and COX-2/5-LOX
inhibitory activity, compound A33 was selected by using Annexin V/PI double
staining assay to detect whether it induced apoptosis of F10 cells. As shown in Figure
5, after treatment of F10 cells with compound A33 at different concentrations (0,
3.125, 6.25, 12.5 and 25 µM) for 48 hours, the apoptotic rate increased significantly
from 5.81% to 46.91% and the percentage of apoptosis cells increased in a
dose-dependent manner. Subsequently, z-VAD-fmk, a commonly used inhibitor of
apoptosis, was used to detect the apoptosis-inducing effect of compound A33 on F10
cells. The therapeutic concentrations of compound A33 were the same as above,
except that 10 µM of z-VAD-fmk was added while adding various concentrations of
compound A33. As shown in Figure 6A, after treating 10 µM z-VAD-fmk and
different concentrations of compound A33, the apoptotic rates were 6.96%, 8.83%,
11.08%, 15.91% and 20.4%, respectively. Compared with the addition of the
compound A33 alone, the addition of z-VAD-fmk significantly inhibited the
apoptosis of F10 cells caused by the compound A33 (Figure 6B). Taken together, it
1
8

ACCEPTED MANUSCRIPT
could be concluded that compound A33 could induce F10 cells apoptosis in a
dose-dependent manner.
2
2
90
91
2
2
92
93
Fig. 5. Compound A33 induces apoptosis in F10 cell for 48h. A): Flow cytometry analysis of
apoptotic F10 cells; B): Apoptotic ratio, Data are shown as mean ± SD of three independent
experiments; **p < 0.01.
2
2
94
95
2
96
2
2
2
3
3
97
98
99
00
01
Fig. 6. z-VAD-fmk inhibits apoptosis of F10 cells induced by compound A33. A): F10 cell were
treated with 0, 3.125, 6.25, 12.5, 25 µM of compound A33 and z-VAD-fmk (10 µM) for 48h, then
apoptotic ratio was analyzed by flow cytometry; B): Comparison of apoptotic ratio between
treated with and without z-VAD-fmk. Data are shown as mean ± SD of three independent
experiments;
3
3
3
3
02
03
04
05
2.6. Compound A33 induced cell cycle arrest
Next, for investigation of the cellular mechanism of anti-proliferation of compound
A33, further assessment was carried out to check whether compound A33 could
induce the cell cycle arrest of F10 cells by flow cytometry. F10 cells were treated with
1
9

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
06
07
08
09
10
11
different concentrations (0, 3.125, 6.25,12.5, 25 µM) of Compound A33 for 48 hours.
The test results were collected in Figure 7. Compared with control group, the
percentage of cells in G2 phase was increased from 22.43 to 33.60% with the dose of
compound A33, while the percentage of cells in G1 phase was decreased from 59.73
to 50.93%. These results indicated that compound A33 could induce cell cycle arrest
in G2 phase with a dose-dependent manner.
3
3
12
13
Fig. 7. A). Influence of compound A33 on F10 cell cycle for 48 h. B). The percentage of cells in
3
3
14
15
G2 graph. Data are shown as mean ± SD of three independent experiments; *p < 0.05.
2.7. Compound A33 inhibited the production of PGE2 in F10 cells
3
3
3
3
3
3
3
3
16
17
18
19
20
21
22
23
Studies have shown that AA produces prostaglandins under the catalyzed by
cyclooxygenase, which is associated with tumor progression. Thus, we tested the
ability of compound A33 to inhibit the production of PGE from AA. After the F10
2
cells were treated with different doses of lipopolysaccharide (LPS), celecoxib and
compound A33 for 24 hours, the supernatant was collected by centrifugation to detect
the expression of PGE with a PGE Enzyme Immunoassay kit. As shown in Figure 8,
2
2
compared with the untreated control group, LPS (1µg/ml) could significantly increase
the secretion of PGE in F10 cells, while that was obviously reduced when treated
2
2
0

ACCEPTED MANUSCRIPT
3
3
24
25
with celecoxib and compound A33. These results indicated that compound A33 could
reduce the production of PGE through COX-2/PGE pathway.
2
2
3
26
3
3
3
27
28
29
Fig. 8. The effect of LPS (1 µg/ml), Celecoxib (6.25 µM), A33 (6.25 µM) on PGE secretion in
2
F10 cells. PGE levels were determined by ELISA assay. Data are shown as mean ± SD of three
2
independent experiments; **p < 0.01.
3
3
3
3
3
3
3
3
3
30
31
32
33
34
35
36
37
38
2.8. Anti-tumor activity in a xenograft model in vivo
Based on the potent anti-proliferative effect and excellent COX-2/5-LOX inhibitory
activity of compound A33 in vitro, its antitumor activity was further tested in vivo.
6
F10 cells (5×10 ) were injected subcutaneously into the right flank of nude mice to
establish xenograft model. When the tumor mass was visible and the tumor size was
3
approximately 100 mm , twenty-four tumor-bearing mice were randomly divided into
four groups: vehicle, celecoxib (20 mg/kg), compound A33 (20 mg/kg) and
compound A33 (40 mg/kg), administered intraperitoneally once every two days. Mice
tumor volume was recorded every other day and the whole treatment period lasted for
2
1

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
two weeks. As shown in Figure 9A, the tumor volume in the vehicle group increased
rapidly, while the treated groups showed a significant inhibitory effect on the tumor
volume. Among them, compound A33 (20 mg/kg) treated groups did not significantly
differ from celecoxib (20 mg/kg) treated group, after 14 days of treatment, the final
3
tumor volumes for these two groups were 1076.58 and 978.35 mm , respectively.
After the last treatment, the tumors were removed and weighed (Figure 9B and 9D).
Compared with the average tumor weight of the vehicle group of 1.24g, other three
treated groups showed a marked decrease, wherein compound A33 (40mg/kg) showed
the lightest tumor weight (0.48 ± 0.06 g). Meanwhile, no obvious change was
observed in the body weight of treated groups, indicating that the compound A33 was
not toxic for these mice. Conversely, the body weight of the vehicle group increased
slightly in the later stage of the treatment period (Figure 9C). Taken together, these
data indicated that compound A33 has excellent anti-tumor activity in vivo and is
worthy of further study as a promising compound for the development of
COX-2/5-LOX dual inhibitors for cancer therapy.
2
2

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
54
55
56
57
58
59
Fig. 9. Compound A33 effectively inhibited tumor growth in xenografts in vivo.(A) Tumour
volumes, Data were measured every other day by using a Vernier caliper and calculated as 0.5 ×
2
3
length × width (mm ). (B) Weight of the excised tumors from each group; (C) body weights of
mice from each group. (D) Photograph of the excised tumors from each group after treatment. **p
< 0.01.
3
3
3
3
3
3
3
3
60
61
62
63
64
65
66
67
2.9. Compound A33 pharmacokinetics in tumor-bearing nude mice
We further studied the metabolic stability of compound A33 after administration in
mice. After 24 h of intraperitoneal injection, blood sampling in mice eyes, obtained
plasma after centrifugation. The plasma was extracted with ethyl acetate. Then treated
sample and compound A33 were detected by HPLC. As shown in Figure 10A, the
peak time of Compound A33 was 39.654 min, injected it into mice for 24 hours.
Compound A33 was detected at 39.950 min (Figure 10B). The results showed that
compound A33 was stable in vivo, and the amide linkage was not easily cleaved.
2
3

ACCEPTED MANUSCRIPT
3
68
3
3
69
70
Fig. 10. The image of the test samples by HPLC. (A) Compound A33. (B) Extracted from plasma
with ethyl acetate.
3
3
3
3
3
3
3
3
3
3
3
71
72
73
74
75
76
77
78
79
80
81
3. Conclusions
In order to further search for COX-2/5-LOX dual inhibitors that increase cancer
treatment while reducing side effects, a series of novel hybrids of diaryl-1,5-diazoles
and morpholine derivatives was designed as COX-2/5-LOX dual inhibitors for cancer
treatment. Most of the hybrid compounds showed potent inhibitory activity, among
1
which the compound A33 with CF substituted on the benzene ring (R ) displayed the
3
most potent activity, and its IC values against F10, HeLa, A549, MCF-7 cells were
5
0
6.43, 8.08, 10.97, 7.65 µM, respectively. At the same time, anti-proliferation and in
vitro COX-2/5-LOX inhibition experiments showed that the introduction of
morpholine derivatives contributed significantly to the reduction of cytotoxicity and
the increase of antiproliferative activity. Especially, the representative compound A33
2
4

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
exhibited excellent ability to inhibit 5-LOX with the IC value of 0.68 µM. Further
5
0
molecular docking studies revealed several interactions between compound A33 and
the active sites of COX-2 and 5-LOX, which might be beneficial for its
antiproliferative effects. In mechanistic studies, compound A33 was observed to
induce apoptosis of F10 cells and G2 phase cell arrest in a dose-dependent manner.
Furthermore, the in vivo anti-tumor activity of compound A33 was confirmed on
F10-xenograft mouse model and pharmacokinetic studies had also demonstrated its
satisfying stability in vivo. In conclusion, compound A33 can further be optimized to
find innovative anti-tumor drug candidates as COX-2/5-LOX dual inhibitors.
4. Experimental section
4.1. Materials and measurements
All commercial reagents and solvents, purchased from Aladdin (China) and Xiya
Reagent, were used in analytical grade. Melting points of all the compounds were
1
determined with an X4 MP apparatus (Jingsong Corp, Shanghai, China). The H
spectra were both recorded in DMSO-d6 and CDCl3 using Bruker
(Rhenistetten-Forchheim, Germany) AM 600 MHz and Bruker AM 400 MHz, using
1
13
TMS as an internal standard. H NMR and C NMR chemical shifts are reported in
δ/ppm and coupling constants in Hz. Thin layer chromatography (TLC) plates coated
with Merck silica gel 60 GF254 monitored the chemical reactions and the purifying of
the products whose spots were visualized under 254/365 nm light. In vitro biological
evaluation of the synthesized compounds was conducted at State Key Laboratory of
Pharmaceutical Biotechnology, Nanjing University, Nanjing.
2
5

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
The COX-1 (human) Inhibitor Screening Assay Kit (catalog No.70117), COX-2
(human) Inhibitor Screening Assay Kit (catalog No.701180), 5-LOX Inhibitor
Screening Assay Kit (catalog No.520111) and PGE enzyme immunoassay (EIA)
2
kit-monoclonal (catalog No.514010) were purchased from Cayman Chemical (MI,
USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased
from Beyotime Institute of Biotechnology (Haimen, China). Annexin V-FITC cell
apoptosis assay kit (catalog No.BA11100) was purchased from BIO-BOX (Nanjing,
China). Caspase Inhibitor Z-VAD-FMK was purchased from Beyotime (Nanjing,
China).
4.2. General procedure for the synthesis of compounds 2a-2k
Sodium methoxide (60 mmol) was added slowly to a stirred solution of anhydrous
methanol (35 mL) at 0 � , solution of dimethyl oxalate (40 mmol) and the substituted
acetophenone (20 mmol) in anhydrous methanol (35 mL) was then gradually added to
the mixture with constant stirring, the reaction mixture was then heated at reflux for 6
h. After cooling to room temperature, it was poured into water (200 mL) and acidified
with the hydrochloric acid solution (1 mol/L) to PH = 3, then a solid product was
immediately formed which was filtered, washed with distilled water. The crude
products were purified by recrystallization with ethanol, ethyl acetate and petroleum
ether (VEtoh: VEtoAc: V = 1: 1: 0.5) washed by ice-water for three times to give pure
PE
intermediate products 2a-2k.
4.3. General procedure for the synthesis of compounds 3a-3k
A mixture of 2a-2k (10 mmol) and 4-hydrazinylbenzenesulfonamide (10 mmol) in
2
6

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
anhydrous methanol (40 mL) was heated at reflux for 6 h. After cooling to room
temperature and pouring it into the water, the precipitate was filtered and washed with
ethanol, the solid compounds 3a-3k were crystallized from ethyl acetate.
4.4. General procedure for the synthesis of compounds 4a-4k
Potassium hydroxide (KOH) (20 mmol) was successively added to a solution of
compounds 3a-3k (4 mmol) in anhydrous methanol, added a few drops of water and
the mixture was stirred at 70 � for 2 hours. After cooling, the mixture solution was
poured into water and acidified with the hydrochloric acid solution (1 mol/L) to PH=3,
extracted three times with ethyl acetate (3×100 mL) and discarded the aqueous layer.
The combined organic extracts were dried with Na SO after vacuum evaporation to
2
4,
obtain a solid product 4a-4k.
4.5. General procedure for the synthesis of compounds A1-A41
To a solution of 4a-4k (1mmol) in CH Cl (20 mL), 1-(3-Dimethylaminopropyl)-
2
2
3-ethylcarbodiimide hydrochloride (EDC·HCl) (1.2 mmol), 1-Hydroxybenzotriazole
(HOBt) (1.2 mmol) and 4-(dimethylamino)pyridine (DMAP) (0.5 mmol) were added
in sequence at 0 ˚C. After half an hour of activation, morpholine and substituted
morpholine (1.2 mmol) was added and stirred overnight at room temperature. The
reaction mixture was washed with the hydrochloric acid solution (1 mol/L, 10 mL),
saturated Na CO solution (10ml), distilled water (10 mL) three times. The combined
2
3
organic extracts were dried with Na SO The crude product was purified by column
2
4,
chromatography (VAcOEt/VPE = 1/1) to give compounds A1-A41.
4-(5-(3-Fluorophenyl)-3-(thiomorpholine-4-carbonyl)-1H-pyrazol-1-yl)benzenesu
27

ACCEPTED MANUSCRIPT
4
48
lfonamide（A1）
1
4
4
4
4
4
49
50
51
52
53
White solid, yield 64.1%, m.p. 207.6-208.1 ℃，H NMR (400 MHz, DMSO-d ) δ:
6
7.87 (d, J = 8.6 Hz, 2H, ArH), 7.55 – 7.46 (m, 6H, ArH), 7.33 (d, J = 8.6 Hz, 2H,
-SO NH ), 7.01 (s, 1H, =CH), 4.13 (d, J = 5.6 Hz, 2H, -CH ), 3.92 (t, J = 4.9 Hz, 2H,
2
2
2
+
-CH ), 2.70 (t, J = 4.2 Hz, 4H, -CH ). MS(ESI): 447.52 [M + H] . Anal. Calcd for
2
2
C H FN O S : C, 53.80; H, 4.25; N, 12.55%; Found: C, 54.01; H, 4.24; N, 12.49.
20
19
4
3 2
4
4
54
55
4-(5-(3-Fluorophenyl)-3-(morpholine-4-carbonyl)-1H-pyrazol-1-yl)benzenesulfon
amide(A2)
1
4
4
4
4
4
4
4
4
56
57
58
59
60
61
62
63
White solid, yield 71.6%, m.p. 177.2-177.9 ℃. H NMR (600 MHz, DMSO-d ) δ:
6
7.89 – 7.85 (m, 2H, ArH), 7.53 (d, J = 8.7 Hz, 2H, ArH), 7.51 – 7.47 (m, 4H, ArH),
7.33 (d, J = 8.6 Hz, 2H, -SO NH ), 7.03 (s, 1H, =CH), 3.97 (t, J = 4.8 Hz, 2H, -CH ),
2
2
2
1
3
3.67 (s, 4H, -CH ), 3.63 (t, J = 4.8 Hz, 2H, -CH ). C NMR (151 MHz, DMSO-d ) δ
2
2
6
161.75, 148.04, 143.95, 142.94, 141.78, 134.34, 131.05, 129.38, 128.28, 127.32,
127.30, 126.18, 126.16, 111.05, 67.00, 66.63, 47.69, 42.84. MS(ESI): 431.45 [M +
+
H] . Anal. Calcd for C H FN O S: C, 53.81; H, 4.45; N, 13.02%; Found: C, 53.59;
2
0
19
4
4
H, 4.43; N, 13.07.
4
4
64
65
4-(5-(3-Chlorophenyl)-3-(thiomorpholine-4-carbonyl)-1H-pyrazol-1-yl)benzenesu
lfonamide(A3)
1
4
4
4
4
4
4
66
67
68
69
70
71
White solid, yield 17%, m.p. 22.1-224.8 ℃. H NMR (600 MHz, DMSO-d ) δ:
6
7.88 (d, J = 6.9 Hz, 2H, ArH), 7.62 (d, J = 6.6 Hz, 2H, ArH), 7.54 (d, J = 8.4 Hz, 2H,
ArH), 7.51 (s, 2H, -SO NH ), 7.34 (d, J = 8.6 Hz, 1H, ArH), 7.22 (d, J = 6.9 Hz, 1H,
2
2
ArH), 7.06 (s, 1H, =CH), 4.12 (t, J = 5.0 Hz, 2H, -CH ), 3.92 (t, J = 5.0 Hz, 2H,
2
+
-CH ), 2.70 (q, J = 5.2 Hz, 4H, -CH ). MS(ESI): 463.97 [M + H] . Anal. Calcd for
2
2
C H ClN O S : C, 51.89; H, 4.14; N, 12.10%; Found: C, 51.69; H, 4.15; N, 12.05.
20
19
4
3 2
4
72
4-(5-(3-Chlorophenyl)-3-(morpholine-4-carbonyl)-1H-pyrazol-1-yl)benzenesulfon
4
4
73
74
amide(A4)
1
Light yellow solid, yield 87.2%, m.p. 201.0-201.5 ℃. H NMR (DMSO-d , 600
6
2
8