Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic acid as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase for cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2015.11.013

Inflammation plays a key role in cancer initiation and propagation. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), two important enzymes in inflammatory responses are up-regulated in various tumor types. Dual inhibition of COX-2 and 5-LOX constitutes a rational concept for the design of more efficacious anti-tumor agents with an improved safety profile. We have previously reported a series of diaryl-1,2,4-triazole derivatives as selective COX-2 inhibitors. Herein, we hybridized the diaryl-1,2,4-triazoles with caffeic acid (CA) which was reported to display 5-LOX inhibitory and anti-tumor activities, affording a novel class of COX-2/5-LOX dual inhibitors as anti-tumor drug candidates. Most of these compounds exhibited potent COX-2/5-LOX inhibitory and antiproliferative activities in vitro. And the most potent compound 22b could significantly inhibit tumor growth in vivo. Furthermore, mechanistic investigation showed that the representative compound 15c blocked cell cycle in G2 phase and induced apoptosis in human non-small cell lung cancer A549 cells in a dose-dependent manner. Our preliminary investigation results would provide new clues for the cancer theatment with COX-2/5-LOX dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2015.11.013

Accepted Manuscript
Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic acid as dual inhibitors
of cyclooxygenase-2 and 5-lipoxygenase for cancer therapy
Hao Cai, Xiaojing Huang, Shengtao Xu, Hao Shen, Pengfei Zhang, Yue Huang,
Jieyun Jiang, Yijun Sun, Bo Jiang, Xiaoming Wu, Hequan Yao, Jingyi Xu
PII:
S0223-5234(15)30350-0
10.1016/j.ejmech.2015.11.013
EJMECH 8203
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2015
Revised Date: 16 October 2015
Accepted Date: 6 November 2015
Please cite this article as: H. Cai, X. Huang, S. Xu, H. Shen, P. Zhang, Y. Huang, J. Jiang, Y. Sun, B.
Jiang X. Wu, H. Yao, J. Xu, Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic acid as dual
inhibitors of cyclooxygenase-2 and 5-lipoxygenase for cancer therapy, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic
acid as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase
for cancer therapy
Hao Caia,b, Xiaojing Huanga,b, Shengtao Xua,b, Hao Shena,b, Pengfei Zhanga,b, Yue Huanga,b, Jieyun
Jiangc, Yijun Sund, Bo Jiang a,b, Xiaoming Wua,b,*, Hequan Yaoa,b, Jingyi Xua,b,
*
a
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
P. R. China.
c
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky
College of Medicine, Lexington, KY 40536, USA
d Drug Screening Center, Nanjing KeyGen Biotech. Co. Ltd., Nanjing 210012, China
* Corresponding authors. Department of Medicinal Chemistry, China Pharmaceutical University,
24 Tong Jia Xiang, Nanjing 210009, P R China.
E-Mail addresses: xmwu@cpu.edu.cn (X. Wu), jinyixu@china.com (J. Xu).
Abstract: Inflammation plays a key role in cancer initiation and propagation. Cyclooxygenase-2
(COX-2) and 5-lipoxygenase (5-LOX), two important enzymes in inflammatory responses are
up-regulated in various tumor types. Dual inhibition of COX-2 and 5-LOX constitutes a rational
concept for the design of more efficacious anti-tumor agents with an improved safety profile. We
have previously reported a series of diaryl-1,2,4-triazole derivatives as selective COX-2 inhibitors.
Herein, we hybridized the diaryl-1,2,4-triazoles with caffeic acid (CA) which was reported to
display 5-LOX inhibitory and anti-tumor activities, affording a novel class of COX-2/5-LOX dual
inhibitors as anti-tumor drug candidates. Most of these compounds exhibited potent
COX-2/5-LOX inhibitory and antiproliferative activities in vitro. And the most potent compound
22b could significantly inhibit tumor growth in vivo. Furthermore, mechanistic investigation
showed that the representative compound 15c blocked cell cycle in G2 phase and induced
apoptosis in human non-small cell lung cancer A549 cells in a dose-dependent manner. Our
preliminary investigation results would provide new clues for the cancer theatment with
COX-2/5-LOX dual inhibitors.
Keywords: Cyclooxygenase-2; 5-Lipoxygenase; Diaryl-1,2,4-Triazoles; Caffeic acid; Anti-tumor
activity.
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1. Introduction
Inflammation is a complex process that involves widespread changes in cellar and molecular
components of physiology. Controlled inflammation is necessary for a series of protective process
including wound healing, tissue repair and defense against pathogens. However, prolonged
chronic inflammation is mostly detrimental and has been linked to a number of diseases, including
cancer [1-3]. Recent studies have validated the postulation that within the tumor
microenvironment, a network of various pro-inflammatory mediators participate in complex
signaling process that facilitates extravasations of tumor cells through the stroma promoting the
development of carcinogenesis [4].
Rarchidonic acid (AA), an essential polyunsaturated fatty acid with 20 carbons is the major
precursor of several classes of signal molecules which are important mediators in inflammatory
responses. The two main metabolic pathways of AA are cyclooxygenase (COX) and lipoxygenase
(LOX) pathways. AA is converted to prostaglandins, prostacyclins and thromboxanes by COX and
to hydroxyeicosatetraenioc acids (HETEs) or leukotrienes (LTs) by LOX. Recent studies regarding
the relationship between AA metabolic process and carcinogenesis reveal novel molecular target
for cancer treatment [5].
It has been reported that COX-2 is up-regulated in various tumor types, such as pancreatic,
prostate and colorectal cancers [6-8], resulting in elevation of downstream prostaglandin E2 (PGE2)
levels [9]. Lots of results indicate that PGE2 could increase the motility and metastic potential of
tumor cells, promote tumor angiogenesis, induce local immunosuppression and inhibit apoptosis
[5, 10]. Besides PGE2 mechanism, COX-2 itself could also conduces to carcinogens. COX-2
peroxidase activity is able to transform many procarciogens into ultimate carcinogens which can
active many genes involved in cell proliferation [5, 11]. Furthermore, COX-2 could also promote
cancer cell survival through lowering the levels of unesterified AA [12]. Inhibition of COX-2
activity has been shown to induce apoptosis and inhibit proliferation and angiogenesis [13, 14].
Similar to COX-2, the expression and activity of 5-LOX have been found to be up-regulated
in many cancer cell lines [15-19], and closely related to tumor size, depth and vessel invasion [20].
It is evident from recent studies that 5-LOX and its downstream products leukotriene B4 (LTB4)
and 5-hydroxyeicosatetranoic acid (5-HETE) could enhance cell proliferation and suppress
apoptosis, thereby promoting the development of carcinogenesis [15, 18, 19, 21-24]. It seems
likely that COX-2 and 5-LOX may represent an integrated system that regulates the proliferation,
metastatic and proangiogenic potential of cancer cells [5]. Considering the frequent co-expression
of these two enzymes and the striking analogy of their biological functions, dual inhibitors of
COX-2 and 5-LOX may present a superior anticancer profile in carcinogenesis. And notably, there
is a cross-talk between COX-2 and 5-LOX pathways, inhibition of only one of them would shunt
AA metabolism to the other pathway, thereby inducing potential side effects [25]. Hence the dual
COX-2/5-LOX inhibitors would also be safer. Overall, dual inhibition of COX-2 and 5-LOX
constitutes a rational concept for the design of more efficacious anti-tumor agents with an
improved safety profile.
Triazole heterocycle is a building block of great value in drug candidates[26, 27]. Recently,
we have identified a series of 1,5-diaryl-1,2,4-trizole derivatives as selective COX-2 inhibitors,
among which compound 1 (Fig. 1) displayed potent and selective COX-2 inhibitory activity (IC50
= 0.37 ꢀM, SI = 0.018), equipotent to that of celecoxib (IC50 = 0.26 ꢀM, SI = 0.015) [28].
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Successively, we developed a series of diaryl-1,2,4-triazoles bearing N-hydroxyurea moiety as
COX-2/5-LOX dual inhibitors for anti-inflammation [29]. Inspired by the obtained interesting
results of our previous studies and in continuation of our endeavor for novel anticancer drugs, we
became interest in exploring dual COX-2/5-LOX inhibitors as anti-tumor candidates with novel
structural characteristics, better anticancer effects and improved safety profiles. Meanwhile, we
noticed that the caffeic acid (CA), a widespread phenolic compound from the group of
hydroxycinnamates, selectively inhibits 5-LOX [30] and a novel series of triazole-containing
caffeic acid analogues was reported as 5-LOX inhibitors recently[31]. Moreover, CA and its
derivatives, for instance, phenethyl caffeate (CAPE) could also inhibit the growth and
differentiation of various cancer cells, promote apoptosis and prevent chemical carcinogenesis
[32-35]. However, it has been found that CA has a stimulatory effect on prostaglandin synthase,
which may result from its stimulation on COX pathway [30]. Accordingly, we hybridized the CA
scaffold with the selective COX-2 inhibitory moiety of compound 1 (Fig. 2) and designed a series
of 1,5-diarylsubstituted-1,2,4-triazole derivatives as COX-2/5-LOX dual inhibitors for anticancer
candidates. Herein we report the synthesis, in vitro and in vivo biological evaluation and docking
studies of these novel hybrids.
Fig. 1. 1,5-diaryl-1,2,4-triazole derivatives as selective COX-2 inhibitors.
Fig. 2. Hybrids of CA and diaryl-1.2.4-triazoles as dual COX-2/5-LOX dual inhibitors.
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2. Results and discussion
2.1 Chemistry
The 1,5-diphenyl-1,2,4-triazole-3-thiol (6a-i) were prepared from corresponding phenyl
hydrazine and benzoyl chloride as described in our previous report [28] (Scheme 1). Subsequent
treatment of the intermediate 6a-g with ethyl bromoacetate or ethyl 3-bromopropionate in the
presence of K2CO3, followed by reduction using LiAlH4, afforded the respective
1,5-diphenyl-1,2,4-triazol-3-thioalkylol (8a-f). Then the hydroxyl group of 8a-e was transformed
into triazo group. And the triazo group could be reduced to amino group through Staudinger
reaction, yielding the intermediates 11a-e (Scheme 2).
The synthesis of CA esters is outlined in Scheme 3. Acetylation of hydroxyl groups of CA
provided corresponding ester 12, which was subsequently reacted with 1,2-dibromoethane or
1,3-dibromopropane, respectively, in presence of Et3N in acetone, to produce intermediates 13a-b.
Condensation of 13a-b with 1,2,4-triazole-3-thiol 6a-b, 6d-e, 6g-i in the presence of K2CO3
afforded compounds 14a-n. Deprotection of 14b,d,e,k,o using 30% MeONa in MeOH yielded
target compounds 15a-e. The target compounds bearing p-NH2SO2 moiety at C-5 phenyl ring were
obtained by deprotection of tert-butyl in CF3COOH–PhOCH3 solution.
And the synthesis route of CA amide derivatives is shown in Scheme 4.Protection of the
hydroxyl groups of CA as an acetate followed by reaction with thionyl chloride afforded the acyl
chloride 13. Condensation of 13 with 1,2,4-triazole-3-thioalkylamine (11a-e) in the presence of
TEA given corresponding amides 17a-g. Subsequently, 17c was deprotected in condition of
MeONa providing the target product 18.
Besides, we substituted the meta-hydroxyl group of CA phenyl ring with methoxyl group and
obtained a series of ferulaic acid esters. Different from CA, the ferulaic acid was protected with
Ac2O in the presence of concentrated H2SO4 (Scheme 5) and the other operation steps were similar
to those in Scheme 3.
At last, both of the hydroxyl groups of CA were removed resulting in cinnamic acid ester
derivatives. These compounds (23a, b) could be directly prepared by treating
1,2,4-triazol-3-thioalkylol (8d of 8f) with cinnamic acid in the presence of DCC and DMAP in
dichloromethane (Scheme 6). And deprotection of 23b afforded the target compound 23c (Scheme
6).
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Scheme 1. Synthesis of 1,5-diphenyl-1H-1,2,4-triazole-3-thiol (6a-i). Reagent and conditions: (i)
KSCN, conc. HCl, anhydrous alcohol, reflux, 5 h; (ii) Et3N, acetone, reflux, 2 h; (iii) a.10% NaOH,
MeOH, reflux, 2 h; b.10% HCl.
Scheme 2. Synthesis of Synthesis of 1,5-diphenyl-1H-1,2,4-triazole-3-thioalkylamine (11a-e).
Reagents and conditions: (i) ethyl bromoacetate or ethyl 3-bromopropionate, K2CO3, acetone,
o
reflux, 3 h; (ii) LAH, dry THF, reflux, 3 h; (iii) MsCl, DCM, 0 C, 2 h; (iv) sodium azide, DMF,
80 oC, 3 h; (v) triphenylphosphine, H2O, THF, r.t., 6 h.
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Scheme 3. Synthesis of affeic acid esters. Reagent and conditions: (i) a. DMAP, DCM, reflux, 5
min; b. acetic anhydride, TEA, reflux 4 h; (ii) 1,2-dibromoethane or 1,3-dibromopropane, TEA,
acetone, reflux, 24 h; (iii) 6a-b, 6d-e, 6g-i, K2CO3, acetone, r.t., 5-10 h; (iv) TFA/methylanisole, 0
oC-r.t., 24 h; (v)MeONa/MeOH (30 %), 0 oC-r.t., 15-30 min.
Scheme 4. Synthesis of CA amides. Reagent and conditions: (i) a. DMAP, DCM, reflux, 5 min; b.
acetic anhydride, TEA, reflux 4 h; (ii) thionyl chloride, reflux, 3 h; (iii) 11a-e TEA, DCM, 0oC-r.t.,
8 h; (iv) TFA/methylanisole, 0 oC-r.t., 24 h; (v) MeONa/MeOH (30 %), 0 oC-r.t., 15-30 min.
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Scheme 5. Synthesis of ferulaic acid esters. Reagent and conditions: (i) Ac2O, conc. H2SO4, r.t.,
10-15 min; (ii) 1,2-dibromoethane, TEA, acetone, reflux, 24 h; (iii) 6a-c or 6h-i, K2CO3, acetone,
r.t., 5-10 h; (iv) MeONa/MeOH (30 %), 0 oC-r.t., 15-30 min.
Scheme 6. Synthesis of cinnamic acid esters. Reagent and conditions: (i) 8b or 8f, DCC, DMAP,
DCM, r.t., overnight; (ii) TFA/methylanisole, 0 oC-r.t., 24 h.
2.2 In Vitro COX-2 and 5-LOX inhibitory activities
The obtained hybrids of CA and diaryl-1.2.4-triazoles were evaluated in vitro to determine
their COX-2 and 5-LOX inhibitory potencies. Standard compounds celecoxib (COX-2 inhibitor)
and zileuton (5-LOX inhibitor) were evaluated in parallel. As summarized in Table 1, all of these
compounds exhibited COX-2 and 5-LOX inhibitory activities. Among them, CA amides having a
F or Br in the para-position of N-1 phenyl ring (R1) exhibited potent COX-2 inhibitory activity
(17a-c and 18 , IC50 = 0.12-0.17 ꢀM) that compared favorably to the positive control celecoxib
(IC50 = 0.14 ꢀM). It is notable that substitution of R1 with electron-withdrawing group enhanced
the COX-2 inhibitory activity. For instance, the fluorin substituted compound 14a exhibited potent
COX-2 inhibition (IC50 = 0.18 ꢀM) that was 5-fold more potent than 14b with a hydrogen in R1
position (IC50 = 0.92 ꢀM). In addition, the hybride compounds linking with amide linkage were
more potent in COX-2 inhibition than those linking with ester-bounds. Meanwhile, the compounds
substituting R4 with a hydroxyl group exhibited potent 5-LOX inhibitory activity (15a-e, 18 and
22a-b, IC50 = 0.71-0.87 ꢀM) comparable to that of Zileuton (IC50 = 0.80 ꢀM), which was
consistent with reported conclusion that the hydroxyl group in R4 position of CA is necessary for
the inhibitory effect on 5-LOX [30]. The R3 substituents also affected the 5-LOX inhibition
slightly (5-LOX inhibition: OH, OCH3 > OAc > H). The structure-activity relationship was
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summarized in Fig. 3. Based on the favorable results of COX-2 and 5-LOX inhibition, compounds
18, 15c, 22b and 23a were chosen to evaluate the selectivity between COX-1 and COX-2. The
experiments suggested that these compounds showed good COX-1/COX-2 selectivity (Table 2).
Table 1. COX-2 and 5-LOX inhibitory activities of target compounds
COX-2
5-LOX
Compd.
R1
R2
n
X
R3
R4
IC50 (ꢀM)a
0.18±0.03
0.92±0.15
0.19±0.02
0.29±0.04
0.31±0.13
0.39±0.04
0.85±0.16
0.36±0.02
0.91±0.17
0.67±0.12
0.37±0.04
0.18±0.06
0.62±0.07
0.64±0.16
0.16±0.09
0.24±0.05
0.17±0.01
0.18±0.02
0.63±0.11
0.12±0.03
0.29±0.05
0.24±0.01
0.87±0.08
0.62±0.23
0.23±0.06
0.58±0.17
0.21±0.05
0.42±0.04
0.67±0.17
0.14±0.03
—
IC50 (ꢀM)a
1.05±0.21
1.21±0.19
1.06±0.32
0.99±0.08
1.05±0.14
1.06±0.23
1.11±0.17
1.03±0.34
1.07±0.12
0.85±0.23
0.79±0.18
0.71±0.09
0.80±0.14
0.77±0.08
0.95±0.16
0.99±0.16
0.98±0.08
0.99±0.15
0.97±0.19
0.71±0.17
0.94±0.21
0.92±0.19
0.98±0.36
0.96±0.19
0.92±0.06
0.84±0.05
0.81±0.14
1.30±0.25
1.28±0.12
—
F
H
CH3SO2
CH3SO2
CH3SO2
CH3SO2
NH2SO2
NH2SO2
NH2SO2
NH2SO2
NH2SO2
CH3SO2
CH3SO2
CH3SO2
NH2SO2
NH2SO2
CH3SO2
CH3SO2
CH3SO2
NH2SO2
NH2SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
CH3SO2
NH2SO2
2
2
2
3
2
2
2
3
3
2
3
3
2
3
2
3
3
2
3
3
2
2
2
2
2
2
2
3
2
O
O
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OH
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OH
14a
14b
14c
CF3
Br
Br
F
O
O
14d
14g
O
O
14i
H
O
14k
14m
14o
F
O
H
O
H
O
15a
Br
F
O
OH
OH
15b
15c
O
OH
OH
H
O
OH
OH
15d
15e
H
O
OH
OH
F
NH
NH
NH
NH
NH
NH
O
OAc
OAc
OAc
OAc
OAc
OH
OAc
OAc
OAc
OAc
OAc
OH
17a
Br
F
17b
17c
Br
H
17e
17g
F
18
Br
F
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OAc
OAc
OAc
OAc
OAc
OH
21a
O
21b
21c
CH3
H
O
O
21d
21e
CF3
H
O
O
22a
CF3
Br
Br
O
OH
22b
23a
O
H
O
H
H
23c
Celecoxib
Zileuton
0.80±0.07
a Values are means of four determinations.
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Fig. 3. Structure-activity relationships of hybrids of CA and diaryl-1.2.4-triazoles.
Table 2. In vitro COX-1/COX-2 inhibitory activity for compounds 18, 15c,22b,23a
IC50 (ꢀM) a
Compd.
Selectivity Index b (SI)
COX-1
COX-2
12.6±1.07
13.2±0.45
20.5±1.52
45.7±3.61
8.2±0.69
0.18±0.06
0.12±0.03
0.21±0.05
0.42±0.04
0.14±0.03
0.014
0.009
0.010
0.009
0.017
15c
18
22b
23a
Celecoxib
aThe test compound concentration required to produce 50% inhibition of COX-1/COX-2 is the
mean of four determinations.
bIn vitro COX-2 selectivity index (COX-2 IC50 / COX-1 IC50).
2.3 Molecular modeling (docking) studies
To investigate the binging interactions, molecular docking studies were carried out between
the most potent compound 18 which exhibited balanced COX-2/5-LOX inhibitory activities and
the mammalian COX-2 and 5-LOX enzymes. The COX active site has been detailedly described
with high-resolution structural information on COX-inhibitor complexes [37-39]. It consists of a
long narrow hydrophobic channel extending from the membrane-binding domain to the heme
cofactor. Despite their similarity, Ile in COX-1 is exchanged for Val in COX-2 at positions 434
and 523, which opens an side pocket off the main channel [40, 41]. Another essential factor for
selectivity is Arg-513 in the side pocket, in place of a His in COX-1 [41]. As illustrated in Fig. 4 a)
and b), the C-5 p-CH3SO2-phenyl moiety inserts into the side pocket, which is crucial for COX-2
selectivity. Moreover, the two oxygen atoms of SO2CH3 form hydrogen bonds with Arg-513
(NH···O, d = 2.4 Å) and His-89 (NH···O, d = 2.1 Å), increasing the stability of the complex.
On the other hand, the fluoro-substituted phenyl is oriented towards the hydrophobic channel
formed by Phe-518, Trp-387, Leu-352, Tyr-385 and Tyr-348. These interactions seem critical to
the optimal inhibition of the enzyme. Besides, the secondary amine of the amide in linkage
offers hydrogen bond to the oxygen atom of Tyr-355 (NH···O, d = 2.4 Å), which may contribute
to the increase of COX-2 inhibitory activity upon replacing the ester to amide (e.g. 15c COX-2
IC50 = 0.29 ꢀM; 18 COX-2 IC50 = 0.12 ꢀM).
Although the crystal structure of human 5-LOX has been reported in 2011 [42], it only gave
an apo-structure with no inhibitor binding. And docking studies with known 5-LOX inhibitors
showed that the correlation coefficient between the predicted and experimental IC50 was negative,
reveling the crystal structure may not be used directly for drug design [43]. Hence 5-LOX
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comparative model was built on the closed model of rabbit 15-LOX (PDB entry 2P0M, chain B,
identity 38.6%) and coral 8R-LOX (PDB entry 2FNQ, identity 38.8%) for studying [43]. As
shown in Fig 4 c) and d), the compound 18 is positioned in the center of the active site. The 4-
fluoro phenyl is buried deep into the bottom channel of the hydrophobic pocket formed by
Leu-368, His-372, Leu-414 and Phe-421, and the fluoro atom forms a hydrogen bond with
His-372 (NH···F, d = 2.2 Å). Notably, the para-hydroxyl of CA could form hydrogen bond with
Asn-180 (OH···O, d = 2.3 Å), which may result in the increase of 5-LOX inhibitory activity. The
other three hydrogen bonds formed with Asn-180 (NH···O, d = 2.8 Å), Gln-413 (NH···O, d =
2.4 Å) and Asn-425 (NH···O, d = 2.3 Å) also play important role in 5-LOX inhibition. Altogether,
the representative compound 18 binds with both active sites of COX-2 and 5-LOX via
hydrophobic interactions. And the binding conformation is stabilized by hydrogen bonds. These
docking studies revealed possible binding mode of these compounds, which may provide insight
into the further modification of the hybrids of CA and diaryl-1.2.4-triazoles as COX-2/5-LOX
dual inhibitors.
Fig. 4. Docking of compound 18 into the COX-2 (entry code: 4FM5) and 5-LOX (comparative
model, templet: 15-LOX: 2P0M and 8R-LOX: 2FNQ) active site. a) H bonding interaction
between 18 and COX-2; b) Hydrophobicity of the active site of COX-2; c) H bonding interaction
between 18 and 5-LOX; d) Hydrophobicity of the 5-LOX pocket. Yellow lines represent hydrogen
bonds.
2.4 MTT assay
According to COX-2/5-LOX inhibitory activities and structural characteristics, 22
compounds were selected for in vitro antiproliferative activity determination using MTT assay
against four different cell lines (A549: human lung cancer, Caco-2: human colon cancer, PC-3:
human prostate cancer and B16-F10: murine melanoma). Assays with cisplatin (CDDP), CAPE,
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CA and compound 1 as reference compounds were included for comparison. As shown in Table 3,
most test compounds showed potent inhibitory effects against these four cell lines, superior to the
parent compounds (CA and compound 1) and almost equivalent to CAPE. And the most potent
compound 22b exhibited equivalent antiproliferative activity to that of CDDP (IC50: 6.78~9.05
ꢀM of 22b vs. 6.93~9.71 ꢀM of CDDP). Interestingly, the amide derivatives 17a-c, 17e, 17g and
18 with superior COX-2 inhibition activities showed weaker effect than ester derivatives on cell
growth (e.g. COX-2: 0.12 ꢀM of 18 vs. 0.29 ꢀM of 15c; cellular activity: 18.72~23.09 ꢀM of 18
vs.9.52~11.16 ꢀM of 15c). It may result from the hydrophobicity of the compounds which may
affect the drug permeation through biological membrane. Moreover, we found that the
antiproliferative activity would increase as the para-position of N-1 phenyl ring (R1) was
substituted with electron withdrawing groups.
Table 3. Antiproliferative activities of selected compounds against different cancer cell lines.
Cell lines (IC50a,b, µM ± SD)
Compd.
A549
Caco-2
PC-3
B16-F10
9.71±0.11
10.68±0.06
>100
CDDPc
CAPE
CA
6.93±0.01
9.72±0.09
>100
9.20±0.23
11.35±0.14
>100
7.69±0.04
10.15±0.29
>100
1
29.53±0.31
11.78±0.83
10.19±0.15
12.86±0.05
12.04±0.11
9.92±0.22
13.80±0.49
11.19±3.16
9.52±3.16
14.07±0.37
17.01±0.35
23.30±0.18
21.82±0.12
18.74±0.97
32.72±1.26
22.14±0.13
11.05±0.22
9.61±0.14
15.01±0.36
9.65±0.17
6.78±0.21
22.34±0.31
19.74±0.25
41.25±0.24
12.81±0.36
11.30±0.27
11.71±0.09
10.97±0.32
11.45±0.19
14.75±0.23
12.42±0.19
11.16±0.19
15.01±0.40
15.08±0.41
26.14±0.62
20.77±0.85
16.71±1.31
31.03±1.74
20.92±0.26
12.07±0.25
10.81±0.36
14.09±0.21
10.77±0.41
9.05±0.07
24.56±0.09
37.47±0.53
15.65±0.77
14.51±0.13
14.88±0.53
9.84±0.47
10.52±0.38
14.56±0.12
12.35±4.08
10.11±4.08
15.02±0.24
18.03±0.63
23.90±0.21
17.56±0.13
17.04±0.29
35.21±1.56
23.09±0.15
13.14±0.19
12.77±0.21
15.53±0.37
9.81±0.73
7.46±0.49
21.92±1.61
20.05±0.18
32.28±0.17
14.21±0.92
11.46±0.33
12.84±1.2
11.03±0.15
10.90±0.26
14.97±0.44
13.13±2.16
10.43±2.16
14.98±0.52
14.62±0.19
16.37±0.21
15.23±0.12
15.16±0.43
29.06±1.31
18.72±0.19
12.44±0.80
11.18±0.43
15.34±0.72
10.47±0.11
8.65±0.55
19.44±0.51
21.16±0.74
14a
14c
14d
14i
14m
15a
15b
15c
15d
17a
17b
17c
17e
17g
18
21a
21b
21c
21e
22b
23a
23c
20.86±0.33
a IC50: Concentration inhibits 50% of cell growth. Results are expressed as the mean ± S.D. of
three independent experiments. c CDDP: cis-Diammineplatinum(II) dichloride.
b
11

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2.5 Induction of apoptosis by compound 15c
Considering the antiproliferative and COX-2/5-LOX inhibitory activity, the compound 15c
was selected for flow cytometry analysis. As shown in Fig. 5, compound 15c could induce
apoptosis in human non-small cell lung cancer A549 cells in a dose-dependent manner. Treatment
of the A549 cells with compound 15c at concentrations of 1 ꢀM, 5 ꢀM and 25 ꢀM for 3 days
resulted in cell apoptosis at ratios of 12.86%, 30.05% and 43.35% respectively, as compared with
7.51% in the vehicle control group.
Fig. 5. Compound 15c induces apoptosis in A549 cells. a): Flow cytometry analysis of apoptotic
A549 cells; b): Apoptotic ratio.
2.6. Cell cycle studies of compound 15c
Furthermore, cell cycle progression experiments were designed to examine the cytostatic
effects of 15c on A549 cells. In control groups, the G1, S and G2 populations represented 54, 42
and 4% of the cells respectively. After incubation with 15c, a G2-block was observed in a
dose-dependent manner (Fig. 6). Similar results were reported by other researcher [40, 44].
12

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Recently, Dong et al. [45] reported that EGFR and COX-2 were co-overexpressed and
co-localized with each other and inhibition of COX-2 decreased levels of EGFR in cancer cells.
And it is reported that inhibition of EGFR may give rise to G2/M phase cell cycle arrest [46, 47].
Taken together, It could suggest that the G2-block caused by compound 15c, at least partially,
assigned to its capacity to decrease EGFR expression. Although further experiments are needed to
extend understanding of the mechanisms, these diaryl-1,2,4-triazole derivatives are expected to be
promising candidates for the development of anticancer agents considering their remarkable
biological profile and novel structure scaffold.
Fig. 6. Influence of compound 15c on A549 cell cycle.
2.7 In vivo anti-tumor activity
Based on the results of cellar activities, we further tested the in vivo antitumor activity of the
most potent compound 22b against mice bearing B16-F10 melanoma. As illustrated in Table 4,
22b inhibited the tumor growth in ratio of 58.9% at the dose of 40 mg/kg exhibiting potent
antitumor activity. Preliminary biological evaluation in vivo indicated that compound
22b was worthy of further study as a potential lead for development of
13

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COX-2/5-LOX dual inhibitors for cancer therapy.
Table 4. In vivo antitumor activity of compound 22b against mice bearing B16-F10 melanoma.
Number of
mice
Weight of
tumor
Ratio of
inhibition
(%)
Weight of mice (g)
P
Drugs
Dose
value
X±SD (g)
start
10
end
10
Start
18.4 ± 1.1 28.1 ± 1.7
18.6 ± 0.5
18.7 ± 0.9 26.0 ± 3.3
End
Saline
22b
0.4 ml/mouse
40 mg/kg
1.92 ± 0.61
10
10
10
10
26.3 ± 2.4 0.79 ± 0.29
0.61 ± 0.51
58.9
68.2
< 0.01
< 0.01
5-FUa
20 mg/kg
a 5-FU: 5-Fluorouracil.
3. Conclusion
In continuation of our endeavor for novel anticancer candidates, we designed a new class of
COX-2/5-LOX dual inhibitors through a pharmacophore hybrid approach followed by SAR
studies. Twenty-nine derivatives were synthesized by hybridizing diaryl-1,2,4-triazoles with CA
scaffold. All target compounds exhibited potent COX-2/5-LOX inhibitory activities and the
selected compounds 18, 15c, 22b and 23a displayed excellent COX-1/COX-2 selectivity. The
docking studies revealed possible binding mode of these compounds, which may provide insight
into the further modification. Antiproliferative activity evaluation suggested that most of these
compounds process potent antiproliferative activities. In mechanistic studies, the representative
compound 15c was found to induce apoptosis and G2/M phase cell cycle arrest in human lung
cancer A549. Moreover, in vivo anti-tumor efficiency evaluation showed that the most potent
compound 22b could significantly inhibit tumor growth in mice. Although detailed mechanisms
remain to be elucidated, compound 22b could warrant further optimization to find novel
anti-tumor drug candidates.
4. Experimental section
4.1 Chemistry
Most chemicals and solvents were purchased from commercial sources. Further purification
and drying by standard methods were employed when necessary. Melting points were determined
on an XT-4 micro melting point apparatus and uncorrected. IR spectra were recorded in CDCl3 or
1
KBr pellets on a Nicolet Impact 410 spectrophotometer. H NMR and 13C NMR spectra were
recorded with a Bruker AV-300 spectrometer in the indicated solvents (TMS as internal standard):
the values of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in
Hz. Purity of all tested compounds was ≥95%, as estimated by HPLC analysis. The major peak of
the compounds analyzed by HPLC accounted for ≥95% of the combined total peak area when
monitored by a UV detector at 210 nm. EI-MS spectra were recorded on an Agilent1100-
14

ACCEPTED MANUSCRIPT
LC-MSD-Trap/SL spectrometer and High-resolution mass spectra were recorded using an Agilent
QTOF 6520.
4.1.1 General procedure for synthesis of compounds 7a–f. The compound 6a-b, d, g
(1.46 mmol) and K2CO3 (1.46 mmol) were suspended in anhydrous acetone(12 mL) . The reaction
o
mixture was stirred for 0.5 h at 50 C, then ethyl bromoacetate or ehthyl 3-bromopropionate
(2.08 mmol) was added and heat under reflux for 3 h. The reaction solution was then cooled down,
filtered and concentrated under reduced pressure. The residue was brown oil and purified by silica
gel column chromatography using petroleum ether/Ethyl acetate (3/1, V/V) to give 7a-f (66-88%
yield) as a white solids.
4.1.2 General procedure for synthesis of compounds 8a–f. To a solution of acetate 7a-f
(0.67 g, 1.35 mmol) in dry DMF (13 mL) was added LAH (0.102 g, 2.68 mmol) in batches at
room temperature in 30 min, and heated under reflux for 3 h. Then the reaction mixture was
cooled to room temperature and 6 mL ethyl acetate and 6 mL water were added. The layers were
allowed to separate and the aqueous layer was extracted three times with ethyl acetate. The
organic layer was combined, washed with a saturated sodium chloride aqueous solution and dried
over anhydrous sodium sulfate then concentrated under reduced pressure. The residue purified by
silica gel column chromatography using petroleum ether/ ethyl acetate (3/1, V/V) as eluent to give
8a-f ( 50-78% yield) as solids.
4.1.3 General procedure for synthesis of compounds 9a–e. To a solution of 8a-e (0.24
mmol) in dry dichloromethane (5 mL) were added TEA (0.091 mL, 0.66 mmol) and
methylsulfonyl chloride (0.055 mL, 0.71 mmol) dropwise under ice bath and stirred for 2 h. The
reaction mixture was diluted with dichloromethane and washed with water twice. The organic
layer was dried over anhydrous sodium sulfate then concentrated under reduced pressure. The
obtained colorless oil was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (3/1, V/V) as eluent to give 9a-e (69-87% yield) as solids.
4.1.4 General procedure for synthesis of compounds 10a-e. To a solution of sulfonate 9a-e
(0.26 mmol) in dry DMF (10 mL) was added sodium azide (0.044 g, 0.68 mmol), then heat to 90
oC for 3 h. The reaction mixture was cooled to room temperature and extracted with ethyl acetate
three times. The organic layer was combined and washed with saturated sodium chloride aqueous
solution, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The
obtained yellow oil was purified by silica gel column chromatography using petroleum ether/
ethyl acetate (4/1, v/v) as eluent to give 10a-e (67-89% yield) as solids.
4.1.5 General procedure for synthesis of compounds 11a-e. To a solution of 10a-e (0.25
mmol) in THF (5 mL) was added triphenylphosphine (0.399 g, 1.52 mmol). The mixture was
stirred at room temperature overnight, then concentrated under reduced pressure. The residue was
diluted with 5 mL of dichloromethane, then was washed with water and saturated sodium chloride
aqueous solution successively. The organic layer was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The obtained crude product was purified by silica gel
column chromatography using dichloromethane/methanol/triethylamine (150/1/0.05, v/v/v) as
eluent to give 11a-e (57-80% yield) as solids.
4.1.6 Procedure for synthesis of compound 12((E)-3-(3,4- diacetoxyphenyl) acrylic
acid).To a solution of CA (0.122 g, 0.68 mmol) suspended in dichloromethane (10 mL), DMAP
(0.015 g, 0.12 mmol) was added. The reaction mixture was heated to reflux, and Ac2O (0.20 mL,
2.13 mmol) and TEA (0.29 mL, 2.09 mmol) were added dropwise 5 min later, then refluxed for 4
15

ACCEPTED MANUSCRIPT
h. The mixture was concentrated under reduced pressure and diluted with dichloromethane, then
washed successively with 1N HCl, water and saturated sodium chloride aqueous solution. The
organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
The obtained white solid was purified by silica gel column chromatography using
dichloromethane/methanol (100/1, v/v) as eluent to give 12 (0.149 g, 83.3% yield) as a white
solid. ESI-MS m/z: 265.1 [M+H]+
.
4.1.7 General procedure for synthesis of compounds 13a, b. To a solution of 12 (0.919 g,
3.48 mmol) suspended in dry acetone (25 mL) were added TEA (1.45 mL, 10.4 mmol) and
1,2-dibromoethane (0.90 mL, 10.4 mmol) or 1,3-dibromopropane dropwise at room temperature.
The reaction mixture was heated to reflux and stirred for 24 h and then filtered. The filtrate was
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using petroleum ether/ethyl acetate (5/1, v/v) as eluent to give 13a,b ( 31.8% and
42.6% yield respectively) as white solids.
4.1.8 General procedure for synthesis of compounds 14a-f, 14h, 14j, 14l and 14n. To a
solution of 13a or 13b (0.27 mmol) in dry acetone (8 mL) were added K2CO3 (0.045 g, 0.33
mmol) and 6a-b, 6d-e or 6g-I (0.30 mmol). The reaction mixture was stirred for 8 h at room
temperature then filtered. The filtrate was concentrated under reduced pressure and the residue
was purified by silica gel column chromatography using dichloromethane/methanol (300/1, v/v) as
eluent to give 14a-f, 14h, 14j, 14l and 14n (26-53% yield) as white solids or colorless oils.
(E)-4-(3-(2-((1-(4-fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-triazol-3
o
1
-yl)thio)ethoxy)-3-oxoprop-1-en-1-yl)-1,2-phenylene diacetate (14a) m.p. 66-70 C. H-NMR
(CDCl3, 300 MHz) δ: ppm 7.91 (2H, d, J = 8.3 Hz, ArH), 7.67 (2H, d, J = 8.3 Hz, ArH), 7.59
(1H, d, J = 17.6 Hz, -CH=), 7.25ꢀ7.34 (4H, m, ArH), 7.11ꢀ7.22 (3H, m, ArH), 6.35 (1H, d, J =
16.0 Hz, -CH=), 4.58 (2H, t, J = 6.4 Hz, OCH2), 3.53 (2H, t, J = 6.4 Hz, SCH2), 3.06 (3H, s,
SO2CH3), 2.30 (6H, s, OAc); 13C NMR (75 MHz, CDCl3) δ: 168.02, 166.23, 164.45, 161.79,
161.24, 153.07, 142.15, 141.64, 139.35, 139.24, 133.59, 133.55, 131.76, 129.63, 127.76, 127.32,
127.20, 126.63, 125.85, 122.31, 121.98, 117.05, 116.74, 114.88, 63.51, 44.17, 33.85, 20.59, 20.56;
IR (KBr, cm-1) : 3462, 3134, 1774,1710, 1638, 1511, 1400, 1317, 1206, 1150, 1091, 988, 849,
+
780, 544; ESI-MS m/z: 640.1 [M+H]+; HRMS (ESI): Calculated for C30H27FN3O8S2 [M+H]+:
640.1218, found: 640.1210.
4.1.9 General procedure for synthesis of compounds 14g, 14i, 14k, 14m, and 14o. To a
solution of 14f, 14h, 14j, 14l or 14n (0.13 mmol) in TFA (3 mL) were added 3 drops of anisole
under ice bath. The reaction mixture was slowly heated to room temperature and stirred for 24 h.
The mixture was concentrated under reduced pressure. The obtained residue was purified through
silica gel column chromatography using dichloromethane/methanol (300/1, v/v) as eluent to give
14g, 14i, 14k, 14m and 14o (28-53% yield) as white solids.
4.1.10 General procedure for synthesis of compounds 15a-e, 18, 22a-b. To a solution of
14b-e, 14k, 14o, 17c, or 21d-e (0.17 mmol) in methanol (3 mL) was added 30% sodium
methylate MeOH solution dropwise until the PH value to 9. The reaction mixture was stirred at
room temperature for 15 min then concentrated. Hydrochloric acid (10%) was added to give pH
5~6. The mixture was concentrated under reduced pressure and diluted with water (15 mL), then
extracted with dichloromethane (10 mL×3) and washed with a saturated aqueous solution of
sodium chloride. The organic layer was dried over anhydrous sodium sulfate and concentrated
under reduced pressure. The residue was purified by silica gel column chromatography using
16

ACCEPTED MANUSCRIPT
dichloromethane/methanol (100/1, v/v) as eluent to give target compounds (37-47% yield) as
white or yellow solids.
(E)-2-((5-(4-(methylsulfonyl)phenyl)-1-phenyl-1H-1,2,4-triazol-3-yl)thio)ethyl
3-(3,4-dihydroxyphenyl)acrylate (15a): m.p. 196-198 oC. 1H-NMR (DMSO-d6, 300 MHz) δ: ppm
9.62 (1H, s, OH), 9.14 (1H, s, OH), 7.92 (2H, d, J = 8.5 Hz, ArH), 7.64 (2H, d, J = 8.5 Hz, ArH),
7.45 (1H, d, J = 15.6 Hz, -CH=), 7.46ꢀ7.51 (3H, m, ArH), 7.39ꢀ7.46 (2H, m, ArH), 7.00 (1H, s,
ArH), 6.96 (1H, d, J = 8.1 Hz, ArH), 6.73 (1H, d, J = 8.0 Hz, ArH), 6.22 (1H, d, J = 15.9 Hz,
-CH=), 4.47 (2H, t, J = 5.9 Hz, OCH2), 3.51 (2H, t, J = 5.8 Hz, SCH2), 3.25 (3H, s, CH3SO2); 13C
NMR (75 MHz, DMSO-d6) δ 166.66, 160.04, 153.56, 148.93, 146.00, 145.91, 142.49, 137.56,
132.08, 130.08, 130.04, 129.97, 127.59, 126.15, 125.81, 121.88, 116.15, 115.25, 113.93, 63.04,
43.61, 30.49; IR (KBr, cm-1): 3415, 3133, 2360, 1628, 1517, 1499, 1400, 1290, 1142, 1086, 996,
+
857, 820, 735, 528; ESI-MS m/z: 538.1 [M+H]+; HRMS (ESI): Calculated for C26H24N3O6S2
[M+H]+: 538.1101, found: 538.1103.
(E)-3-(3,4-dihydroxyphenyl)-N-(3-((1-(4-fluorophenyl)-5-(4-(methylsulfonyl)ph-
enyl)-1H-1,2,4-triazol-3-yl)thio)propyl)acrylamide (18): m.p. 80-82 oC; 1H-NMR (DMSO-d6, 300
MHz) δ: ppm 9.21 (2H, brs, OH), 8.09 (1H, t, J = 5.2 Hz, NH), 7.94 (2H, d, J = 8.6 Hz, ArH),
7.68 (2H, d, J = 8.6 Hz, ArH), 7.51ꢀ7.59 (2H, m, ArH), 7.33ꢀ7.39 (2H, m, ArH), 7.23 (1H, d, J
= 15.7 Hz, -CH=), 7.93 (1H, s, ArH), 6.82 (1H, d, J = 8.3 Hz, ArH), 6.74 (1H, d, J = 8.0 Hz,
ArH), 6.30 (1H, d, J = 15.8 Hz, -CH=), 3.67 (2H, t, J = 6.6 Hz, SCH2), 3.24 (3H, s, SO2CH3),
3.17 (2H, t, J = 5.8 Hz, NHCH2); 13C NMR (75 MHz, DMSO) δ: 167.70, 164.61, 161.41, 152.84,
145.96, 143.64, 140.59, 139.62, 133.73, 129.69, 129.47, 127.88, 127.46, 127.34, 124.71, 122.16,
122.12, 121.78, 118.73, 116.90, 116.59, 115.43, 44.17, 40.21, 32.42, 29.60. IR (KBr, cm-1): 3458,
3135, 1655, 1510, 1400, 1262, 1147, 1090, 989, 843, 804, 776, 541; ESI-MS m/z: 569.2 [M+H]+;
HRMS (ESI): Calculated for C27H25FN4NaO5S2+ [M+Na]+: 591.1149, found: 591.1158.
(E)-2-((5-(4-(methylsulfonyl)phenyl)-1-phenyl-1H-1,2,4-triazol-3-yl)thio)ethyl 3-(4-hydroxy
o
1
-3-methoxyphenyl)acrylate (22a): m.p. 94-98 C; H-NMR (CDCl3, 300 MHz) δ: ppm 7.86 (2H,
d, J = 8.5 Hz, ArH), 7.67 (2H, d, J = 8.5 Hz, ArH), 7.60 (1H, d, J = 15.9 Hz, -CH=), 7.44ꢀ7.47
(3H, m, ArH), 7.30ꢀ7.33 (2H, m, ArH), 6.99ꢀ7.04 (2H, m, ArH), 6.89 (1H, d, J = 8.1 Hz, ArH),
6.28 (1H, d, J = 15.9 Hz, -CH=), 5.96 (1H, s, OH), 4.58 (2H, t, J = 6.4 Hz, OCH2), 3.90 (3H, s,
OCH3), 3.54 (2H, t, J = 6.4 Hz, SCH2), 3.06 (3H, s, SO2CH3); 13C NMR (75 MHz, CDCl3) δ
166.85, 160.87, 152.86, 148.04, 146.74, 145.24, 141.74, 137.35, 132.44, 129.71, 129.66, 129.55,
127.57, 126.82, 125.31, 123.05, 114.99, 114.69, 109.36, , 62.98, 55.92, 44.28, 30.52; IR (KBr,
cm-1): 3454, 3143, 1693, 1637, 1604, 1515, 1501, 1400, 1309, 1286, 1267, 1147, 1092, 780, 698,
599, 578, 531; ESI-MS m/z: 552.1 [M+H]+; HRMS (ESI): Calculated for C27H26N3O6S2+ [M+H]+:
552.1258, found: 552.1261.
4.1.11 Procedure for synthesis of compound 16((E)-3-(3,4- diacetoxyphenyl) acryloyl
chloride). To a solution of acid 12 (0.220 g, 0.83 mmol) in thionyl chloride (8mL) was added one
drop of DMF as catalyst. The mixture was heated to reflux for 3 h, then was concentrated under
reduced pressure to give 16 (0.219 g, 93.0% yield) as a brownness solid. ESI-MS m/z: 282.0
[M+H]+
.
4.1.12 General procedure for synthesis of compounds 17a-d, 17f. To a solution of amine
11a-e (0.38 mmol) in dry DMF (7 mL) were added TEA (0.078 mL, 0.56 mmol) and 16 (0.156 g,
0.56 mmol) under ice bath. The reaction mixture was slowly heated to room temperature and
stirred for another 8 h. The mixture was diluted with dichloromethane, then washed with water
17

ACCEPTED MANUSCRIPT
and saturated aqueous solution of sodium chloride successively. The organic layer was dried over
anhydrous sodium sulfate and concentrated under reduced pressure. The obtained yellow oil was
purified by silica gel column chromatography (eluent: dichloromethane/methanol = 200:1) to give
17a-d or 17f (37-76% yield) as white or yellow solids.
(E)-4-(3-((2-((1-(4-fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-triaz-
o
ol-3-yl-)thio)ethyl)amino)-3-oxoprop-1-en-1-yl)-1,2-phenylene diacetate (17a): m.p. 80-84 C.
1H-NMR (CDCl3, 300 MHz) δ: ppm 7.88 (2H, d, J = 8.4 Hz, ArH), 7.55 (2H, d, J = 8.3 Hz, ArH),
7.25ꢀ7.29 (6H, m, ArH, -CH=), 7.20 (3H, m, ArH), 6.48 (1H, d, J = 15.9 Hz, -CH=), 3.67 (2H,
m, SCH2), 3.32 (2H, t, J = 5.9 Hz, NHCH2), 3.06 (3H, s, SO2CH3), 2.25 (6H, s, OAc); 13C NMR
(75 MHz, CDCl3) δ: 168.03, 165.53, 164.43, 161.22, 153.06,142.12, 141.62, 139.22, 139.10,
133.56, 131.91, 129.60, 127.75, 127.32, 127.20, 125.86, 123.82, 122.29, 121.64, 117.08, 116.77,
44.17, 40.13, 31.78, 20.61, 20.57; IR (KBr, cm-1): 3461, 3133, 2925, 2854, 1774, 1637, 1510,
1400, 1206, 1106, 987, 841, 771, 544; ESI-MS m/z: 639.0 [M+H]+; HRMS (ESI): Calculated for
C30H27FN4NaO7S2+ [M+Na]+: 661.1197, found: 661.1200.
4.1.13 General procedure for synthesis of compounds 17e, 17g ans 23c. To a solution of
17d ,17f or 23b (0.09 mmol) in TFA (2.5 mL) was added two drops of benzaldehyde under ice
bath. The reaction mixture was slowly heated to room temperature and stirred for 24 h. Then the
mixture was concentrated under reduced pressure. The obtained brownish red oil was purified by
silica gel column chromatography using dichloromethane/methanol (150/1, v/v) as eluent to give
17e,17g or 23b (45-83% yield) as white bubble solids.
2-((1-(4-bromophenyl)-5-(4-sulfamoylphenyl)-1H-1,2,4-triazol-3-yl)thio)ethyl
cinnamate
o
1
(23c): m.p. 78-82 C; H-NMR (CDCl3, 300 MHz) δ: ppm 7.86 (2H, d, J = 8.3 Hz, ArH), 7.67
(1H, d, J = 16.0 Hz, -CH=), 7.52ꢀ7.59 (4H, m, ArH, -CH=), 7.46ꢀ7.51 (2H, m, ArH), 7.36ꢀ
7.38 (3H, m, ArH), 7.17 (2H, d, J = 8.6 Hz, ArH), 6.40 (1H, d, J = 16.0 Hz, -CH=), 5.28 (2H, s,
SO2NH2), 4.57 (2H, t, J = 6.3 Hz, OCH2), 3.52 (2H, t, J = 6.3 Hz, SCH2); 13C NMR (75 MHz,
CDCl3) δ: 166.63, 161.12, 153.12, 145.25, 143.55, 136.28, 134.20, 132.86, 131.13, 130.41,
129.50, 128.88, 128.06, 126.78, 126.62, 125.31, 123.33, 117.58, 63.11, 30.41; IR (KBr, cm-1):
3416, 3134, 1637, 1492, 1400, 1166, 1095, 986, 833, 767, 619, 543; ESI-MS m/z: 585.0 [M+H]+;
HRMS (ESI): Calculated for C25H22BrN4O4S2+ [M+H]+: 585.0260, found: 585.0268.
4.1.14 Procedure for synthesis of compound 19 ((E)-3-(4-acetoxy-3-methoxyphenyl)acrylic
acid). To a solution of ferulic acid (0.490 g, 2.52 mmol) suspended in acetic anhydride (5 mL)
was added one drop of concentrated sulfuric acid. The reaction mixture was stirred 12 minutes at
room temperature. The mixture was poured into 5 mL of ice water to give white crystal, filtered
and collected the solid . The residue was dissolved in saturated potassium carbonate solution and
filtered again. 2N HCl was added to the filtrate to adjust the pH(≈4) and a lot of white solid was
separated out. Then the mixture was filtered, and the residue was washed with water before drying
to give 19 (0.536 g,89.9% yield) as a white solid.
4.1.15
Procedure
for
synthesis
of
compound
20
((E)-2-bromoethyl
3-(4-acetoxy-3-methoxyphenyl)acrylate). To a solution of 19 (0.536 g, 2.27 mmol) in dry acetone
(6 mL) were added TEA (0.95 ml, 6.84 mmol) and 1,2-dibromoethane (0.59 ml, 6.85 mmol) . The
reaction mixture was heated to reflux and stirred for another 24 h and filtered, then the filtrate was
concentrated under reduced pressure to give brown oil residue. The residue was purified by silica
gel column chromatography using petroleum ether/ ethyl acetate (5/1, v/v) as eluent to give 20
(0.481 g, 61.8% yield) as a white solid. 1H-NMR (CDCl3, 300 MHz) δ: ppm 7.69 (1H, d, J = 16.0
18

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Hz, -CH=), 7.12ꢀ7.15 (2H, m, ArH), 7.06 (1H, d, J = 7.9 Hz, ArH), 6.41 (1H, d, J = 16.0 Hz,
-CH=), 4.52 (2H, t, J = 6.1 Hz, OCH2), 3.87 (3H, s, OCH3), 3.59 (2H, t, J = 6.1 Hz, CH2Br), 2.32
(3H, s, OAc); ESI-MS m/z: 362.4 [M+NH4]+.
4.1.16 General procedure for synthesis of compounds 21a-e. To a solution of 20 (0.100 g,
0.29 mmol) in dry acetone (8 mL) were added K2CO3 (0.048 g, 0.35 mmol) and 6a-c or 6h-i (3.49
mmol) . The reaction mixture was stirred for 5 h at room temperature then filtered. The filtrate
was concentrated under reduced pressure to give brown bubble solid. The residue was purified by
silica gel column chromatography using dichloromethane/methanol (300/1, v/v) as eluwnt to give
21a-e (42-57% yield) as white solids.
(E)-2-((1-(4-bromophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-triazol-3-yl)thio)
ethyl
o
1
3-(4-acetoxy-3-methoxyphenyl)acrylate (21a): m.p. 160-164 C. H-NMR (CDCl3, 300 MHz) δ:
ppm 7.93 (2H, d, J = 8.3 Hz, ArH), 7.56ꢀ7.69 (5H, m, ArH, -CH=), 7.19 (2H, d, J = 8.6 Hz,
ArH), 7.02ꢀ7.06 (3H, m, ArH), 6.36 (1H, d, J = 16.0 Hz, -CH=), 4.59 (2H, t, J = 6.2 Hz, OCH2),
3.84 (3H, s, OCH3), 3.53 (2H, t, J = 6.2 Hz, SCH2), 3.07 (3H, s, SO2CH3), 2.32 (3H, s, OAc); 13C
NMR (75 MHz, CDCl3) δ: 169.05, 166.76, 161.03, 152.96, 151.43, 146.06, 144.59, 141.85,
141.77, 133.23, 132.71, 132.53, 129.77, 128.82, 125.36, 122.67, 123.25, 121.36, 117.95, 111.30,
63.20, 56.07, 44.30, 30.42, 20.63; IR (KBr, cm-1): 3461, 3133, 2922, 2835, 1638, 1400, 1147,
1089, 988, 833, 780, 527; ESI-MS m/z: 672.0 [M+H]+; HRMS (ESI): Calculated for
C29H27BrN3O7S2+ [M+H]+: 672.0468, found: 672.0455.
4.1.17 General procedure for synthesis of compounds 23a-b. To a solution of 8b or 8F
(0.40 mmol) in dry dichloromethane (12 mL) were added DCC (0.080 g, 0.58 mmol), DMAP
(0.045 g, 0.37 mmol) and Cinnamic acid (0.072 g, 0.49 mmol) . The reaction mixture was stirred
overnight at room temperature and filtered, then the filtrate was concentrated under reduced
pressure.The residue was purified by silica gel column chromatography using petroleum ether/
ethyl acetate (3/1, v/v) as eluent to give 23a-b (0.118 g, 49% and 53% yield respectively) as white
solids.
3-((1-(4-bromophenyl)-5-(4-(methylsulfonyl)phenyl)-1H-1,2,4-triazol-3-yl)thio)propyl
o
1
cinnamate (23a): m.p. 64-68 C. H-NMR (CDCl3, 300 MHz) δ: ppm 7.91 (2H, d, J = 8.5 Hz,
ArH), 7.65ꢀ7.69 (3H, m, ArH, -CH=), 7.55 (2H, d, J = 8.7 Hz, ArH), 7.48ꢀ7.51 (2H, m, ArH),
7.38ꢀ7.40 (3H, m, ArH), 7.21 (2H, d, J = 11.5 Hz, ArH), 6.41 (1H, d, J = 16.0 Hz, -CH=), 4.38
(2H, t, J = 6.1 Hz, OCH2), 3.35 (2H, d, J = 6.2 Hz, SCH2), 3.04 (3H, s, SO2CH3), 2.25 (2H, m,
-CH2-); 13C NMR (75 MHz, CDCl3) δ: 166.79, 161.56, 153.95, 144.99, 143.52, 141.69, 134.75,
132.57, 132.45, 129.97, 129.75, 129.51, 128.86, 126.38, 125.30, 122.83, 122.08, 63.85, 44.29,
29.75, 28.25;
IR (KBr, cm-1): 3418, 3134, 2927, 2852, 1637, 1492, 1400, 1312, 1148, 1089, 986,
837, 776, 597, 531, 484, 453, 433, 412; ESI-MS m/z: 598.1 [M+H]+; HRMS (ESI): Calculated for
C27H25BrN3O4S2+ [M+H]+: 598.0464, found: 598.0470.
All the analytical data of other compounds are shown in supporting information.
4.2 In vitro COX and 5-LOX inhibition assay
The ability of the synthesized compounds to inhibit COX-1 was determined by
6-keto-PGF1αassay. Endotheliocyte was gained from the aorta pectoralis of neogenesis calf in vitro.
The cells (1.0 × 106 cells) in 1 mL of DMEM supplemented with 10% heated-inactivated fetal
19

ACCEPTED MANUSCRIPT
bovine serum, 2 mM glutamine, 60 mg/L penicillin and 100 mg/L streptomycin were plated in a
24-well culture plate and incubated at 37 oC under an atomosphere of 5% CO2 for 24 h. Then the
cells were treated with test compounds, DMSO and celecoxib, incubated for 20 min. Then
arachidonic acid (10 ꢀM) was added. After incubation for another 20 min, the culture was
collected for 6-keto-PGF1α determination. 6-keto-PGF1α was measured by using a radioimmunity
assay kit according to the manufacturer’s instructions.
The COX-2 inhibitory activity was determined by PGE2 production assay in rat abdominal
macrophages. Cells in 1 mL of RPMI 1640 containing 5% FCS were plated in a 24-well plate at a
o
density of 1.0 × 106cells per well. Incubated for 24 h at 37 C under an atomosphere of 5% CO2,
then aspirin (1 mM) was added to inactivate COX-1. Replaced the culture media with fresh meida
containing LPS (1 ꢀg/mL) to induce COX-2 expression. After incubation for 6 h, the cell were
treated with the test compounds , celecoxib and DMSO and incubated for 30 min. Added
arachidonic acid (10 ꢀM) and after incubation for another 20 min, the culture were collected for
PGE2 determination using an enzyme immunoassay (EIA) kit (Cat-lot no.414010, 96-well,
Cayman Chemical Company) according to the manufacturer’s instructions.
The inhibitory potency of target compounds on 5-LOX was determined by the production of
LTB4 under the stimulation of calcium ionophore A23187. Leukocytes were obtained from the
abdominal cavity of Sprague-Dawley rats which have been injected intraperitoneally with 20
mL/kg of a 0.2% (w/v) glycogen solution. The cell suspensions were collected with Hanks
solution and plated in a 24-well plate at the density of 1.0 × 106cells per well. After incubation for
10 min at 37 oC under an atomosphere of 5% CO2, the cells were treated with L-cysteine (10 mM),
indomethacin (1 mg/L), DMSO, celecoxib and test compounds in turn and incubated for another
30 min. Added calcium ionophore A23187 (5 ꢀmol) to initiate LTB4 production and incubated for
another 30 min. After being clarified by centrifugation, the supernatant was plated in a 96-well
o
plate and incubated overnight at 4 C
.
Added chromogen and retained for 90 min. The 5-LOX
activity was determined by using an EIA kit (Cat-lot no. 520111, 96-well, Cayman Chemical
Company) according to the manufacturer’s instructions. Percent inhibition was calculated
according to the following formula:
Inhibition (%) = 100 × (OD blank – OD comp)/OD blank.
The IC50 values were determined as the maximal inhibition (100%) by Bliss method.
4.3 Molecular modeling (docking) study
Molecular docking studies with flexible ligand and rigid receptor were performed with the
AutoDock Vina program[48]. Structures of the proteins and ligand were prepared using
AutoDockTools GUI for docking preparation. The pictures were generated using Pymol and
Discover Studio Visualizer 4.0 software. For COX-2, crystallographic structure in complex with
(2-fluorobiphenyl-4-yl)acetic acid (PDB ID: 4FM5) were used for docking. The volume chosen
for the grid box was 24 Å × 24 Å × 24 Å, center point (x = 19.137, y = -14.174, z = -46.89).
Exhaustiveness was increase to 20, and 14 ligand poses were generated. The structure of 5-LOX
was comparatively modeled as descriped in previous report [39]. The grid box was defined to
include residues of the active site with the grid size of 24 Å × 26 Å × 26 Å. Exhaustiveness was
increase to 20 and 9 ligand poses were generated.
20

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4.4 MTT assay in vitro
Test cell lines were plated on 96-well plates at the density of 5 × 104/well and incubated for
o
24 h at 37 C under an atomosphere of 5% CO2. All of the test compounds were dissolved in
DMSO at different concentrations and treated to the cells. Incubated for another 72 h following by
the addition of MTT (5mg/mL in PBS), After incubating for 4 h, the optical density was detected
with a microplate reader at 570 nm. The IC50 was calculated according to the dose-dependent
curves and expressed as mean ± S.D. of three independent experiments.
4.5 Analysis of cellular apoptosis
A549 cells were seed into 6-well plates and incubated for 24 h at 37 oC under an atomosphere
of 5% CO2. Then test compounds were added in a certain concentration and negative control
which was treated with DMSO were included. After incubation for 72 h, the treated cells were
trypsinized. Washed the cells with PBS twice and centrifuged at 2000 rpm to collect the cells (5 ×
105). 500 ꢀL of binding buffer were added to suspend the cells. Then 5 ꢀL of Annexin V-APC and
7-AAD were added successively and mixed well. Reacted for 5~15 min at room temperature in
dark and analyzed the cell apoptosis with flow cytometry (FACS Calibur Bectone-Dickinson).
4.6 Analysis of cell cycle arrest
The A549 cells were incubated with the test compounds as described above. After incubating
for 72 h, the treated cells were trypsinized, washed with PBS and centrifuged at 2000 rpm. The
collected cells were fixed by adding cold ethanol (4 oC, overnight) and incubated for 30 min in
PBS containing 100 ꢀL RNase A of RNase and 400ꢀL of propidium iodide. Cell DNA content
was measured using flow cytometry (FACS Calibur Bectone-Dickinson) for cell cycle distribution
analysis.
4.7 In vivo antitumor assay
Antitumor activity against mice bearing B16-F10 melanoma was performed as described by
Cao et al. with a slight modification [49]. Mice (from Institute of Cancer Research) with body
weight 18-22 g were transplanted with B16-F10 cells on the right oxter subcutaneously. 7 days
later, mice were weighted and divided randomly in to 3 groups (10 mice/group). Treated the three
groups of mice with saline (containing 1% DMSO/2% poloxamer, vehicle), test compound 22b
(40 mg/kg) and 5-FU (20 mg/kg, positive control) through intravenous injection respectively. The
mice were sacrificed at the 25th day after tumor transplantation. Excised the tumors and weighted.
The inhibition was calculated as follows: Tumor inhibition ratio (%) = (1-average tumor weight of
the drug treated group/average tumor weight of vehicle group) × 100%.
21

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Acknowledgement
This study was supported by the grants from State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. SKLNMZZCX201404), Natural Science Foundation of
Jiangsu Province (No. BK20130645 ), Huahai Pharmaceutical Graduate Innovation Fund (No.
CX14B-005HH) and Innovation project of Jiangsu Province (No. KYLX15-0636). We appreciate
Prof. Luhua Lai of Peking University for direction of comparative modeling of 5-LOX.
Conflicts of interest
The authors declare no conflict of interest.
Supporting Information
Detailed experimental procedures including spectroscopic and analytical data.
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Highlights (for review)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Novel COX-2/5-LOX dual inhibitors were designed via pharmacophore hybrid approach.
Most compounds showed COX-2/5-LOX inhibitory and anti-proliferative activities.
The compound 15c was found to induce apoptosis and G2/M phase cell cycle arrest.
The most potent compound 22b significantly inhibited tumor growth in vivo.
The docking studies revealed possible binding mode of these compounds.