Neuroprotective effect of novel celecoxib derivatives against spinal cord injury via attenuation of COX-2, oxidative stress, apoptosis and inflammation

Article abstract of DOI:10.1016/j.bioorg.2020.104044

A novel series of celecoxib derivatives were synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities for benefit in spinal cord injury (SCI). The title compounds were synthesized by conventional methods in good yields and subsequently tested for inhibitory activity against COX-1/COX-2. The most potent COX-2 inhibitor among the tested derivatives was further assayed for protective effect against experimental SCI of Sprague-Dawley rats. The designed compounds showed considerable inhibition of COX-2 as compared to COX-1 revealing compound 7m as most potent inhibitor of COX-2 isoenzyme (IC₅₀ = 0.04 μM). The expression of mitochondrial apoptotic genes (Bcl-2 and Bax) together with COX-2 and iNOS was restored near to normal as evidenced by western blot analysis in SCI rats. Taken altogether, compound 7m was identified as most potent inhibitor of COX-2. It also showed protective action against SCI via attenuation of COX-2, oxidative stress and apoptosis and inflammation in Male Sprague-Dawley rats.

Full text of DOI:10.1016/j.bioorg.2020.104044

BioorganicChemistry101(2020)104044
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Neuroprotective effect of novel celecoxib derivatives against spinal cord
injury via attenuation of COX-2, oxidative stress, apoptosis and
inflammation
Yan An, Jianing Li, Yajun Liu⁎, Mingxing Fan
Department of Spine Surgery, Beijing Jishuitan Hospital, Beijing 100035,China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel series of celecoxib derivatives were synthesized and evaluated for cyclooxygenase (COX-1/COX-2) in-
hibitory activities for benefit in spinal cord injury (SCI). The title compounds were synthesized by conventional
methods in good yields and subsequently tested for inhibitory activity against COX-1/COX-2. The most potent
COX-2 inhibitor among the tested derivatives was further assayed for protective effect against experimental SCI
of Sprague-Dawley rats. The designed compounds showed considerable inhibition of COX-2 as compared to COX-
1 revealing compound 7m as most potent inhibitor of COX-2 isoenzyme (IC50 = 0.04 µM). The expression of
mitochondrial apoptotic genes (Bcl-2 and Bax) together with COX-2 and iNOS was restored near to normal as
evidenced by western blot analysis in SCI rats. Taken altogether, compound 7m was identified as most potent
inhibitor of COX-2. It also showed protective action against SCI via attenuation of COX-2, oxidative stress and
apoptosis and inflammation in Male Sprague-Dawley rats.
Celecoxib
COX-2
Inflammation
Oxidative stress
Apoptosis
1. Introduction
agent/drugs proved effective till now against SCI which has put selec-
tive pressure to discover novel agents/molecules which can mitigate the
The loss of motor and sensory function in a person due to spinal
cord injury (SCI) is major cause of disability and mortality across the
world. The physiological, biochemical and structural alterations cause
irreversible damage to the spinal cord followed by SCI [1]. According to
estimates, around more than 2 million people live with SCI and more
than million new cases reported each year in the worldwide. This large
community affected by SCI have huge, life-changing impacts and fi-
nancial implication on the associated families. The studies have con-
firmed that, the etiology of SCI is multi-factorial in nature involving
multiple mechanisms [2,3]. Moreover, the damage caused by SCI to the
spine categorised specifically in two distinct forms, which are inter-
related to each other, such as, primary and secondary. The primary SCI
describes immediate damage by mechanical trauma causing alteration
of cellular membrane, micro-vasculature system which enhances cel-
lular permeability and loss of integrity of myelin and axons. This milieu
initiates secondary injury concomitantly which are multi-faceted and
can last for years. The secondary injuries consist of numerous sub-
phases which causing loss of neuronal activity due oxidative stress,
inflammation, apoptosis including other physiological changes [4–7].
Therefore, many of the therapeutic modalities against SCI are targeting
complication arises due to secondary injuries, However, none of the
consequences of SCI via multiple mechanisms (see Scheme 1).
Inflammation is the characteristic hallmark of SCI. It induces se-
cretion of cytokines released by CNS or inflammatory cells [tumour
necrosis factor (TNF)-α, Interleukin (IL)-1β and IL-6] and play key role
in the regulation of the various steps of inflammatory reactions, i.e.,
recruitment and activation, but also in the downregulation of in-
flammatory cells [8,9]. This increase in inflammation in SCI is fre-
quently coupled with oxidative stress due to loss of antioxidant capa-
city. Oxidative stress is defined as imbalance between oxidants and
antioxidants, ultimately resulting in necrosis and lipid peroxidation.
Malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase
(SOD) are important biomarkers of oxidative stress after SCI [10–12].
Apoptosis is another prominent clinical feature in the spinal cord post
SCI after inflammation. It is known as programmed cell death and
mainly controlled by the genes of Bcl-2 family (Bcl2 and Bax). The
proportion of Bcl-2/Bax determines whether cells undergoing apoptosis
or not. Besides, caspase associated cascade reaction is the important
pathway for apoptosis, and caspase-3 is the key protease in the process
of apoptosis [13]. The cyclooxygenase-2 (COX-2) is an inducible en-
zyme which produces leukotrienes and thromboxanes and reported to
induce inflammatory cascade. Experimental evidences suggests that
⁎ Corresponding author.
E-mail address: ay_jstspine@vip.163.com (Y. Liu).
https://doi.org/10.1016/j.bioorg.2020.104044
Received 13 May 2020; Received in revised form 25 May 2020; Accepted 22 June 2020
Availableonline25June2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y. An, et al.
BioorganicChemistry101(2020)104044
Scheme 1. . Synthesis of title analogues 7 (a-m). Where, (a) CF3COOEt, NaOMe/MeOH, reflux, 95%; (b) EtOH/HCl, reflux, 82%; (c) PCl5, CH2Cl2, reflux, 74% (d)
Various aniline 6(a-m), Na2CO3, 4–6 h, room temperature.
following to SCI, the level of COX-2 was found overexpressed primarily
in brain and activated macrophages in response to inflammatory stimuli
or lipopolysaccharides [14]. It has been found that improving anti-
oxidative capacity and inhibiting inflammation and apoptosis could
ameliorate neurological impairment in SCI. Therefore, present study
discusses the development of novel COX-2 inhibitors against SCI.
Pyrazole is well know heterocyclic molecule endowed with limitless
therapeutic roles such, as anti-cancer [15–17], anti-viral [18–21], anti-
bacterial [22–25], anti-fungal [26,27], anti-diabetic [28,29] and many
more [30]. Celecoxib, a well known pyrazole based NSAID (Non-ster-
oidal anti-inflammatory drug) and selective COX-2 inhibitor used to
treat arthritis, acute pain, menstrual cramps and in familial adenoma-
tous polyposis [31,32]. Moreover, lot of NSAIDS are derived from
pyrazole nucleus as shown in Fig. 1. The potent activity of Celecoxib led
to the discovery of various derivatives and pro-drugs in search of novel
anti-inflammatory agents [33]. Moreover, Celecoxib has shown to
ameliorate after effects of SCI alone or in combination of agents [34].
Thus, prompted by the above, the present study deals with the dis-
covery and development of novel derivatives of Celecoxib as potent
COX-2 inhibitor and their protective effect against experimentally in-
duced spinal cord injury in rats.
2.2. In-vitro COX inhibitory activity
The newly developed Celecoxib derivatives were tested for in-vitro
COX-1/COX-2 isozyme inhibition studies and the results are presented
in Table 1. The compounds were analysed in comparison with template
molecule of synthesis Celecoxib as standard drug. On closely inspecting
the inhibitory activity chart, it was found that, entire set of compounds
showed mild to significant inhibitory activity against COX-2 than COX-
1. For instance, compound 7a, with no-substitution on the flanked
phenyl ring showed least inhibitory activity against both the tested
isoenzyme. The activity was significantly increased upon insertion of
para-methyl group at the phenyl against both COX-1 and COX-2 with
enhanced activity against COX-2 isoenzyme (7b). On changing position
of methyl group from para to ortho (7c), the activity was significantly
decreased. The inhibitory activity was further dropped in the case of
compound 7d and 7e having para and ortho methoxy group, respec-
tively. To our surprise, the inhibitory activity against COX-2 as com-
pared COX-1 was significantly increased on insertion of para-nitro
group with drastic improvement in SI, 7f. The changing of substitution
pattern of para-nitro group to ortho showed marked reduction in in-
hibitory activity against both the tested isoenzyme together with drop
in SI, 7g. The activity was further dropped in the case of compound 7h
and 7i having para and ortho substituted chloro atom, respectively. The
replacement of chloro with fluoro atom (7k and 7l) showed drastic
improvement in inhibitory activity and SI. In the next instance, the
activity was considerably improved upon introduction of tri-fluro me-
thyl group (7l and 7m). Out of which, compound 7m containing para-
trifluoro methyl group showed most potent inhibitory (IC50 = 0.04 µM)
activity against COX-2 than COX-1 among all the tested derivatives
with highest selectivity against COX-2.
2. Results and discussion
2.1. Chemistry
The compounds were synthesized by claisen condensation of the
commercially available p-methyl-acetophenone (1) and trifluoroacetic
acid ethyl ester gave diketone (2) in good yield. Compound 2 was then
reacted with the commercially available p-hydrazino-benzenesulfonic
acid (3) in refluxing ethanol to afford sulfonic acid derivative 4 (yield
over 90%). This reaction is a regioselective transformation and the 1,5-
diarylpyrazole could be generated almost exclusively by carrying out
the condensation in the presence of the hydrochloride salt of the
phenyl-hydrazine. The intermediate 4 was converted into its sulfonyl
chloride 5 with phosphorus pentachloride in dry dichloromethane. It
was later allowed to react with substituted anilines (6a-n) to afford
target compounds (7a-m) in excellent yields after recrytallisation. The
structures of compounds were ascertained by FT-IR, NMR, mass and
elemental analysis.
The structure-activity relationship (SAR) studies suggest that minor
changing of substitution pattern have marked influence on the activity.
It has been found that compounds containing electron withdrawing
substituent showed significant inhibition of COX-2 as compared to
COX-1. Moreover, compounds containing electron donating group
showed least activity against COX-1. Among the pattern of substituent,
para is more favourable for inhibitory activity than ortho-substituted
counterparts. This has also strong influence on the SI.
2

Y. An, et al.
BioorganicChemistry101(2020)104044
Fig. 1. Some examples of pyrazole based NSAID and design of target compounds.
7m was assessed on Adult male Sprague-Dawley rats via BBB scores on
1, 4, 7, 10 and 14 day post-SCI. The results are presented in Fig. 2. The
sham group showed no alteration in motor function, whereas, in sur-
gery group the motor function was altered significantly as compared to
sham. The 7m treated rats showed dose-dependent improvement of
motor function near to normal as compared to SCI group.
Table 1
. Inhibitory activity of compound 7 (a-m) against COX-1 and COX-2 isoenzyme.
Compound
R1
IC50 (in µM)
Selectivity Index (SI)a
COX-1
COX-2
7a
H
42.16
23.35
20.30
22.34
22.20
22.32
26.43
32.05
36.40
17.22
20.61
27.13
11.51
13.37
28.45
15.71
18.25
19.74
20.91
1.23
1.48
7b
4-CH3
2-CH3
4-OCH3
2-OCH3
4-NO2
2-NO2
4-Cl
1.48
7c
1.11
2.3.2. Effect of compound 7m on neurons in SCI rats
7d
1.13
The loss of neuronal normal architecture is well-evidenced in nu-
merous clinical and non-clinical studies reporting SCI [39]. Therefore,
in the next part, we investigate the effect of compound 7m on the
histopathology of neurons via H and E and Nissl staining. As evidence
by Fig. 3, the Sham treated rats showed normal neuronal architecture,
which was later found deteriorated in SCI treated group due to the
presence of edema, necrosis and enhanced infiltration. On the other
hand, 7m treated rats showed reversal of these features near to normal
in concentration-dependent manner as compared to SCI group. The
result suggests that compound 7m showed protective effect against SCI
might be due to restoration of integrity of neurons lost after SCI.
7e
1.06
7f
18.14
12.35
9.56
7g
2.14
7h
3.35
7i
2-Cl
5.62
6.47
7j
4-F
0.78
22.07
21.24
4.03
7k
2-F
0.97
7l
2-CF3
4-CF3
1.14
7m
Celecoxib
0.04
287.75
148.55
0.09
a
SI: (COX-1 IC50/COX-2 IC50).
2.3. In-vivo pharmacological activity
2.3.3. Effect of compound 7m on oxidative stress in SCI rats
The production of reactive oxygen species (ROS) due to impaired
oxidative defense mechanism leads to the development of oxidative
stress. This oxidative stress showed to be very detrimental to the neu-
rons after SCI due to cyto-toxic effects and enhanced lipid-peroxidation.
The central nervous system consists of large amount of lipids, thus it
became very labile to the damage caused by oxidative stress [40].
Therefore, in the next part of the study we examined the effect 7m on
the endogenous biomarkers of the oxidative stress and the results are
presented in Fig. 4. It was found that, compound 7m decreases the MDA
level and enhances the activity of SOD and GSH level as compared to
SCI-treated group. It has been suggested that, compound 7m
The in-vitro activity of compound against COX isoenzyme disclosed
compound 7m as highly potent inhibitor of COX-2. Therefore, it is
worthwhile to assess the pharmacological activity of compound 7m
against experimentally induced SCI in rats.
2.3.1. Effect of compound 7m on the motor function of SCI rats
The loss of motor function is a characteristic hallmark of the SCI
[35]. In many experimental studies, the affected motor function was
assessed by Basso-Beattie-Bresnahan (BBB) scores and is widely ac-
cepted [36–38]. Therefore, in the present study, the effect of compound
3

Y. An, et al.
BioorganicChemistry101(2020)104044
Fig. 2. Effect of compound 7m on motor function as determined by (A) BBB Score and (B) ratio of wet/dry spinal cord. Values represent the mean
SD and are
representative of three independent experiments. ##P < 0.05 vs sham; *P < 0.05, **P < 0.01 vs. SCI, one-way analysis of variance followed by a Tukey’s post hoc
test.
significantly reduces the burden of oxidative stress caused by SCI,
which might be accountable for its neuro-protective effect against SCI.
[41]. Therefore, the effect of compound 7m was investigated on various
pro-inflammatory cytokines and interleukins and results are presented
in Fig. 5. The level of TNF-α, IL-1β and IL-6 were found to be enhanced
in the injured spinal cord of the rats. Whereas, in 7m treated rats, the
level of these tested cytokines were considerably reduced in dose-de-
pendent manner with maximum in 10 mg/kg treated group. Thus, it
2.3.4. Effect of compound 7m on inflammation SCI rats
Inflammation was markedly reported in patients affected by SCI
followed by oxidative stress which leads to the progression of disease
Fig. 3. Effect of compound 7m on the neurons after SCI. (A) H & E and (B) Nissl staining. Values represent the mean
SD and are representative of three
independent experiments. ##P < 0.05 vs sham; *P < 0.05, **P < 0.01 vs. SCI, one-way analysis of variance followed by a Tukey’s post hoc test.
4

Y. An, et al.
BioorganicChemistry101(2020)104044
Fig. 4. . Effect of compound 7m on the oxidative stress of spinal cord of rats after SCI induction (A) MDA, (B) GSH and (C) SOD. Values represent the mean
SD and
are representative of three independent experiments. ##P < 0.05 vs sham; *P < 0.05, **P < 0.01 vs. SCI, one-way analysis of variance followed by a Tukey’s post
hoc test.
Fig. 5. . Effect of compound 7m on the proinflammtory cytokines as determined by ELISA analysis (A) TNF-α, (B) IL-1β and (C) IL-6. Values represent the
mean
SD and are representative of three independent experiments. ##P < 0.05 vs sham; *P < 0.05, **P < 0.01 vs. SCI, one-way analysis of variance followed
by a Tukey’s post hoc test.
could be suggested that compound 7m causes attenuation for in-
flammation which might be suggested as its plausible mechanism un-
derlying neuro-protective effect against SCI.
3. Material and methods
3.1. Chemistry
3.1.1. Synthesis of target molecules
2.3.5. Effect of compound 7m on apoptosis in SCI rats
The chemicals used in the current work were obtained from Sigma
Aldrich (USA). 1H NMR spectra were recorded in d6-DMSO on a Bruker
Avance-400 NMR spectrometer with TMS as the internal reference. 13C
NMR spectra were recorded on a Bruker Avance-100 NMR spectrometer
in d6-DMSO on the same spectrometers with TMS as the internal re-
ference. The multiplicity of a signal is indicated as: s – singlet, d –
doublet, t – triplet, q – quartet, m – multiplet, br – broad, dd – doublet
of doublets, etc. Coupling constants (J) are quoted in Hz and reported to
the nearest 0.1 Hz. Infrared spectra were recorded as a neat thin film on
a Perkin-Elmer Spectrum One FT-IR spectrometer using Universal ATR
sampling accessories. Letters in the parentheses refer to the relative
absorbency compared to the most intense peak: w – weak, < 40%; m –
medium, 40–75%; s – strong, greater than 75%. Melting points were
obtained using an OptiMelt automated melting point system. MS
spectra were recorded on an Agilent 1100 LC/MS.
Apoptosis promoted by inflammation is the critical pathway which
is found to be significantly altered during SCI. It is believed to play key
a role in the pathogenesis of acute and chronic SCI via promoting ne-
crosis of neurons and oligodendrocytes which ultimately reduces the
function of neurons [42]. Therefore, it is suggested that, limiting the
inflammation and oxidative stress in SCI delay the apoptosis and in-
creases the chances of neuronal regeneration. Encouraged by the ex-
cellent anti-inflammatory and antioxidant activity of compound 7m, it
is worthwhile to determine its effects on apoptotic biomarkers. Various
studies showed that mitochondrial genes have momentous impact on
the apoptotic pathway, such as the Bcl-2 and Bax which is considered as
anti-apoptotic and pro-apoptotic gene, respectively. The delicate bal-
ance between these two genes governs apoptosis, and any imbalance
causes defect in apoptotic pathway. In western blot analysis (Fig. 6), the
level of Bcl2 was found to be decreased together with increased Bax
expression in SCI group as compared to control. Whereas upon treat-
ment with compound 7m, the level of these mitochondrial genes was
restored near to normal. Concerning the role of COX-2 in the SCI and
excellent in-vitro inhibitory activity of compound 7m against COX-2,
the next part of the study was aimed to assess whether 7m could able to
inhibit COX-2 in-vivo or not. As shown in Fig. 7, compound 7m causes
does-dependent decrease in expression of iNOS and COX-2 after SCI as
determined by western blot analysis.
Synthesis of compound 2, 4 and 5 was performed with earlier re-
ported procedure given elsewhere, and identities of the compounds
were ascertained with the help of melting point, mass and elemental
analysis [43].
3.2. General synthesis of compounds 7 (a-m)
Compound 5 (12 mmol) was added over a period of time of 15 min
5

Y. An, et al.
BioorganicChemistry101(2020)104044
Fig. 6. . Effect of 7m on the (a) expression of Bcl-2 and Bax in spinal cord of SCI rats via western blot analysis; and representative quantitative analysis of (b) Bcl2 and
(c) Bax Values represent the mean
SD and are representative of three independent experiments. ##P < 0.05 vs sham; *P < 0.05, **P < 0.01 vs. SCI, one-way
analysis of variance followed by a Tukey’s post hoc test.
to an aqueous mixture of corresponding anilines 6 (a-m) (10 mmol) and
Na2CO3 (12 mmol) in water (50 mL) and temperature was maintained
at 0 °C. The resulting mixture was then continuously stirred for 4–6 h at
room temperature and then acidified with 10% HCl at 0 °C. The target
product 7 (a-m) was obtained as precipitate after filtration, washed
with water, dried, and then recrystallized.
21.3; Mass: 458.53 (M+H)+; Elemental analysis for C23H18F3N3O2S:
Calculated: C, 60.39; H, 3.97; N, 9.19. Found: C, 60.42; H, 3.95; N,
9.21.
3.2.2. N-(p-tolyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (7b)
Yield: 82%; M.p: 205–206 °C; MW: 471.50 ; Rf: 0.76; FTIR (νmax
;
3.2.1. N-phenyl-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
cm−1 KBr): 3251 (NeH stretching), 3087 (CeH broad), 2952 (alkyl CeH
stretching), 1687 (C]N aromatic), 1649 (C]C), 1329 (aromatic CF3
stretching), 1152 (SO2 stretching), 1075 (CeN stretching), 955 (SeN
stretching), 816 (CeS stretching), 731; 1H NMR (400 MHz, DMSO‑d6,
TMS) δ ppm: 7.85 (d, 2H, J = 1.90 Hz, AreH), 7.81 (d, 2H,
J = 1.84 Hz, AreH), 7.58 (d, 2H, J = 1.42 Hz, AreH), 7.16 (d, 2H,
J = 1.45 Hz, AreH), 7.02 (d, 2H, J = 1.29 Hz, AreH), 6.96 (d, 2H,
J = 1.32 Hz, AreH), 6.51 (s, 1H, pyrazole-H), 3.97 (s, 1H, NH-SO2-),
2.34 (s, 3H, CH3), 2.32 (s, 3H, CH3); 13C NMR (100 MHz, DMSO) δ
ppm:153.5, 143.6, 142.9, 137.3, 134.7, 131.8, 131.2, 130.1, 129.8,
129.5, 127.9, 125.8, 123.1, 119.5, 117.3, 107.5, 21.3; Mass: 472.59 (M
+H)+; Elemental analysis for C24H20F3N3O2S: Calculated: C, 61.14; H,
4.28; N, 8.91. Found: C, 61.18; H, 4.28; N, 8.94.
benzenesulfonamide (7a)
Yield: 79%; M.p: 193–194 °C; MW: 457.47 ; Rf: 0.72; FTIR (νmax
;
cm−1 KBr): 3249 (NeH stretching), 3083 (CeH broad), 2959 (alkyl CeH
stretching), 1681 (C]N aromatic), 1646 (C]C), 1328 (aromatic CF3
stretching), 1154 (SO2 stretching), 1079 (CeN stretching), 954 (SeN
stretching), 814 (CeS stretching), 735; 1H NMR (400 MHz, DMSO-d6,
TMS) δ ppm: 7.86 (d, 2H, J = 1.91 Hz, AreH), 7.80 (d, 2H,
J = 1.86 Hz, AreH), 7.59 (d, 2H, J = 1.43 Hz, AreH), 7.18 (d, 2H,
J = 1.63 Hz, AreH), 7.15 (d, 2H, J = 1.46 Hz, AreH), 6.82 (t, 1H,
J = 1.26 Hz, AreH), 6.80 (d, 2H, J = 1.35 Hz, AreH), 6.51 (s, 1H,
pyrazole-H), 3.98 (s, 1H, NHeSO2e), 2.35 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO) δ ppm:153.6, 143.5, 142.9, 137.2, 131.7, 130.1,
129.5, 129.2, 128.9, 127.9, 125.7, 123.1, 122.4, 119.4, 117.3, 107.5,
6

Y. An, et al.
BioorganicChemistry101(2020)104044
Fig. 7. . Effect of compound 7m on COX-2
and iNOS expression in spinal cord of SCI
rats via western blot analysis; and re-
presentative quantitative analysis of (b)
iNOS and (c) COX-2. Values represent the
mean
SD and are representative of three
independent experiments. ##P
< 0.05 vs
sham; *P < 0.05, **P < 0.01 vs. SCI, one-
way analysis of variance followed by
a
Tukey’s post hoc test.
3.2.3. N-(o-tolyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide (7c)
Calculated: C, 59.13; H, 4.14; N, 8.62. Found: C, 59.12; H, 4.18; N,
8.65.
Yield: 78%; M.p: 212–213 °C; MW: 471.50 ; Rf: 0.82; FTIR (νmax
;
cm−1 KBr): 3256 (NeH stretching), 3085 (CeH broad), 2954 (alkyl CeH
stretching), 1689 (C]N aromatic), 1648 (C]C), 1326 (aromatic CF3
stretching), 1153 (SO2), 1078 (CeN stretching), 956 (SeN stretching),
815 (CeS stretching), 728; 1H NMR (400 MHz, DMSO‑d6, TMS) δ ppm:
7.86 (d, 2H, J = 1.92 Hz, AreH), 7.79 (d, 2H, J = 1.82 Hz, AreH),
7.59 (d, 2H, J = 1.41 Hz, AreH), 7.15 (d, 2H, J = 1.47 Hz, AreH),
7.08–6.71 (m, 4H, CHx4, AreH), 6.52 (s, 1H, pyrazole-H), 3.97 (s, 1H,
NHeSO2e), 2.35 (s, 3H, CH3), 2.13 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO) δ ppm:153.5, 143.4, 142.9, 137.2, 136.1, 133.8, 131.7, 131.4,
130.2, 129.5, 127.9, 126.5, 125.7, 124.7, 123.9, 123.1, 117.4, 107.5,
3.2.5. N-(2-methoxyphenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl)benzenesulfonamide (7e)
Yield: 71%; M.p: 215–216 °C; MW: 487.50 ; Rf: 0.81; FTIR (νmax
;
cm−1 KBr): 3254 (NeH stretching), 3085 (CeH broad), 2952 (alkyl CeH
stretching), 2821 (OCH3 stretching), 1687 (C]N aromatic), 1645 (C]C),
1323 (aromatic CF3 stretching), 1156 (SO2 stretching), 1078 (CeN
stretching), 955 (SeN stretching), 816 (CeS stretching), 704; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.86 (d, 2H, J = 1.92 Hz, AreH),
7.82 (d, 2H, J = 1.83 Hz, AreH), 7.58 (d, 2H, J = 1.45 Hz, AreH),
7.14 (d, 2H, J = 1.3 Hz, AreH), 7.36–6.82 (m, 4H, CHx4, AreH), 6.52
(s, 1H, pyrazole-H), 3.99 (s, 1H, NHeSO2e), 3.83 (s, 3H, OCH3), 2.34
(s, 3H, CH3); 13C NMR (100 MHz, DMSO) δ ppm:153.6, 147.4, 143.5,
142.9, 137.2, 131.7, 130.1, 129.5,128.4, 127.9, 127.1, 125.7, 123.2,
21.3, 17.3; Mass: 472.54 (M+H)+
;
Elemental analysis for
C
24H20F3N3O2S: Calculated: C, 61.14; H, 4.28; N, 8.91. Found: C, 61.15;
H, 4.26; N, 8.89.
122.6, 122.3, 117.3, 113.4, 107.5, 55.8, 21.3 ; Mass: 488.56 (M+H)+
;
3.2.4. N-(4-methoxyphenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl)benzenesulfonamide (7d)
Elemental analysis for C24H20F3N3O3S: Calculated: C, 59.13; H, 4.14; N,
8.62. Found: C, 59.14; H, 4.15; N, 8.64.
Yield: 73%; M.p: 217–218 °C; MW: 487.50 ; Rf: 0.85; FTIR (νmax
;
cm−1 KBr): 3258 (NeH stretching), 3083 (CeH broad), 2955 (alkyl CeH
stretching), 2821 (OCH3 stretching), 1686 (C]N aromatic), 1642 (C]C),
1325 (aromatic CF3 stretching), 1152 (SO2 stretching), 1073 (CeN
stretching), 956 (SeN stretching), 813 (CeS stretching), 709; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.84 (d, 2H, J = 1.94 Hz, AreH),
7.81 (d, 2H, J = 1.84 Hz, AreH), 7.57 (d, 2H, J = 1.43 Hz, AreH),
7.16 (d, 2H, J = 1.4 Hz, AreH), 6.98 (d, 2H, J = 1.52 Hz, AreH), 6.80
(d, 2H, J = 2.25 Hz, AreH), 6.51 (s, 1H, pyrazole-H), 3.98 (s, 1H,
NHeSO2e), 3.82 (s, 3H, OCH3), 2.35 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO) δ ppm:153.5, 153.2, 143.5, 142.9, 137.2, 131.8, 130.1, 130.0,
129.5, 127.9, 125.7, 124.5, 123.1, 117.4, 115.1, 107.5, 55.8, 21.3;
3.2.6. N-(4-nitrophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl)benzenesulfonamide (7f)
Yield: 84%; M.p: 228–229 °C; MW: 502.47 ; Rf: 0.86; FTIR (νmax
;
cm−1 KBr): 3259 (NeH stretching), 3084 (CeH broad), 2954 (alkyl CeH
stretching), 1685 (C]N aromatic), 1643 (C]C), 1534 (NO2 stretching),
1327 (aromatic CF3 stretching), 1158 (SO2 stretching), 1077 (CeN
stretching), 957 (SeN stretching), 816 (CeS stretching), 702; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.85 (d, 2H, J = 1.93 Hz, AreH),
7.81 (d, 2H, J = 1.84 Hz, AreH), 7.57 (d, 2H, J = 1.44 Hz, AreH),
7.16 (d, 2H, J = 1.5 Hz, AreH), 7.92 (d, 2H, J = 1.85 Hz, AreH), 7.06
(d, 2H, J = 2.24 Hz, AreH), 6.51 (s, 1H, pyrazole-H), 3.97 (s, 1H,
Mass: 488.52 (M+H)+
;
Elemental analysis for C24H20F3N3O3S:
NHeSO2e), 2.35 (s, 3H, CH3); 13C NMR (100 MHz, DMSO)
δ
7

Y. An, et al.
BioorganicChemistry101(2020)104044
ppm:153.6, 143.8, 143.5, 142.9, 137.8, 137.2, 131.7, 130.1, 129.5,
127.8, 125.7, 124.7, 123.1, 119.1, 117.3, 107.5, 21.3; Mass: 502.49 (M
+H)+; Elemental analysis for C23H17F3N4O4S: Calculated: C, 54.98; H,
3.41; N, 11.15. Found: C, 54.93; H, 3.45; N, 11.13.
(d, 2H, J = 1.87 Hz, AreH), 6.51 (s, 1H, pyrazole-H), 3.96 (s, 1H,
NHeSO2e), 2.35 (s, 3H, CH3); 13C NMR (100 MHz, DMSO)
δ
ppm:157.3, 153.7, 143.4, 142.9, 137.2, 133.4, 131.7, 130.1, 129.5,
127.9, 125.7, 123.1, 117.9, 117.3, 116.3, 107.5, 21.3; Mass: 476.48 (M
+H)+; Elemental analysis for C23H17F4N3O2S: Calculated: C, 58.10; H,
3.60; N, 8.84. Found: C, 58.12; H, 3.63; N, 8.85.
3.2.7. N-(2-nitrophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl)benzenesulfonamide (7g)
Yield: 78%; M.p: 223–224 °C; MW: 502.47 ; Rf: 0.82; FTIR (νmax
;
3.2.11. N-(2-fluorophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-
cm−1 KBr): 3257 (NeH stretching), 3089 (CeH broad), 2956 (alkyl CeH
stretching), 1687 (C]N aromatic), 1645 (C]C), 1537 (NO2 stretching),
1329 (aromatic CF3 stretching), 1156 (SO2 stretching), 1076 (CeN
stretching), 953 (SeN stretching), 817 (CeS stretching), 712; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.86 (d, 2H, J = 1.92 Hz, AreH),
7.83 (d, 2H, J = 1.85 Hz, AreH), 7.59 (d, 2H, J = 1.46 Hz, AreH),
7.14 (d, 2H, J = 1.4 Hz, AreH), 8.09–7.06 (m, 4H, CHx4,AreH), 6.52
(s, 1H, pyrazole-H), 3.98 (s, 1H, NHeSO2e), 2.34 (s, 3H, CH3); 13C
NMR (100 MHz, DMSO) δ ppm:153.7, 143.5, 142.9, 137.2, 137.1,
135.6, 134.1, 131.7, 130.1, 129.5, 127.8, 125.9, 125.7, 123.1, 119.8,
119.6, 117.3, 107.5, 21.3; Mass: 502.52 (M+H)+; Elemental analysis
for C23H17F3N4O4S: Calculated: C, 54.98; H, 3.41; N, 11.15. Found: C,
54.97; H, 3.40; N, 11.17.
1-yl)benzenesulfonamide (7k)
Yield: 70%; M.p: 220–221 °C; MW: 475.46; Rf: 0.83; FTIR (νmax
;
cm−1 KBr): 3258 (NeH stretching), 3089 (CeH broad), 2956 (alkyl CeH
stretching), 1687 (C]N aromatic), 1645 (C]C), 1327 (aromatic CF3
stretching), 1158 (SO2 stretching), 1154 (C-F stretching), 1075 (CeN
stretching), 952 (SeN stretching), 811 (CeS stretching),696; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.87 (d, 2H, J = 1.98 Hz, AreH),
7.78 (d, 2H, J = 1.89 Hz, AreH), 7.62 (d, 2H, J = 1.46 Hz, AreH),
7.14 (d, 2H, J = 1.4 Hz, AreH), 6.94–6.63 (m, 4H, CHx4,AreH), 6.54
(s, 1H, pyrazole-H), 3.98 (s, 1H, NHeSO2e), 2.34 (s, 3H, CH3); 13C
NMR (100 MHz, DMSO) δ ppm:157.5, 153.6, 143.5, 142.9, 137.3,
133.7, 131.7, 130.1, 129.5, 127.9, 125.7, 125.1, 123.4, 123.1, 117.9,
117.4, 116.3, 107.5, 21.3; Mass: 476.52 (M+H)+; Elemental analysis
for C23H17F4N3O2S: Calculated: C, 58.10; H, 3.60; N, 8.84. Found: C,
58.14; H, 3.59; N, 8.83.
3.2.8. N-(4-chlorophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl)benzenesulfonamide (7h)
Yield: 85%; M.p: 231–232 °C; MW: 491.91 ; Rf: 0.80; FTIR (νmax
;
3.2.12. 4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-(2-
cm−1 KBr): 3254 (NeH stretching), 3083 (CeH broad), 2957 (alkyl CeH
stretching), 1689 (C]N aromatic), 1642 (C]C), 1327 (aromatic CF3
stretching), 1159 (SO2 stretching), 1078 (CeN stretching), 957 (SeN
stretching), 812 (CeS stretching), 792 (CeCl stretching), 701; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.87 (d, 2H, J = 1.92 Hz, AreH),
7.79 (d, 2H, J = 1.87 Hz, AreH), 7.56 (d, 2H, J = 1.42 Hz, AreH),
7.14 (d, 2H, J = 1.4 Hz, AreH), 7.32 (d, 2H, J = 1.51 Hz, AreH), 7.12
(d, 2H, J = 1.84 Hz, AreH), 6.53 (s, 1H, pyrazole-H), 3.99 (s, 1H,
(trifluoromethyl)phenyl)benzenesulfonamide (7l)
Yield: 77%; M.p: 245–246 °C; MW: 525.47; Rf: 0.88; FTIR (νmax
;
cm−1 KBr): 3256 (NeH stretching), 3086 (CeH broad), 2959 (alkyl CeH
stretching), 1689 (C]N aromatic), 1643 (C]C), 1329 (aromatic CF3
stretching), 1157 (SO2 stretching), 1152 (C-F stretching), 1078 (CeN
stretching), 956 (SeN stretching), 814 (CeS stretching), 698; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.88 (d, 2H, J = 1.94 Hz, AreH),
7.83 (d, 2H, J = 1.84 Hz, AreH), 7.57 (d, 2H, J = 1.42 Hz, AreH),
7.16 (d, 2H, J = 1.5 Hz, AreH), 7.46–6.73 (m, 4H, CHx4,AreH), 6.52
(s, 1H, pyrazole-H), 3.97 (s, 1H, NHeSO2e), 2.35 (s, 3H, CH3); 13C
NMR (100 MHz, DMSO) δ ppm:153.6, 143.6, 142.8, 137.1, 132.8,
132.2, 131.8, 130.1, 129.5, 127.3, 127.8, 125.7, 125.2, 125.1, 123.1,
119.1, 117.3, 115.1 107.5, 21.3; Mass: 526.49 (M+H)+; Elemental
analysis for C24H17F6N3O2S: Calculated: C, 54.86; H, 3.26; N, 8.00.
Found: C, 54.89; H, 3.24; N, 8.02.
NHeSO2e), 2.33 (s, 3H, CH3); 13C NMR (100 MHz, DMSO)
δ
ppm:153.6, 143.5, 142.8, 137.3, 135.8, 131.7, 130.1, 129.7, 129.5,
127.8, 127.6, 125.8, 123.1, 122.1, 117.4, 107.4, 21.4; Mass: 492.94 (M
+H)+; Elemental analysis for C23H17ClF3N3O2S: Calculated: C, 56.16;
H, 3.48; N, 11.59. Found: C, 56.18; H, 3.52; N, 11.57.
3.2.9. N-(2-chlorophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl)benzenesulfonamide (7i)
Yield: 81%; M.p: 228–229 °C; MW: 491.91; Rf: 0.79; FTIR (νmax
;
3.2.13. 4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-(4-
cm−1 KBr): 3257 (NeH stretching), 3085 (CeH broad), 2959 (alkyl CeH
stretching), 1684 (C]N aromatic), 1645 (C]C), 1329 (aromatic CF3
stretching), 1157 (SO2 stretching), 1079 (CeN stretching), 953 (SeN
stretching), 815 (CeS stretching), 789 (CeCl stretching), 693; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.85 (d, 2H, J = 1.94 Hz, AreH),
7.82 (d, 2H, J = 1.82 Hz, AreH), 7.60 (d, 2H, J = 1.41 Hz, AreH),
7.16 (d, 2H, J = 1.5 Hz, AreH), 7.38–6.74 (m, 4H, CHx4,AreH), 6.53
(s, 1H, pyrazole-H), 3.97 (s, 1H, NH-SO2-), 2.35 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO) δ ppm:153.7, 143.5, 142.9, 137.2, 131.8, 130.7,
130.1, 129.5, 127.9, 127.7, 127.6, 125.8, 125.4, 125.2, 123.1, 122.6,
117.3, 107.5, 21.3; Mass: 492.90 (M+H)+; Elemental analysis for
(trifluoromethyl)phenyl)benzenesulfonamide (7m)
Yield: 79%; M.p: 241–242 °C; MW: 525.47; Rf: 0.85; FTIR (νmax
;
cm−1 KBr): 3259 (NeH stretching), 3087 (CeH broad), 2958 (alkyl CeH
stretching), 1687 (C]N aromatic), 1645 (C]C), 1327 (aromatic CF3
stretching), 1159 (SO2 stretching), 1151 (CeF stretching), 1075 (CeN
stretching), 956 (SeN stretching), 817 (CeS stretching), 696; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.87 (d, 2H, J = 1.94 Hz, Ar-H),
7.81 (d, 2H, J = 1.79 Hz, AreH), 7.60 (d, 2H, J = 1.45 Hz, AreH),
7.15 (d, 2H, J = 1.4 Hz, AreH), 7.35 (d, 2H, J = 1.82 Hz, AreH), 7.31
(d, 2H, J = 1.28 Hz, AreH), 6.52 (s, 1H, pyrazole-H), 3.98 (s, 1H,
NHeSO2e), 2.34 (s, 3H, CH3); 13C NMR (100 MHz, DMSO)
δ
C
23H17ClF3N3O2S: Calculated: C, 56.16; H, 3.48; N, 11.59. Found: C,
ppm:153.7, 143.5, 142.8, 141.2, 137.2, 131.8, 130.1, 129.6, 127.8,
126.5, 125.9, 125.7, 124.1, 123.1, 119.8, 117.3, 107.5, 21.3; Mass:
526.53 (M+H)+; Elemental analysis for C24H17F6N3O2S: Calculated: C,
54.86; H, 3.26; N, 8.00. Found: C, 54.85; H, 3.28; N, 8.01.
56.14; H, 3.51; N, 11.61.
3.2.10. N-(4-fluorophenyl)-4-(5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl)benzenesulfonamide (7j)
Yield: 75%; M.p: 223–224 °C; MW: 475.46; Rf: 0.87; FTIR (νmax
;
3.3. In-vitro COX inhibitory activity
cm−1 KBr): 3259 (NeH stretching), 3087 (CeH broad), 2954 (alkyl CeH
stretching), 1686 (C]N aromatic), 1643 (C]C), 1328 (aromatic CF3
stretching), 1159 (SO2 stretching), 1152 (C-F stretching), 1074 (CeN
stretching), 952 (SeN stretching), 817 (CeS stretching), 698; 1H NMR
(400 MHz, DMSO‑d6, TMS) δ ppm: 7.89 (d, 2H, J = 1.96 Hz, AreH),
7.82 (d, 2H, J = 1.81 Hz, AreH), 7.58 (d, 2H, J = 1.49 Hz, AreH),
7.17 (d, 2H, J = 1.5 Hz, AreH), 6.98 (d, 2H, J = 1.43 Hz, AreH), 6.78
The inhibitory activity against COX-1/2 of entire target compounds
was measured using colorimetric COX (ovine) Inhibitor Screening Assay
Kit (catalog number 560131, Cayman Chemical, MI, USA) as per the
manufacturer’s instruction. This method allows to estimate the perox-
idase activity of COX by colorimetric monitoring of presence of the
oxidized form of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at
8

Y. An, et al.
BioorganicChemistry101(2020)104044
590 nm. The test is based on the oxidation of TMPD during the re-
duction of PGG2 (prostaglandin G2) to PGH2, which is reflected by a
change in color, measured spectrophotometrically (Victor2 micro-
spectrophotometer, PerkinElmer Waltham, MA, USA). The assay uses
Tris-HCl buffer
ELISA kits as per manufacturer’s instructions.
3.4.7. Terminal dexynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) staining
The spinal cord sections were stained using a TUNEL apoptosis
detection kit (Wuhan Boster Biological Engineering Co., Ltd., Wuhan,
China) according to the manufacturer's instruction.
(0.1 M assay buffer, pH 8.0), a solution of heme in dimethylsulf-
oxide (DMSO), enzymes (COX-1, COX-2), arachidonic acid (100 μM),
KOH (0.1 M) and a solution of TMPD. The assay mixture contains:
150 μL of assay buffer, 10 μL of heme, and 10 μL of COX-1 or COX-2. To
the wells for determination of 100% enzyme activity (each COX sample
was assayed in triplicate) were added 10 μL of the substances used as
solvents. To the other wells were added 10 μL of tested inhibitors at
10 µM. To all wells were added 20 μL of TMPD. The reaction was in-
itiated by addition of arachidonic acid. The effect of tested inhibitors on
COX-1 and COX-2 enzyme activity was measured by assaying the rate of
TMPD oxidation within 2 min in a spectrophotometer at 590 nm. We
determined the activity factor at 2 min of incubation with the tested
compounds in comparison to the initial activity of the enzyme. This
enabled the calculation of IC50 values (concentrations at which 50%
inhibition of enzyme activity occurred).
3.4.8. Western blot assay
10% SDS-PAGE was carried out to separate the proteins and trans-
ferred on PVDF membranes (Millipore, Bedford, MA, USA). The mem-
brane were incubated primary antibodies (1:1000; CST or Abcam, USA)
after blocking for overnight. The corresponding secondary antibodies
(1:2000; Abcam, USA) were further incubated for 2 h. The proteins
bands were observed using enhanced chemiluminescence substrate
(Millipore, Billerica, MA, USA) and photographed with Image Quant
LAS 4000 (GE, USA) and was analyzed by Image J software (NIH,
Bethesda, MD, USA).
3.5. Statistical analysis
3.4. In vivo pharmacology
All data are recorded as mean
SD of three independent experi-
ments. Data were statistically analyzed by one-way analysis followed by
the by a Tukey’s post hoc test using statistical software GraphPad Prism
5.0 (California, USA). The P value < 0.05 was considered as statisti-
cally significant.
3.4.1. Animals
The Sprague-Dawley rats (male adult, 230–260 gm) rats after pro-
curing from animal house and were kept in polypropylene cages with ad
libitum supply of food and water at controlled humidity and tempera-
ture with alternate, dark and light cycle.
4. Conclusion
3.4.2. Establishment of spinal cord injury (SCI)
Our study has shown the development of novel celecoxib derivatives
via facile synthetic route as COX-2 inhibitor. Among the synthesized
derivatives, compound 7m, a highly potent COX-2 inhibitor showed
protective action against SCI via attenuation of COX-2, oxidative stress,
apoptosis and inflammation in Male Sprague-Dawley rats.
The SCI injury was induced in the rats as per Allens method with
modifications [32]. Initially, a 2 cm midline incision was made on skin
targeting the eighth thoracic segment (T8) along the vertebrae. The
laminectomy was performed by removing vertebral lamina of T8. The
SCI injury was induced dropping 10 g rod from the distance of 5 cm on
the spinal cord of rats and rats were put on rest for 3 min. The sham
treated rats observe only laminectomy. The surgical site was sutured
together, and intramuscular injection of penicillin (200,000 U) was
administered for three consecutive days after surgery. Compound 7m
after suspending in 5% CMC was administered once a day for 14 days to
rats in graded dose of 2.5 mg/kg; 5 mg/kg; and 10 mg/kg via i.p. im-
mediately after surgery.
Ethics approval and consent to participate
The animal use protocol listed below has been reviewed and ap-
proved by the Animal Ethical and Welfare Committee of Beijing
Jishuitan Hospital, Beijing, China.
Declaration of Competing Interest
3.4.3. Evaluation of motor function
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
The effect of compound 7m on rats motor function was determined
using Basso, Beattie and Bresnahan (BBB) locomotor rating scale be-
tween 0 and 21 at the day 1, 4, 7, 10 and 14 day post-SCI. The effect
was evaluated by person who was blinded to the treatment group, and
the mean of the three measurements was recorded [44].
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104044.
3.4.4. Histological examinations
The segments of spinal cord in the vicinity of the lesion epicenter
were collected and 5 μm-thick sections was obtained after embedded in
paraffin for transverse sectioning. Sections were stained with hema-
toxylin and eosin together with cresyl violet for HE staining and Nissl
staining, respectively.
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