Synthesis and Biological Evaluation of 1,3,5-Trisubstituted 2-Pyrazolines as Novel Cyclooxygenase-2 Inhibitors with Antiproliferative Activity

Article abstract of DOI:10.1002/cbdv.202000832

A new series of 1,3,5-trisubstituted 2-pyrazolines for the inhibition of cyclooxygenase-2 (COX-2) were synthesized. The designed structures include a COX-2 pharmacophore SO₂CH₃ at the para-position of the phenyl ring located at C-5 of a pyrazoline scaffold. The synthesized compounds were tested for in vitro COX-1/COX-2 inhibition and cell toxicity against human colorectal adenocarcinoma cell lines HT-29. The lead compound (4-chlorophenyl){5-[4-(methanesulfonyl)phenyl]-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl}methanone (16) showed significant COX-2 inhibition (IC₅₀=0.05±0.01 μM), and antiproliferative activity (IC₅₀=5.46±4.71 μM). Molecular docking studies showed that new pyrazoline-based compounds interact via multiple hydrophobic and hydrogen-bond interactions with key binding site residues of the COX-2 enzyme.

Full text of DOI:10.1002/cbdv.202000832

DOI: 10.1002/cbdv.202000832
FULL PAPER
Synthesis and Biological Evaluation of 1,3,5-Trisubstituted 2-
Pyrazolines as Novel Cyclooxygenase-2 Inhibitors with
Antiproliferative Activity
Teymour Vahedpour,a, b Jatinder Kaur,c, d Salar Hemmati,e Maryam Hamzeh-Mivehroud,a, b
Ali Akbar Alizadeh,a, f Frank Wuest,c, d, g and Siavoush Dastmalchi*a, b, h
a
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz 5165665811, Iran
b
Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz
5165665811, Iran, e-mail: dastmalchi.s@tbzmed.ac.ir
Department of Oncology, University of Alberta, Edmonton, AB, T6G 1Z2, Canada
c
d
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, T6G 2H7, Canada
e
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 5165665811, Iran
f
Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz 5165665811, Iran
g
Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada
h
Faculty of Pharmacy, Near East University, Po.Box: 99138, Nicosia, North Cyprus, Mersin 10, Turkey
A new series of 1,3,5-trisubstituted 2-pyrazolines for the inhibition of cyclooxygenase-2 (COX-2) were
synthesized. The designed structures include a COX-2 pharmacophore SO2CH3 at the para-position of the phenyl
ring located at C-5 of a pyrazoline scaffold. The synthesized compounds were tested for in vitro COX-1/COX-2
inhibition and cell toxicity against human colorectal adenocarcinoma cell lines HT-29. The lead compound (4-
chlorophenyl){5-[4-(methanesulfonyl)phenyl]-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl}methanone (16) showed sig-
nificant COX-2 inhibition (IC50 =0.05�0.01 μM), and antiproliferative activity (IC50 =5.46�4.71 μM). Molecular
docking studies showed that new pyrazoline-based compounds interact via multiple hydrophobic and
hydrogen-bond interactions with key binding site residues of the COX-2 enzyme.
Keywords: COX-2 inhibitors, 2-pyrazolines, anti-inflammatory agents, anticancer drugs.
1. Introduction
COX-1 isoform is constitutively expressed and linked
The biochemical origin of inflammation is linked with with the production of PGs to maintain homeostasis.[8]
the metabolic conversion of arachidonic acid (AA) to a COX-2 is the inducible isoform and is known to be
set of prostanoids, including prostaglandins (PGs) and expressed mainly at inflammation sites and during the
thromboxane.[1–4] Prostanoids metabolism is involved progression of several types of cancers.[9–13] COX-3 is
in several physiological and pathological processes described as a different COX-1 splice variant.[14] Both
such as inflammation, ovulation, renal protection, COX-1 and COX-2 have a degree of close structural
thrombosis, fever, and angiogenesis.[5]
and sequence similarity. However, these isoforms differ
Cyclooxygenases (COXs) are homodimeric, rate- by the existence of an additional sub-pocket (known
limiting enzymes for the metabolism of AA to yield as secondary pocket) in the COX-2 binding site,
various prostanoids. COXs are known to exist in three making its active site approximately 25% larger than
isoforms, COX-1, COX-2, and COX-3.[6–8] Among these, that of COX-1.[4] The ulcerogenic and gastrointestinal
side effects of classical non-steroidal anti-inflammatory
drugs (aspirin, ibuprofen, and naproxen) are believed
to be linked with non-selective inhibition of both COX-
1 and COX-2 isoforms. Therefore, selective COX-2
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202000832
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Chem. Biodiversity 2021, 18, e2000832
inhibitors (celecoxib, rofecoxib, valdecoxib) were de- also been achieved by introducing substitution other
veloped to overcome the side effects associated with than the diaryl groups. For example, Rathish et al.
the use of classical non-steroidal anti-inflammatory developed a series of compounds having extra
drugs. Unfortunately, some of the selective COX-2 substituted phenyl moiety on the central ring (Com-
inhibitors, like rofecoxib, were removed from the pound 3) with potent anti-COX-2 activity and in vivo
market due to an increased risk of cardiovascular anti-inflammatory effects.[19] Wuest and co-workers
complications. In recent years, several new classes of prepared a series of diaryl-substituted triazoles, where
COX-2 inhibitors have been developed, including the the linker between the arylsulfonamide group and
modification of known COX inhibitors to nitric oxide- central triazole ring was modified (Compound 4).[20]
releasing prodrugs for eliminating cardiovascular The similarity of these structures to that of the
complications.[15,16] The different roles of COX enzymes renowned selective COX-2 inhibitors is evident, as
in the development and progression of cancer also illustrated in Figure 1.
prompted the use of COX inhibitors as anticancer
Several classes of biologically active 2-pyrazolines
(or 4,5-dihydropyrazoles) have been developed with
drugs.[17]
Selective COX-2 inhibitors are frequently comprised anti-inflammatory,[21]
anticancer,[22,23]
antidepres-
of a vicinal diaryl system with a central heterocyclic sant,[24] anticonvulsant,[25] antibacterial,[26,27] and anti-
ring, and the presence of either SO2CH3 or SO2NH2 malarial activities.[27] There are also some studies
groups, which play a crucial role for selective binding showing that compounds with 2-pyrazoline scaffold
to the secondary pocket of the COX-2 isoform.[15,18] have selective COX-2 inhibitory effects.[19,28–31]
Differing from this general chemical arrangement,
The suggested compounds in this study were
several other classes of selective COX-2 inhibitors have designed by introducing variations in the template
also been developed (Figure 1). As shown in the Figure, structures in order to improve the inhibitory effects
the central five-membered ring can be exemplified by through increased interaction with the COX-2 enzyme.
different structures such as pyrazole, 2-furanone, 2- The variations included modification of the linker
pyrazoline, 1,2,3-triazole, and 2-pyranone present in between the aryl and central 2-pyrazoline ring, the
celecoxib (1), rofecoxib (2), compounds 3, 4, and 5, introduction of the third aryl group on the central
respectively. The variations on the central ring have
Figure 1. Structure of selective COX-2 inhibitors and novel pyrazoline-based compounds. The title compounds were designed
based on celecoxib, rofecoxib, and structures 3,[19] 4,[20] and 5[32] with known selective COX-2 inhibitory effect. The pharmacophores
are color coded for easy tracking the origin of the different structural elements in the designed scaffold.
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Chem. Biodiversity 2021, 18, e2000832
ring, and rearrangement of the substitutions on the 2. Results and Discussion
central five-membered ring (Figure 1).
2.1. Chemistry
We prepared a series of tri-aryl/alicyclic substituted
2-pyrazoline-based compounds as novel tandem anti- The synthetic route for the preparation of pyrazoline-
inflammatory and anticancer drugs (compounds 12– based compounds is depicted in Scheme 1. Briefly,
24). All compounds were tested for the COX-1/2 starting from compound 6, compounds 7 and 8 were
inhibitory potency and their antiproliferative activity in synthesized sequentially by using protection and
human colorectal adenocarcinoma cells.
OXONE-mediated oxidation, respectively. Then the key
building block 4-(methylsulfonyl)benzaldehyde (9) was
prepared by deprotection of 8 under acidic reaction
conditions. Next, Claisen-Schmidt reaction between 4-
(methylsulfonyl)benzaldehyde (9) and acetophenone
gave compound 10, which was refluxed with
hydrazine hydrate to afford pyrazoline compound 11.
Scheme 1. Synthesis route for the preparation of pyrazoline-based compounds 12–24.
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Pyrazoline compound 11 was reacted with a series of isoform, compound 18 with two SO2CH3 groups
substituted benzoyl chlorides and isocyanates to showed only micromolar inhibitory potency (8.6 μM).
prepare tri-aryl/alicyclic substituted 2-pyrazolines 12– Evaluation of the second series of compounds 19–24
24. The spectroscopic data of compounds 12–24 can carrying an amide linker revealed that the incorpora-
be found in Supporting Information.
tion of a para-substituent on one of the phenyl rings
resulted in an overall improvement in COX-2 inhibition
activity and selectivity profile. Within this series, the 5-
[4-(methylsulfonyl)phenyl]-3-phenyl-N-(p-tolyl)-4,5-di-
2.2. In Vitro Cyclooxygenase (COX) Inhibition Studies
COX-1 and COX-2 inhibitory potencies of compounds hydro-1H-pyrazole-1-carboxamide (20) showed appre-
11–24 were determined in triplicate using a COX ciable submicromolar COX-2 inhibition and a good
inhibitor screening assay kit. The results are summar- selectivity profile over COX-1 (COX-2 IC50 =0.80 μM,
ized in Table 1. Among all tested pyrazolines 11–24, SI=116). Interestingly, replacement of the phenyl ring
(4-chlorophenyl){5-[4-(methanesulfonyl)phenyl]-
with a cyclohexyl group in compound 24 led to a
3-phenyl-4,5-dihydro-1H-pyrazol-1-yl}methanone (16) comparable submicromolar COX-2 inhibitory (IC50 =
showed very good COX-2 inhibitory potency (IC50 = 0.78 μM) and selectivity profile (SI >128).
0.05 μM), which is in the same range of the reference
A closer evaluation revealed that the physico-
compound celecoxib (IC50 =0.01 μM). However, com- chemical features of para-substitutions such as elec-
pound 16 also displayed considerable inhibition tron-withdrawing (Hammett σ constant),[33] lipophilic-
against COX-1 isoform (IC50 =0.16 μM). Among first ity (Hansch π constant),[34] and steric (Taft Es constant
series of compounds 12–18 with carbonyl linker, and vdW volume) properties influence the COX-2
compound 14 (X=OCH3) also showed appreciable inhibition potency of the first series of compounds 12
inhibition of both COX-1 and COX-2 isoforms (COX-2 to 18.[35] The results showed that COX-2 activity
IC50 =0.21 μM, COX-1 IC50 =0.70 μM). Compounds 12 increases as the lipophilicity of the para-substitution
(X=H) and 13 (X=CH3) displayed moderate COX-2 increases (R2 =0.82, Table S1 and Figure S71, Support-
inhibition of 0.81 and 0.78 μM, and a COX-2 selectivity ing Information). However, the activity decreases as
index (SI) of 12 and 23, respectively. Although SO2CH3 the electron withdrawing effect and size of the
group is known to support binding to the COX-2 substituent increase (Table S1 and Figure S71, Support-
Table 1. In vitro COX-1 and COX-2 enzyme inhibition and cytotoxicity data of compounds 11–24.
Compd.
IC50 (μM)[a]
COX-2 SI[b]
Cytotoxicity[c] (IC50, μM)
COX-1
2.44
COX-2
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Celecoxib
Cisplatin
0.36
0.81
0.78
0.21
3.44
0.05
3.02
8.58
9.40
0.80
1.95
0.69
0.58
0.78
0.01
–
6.78
12.12
22.77
3.33
0.35
219.00�26.77
22.64�12.87
9.53�3.28
9.82
17.76
0.70
1.21
0.16
61.71�26.46
344.40�127.85
5.46�4.71
3.20
>100
>100
8.90
93.39
>100
3.17
3.06
>100
>33.11
>11.66
0.95
116.74
>51.28
4.59
314.70�44.87
72.94�24.23
23.79�12.42
57.41�12.01
117.90�23.01
121.20�33.57
>500
5.28
>128.21
>10000.00
–
44.18�9.05
40.59�1.74
19.85�2.79
>100
–
[a]
Assays were conducted as described in the Methods section. IC50, half-maximal inhibitory concentration. The in vitro test
compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, μM) is
the mean of three determinations acquired using the enzyme assay kit (Cayman Chemical, Ann Arbor, USA; item number: 700100)
and the deviation from the mean is <10% of the mean value. [b] COX-2 SI, in vitro COX-2 selectivity index: [(COX-1 IC50)/(COX-2 IC50)].
[c] In vitro cytotoxicity MTT assay. IC50 values are presented as mean�SD of three independent experiments and the values greater
than 100 μM are extrapolations obtained from curve fitting method.
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Chem. Biodiversity 2021, 18, e2000832
ing Information). It seems that para-Cl group has
appropriate lipophilicity, electronic effect and size
required for the observed high activity. For instance,
the higher COX-2 inhibitory potency of para-Cl com-
pound 16 than that of para-F 15 and para-NO2 17
could be attributed to its higher lipophilicity. More-
over, its steric and electron-withdrawing effects are
smaller than those of para-NO2, which may also
indicate its higher potency. Although compared to
para-F group, para-Cl has unfavorable steric and
electron withdrawing effects, its ultimate influence on
COX-2 activity is positive, which may have brought
about due to the summed impact of all properties.
The medium activity of para-CH3 of 18 and low activity
of para-SO2CH3 of 18 derivatives could also be
rationalized based on the above drawn structure-
activity relationship (SAR). However, relatively good
anit-COX-2 activity of para-OCH3 14 cannot be
explained by its low hydrophobicity and relatively
bigger size. It seems that the more electron donating
the para-substitution, the higher the activity (Table S1
and Figure S71, Supporting Information). Considering
that para-OCH3 is an electron donating group, and its
size is not that big, the observed sub-micro molar IC50
for COX-2 inhibition may be justified.
Figure 2. Correlation between COX-2 inhibitory potency and
antiproliferative activity against HT-29 cells for synthesized
compounds 11–24. Compounds 18 and 19 are outliers, and
compound 23 was not included in the correlation due to lack
of cytotoxicity at the used concentrations.
human COX-2 structure (PDB code: 5KIR). The analysis
of the obtained results demonstrated that (S)-enan-
2.3. In Vitro Cell Toxicity Effect
To investigate the cytotoxic effect of compounds 11– tiomers were docked with much better consistency
24, the MTT assay was conducted using human into the active site of COX-2 enzyme when compared
colorectal adenocarcinoma cells HT-29. The cytotox- to (R)-enantiomers. For example, the results of docking
icity effect of the compounds, celecoxib, and cisplatin calculations showed that the para-methylsulfonyl
is summarized in Table 1. Among all tested com- moiety of the phenyl ring on pyrazoline C5 position as
pounds, the most potent COX-2 inhibitor (4- the predominant pharmacophore of the studied
chlorophenyl){5-[4-(methanesulfonyl)phenyl]-3-phenyl- compounds enters the secondary pocket (also named
4,5-dihydro-1H-pyrazol-1-yl}methanone (16) showed side pocket). The interactions of all (S)-enantiomers
the highest cytotoxicity activity against HT-29 cells docked into the active site of COX-2 were analyzed
(IC50 =5.46 μM). The observed cytotoxicity effect of using the LigPlot+ (Version v.2.1) and the most
compound 16 was higher than that of celecoxib and commonly observed interactions were identified as
cisplatin.
follows: a hydrogen bond between SO2CH3 group of
A correlation between the COX-2 inhibition activity the studied compounds and side chain of Arg513
and cytotoxicity against HT-29 cells is presented in residue as well as hydrophobic contacts between the
Figure 2. Except for compounds 18, 19, and 23, all compounds and His90, Val116, Gln192, Val349, Leu352
tested compounds showed a good correlation.
,
,
Ser353, Tyr385, Trp387, Ala516, Phe518, Met522, Val523
Gly526, Ser530, and Leu531. Figure 3 shows the predicted
interactions between compound 16 (the most potent
derivative) and COX-2 enzyme. In vitro COX-2 inhib-
2.4. Molecular Docking
To predict the mode of interactions between the ition assay showed that compound 16 with a para-Cl
synthesized compounds and COX-2 isoenzyme, molec- substitution is the most active derivative. The activity
ular docking studies were conducted using GOLD enhancing effect of this substitution was related to its
program. Both (R)- and (S)-enantiomers of the studied optimum physico-chemical properties explained
compounds were docked into the binding site of above. Furthermore, the results of docking analyses
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Figure 3. Molecular docking of lead compound 16 in COX-2. (A) The side chains of important residues of the enzyme were shown
as lines, while 16 was shown as ball-and-stick representation. The H-bond between side chain guanidine N atom of Arg513 (donor)
and oxygen atom (acceptor) of SO2CH3 group of 16 was highlighted with red dashed line. The measured distance between these
heavy atoms involved H-bond was 3.02 Å. (B) The two-dimensional schematic LigPlot view of the interaction between 16 and COX-
2 obtained by docking.
were also in agreement with the in vitro COX-2 assay. group is less than Cl group and hence may involve in
Investigation of docking results showed that residues a weaker hydrophobic interaction with the enzyme.
in the binding pocket of COX-2 enzyme surrounding On the other hand, it size is bigger than Cl and once
the para-substituted benzoyl moiety of the synthe- again may not be accommodated in the binding
sized compounds are Val116, Arg120, Val349, Tyr355
,
subsite as perfectly as does the Cl group. However, the
Leu359, Ala527 and Leu531 with predominantly hydro- differences between CH3 and Cl groups are not as
phobic side chains forming mainly hydrophobic inter- substantial as those of F, NO2 and SO2CH3 relative to
actions with the para-substituted benzoyl part of the Cl group. This might be the reason for sub-micromolar
compounds. This hydrophobic subsite perfectly ac- IC50 value of compound 13 in COX-2 inhibition assay.
commodates the hydrophobic and medium-size Cl Collectively, one may conclude that presence of a
group of compound 16, but less hydrophobic and para-substitution on benzoyl moiety of the synthe-
relatively small F group (compound 15) may not sized compounds with appropriate size capable of
suitably form appropriate interaction, and hence show forming hydrophobic interaction with COX-2 enzyme
less activity. Substitution of para-NO2 group (com- may lead to stronger inhibition.
pound 17) with much bigger size and very hydrophilic
nature relative to Cl has led to decreased COX-2
inhibition activity most probably due to steric hin-
drance and inability to form favorable hydrophobic
3. Conclusions
interaction. Presence of para-SO2CH3 group in 18 In this study, we synthesized two series of 2-pyrazo-
instead of para-Cl as in 16 on benzoyl moiety line-based derivatives with COX-2 inhibitory activity,
diminished COX-2 inhibition potency most likely due among which compounds 16 was identified as the
to its bigger size and hydrophilic properties. The most potent COX-2 inhibitor. The binding interactions
compound 13 with para-CH3 group demonstrated an were analyzed using molecular docking studies. In
activity less than that of 16 (para-Cl), but higher than addition, cytotoxic effects of the studied compounds
those shown for 15 (para-F), 17 (para-NO2) and 18 were evaluated on colorectal adenocarcinoma cells
(para-SO2CH3). Lipophilicity of the methyl functional and the findings revealed that compound 16 is also a
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Chem. Biodiversity 2021, 18, e2000832
potent cytotoxic agent. The results suggest that 16 dimethyl-2-(4-(methylthio)phenyl)-1,3-dioxane). White
°
can serve as a dual anti-inflammatory and antiprolifer- crystal. Yield: 93%; M.p. 74–75 C.
ative agent.
(b) Oxidation Step
Experimental Section
Compound 7 (10 g, 41.95 mmol) was dissolved in
tetrahydrofuran (THF). A solution of OXONE® (MW=
307.38) (31 g, 0.1 mol) in distilled water (130 mL) was
Materials and Measurements
All chemicals and solvents were purchased from the then added dropwise and the reaction mixture was
Sigma-Aldrich or Merck companies. Solvents were stirred at room temperature for 6 h. After completion
dried and purified according to the literature if of the reaction, determined by TLC, the acidic environ-
required. The progress of the reactions was monitored ment of the reaction mixture was neutralized with
by thin-layer chromatography (TLC) using pre-coated Na2CO3 followed by removing of solvent mixture by a
plates of silica gel 60 F254 (Merck) and visualized under rotary evaporator under reduced pressure at room
UV light (254 nm). Melting points were determined temperature. The solid residue was dissolved in a
using Electrothermal melting point apparatus (Cat. No. mixture of water and ethyl acetate, and transferred to
IA 9200). Infrared spectra were recorded on a a separating funnel. The organic ethyl acetate phase
Shimadzu FT-IR-8400S spectrophotometer using KBr was separated and dried with Na2SO4, and subse-
disk sample preparation method. The 1H- and 13C-NMR quently filtered and evaporated to afford the crude
spectra were recorded on Bruker operating at 400 MHz product 8 (5,5-dimethyl-2-(4-(methylsulfonyl)phenyl)-
and 100 MHz, respectively, using (D6)DMSO as solvent 1,3-dioxane). White crystal. Yield: 98%. M.p. 143–
°
and TMS as internal reference. Coupling constant (J) 144 C.
values are estimated in hertz (Hz) and spin multiples
are given as s (singlet), d (double), t (triplet), q
(quartet), m (multiplet), and br. (broad). The elemental
(c) Deprotection Step
analyses were obtained using Costech Elemental Compound 8 (3 g, 11.10 mmol) was mixed with
Combustion System CHNSÀ O (ECS 4010). The purity of 100 mL water and then, 2 mL concentrated HCl (37%)
the compounds was determined using HPTLC (CAMAG was added to the mixture dropwise followed by reflux
system, Switzerland) and reported as percentages.
for 1 h. After that, the reaction mixture was neutralized
by adding K2CO3 crystals while monitoring the pH
using pH paper. The mixture was cooled to room
temperature to obtain white crystals of compound 9,
Synthesis of 4-(Methylsulfonyl)benzaldehyde (9)
Compound 9 was synthesized through three steps which was then collected on a sintered glass filter
including protection, oxidation, and deprotection as under vacuum and rinsed with distilled water. White
°
outlined below.
crystal; Yield: 91%. M.p. 160–161 C.
(a) Protection Step
Synthesis
of
(E)-3-(4-(Methylsulfonyl)phenyl)-
1-phenylprop-2-en-1-one (10)
4-(Methylthio)benzaldehyde (6; 10 g, 65.69 mmol),
neopentyl glycol (7 g, 67.21 mmol), p-toluenesulfonic Compound 9 (4-(methylsulfonyl)benzaldehyde; 2 g,
acid monohydrate (120 mg, 0.63 mmol, as catalyst), 10.86 mmol) was mixed with acetophenone (1.3 g,
and 100 mL dry toluene were mixed in a round 10.86 mmol) in 60 mL alkaline methanol (12 mmol
bottom flask equipped with a dean-stark trap and a NaOH) and stirred at room temperature for 6 h to
reflux condenser. Reaction mixture was refluxed for prepare compound 10. About one half of the solvent
azeotropic dehydration. After completion of dehydra- was evaporated under reduced pressure and the
tion, reaction mixture was cooled and washed with precipitate of 10 was filtered and rinsed with cold
NaHCO3 aqueous solution. The organic phase was methanol. The crude product was recrystallized in
°
dried over Na2SO4 and then, the organic solvent was ethanol. Yellow solid. Yield: 85%. M.p. 122–124 C. IR
removed by evaporator under reduced pressure at (KBr, cmÀ 1): 1143 (S=O, symmetric stretching), 1298
°
40 C. The crude solid residue was crystallized from (S=O, asymmetric stretching), 1602 (C=C, stretching),
hexane to obtain white crystals of compound 7 (5,5- 1670 (C=O, stretching).
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Synthesis
of
5-(4-(Methylsulfonyl)phenyl)-3-phenyl- separated, dried with sodium sulfate, and evaporated.
4,5-dihydro-1H-pyrazole (11)
The crude product was crystallized in ethyl acetate.
To the chalconic compound 10 (3 g, 10.49 mmol)
dissolved in boiling ethanol in a round bottom flask
COX Inhibition Assay
equipped with
a reflux condenser was added The inhibition activity of test compounds against ovine
hydrazine hydrate 80% (2 equiv.) and the reflux was COX-1 and recombinant human COX-2 was deter-
continued. After completion of the reaction after 1 h, mined using a COX inhibitor assay (Cayman Chemical,
monitored by TLC, the mixture was cooled down to Ann Arbor, USA; item number: 700100) following the
room temperature and the precipitate was collected manufacturer’s protocol. Each compound was assayed
on a sintered glass filter and washed with cold in triplicate, and PRISM5 software was used to
ethanol. The crude product was recrystallized in calculate IC50 values. In addition to celecoxib, both
°
ethanol. White crystal. Yield: 90%. M.p. 190–192 C. IR Dup-697 (potent COX-2 inhibitor) and SC-560 (potent
(KBr, cmÀ 1): 1145 (S=O, symmetric stretching), 1299, COX-1 inhibitor) were used as internal controls during
(S=O, asymmetric stretching), 1591 (C=N, stretching), the screening of test compounds. All statistical analy-
3346 (NÀ H, stretching)
ses were performed by Microsoft Excel.
MTT Assay
General Synthesis of (5-(4-(Methylsulfonyl)phenyl)-
3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(aryl)methanone
(12–18)
The antiproliferative activities of the synthesized
pyrazoline-based derivatives against HT29 (human
Pyrazoline 11 and triethylamine were dissolved in dry colorectal adenocarcinoma cells) were evaluated using
dichloromethane and then, appropriate acyl chloride colorimetric MTT assay. Cisplatin and celecoxib were
was added. The reaction mixture was refluxed and used as positive controls. The cells were grown in
monitored by TLC to ensure the absence of significant Dulbecco’s modified Eagle’s medium (DMEM) supple-
amount of pyrazoline 11. After cooling, the reaction mented with 10% fetal bovine serum (FBS), 1% L-
mixture was transferred to separating funnel and glutamine, 100 u/mL penicillin, and 100 μg/L strepto-
°
washed with water solution of sodium carbonate. The mycin at 37 C and 5% CO2 atmosphere in a
organic layer was separated, dried with sodium sulfate, humidified incubator. Then, cells were seeded into
and evaporated. The crude product was crystallized in sterile 96-well plates (7000 cells/well) and incubated
°
ethyl acetate. The pyrazoline ring in these structures overnight at 37 C. All of compounds and controls
contain three protons giving rise to three doublet of were prepared in DMSO with stock concentration of
doublet (dd) peaks in NMR spectra known for the ABC 20 mΜ. Different concentrations of compounds were
system. Two germinal protons located at the C4 prepared using serum free DMEM media. Cells were
position are either in cis or trans geometry relative to treated with compounds in final concentrations rang-
the vicinal proton at C5 position of the pyrazoline ring. ing from 0.032–100 μΜ. After 48 h treatment, MTT
These protons are referred to as 4-Hcis, 4-Htrans, and 5-H solution was added to the wells with a final concen-
in the description of NMR spectra, respectively. In tration of 0.5 mg/mL and incubated for 4 h. Following
some compounds, the peaks for 4-Htrans were hidden the removal of medium from wells, for solubilization
under the peak for methylsulfonyl protons.
of formed formazan crystals, 100 μL of DMSO was
added and incubated for 30 min at room temperature.
The absorbance was then measured at 570 nm using
an ELISA microplate reader. The cell viability was
compared to negative controls where cells were only
exposed to the medium. The maximum concentration
General Synthesis of 5-(4-(Methylsulfonyl)phenyl)-
3-phenyl-N-(aryl/cyclohexyl)-4,5-dihydro-1H-pyrazole-
1-carboxamide (19–24)
Pyrazoline 11 and appropriate isocyanate were dis- of DMSO in the cell culture media was less than 0.5%.
solved in dry dichloromethane in round bottom flask All experiments were performed in triplicates and the
equipped with a reflux condenser. The reaction data were analyzed using PRISM software to calculate
mixture was refluxed until significant amount of IC50 values. Microsoft Excel was used to calculate
pyrazoline 11 disappeared in TLC. After cooling, the mean IC50 and standard deviations.
reaction mixture was transferred to separating funnel
and washed with water. The organic layer was
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References
Molecular Docking Studies
[1] R. Medzhitov, ‘Origin and physiological roles of inflamma-
tion’, Nature 2008, 454, 428–435.
[2] W. L. Smith, Y. Urade, P.-J. Jakobsson, ‘Enzymes of the
cyclooxygenase pathways of prostanoid biosynthesis’,
Chem. Rev. 2011, 111, 5821–5865.
[3] W. A. van der Donk, A. L. Tsai, R. J. Kulmacz, ‘The cyclo-
oxygenase reaction mechanism’, Biochemistry 2002, 41,
15451–15458.
[4] A. L. Blobaum, L. J. Marnett, ‘Structural and Functional Basis
of Cyclooxygenase Inhibition’, J. Med. Chem. 2007, 50,
1425–1441.
[5] D. L. Simmons, R. M. Botting, T. Hla, ‘Cyclooxygenase
isozymes: the biology of prostaglandin synthesis and
inhibition’, Pharmacol. Rev. 2004, 56, 387–437.
[6] W. L. Smith, R. M. Garavito, D. L. DeWitt, ‘Prostaglandin
endoperoxide H synthases (cyclooxygenases)-1 and À 2’, J.
Biol. Chem. 1996, 271, 33157–33160.
[7] R. G. Kurumbail, A. M. Stevens, J. K. Gierse, J. J. McDonald,
R. A. Stegeman, J. Y. Pak, D. Gildehaus, J. M. Iyashiro, T. D.
Penning, K. Seibert, P. C. Isakson, W. C. Stallings, ‘Structural
basis for selective inhibition of cyclooxygenase-2 by anti-
inflammatory agents’, Nature 1996, 384, 644–648.
[8] W. L. Smith, D. L. DeWitt, R. M. Garavito, ‘Cyclooxygenases:
structural, cellular, and molecular biology’, Annu. Rev.
Biochem. 2000, 69, 145–182.
The 3D structures of compounds were generated by
HyperChem software (version 8.0). The initial struc-
tures were energy minimized using molecular mechan-
ics MM+ force field (ref) followed by fully optimization
via AM1 semi-empirical method available in Hyper-
Chem. The optimized structures were converted to
SYBYL mol2 file format using OpenBabel 2.0.2 version
to be used as an acceptable format for docking
procedure. Flexible docking of the studied compounds
into the active site of the COX-2 isoenzyme was
performed using GOLD program (version 5.0) running
under LINUX operating system. For this purpose, the
crystal structure of COX-2 (PDB code: 5KIR) was
obtained from Protein Data Bank. Prior to molecular
docking, hydrogen atoms were added to the protein
in GOLD program. The active site of the enzyme was
determined based on the position of rofecoxib co-
crystallized with COX-2. To this end, geometric center
of the residues involved in the binding to the co-
crystallized inhibitor molecule (i.e., rofecoxib) was
calculated and set as the center of the binding site.
Following the optimization of the docking parameters
by re-docking the rofecoxib into the active site of the
enzyme, the optimized structures were docked into
the COX-2. The best solutions in terms of scoring
function were selected and used for analysis of
interactions using LigPlot+ and PyMOL program.
[9] D. Wang, R. N. Du Bois, ‘The role of anti-inflammatory
drugs in colorectal cancer’, Annu. Rev. Med. 2013, 64, 131–
144.
[10] D. Wang, R. N. Du Bois, ‘Eicosanoids and cancer’, Nat. Rev.
Cancer 2010, 10, 181–193.
[11] J. B. Méric, S. Rottey, K. Olaussen, J. C. Soria, D. Khayat, O.
Rixe, J. P. Spano, ‘Cyclooxygenase-2 as
a target for
anticancer drug development’, Crit. Rev. Oncol. Hematol.
2006, 59, 51–64.
[12] Z. Khan, N. Khan, R. P. Tiwari, N. K. Sah, G. B. Prasad, P. S.
Bisen, ‘Biology of Cox-2: an application in cancer therapeu-
tics’, Curr. Drug Targets 2011, 12, 1082–1093.
[13] D. Wang, R. N. Dubois, ‘The role of COX-2 in intestinal
inflammation and colorectal cancer’, Oncogene 2010, 29,
781–788.
[14] N. V. Chandrasekharan, H. Dai, K. L. Roos, N. K. Evanson, J.
Tomsik, T. S. Elton, D. L. Simmons, ‘COX-3, a cyclooxyge-
nase-1 variant inhibited by acetaminophen and other
analgesic/antipyretic drugs: cloning, structure, and expres-
sion’, Proc. Natl. Acad. Sci. USA 2002, 99, 13926–13931.
[15] N. U. A. Mohsin, M. Irfan, ‘Selective cyclooxygenase-2
inhibitors: A review of recent chemical scaffolds with
promising anti-inflammatory and COX-2 inhibitory activ-
ities’, Med. Chem. Res. 2020, 29, 809–830.
Acknowledgment
The authors would like to thank Biotechnology
Research Center, School of Pharmacy, and Research
Deputy at Tabriz University of Medical Sciences for
providing research facilities and financial support
under the Postgraduate Research Scheme toward the
Ph.D. thesis of TV (Grant number 49292).
Author Contribution Statement
T.V. performed all the experiments. J.K. performed the
COX inhibition assays. S.H. supervised the synthetic
procedure. M.H. helped in molecular docking and data
analysis. A.A.A. assisted in MTT assay. F.W. helped in
COX inhibition assay and data analysis. S.D. designed
and supervised this project. All authors have contrib-
uted to the final version and approved the final
manuscript.
[16] A. Bhardwaj, J. Kaur, E. E. Knaus, ‘Can nitric oxide-releasing
hybrid drugs alleviate adverse cardiovascular risks?’, Future
Med. Chem. 2013, 5, 381–383.
[17] P. Singh, A. Bhardwaj, ‘Mono-, Di-, and Triaryl Substituted
Tetrahydropyrans as Cyclooxygenase-2 and Tumor Growth
Inhibitors. Synthesis and Biological Evaluation’, J. Med.
Chem. 2010, 53, 3707–3717.
[18] A. Bhardwaj, J. Kaur, M. Wuest, F. Wuest, ‘In situ click
chemistry generation of cyclooxygenase-2 inhibitors’, Nat.
Commun. 2017, 8, 1.
www.cb.wiley.com
(9 of 10) e2000832
© 2021 Wiley-VHCA AG, Zurich, Switzerland

Chem. Biodiversity 2021, 18, e2000832
[19] I. G. Rathish, K. Javed, S. Ahmad, S. Bano, M. S. Alam, K. K. and antimicrobial agents’, Bioorg. Med. Chem. 2017, 25,
Pillai, S. Singh, V. Bagchi, ‘Synthesis and anti-inflammatory
activity of some new 1,3,5-trisubstituted pyrazolines bear-
ing benzene sulfonamide’, Bioorg. Med. Chem. Lett. 2009,
19, 255–258.
1949–1962.
[28] M. V. R. Reddy, V. K. Billa, V. R. Pallela, M. R. Mallireddigari,
R. Boominathan, J. L. Gabriel, E. P. Reddy, ‘Design, syn-
thesis, and biological evaluation of 1-(4-sulfamylphenyl)-3-
trifluoromethyl-5-indolyl pyrazolines as cyclooxygenase-2
(COX-2) and lipoxygenase (LOX) inhibitors’, Bioorg. Med.
Chem. 2008, 16, 3907–3916.
[29] R. Fioravanti, A. Bolasco, F. Manna, F. Rossi, F. Orallo, F.
Ortuso, S. Alcaro, R. Cirilli, ‘Synthesis and biological
evaluation of N-substituted-3,5-diphenyl-2-pyrazoline de-
rivatives as cyclooxygenase (COX-2) inhibitors’, Eur. J. Med.
Chem. 2010, 45, 6135–6138.
[30] S. Bano, K. Javed, S. Ahmad, I. G. Rathish, S. Singh, M. S.
Alam, ‘Synthesis and biological evaluation of some new 2-
pyrazolines bearing benzene sulfonamide moiety as
potential anti-inflammatory and anti-cancer agents’, Eur. J.
Med. Chem. 2011, 46, 5763–5768.
[31] B. P. Bandgar, L. K. Adsul, H. V. Chavan, S. S. Jalde, S. N.
Shringare, R. Shaikh, R. J. Meshram, R. N. Gacche, V.
Masand, ‘Synthesis, biological evaluation, and docking
studies of 3-(substituted)-aryl-5-(9-methyl-3-carbazole)-1H-
2-pyrazolines as potent anti-inflammatory and antioxidant
agents’, Bioorg. Med. Chem. Lett. 2012, 22, 5839–5844.
[32] P. N. Rao, M. J. Uddin, E. E. Knaus, ‘Design, synthesis, and
structure-activity relationship studies of 3,4,6-triphenylpyr-
an-2-ones as selective cyclooxygenase-2 inhibitors’, J. Med.
Chem. 2004, 47, 3972–3990.
[20] J. Kaur, A. Bhardwaj, S. K. Sharma, F. Wuest, ‘1,4-Diaryl-
substituted triazoles as cyclooxygenase-2 inhibitors: Syn-
thesis, biological evaluation, and molecular modeling
studies’, Bioorg. Med. Chem. 2013, 21, 4288–4295.
[21] R. Bashir, S. Ovais, S. Yaseen, H. Hamid, M. S. Alam, M.
Samim, S. Singh, K. Javed, ‘Synthesis of some new 1,3,5-
trisubstituted pyrazolines bearing benzene sulfonamide as
anticancer and anti-inflammatory agents’, Bioorg. Med.
Chem. Lett. 2011, 21, 4301–4305.
[22] T. K. Mohamed, R. Z. Batran, S. A. Elseginy, M. M. Ali, A. E.
Mahmoud, ‘Synthesis, anticancer effect and molecular
modeling of new thiazolylpyrazolyl coumarin derivatives
targeting VEGFR-2 kinase and inducing cell cycle arrest
and apoptosis’, Bioorg. Chem. 2019, 85, 253–273.
[23] N. M. Stefanes, J. Toigo, M. F. Maioral, A. V. Jacques, L. D.
Chiaradia-Delatorre, D. M. Perondi, A. A. B. Ribeiro, Á.
Bigolin, I. M. S. Pirath, B. F. Duarte, R. J. Nunes, M. C. Santos-
Silva, ‘Synthesis of novel pyrazoline derivatives and the
evaluation of death mechanisms involved in their anti-
leukemic activity’, Bioorg. Med. Chem. 2019, 27, 375–382.
[24] A. Özdemir, M. D. Altıntop, Z. A. Kaplancıklı, Ö. D. Can, Ü.
Demir Özkay, G. Turan-Zitouni, ‘Synthesis and Evaluation of
New 1,5-Diaryl-3-[4-(methyl-sulfonyl)phenyl]-4,5-dihydro-
1H-pyrazole Derivatives as Potential Antidepressant
Agents’, Molecules 2015, 20.
[33] L. P. Hammett, ‘The Effect of Structure upon the Reactions
of Organic Compounds. Benzene Derivatives’, J. Am. Chem.
Soc. 1937, 59, 96–103.
[34] C. Hansch, A. Leo, R. W. Taft, ‘A survey of Hammett
substituent constants and resonance and field parameters’,
Chem. Rev. 1991, 91, 165–195.
[25] Z. Özdemir, H. B. Kandilci, B. Gümüşel, Ü. Çalış, A. A. Bilgin,
‘Synthesis and studies on antidepressant and anticonvul-
sant activities of some 3-(2-furyl)-pyrazoline derivatives’,
Eur. J. Med. Chem. 2007, 42, 373–379.
[35] R. W. Taft, ‘Linear Free Energy Relationships from Rates of
Esterification and Hydrolysis of Aliphatic and ortho-Sub-
stituted Benzoate Esters’, J. Am. Chem. Soc. 1952, 74,
2729–2732.
[26] A. V. Chate, A. A. Redlawar, G. M. Bondle, A. P. Sarkate, S. V.
Tiwari, D. K. Lokwani, ‘A new efficient domino approach for
the synthesis of coumarin-pyrazolines as antimicrobial
agents targeting bacterial d-alanine-d-alanine ligase’, New
J. Chem. 2019, 43, 9002–9011.
[27] V. K. Mishra, M. Mishra, V. Kashaw, S. K. Kashaw, ‘Synthesis
of 1,3,5-trisubstituted pyrazolines as potential antimalarial
Received October 7, 2020
Accepted February 1, 2021
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