New nimesulide derivatives with amide/sulfonamide moieties: Selective COX-2 inhibition and antitumor effects

European Journal of Medicinal Chemistry 221 (2021) 1 13566

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

New nimesulide derivatives with amide/sulfonamide moieties:

Selective COX-2 inhibition and antitumor effects

a

,

*

,

c

,

¹, Adem Ozleyen ^{b 1}, Yakup Berkay Yılmaz ^{b d}, Pinar Siyah ^e,

ꢀ

€

Tugba Güngor

a

e

d, **

ꢀ

Mehmet Ay , Serdar Durdagı , Tugba Boyunegmez Tumer

^aDepartment of Chemistry, Faculty of Sciences and Arts, Natural Products and Drug Research Laboratory, Çanakkale Onsekiz Mart University, Çanakkale,

17020, Turkey

^bGraduate Program of Biomolecular Sciences, School of Graduate Studies, Canakkale Onsekiz Mart University, 17020, Çanakkale, Turkey

^cSchool of Chemistry, University of Leicester, LE1 7RH, Leicester, United Kingdom

^dDepartment of Molecular Biology and Genetics, Faculty of Arts and Science, Çanakkale Onsekiz Mart University, 17020, Çanakkale, Turkey

^eComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahçes¸ehir University, 34353, Istanbul,

Turkey

a r t i c l e i n f o

a b s t r a c t

Article history:

Seventeen new amide/sulfonamide containing nimesulide derivatives were synthesized and character-

ized by several spectroscopic techniques and primarily investigated for their inhibitory potential on COX

enzymes and other pro-inﬂammatory factors. Experimental analyses showed that among seventeen

compounds, N8 and N10 have remarkable potency and selectivity for the COX-2 enzyme over COX-1 at

very low doses as compared to nimesulide. Moreover, both N8 and N10 selectively reduced the Lipo-

polysaccharide (LPS)-stimulated COX-2 mRNA expression level while the COX-1 level remained stable.

Both PGE₂release and nitric oxide production in macrophage cells were signiﬁcantly suppressed by the

N8 and N10 treatment groups. In silico ADME/Tox, molecular docking and molecular dynamics (MD)

simulations were also conducted. Additionally, all compounds were also screened in a panel of cancer

cell lines for their antiproliferative properties by MTT and SRB assays. Compound N17 exhibited a

Received 27 March 2021

Received in revised form

15 May 2021

Accepted 18 May 2021

Available online 24 May 2021

Keywords:

Nimesulide

Synthesis

Amide

considerable antiproliferative effect on the colon (IC₅₀: 9.24 mM) and breast (IC₅₀: 11.35 mM) cancer cell

Sulfonamide

Anti-inﬂammatory

Anticancer

MD simulations

Docking

lines. N17 exposure for 48 h decreased expression of anti-apoptotic protein BCL-2 and increased the

expression of apoptogenic BAX. Besides, the BAX/BCL-2 ratio was increased with visible ultrastructural

changes and apoptotic bodies under scanning electron microscopy. In order to investigate the structural

and dynamical properties of selected hits on the target structures, multiscale molecular modeling studies

are also conducted. Our combined in silico and in vitro results suggest that N8 and N10 could be further

developed as potential nonsteroidal anti-inﬂammatory drugs (NSAIDs), while cytotoxic N17 might be

studied as a potential lead compound that could be developed as an anticancer agent.

Binary QSAR models

1. Introduction

and thromboxane (TxA) by inhibiting cyclooxygenase isoforms:

COX-1 and COX-2. Unfortunately, long-term NSAID therapy leads to

Daily, over 30 million people with inﬂammatory symptoms are

estimated to have prescription for nonsteroidal anti-

inﬂammatory drugs (NSAIDs) [1]. The primary action mechanism

severe adverse effects in the GI-tract by undesired inhibition of

COX-1 resulting in dramatic reductions in the gastroprotective PGs

levels. COX-1 is the predominant isoform found in the different

parts of the GI tract particularly mucosal epithelium of the gastric

fundus, corpus, antrum, duodenum, jejunum, ileum, cecum, and

colon [2]. Speciﬁcally, PG deﬁciency induced by NSAIDs cause

gastric hypermotility due to inhibition of COX-1 but not COX-2 [3].

The highlighting of the risks of COX-1 inhibition has paved the way

for the design of NSAIDs with COX-2 selectivity.

a

of NSAIDs is based on repressing the release of prostaglandins (PGs)

* Corresponding author.

** Corresponding author.

€

E-mail addresses: tgungor@comu.edu.tr (T. Güngor), tumertb@comu.edu.tr

Selective COX-2 inhibition was thought to be a promising

(T.B. Tumer).

1

T.G. and A.O. contributed equally.

https://doi.org/10.1016/j.ejmech.2021.113566

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

strategy; the inhibition of inducible COX prevents the release of

excess amounts of pro-inﬂammatory PGE₂while spares the gas-

troprotective prostaglandin synthesis mediated by COX-1. In the

beginning, the drugs with high selectivity towards COX-2 exhibited

favorable safety proﬁles as compared to the traditional NSAIDs.

Especially, the class of coxibs including celecoxib, etoricoxib, par-

ecoxib, rofecoxib, valdecoxib, and lumiracoxib gained early success

in clinics as selective COX-2 inhibitors. However, reports relevant to

cardiovascular problems in rofecoxib, valdecoxib, and etoricoxib

users brought about their withdrawal from markets. The underly-

ing reason is said to be potential damage on the balance between

COX-1 mediated prothrombotic TxA₂and COX-2 mediated antith-

rombotic PGI₂production [4]. Unlike other coxibs, celecoxib is still

accessible in pharmacies with a warning label in its prescription

highlighting the potential adverse actions.

conﬁrmed that hepatic reactions caused by nimesulide are in line

with other NSAIDs and generally relevant to the intake of high

doses, which is far from those recommended [9,10]. A compre-

hensive and critical revision of clinical data regarding the efﬁcacy

and safety proﬁle of nimesulide revealed that among other NSAIDs,

it confers a favorable and unchanged beneﬁt/risk assessment over

time [1 1,12].

Apart from its distinctive safety properties, nimesulide has also

unique therapeutic proﬁles that are not shared with other selective

COX-2 inhibitors (Scheme 1). Nimesulide possesses a wide range of

multitarget actions known to be important in the mitigation of

inﬂammatory processes such as inhibition of reactive oxygen spe-

cies (ROS), nitric oxide (NO), peroxynitrite (ONOO^ꢀ), cytokines,

histamine, and elastase releases. It also downregulates the activ-

ities of nitric oxide synthase (NOS) and metalloproteinases (MMPs).

Moreover, it reduces the rate of monocyte death, chemotaxis, and

transendothelial migration [12]. Some in vitro and in vivo animal

model studies demonstrated its inhibitory effects on cancer cell

proliferation and metastasis [13]. Recently, nimesulide is recom-

mended to be repurposed as a potential antidiabetic agent due to

its anti-glycation activity [14]. Very recently, researchers suggested

that nimesulide should be included in the list of the drugs to be

tested for the identiﬁcation of co-adjuvant in the treatment of

COVID-19 [15]. They found that Nimesulide abolishes the transport

function of the B0AT1-ACE-2 supercomplex for SARS-CoV-2 viral

entry [15].

4-NItro-2-phenoxyMEthane-SULphonanilIDE,

NIMESULIDE

(Scheme 1), has been in the markets for a long period as one of the

most commonly used preferential anti-inﬂammatory agents with

potent analgesic and antipyretic effects [5]. While coxibs are 375-

800-fold more selective to COX-2, nimesulide has been shown to

inhibit COX-2 about 21-folds more selectively than COX-1 [6]. In

addition, it has been reported that at higher doses, its selectivity to

COX-2 is lost [7]. For this reason, nimesulide does not lead to severe

cardiovascular problems like other highly selective COX-2 in-

hibitors suppressing the biosynthesis of vascular prostacyclin. As

well as it does not provoke gastric damage like other NSAIDs

inhibiting gastroprotective PGs synthesis, even it is reported to be

protective against NSAIDs-induced ulcers [8].

Multitarget pharmacological proﬁles represented by the chem-

ical entity of nimesulide inspired us for the development of new

derivatives with amide and sulfonamide moieties. For this purpose,

new nimesulide derivatives (NDs) were designed, synthesized, and

The most relevant adverse effect associated with nimesulide is

hepatotoxicity. However, European Medicines Agency (EMA)

Scheme 1. Design process of new amide/sulfonamide derivatives of nimesulide as anti-inﬂammatory and anticancer agents.

2

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

investigated for their anti-inﬂammatory and anticancer effects.

Moreover, in silico studies including molecular docking, molecular

dynamics (MD) simulations, and ADME/Tox investigations were

performed. Target compounds (N1eN17) were designed through

three main steps (Scheme 1). In the ﬁrst step, the phenyl group of

nimesulide was changed with ethyl and isopropyl groups which are

expected to be potentially active in the light of our preliminary

theoretical studies (for compounds N1-12 and N14e16). Also, the

phenyl group was not changed in compounds N13 and N17. The

following step is the reduction of nitro to the amino group. The ﬁnal

step to form the target compounds N1eN17 is amidation or sul-

fonamidation reactions between the amino group and benzoyl

chloride/benzoic acid derivatives or benzenesulfonyl chloride de-

rivatives bearing various functional groups (-NO₂, eF, eOCH₃,

eCONH₂).

Nimesulide is the sole NSAID containing a nitro group. Although

nitro-containing compounds are considered structurally unsafe

molecules, there are many physiologically active therapeutics

having this group in markets such as nilutamide, nitrendipine,

nitrazepam, and nitromide. Indeed, the radical scavenger behavior

of the nimesulide had been attributed to the incorporation of a 4-

nitro group into the sulphoanilide structure [12]. Besides, the pre-

vious structure-activity (SAR) studies including new NDs reported

that compounds without NO₂possess lower antinociceptive ac-

tivity. In our previous studies, a series of the nitro-containing

benzamide compounds were synthesized and investigated for

their anti-inﬂammatory and neuroinﬂammatory effects in lipo-

polysaccharide (LPS)-induced macrophages and microglia cells by

focusing their inhibitory effects on pro-inﬂammatory factors, iNOS,

were synthesized in a convenient and mild approach by stirring N-

(4-amino-2-alkoxyphenyl)methanesulfonamide derivatives and

substituted benzoyl chlorides/benzenesulfonyl chlorides in the

presence of triethylamine in chloroform at room temperature [22]

(Scheme 2). While the reactions of amide derivatives N1-4, N10e11

and N16e17 were completed in short reaction times (2e3 h) with

moderate to good yields (65e83%), the reactions of sulfonamide

derivatives N6-9 and N14e15 were completed in longer reaction

times (12e24 h) with 51e78% yields.

Additionally, amide products N5, N12, and N13 were obtained in

a different amidation way based on benzoic acid derivatives instead

of benzoyl chlorides using coupling reagents such as DCC or

EDCI.HCl (Scheme 3). The 16

h

reaction of 2-nitro-4-

methoxybenzoic acid and N-(4-amino-2-isopropoxyphenyl)meth-

anesulfonamide with DCC in the presence of catalyst DMAP in DCM

at reﬂux temperature gave the compound N5 in 69% yield. In a

similar way, compounds N12 and N13 were obtained at the reaction

condition which is including terephthalamic acid (4-

carbamoylbenzoic acid) as starting material in the presence of

EDCI.HCl, HOBt and triethylamine in DMF at 100e120 ^ꢁC for long

reaction times (24 and 43 h, respectively).

Consequently, target nimesulide compounds (N1eN17) were

synthesized successfully and identiﬁed by melting point, MS, FT-IR,

¹H and ¹³C NMR spectroscopic techniques (See Experimental Part

and Supporting Information). Also, the purity of compounds (>95%)

was checked with HPLC analysis (See Experimental Part and Sup-

porting Information). The structures of known starting materials

were conﬁrmed by the comparison with literature data. Model

compound,

N-(3-isopropoxy-4-(methylsulfonamido)phenyl)-4-

COX-2, IL-1

b

, and TNF-

a

[16,1 7]. The promising results of nitro

methoxy-2-nitrobenzamide (N5) was conﬁrmed by ¹H NMR as

the presence of eNH singlet protons of amide and sulfonamide at

8.72 and 10.59 ppm. The doublet peak with 6H integration at

1.31 ppm, and the septet peak with 1H integration at 4.54 ppm

corresponds to the eCH₃and eCH protons of the isopropyl ether

group, respectively. It was determined that the eCH₃protons of

sulfonamide and arylmethyl ether groups were resonated as singlet

peaks with 3H integration at 2.89 ppm and 3.89 ppm, respectively.

In addition, six aromatic protons were found in the range of

7.72e7.12 ppm. The evaluation of the ¹³C NMR spectrum of com-

pound N5 showed carbonyl C]O signal at the highest chemical

shift (164.07 ppm) and aromatic peaks of two phenyl rings in the

range of 160.98e105.35 ppm. Also, aliphatic peaks were observed

at the expected region of spectrum such as two isopropyl carbons at

22.02 and 70.56 ppm, methyl carbon of sulfonamide at 40.55 ppm

and methyl carbon of arylmethyl ether at 56.78 ppm. FT-IR spec-

trum of N5 showed the NeH stretching of amide and sulfonamide

at 3319, 3246 cm^ꢀ1and the carbonyl stretching of amide at

substituted benzamides encouraged us to expand the scope of

these compounds. Thereupon, in the present study, the design of

new compounds was executed by the combination of nimesulide

and nitrobenzamides of our previous studies. Further, various

functional groups (-F, eOCH₃, eCONH₂) instead of the nitro group

and as well as sulfonamide moiety instead of amide were included

in the product series to investigate the effects of different func-

tional groups or pharmacophores on anti-inﬂammatory and anti-

cancer activities.

2. Results and discussion

2.1. Chemistry

The synthetic strategy for seventeen new NDs (N1eN17) is

outlined in Schemes 2 and 3. Ethoxy or isopropoxy derivatives of N-

(4-amino-2-alkoxyphenyl)methanesulfonamides were synthesized

in three steps (See Supporting Information). Firstly, 2-amino-5-

nitrophenol was reacted with iodoethane or 2-iodopropane to

obtain the etheric intermediates via Williamson ether synthesis

[18e20]. The reaction of intermediates with methanesulfonyl

chloride (MsCl) in the presence of sodium hydride at room tem-

perature afforded dimethanesulfonamide derivatives which were

subsequently converted into methanesulfonamide derivatives at

basic hydrolysis conditions [18,19]. An alternative, one-step syn-

thesis of methanesulfonamides was also performed using MsCl and

pyridine at room temperature for 40 h in DCM with good yields

(>80%) [21]. The nitro functionality was reduced with tin/hydro-

chloric acid to obtain amines in good yields (74 and 87%, ethyl ether

and isopropyl ether derivatives, respectively) in the last step [22].

N-(4-amino-2-phenoxyphenyl)methanesulfonamide as the start-

ing material of compounds N13 and N17 was synthesized beginning

from 2-amino-5-nitrophenol according to the literature methods

[22,23].

1657 cm^ꢀ1. Also, aromatic CeH stretching at 3154-3068 cm^ꢀ1

,

aliphatic CeH stretching at 2980-2850 cm^ꢀ1and CeOeC stretching

of ether at 1162 cm^ꢀ1were observed. The FT-IR, ¹H and ¹³C NMR

spectra of all other ﬁnal products were determined almost in a

similar pattern like compound N5. The MS of all new products

showed the characteristic [M ꢀ H]^-or [M þ H]^þmolecular ion peaks

to their corresponding molecular weight.

2.2. COX inhibition assay

NSAIDs exert their actions by inhibiting the activity of rate-

limiting enzymes, COXs, in the pro-inﬂammatory prostanoids

biosynthesis [24]. Retrospective studies stated that nonselective

inhibition of COX might end up with undesirable side effects,

particularly in the gastrointestinal system due to inhibition of the

COX-1 enzyme [25]. Thus, preferential inhibition of the COX-2

enzyme has been pointed out as a safer therapeutic approach to

healing inﬂammation-caused diseases. Firstly, the inhibitory effects

Final amide and sulfonamide products N1-4, N6-11 and N14e17

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

Scheme 2. Synthesis of N1-4, N6-11 and N14e17 compounds.

Scheme 3. Synthesis of N5, N12 and N13 compounds.

4

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

of seventeen NDs were investigated on the COX-2 enzyme at 1

m

M

various diseases through activated arachidonic acid metabolism

[26]. PGE₂mediates the inﬂammatory cascades and excessive PGE₂

production demonstrates the pathophysiological process in or-

ganisms especially in the kidney [27]. Thus, suppression of PGE₂

release is important for the prevention and treatment of inﬂam-

mation. In the present study, 50 and 100 mM doses of N8 suppressed

the release of PGE₂by 77% and 44%, and N10 suppressed the pro-

duction of PGE₂by 67% and 68%, respectively (Fig. 2B).

dose. The results were illustrated as a percentage of remaining

enzyme activity in Fig. 1. Among the seventeen compounds, N1, N2,

N3, N4, and N6 showed similar or better inhibitory effects as

compared to reference compound nimesulide, however, the best

inhibitory scores were obtained for N8 and N10 with 88.0 and

72.8%, respectively (Fig. 1A). As shown in Fig. 1B, the compounds

with inhibitory effect on COX-2 were also analyzed on the recom-

binant COX-1 enzyme at 10

mM dose. According to results, only N8

Additionally, 50 and 100

mM doses of N8 suppressed the COX-2

and N10 decreased the activity of the COX-1 enzyme by 21.0 and

27.2%, respectively. However, at identical doses, reference com-

pound nimesulide exhibited no inhibitory effect on the enzymatic

activity of COX-1, it decreased the COX-2 activity by 12.1%. The

ﬁndings for N8 and N10 in terms of both enzymes were highly

compatible with the effects of celecoxib (Fig. 1B). According to the

results, N8 and N10 compounds were selected as lead anti-

inﬂammatory compounds and decided to be analyzed further

through cell-based assays.

mRNA expressions 47% and 60% in 1

m

g/mL LPS-induced THP-1

macrophages, respectively. At the same doses, N10 downregulated

the COX-2 mRNA expressions by 53% and 61%, respectively. Sup-

pression on COX-2 levels for both compounds was found to almost

as effective as positive control celecoxib. Furthermore, both com-

pounds did not change the COX-1 mRNA expressions signiﬁcantly

(Fig. 2C). These results suggested that N8 and N10 preferentially

suppressed COX-2 mRNA levels without affecting COX-1 levels

signiﬁcantly similar to celecoxib.

2.3. PGE₂release from LPS induced THP-1 cells and COX-1/COX-2

expression levels

2.4. Screening of pro-inﬂammatory markers

The primary pharmacological action of nimesulide is based on

preferential inhibition of COX-2 thus the production of prosta-

glandins particularly, PGE₂. However, it takes the strength as an

effective anti-inﬂammatory drug from its suppressive effects on

other pro-inﬂammatory factors such as NO/iNOS signaling and

cytokines. In vitro and in vivo studies reported that nimesulide at

THP-1 human monocytic cell line is a good model to investigate

the anti-inﬂammatory effects of the compounds. When they are

challenged with PMA stimulation, they differentiate into adherent

LPS inducible cells with macrophage-like phenotypes (Fig. 2A).

PGE₂production can be stimulated in response to inﬂammation in

Fig. 1. Inhibitory effects of NDs on recombinant COX-2 enzyme at 1 mM (A, B, orange bars) and effective compounds were also screened on COX-1 enzyme at 10 mM (B, black bars). As

positive control nimesulide and celecoxib were examined at same doses. C: Control. *p < 0.02, **p < 0.005, ***p < 0.001.

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European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 2. Morphological changes of THP-1 cells were imaged before and after PMA treatment by using inverted microscope (A). The effects of N8 and N10 on PGE₂release (B) and

mRNA expression of COXs in activated THP-1 (C) at 50 and 100

mM doses. Celecoxib (100

mM): Positive control. C: Control. *p < 0.02, **p < 0.005, ***p < 0.001.

pharmacological concentration effectively inhibits NO release and

production of TNF [28,29]. Previously, Bogaart et al. reported that

cytokine expression, the RAW264.7 cell line was used. Accord-

ingly, murine macrophages were stimulated with 1 mg/mL LPS

a

for NO production PMA-differentiated THP-1 did not give a

response to LPS stimulation while murine macrophages such as

RAW264.7 cell line could be triggered [30]. Bertholet et al. stated

that NO might be released by murine macrophages in response to

cytokine activation but not by human macrophages under the same

conditions, even though iNOS is expressed at both mRNA and

protein levels [31]. Our previous results also support these ﬁndings

[32]. The reason behind this issue is still controversial but it is

thought to be a lack of complete mechanism required for NO syn-

thesis in the THP-1 cellular environment and epigenetic regulations

around the iNOS transcription start site [31,33]. However, Knowles

and Moncada reported 81e93% similarity in NOS genes between

mammalian orthologs and further computational analyses revealed

a high degree of three-dimensional overlap between the oxygenase

domains of human and murine iNOS enzymes [34e37]. This

observation demonstrates the potential utility of the cell-based

murine iNOS assay to identify inhibitors of the human enzyme.

For this reason, in the current study, to screen the effects of N8 and

N10 on LPS induced NO/iNOS signaling and pro-inﬂammatory

treatment in the presence and absence of compounds. As compared

to the LPS-free group, LPS stimulation triggered the NO production

in cells (Fig. 3A). Compound N8 decreased the NO release at 20 and

50

and 50

respectively. In contrast to N8 and N10, reference compound

nimesulide at 20 M slightly increased the NO production as

mM doses by 40.7 and 68.1%, respectively. On the other hand, 20

mM doses of N10 reduced the NO release by 43.1 and 46.4%,

m

compared to the control but the differences were not found to be

statistically signiﬁcant. In the assay, 1400 W was used as a positive

control, which is a very potent and selective iNOS inhibitor, and at

20 mM it decreased the NO release by 62.5%. Besides, the effects of

N8 and N10 on the expression level of LPS-induced iNOS expression

in murine macrophages were determined by qPCR analysis. As

shown in Fig. 3B, the expression level of iNOS at the mRNA level

remained stable in the N8 treated group as compared to the control

group, however, the iNOS expression was decreased by the N10

treatment in a dose-dependent manner. N10 down-regulated the

expression by 40% at 20

pressed at 50 M dose.

mM dose and almost completely sup-

m

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European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 3. The effects of N8 and N10 on NO production in LPS-treated murine RAW 264.7 cells (A). The effects of N8 and N10 on mRNA expression of iNOS (B), IL-1

Nimesulide (20 M): Reference compound. 1400 W (20 M): Positive control. C: Control. ***p < 0.001.

b (C), and TNF-a (D).

m

Overexpression of pro-inﬂammatory markers including IL-1

b

the cytotoxicity of the compounds in two successive steps: 1)

Screening of all the compounds at 100 M by MTT assay. 2) Eval-

and TNF- is associated with the release of exaggerated amounts of

a

m

cytokines that lead to the progression of several inﬂammatory

diseases such as metabolic syndrome, cancer, type II diabetes, and

uating the IC₅₀values of the compounds which resulted in more

than 50% inhibition at the ﬁrst step, by SRB assay. Accordingly,

screening studies resulted in a single effective cytotoxic compound,

the remaining cell viabilities in N17-treated breast, colon, and

prostate cancer cell lines were determined as 22, 33, and 25%,

respectively. These results were found highly compatible with the

effects of positive control doxorubicin for which cell viabilities

remained at 14, 21, and 12%, in the same cell lines (Table 1).

Therefore, N17 was selected for the determination of the half-

maximal inhibitory concentration (IC₅₀) values by performing the

SRB assay. For this purpose, those cells were treated with N17 at

Alzheimer's disease [38]. In this study, 20 and 50

to a reduction in the mRNA expression level of IL-1

respectively. Additionally, at the same doses, N10 reduced the IL-1

mRNA level by 45 and 51% as compared to the control group

(Fig. 3C). The expression of TNF- was also changed by N8 and N10

treatment at 20 and 50 M doses. The treatment of N8 down-

regulated the mRNA expressions of TNF- by 46 and 22%, respec-

mM doses of N8 led

b

by 39 and 22%,

b

a

m

a

tively. Additionally, N10 reduced the LPS-induced TNF-

a mRNA

level by 44 and 57% at the same doses (Fig. 3D).

different ﬁnal concentrations (0e200

Table 2, the IC₅₀values of N17 were found as 9.24 and 11.35

the colon and breast cancer cell lines, respectively. However, the

value was higher than 100 M in prostate cancer cells. To display

the selective cytotoxic characteristic, N17 was also evaluated in

healthy cells, and its IC₅₀value was determined higher than

m

M) for 48 h. As evident from

mM in

2.5. Anticancer properties of compounds N1eN17

m

The synthesized compounds were screened in vitro for their

antiproliferative activity against three different human cancer cell

lines: MCF-7 (breast carcinoma), PC-3 (prostate carcinoma) and HT-

29 (colon carcinoma). Human umbilical vein endothelial cells

(HUVEC) were used as non-cancerous control cells to represent the

selectivity of the compounds for cancerous tissues. We determined

200 mM.

Apart from the cytotoxic properties of N17, its effects on the

long-term survival and ability of the cells to form colonies were also

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European Journal of Medicinal Chemistry 221 (2021) 113566

Table 1

not seen any colony formation in the highest concentration of N17.

To elucidate the mechanism lying behind the antitumor effects

of N17 partially, we investigated the expression of apoptosis-

related proteins BAX (BCL-2-associated X protein) and BCL-2 (B-

cell lymphoma 2) by Western blot analyses. According to the re-

sults, the expression of pro-apoptotic BAX protein was up-

regulated by 24 h N17 treatment in a dose-dependent manner.

Besides, the level of anti-apoptotic BCL-2 was signiﬁcantly down-

regulated in the same treatment group. The increasing ratio of

BAX/BCL-2 shown in Fig. 5 suggested that N17 may lead to

apoptotic cell death by the activation of intrinsic pathway.

To conﬁrm the apoptotic effects of N17, the apoptotic bodies and

ultrastructural changes in cancer cells were examined by SEM an-

alyses. As marked with red arrows, the treated cells appeared to

have irregular shapes and exhibited apoptotic structures including

an increasing number of blebs and apoptotic bodies as compared to

the control cells. While the control cells conserved their typical

morphologies with quite adherent appearance, N17-treated cells

lost their pre-apoptotic structures by elongating and shrinkage

(Fig. 6).

Cytotoxicities of NDs in MCF-7, HT-29, and PC-3 cell lines at 100 mM concentration.

Percentage cell viability was presented as the mean

experiments.

SD of three independent

Compound (100

mM)

Cell viability (%)

MCF-7

HT-29

PC-3

Vehicle

N1

N2

N3

N4

N5

N6

N7

N8

100.0 17.3

122.0 4.6

90.8 14.2

115.7 26.2

100.8 20.1

63.5 6.7

138.7 21.4

127.0 24.5

118.7 22.1

57.9 6.4

100.0 15.6

111.5 27.4

70.4 19.7

114.0 29.7

124.3 40.0

110.6 19.6

133.4 19.9

115.8 20.8

118.4 21.6

75.4 12.9

49.6 24.0

67.9 7.3

79.4 14.8

64.4 17.8

92.9 28.4

101.6 26.9

80.1 6.0

100.0 6.7

100.5 5.8

90.2 11.1

84.8 11.6

81.0 11.2

94.2 10.7

77.9 11.9

75.5 10.6

65.7 27.6

91.4 12.9

79.4 12.9

65.8 5.0

49.8 10.9

88.7 16.3

75.5 7.3

68.6 22.7

90.0 9.7

24.8 6.6

11.8 0.9

N9

N10

N11

N12

N13

N14

N15

N16

N17

Doxorubicin

109.7 15.2

152.8 11.6

61.9 8.3

53.1 8.6

130.4 29.7

130.8 26.5

64.4 7.3

22.2 3.0

14.1 3.8

33.1 3.8

21.0 5.1

2.6. Hybrid in silico studies

The synthesized molecules (N1eN17) were targeted to human

COX-1 and COX-2 target proteins. The interactions occurred be-

tween the ligands and the crucial amino acid residues at the target

proteins were determined by in silico simulations. The pharmaco-

kinetics (absorption, distribution, metabolism and excretion

(ADME)) characteristics of the synthesized molecules were also

determined. The toxicities that molecules could cause were also

estimated. The therapeutic activities against various diseases were

investigated.

Table 2

IC₅₀values of DOX and N17 in the HT-29 colon cancer, PC-3 prostate cancer, MCF-7

breast cancer, and HUVEC healthy cell lines. The values are expressed as the mean

SE of three independent experiments.

Compound (

mM)

HT-29

PC-3

MCF-7

HUVEC

Doxorubicin

N17

0.15 0.06

9.24 1.71

16.86 1.19

>100

0.16 0.08

11.35 1.55

0.10 0.04

>200

2.6.1. Molecular docking studies

determined by clonogenic assay in a dose-dependent manner. As

shown in Fig. 4, the treatment of N17 led to a dose-dependent

decrease in the colony number of breast cancer cells. Even, it was

After ligand and protein preparation, grid boxes were generated

from the ligands at the binding pockets of the target proteins. The

ligands (N1eN17) were docked into these grid boxes of human

Fig. 4. The effect of N17 on colony formation ability of MCF-7 cancer cells. Cells were treated with 10 and 25 mM of the compound N17. ***p < 0.001.

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

show that some of the compounds do not form binding poses or

have low docking scores in COX-1 (Table 3). The obstacle in tar-

geting all these ligands to COX-1 while they form strong binding to

the COX-2 protein may represent target-speciﬁc inhibition of COX-2

by these compounds. In addition, the fact that nimesulide gives

better binding scores to COX-2 than to COX-1 protein conﬁrms both

in silico and in vitro results [23,40].

Apoptosis in a cell is governed by pro-apoptotic and anti-

apoptotic proteins of the BCL-2 family. When BCL-2 family get

unbalanced, it can prevent the cells from undergoing apoptosis and

cause tumor growth. BCL-2 is overexpressed in many human cancer

types and has often been associated with the increase in tumor

development and resistance to cancer treatment. Thus, BCL-2

protein is one of the key targets for cancer treatment [41e43].

Most of the compounds studied showed higher docking scores

against the BCL-2 protein (Table 3) compared to the FDA-approved

BCL-2 inhibitor Venetoclax (Table 4). This could indicate that these

molecules could be promising inhibitors for the BCL-2 target.

2.6.2. Interactions between studied target proteins and hit

molecules investigated by MD simulations

Interactions between COX-1, COX-2 and BCL-2 target proteins

and synthesized compounds were investigated throughout the MD

simulations. Compounds that show successful docking poses at the

target proteins were used in MD simulations. For this aim, top-

docking poses were used. Simulations were run at 310 K for 100

ns in the Desmond program [44]. MM/GBSA analyses were con-

ducted for the assessment of the average binding free energies of

the compounds at the binding sites of the targets. Fig. 7 shows 2D

ligand (N10) interactions diagrams, residue interaction fractions

and N10-COX-1 residue contact maps during the simulations. Re-

sults show that especially Val349, Tyr385, Ser530, and Leu531 have

crucial interactions for the ligand binding, and they mainly

Fig. 5. The effects of N17 on BAX/BCL-2 expression levels in MCF-7 breast cancer cells.

Cells were treated with 10 and 25

mM of the compound N17 for 24 h. C:

Control.***p < 0.001.

COX-1 and COX-2 proteins to estimate their binding energies and

binding poses. All molecular docking studies were performed by a

grid-based docking program (Glide) [39]. Tables 3 and 4 show the

scores of top-docking poses of the studied compounds and refer-

ence molecules (i.e., known inhibitors of studied targets). Results

Fig. 6. The ultrastructural changes of treated colon cancer cells were displayed by SEM analyses. The treatment of N17 (C,D) for 48 h led to the loss of normal morphology and

normal cell size as compared to the untreated cells (A,B). Magniﬁcations: 700x for A; 750x for C; 1,500x for B, D micrographs.

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European Journal of Medicinal Chemistry 221 (2021) 113566

Table 3

Molecular docking scores of the compounds against COX-1, COX-2, and BCL-2 targets.

Compound

COX-1 docking score (kcal/mol)

COX-2 docking score (kcal/mol)

BCL-2 docking score (kcal/mol)

N1

N2

N3

N4

N5

N6

N7

N8

e

ꢀ6.255

ꢀ5.176

ꢀ5.072

ꢀ5.199

ꢀ4.701

ꢀ5.643

ꢀ5.365

ꢀ6.412

ꢀ6.111

ꢀ4.878

ꢀ5.306

ꢀ5.682

ꢀ6.798

ꢀ5.380

ꢀ6.171

ꢀ6.033

ꢀ6.420

ꢀ5.593

ꢀ4.041

e

ꢀ5.725

ꢀ5.740

ꢀ5.565

ꢀ5.580

ꢀ3.406

e

N9

e

N10

N11

N12

N13

N14

N15

N16

N17

ꢀ5.397

e

ꢀ2.472

ꢀ6.447

e

ꢀ5.287

e

ꢀ4.869

e

ꢀ8.648

Table 4

Molecular docking scores of the reference molecules against COX-1, COX-2, and BCL-2 targets.

Reference Molecules Docking Scores (kcal/mol)

Celecoxib

COX-1

COX-2

BCL-2

ꢀ4.255

ꢀ3.641

ꢀ9.742

ꢀ8.014

ꢀ4.770

ꢀ5.594

Nimesulide

Rofecoxib

Nimesulide

Venetoclax

Nimesulide

maintain their contacts with the N10. Hydrophobic interactions

occur with Val116, Val349, Tyr355, Ala527, Leu531 amino acids

while hydrogen bonds are established with Tyr385, Ser530 amino

acids, and water bridges are formed with Tyr348, Tyr355, Tyr385

amino acid residues.

Fig. 8 shows 2D ligand interactions diagram, residue interaction

fractions and COX-2 residue contact maps during the simulations

for N8. MD results show that especially Val116, Arg120, Leu352,

Leu359, Arg513, Phe518, and Val523 form crucial interactions with

the N8. Hydrogen bonds are established with Ser353, Ser359, and

Ser530 residues while hydrophobic interactions occur at Val349,

Tyr355, Leu359, Leu531, and Leu534 amino acids at the binding site.

In addition, water bridges are formed with Leu352, Tyr355, Tyr385,

and Ser530.

Fig. 9 shows ligand interactions diagram, residue interaction

fractions and BCL-2 residue contact maps during the simulations

for N17. Simulation results show that especially Phe101, Asp108,

Phe109, Met112, Gln115, Leu134, and Arg143 at the BCL-2 binding

site construct critical interactions with the N17 and these in-

teractions maintain throughout the simulations. Hydrogen bonds

are formed with Asp108 and Gln115 residues. Furthermore, hy-

drophobic interactions occurred with Phe101, Phe109, Met112,

Leu134, and Arg143 and water bridges are formed with Asp108 and

Glu111.

proteins were prepared, and MD simulations were performed. All

simulation protocols were conducted in the same way using the

same settings as the other studied ligand-protein complex systems

in this study.

Average MM/GBSA scores of Celecoxib, Rofecoxib and Ven-

etoclax were found as ꢀ72.58, ꢀ65.56, and ꢀ81.85 kcal/mol,

respectively. While average MM/GBSA results of selected hit ligands

and reference compounds have similar binding free energy results,

MM/GBSA score of Venetoclax was found higher compared to hit

compounds. The reason could be different molecular sizes of the

hits and Venetoclax. Molecular weight of Venetoclax (868.4 g/mol)

is much higher than studied compounds. Moreover, Nimesulide

was used as the reference compound and its average MM/GBSA

scores were found as ꢀ53.62 and ꢀ36.39 kcal/mol for COX-2 and

BCL-2 proteins, respectively. The MM/GBSA results of the hit mol-

ecules were compared with the values given by the reference

molecules and it was determined that they were in an acceptable

range (Tables 5 and 6). The results of reference compounds were

considered, and molecules with similar or higher docking and MM/

GBSA scores were selected. As a result, N2 and N10 at COX-1; N6,

N7, N8, N9, N13, and N17 at COX-2; N8, N10, N13, and N17 at BCL-2

showed higher interaction energy scores (in absolute values)

compared to other compounds.

Table 5 shows average Molecular Mechanics/Generalized Born

Surface Area (MM/GBSA) results for the selected compounds at

three different targets. In order to compare the binding free energy

results, same MD simulations protocols were also conducted for

known inhibitors of studied targets. Reference complex structures

were used directly from their co-crystallized forms. The known li-

gands Celecoxib, Rofecoxib and Venetoclax that were co-

crystallized with the protein were kept in their targets COX-1

(6Y3C), COX-2 (5KIR) and BCL-2 (4LXD), respectively. These target

2.6.3. Pharmacokinetic properties, toxicity and disease-QSAR

analysis of the molecules

The pharmacokinetic characteristics, toxicity properties, me-

tabolites and therapeutic activities of the molecules were predicted

by MetaCore/MetaDrug platform which is an integrated software

suite of Clarivate Analytics (https://portal.genego.com/). 25 com-

mon diseases and 26 different toxicity quantitative structure-

activity relationships (QSAR) models were used in the analysis of

synthesized compounds (Table 7).

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European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 7. 2D and 3D ligand interactions diagrams, interaction fractions and contact map of N10 at the binding pocket residues of COX-1.

All compounds were screened at the inﬂammation-QSAR model.

The used inﬂammation disease-QSAR model (Training set N ¼ 598,

test set N ¼ 93) has following statistical results: Sensitivity ¼ 0.86,

Speciﬁcity ¼ 0.84, Accuracy ¼ 0.85, MCC ¼ 0.69.

2.7. Structure activity relationship

According to the anti-inﬂammatory and anticancer studies of

NDs, the preliminary structure activity relationship (SAR) was

characterized (Fig. 10). It was determined that compound N8, N-(3-

ethoxy-4-(methylsulfonamido)phenyl)-4-

nitrobenzenesulfonamide and compound N10, N-(3-isopropoxy-4-

(methylsulfonamido)phenyl)-4-ﬂuorobenzamide were showed

better COX-2 inhibition (88.0 and 72.8%, respectively) than nime-

sulide and compatible with celecoxib. N8 and N10 decreased the

activity of COX-1 enzyme (21.0 and 27.2%, respectively) but their

selectivity scores were still better than nimesulide. Likewise,

compound N8 and N10 suppressed the COX-2 mRNA expressions in

Compounds N6, N7, N8, N9, N10, N15, N16 and N17 showed

activity prediction against inﬂammation in binary QSAR models

(Table 7). It was also determined that all molecules obey the

druggability rules and can be promising drug candidates for the

future (Supporting Information, Table S2).

Precited therapeutic activity values of compounds against can-

cer were screened using the binary QSAR models. The used cancer

disease-QSAR model (Training set N ¼ 886, test set N ¼ 167) has

following statistical results: sensitivity ¼ 0.89, speciﬁcity ¼ 0.83,

accuracy ¼ 0.86, MCC ¼ 0.72.

LPS-induced THP-1 macrophages (47 and 53% for 50

mM doses, 60

Molecules with predicted therapeutic activity values higher

than 0.5 were identiﬁed as molecules with therapeutic activity

potential. Accordingly, it is predicted that N6, N7, N8, N9, N12, N13,

N14 and N15 molecules may have anti-cancer effects (Table 8). The

fact that these molecules were found to target the BCL-2 protein

(Table 3), which is often associated with cancer, was also conﬁrmed

by the prediction of cancer therapeutic activities of the molecules

in QSAR studies.

and 61% for 100 M doses, respectively). Furthermore, it was

m

observed that compound N8 and N10 suppressed the release of

PGE₂and NO, effectively. When the other compounds were eval-

uated in detail, nitro containing amide derivatives named as

N1eN4 and 2-nitrophenylsulfonamide derivative N6 showed

similar or better inhibitory effect as compared to the standard

compound, nimesulide. Unlike, compound N7 (3-nitrosubstituted

sulfonamide derivative) and compound N9 (isopropyl ether

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 8. 2D and 3D ligand interactions diagrams, interaction fractions and contact map of N8 at the binding pocket residues of COX-2.

derivative) which are structurally very similar to N8 did not show

signiﬁcant COX-2 inhibitory activity. So, it can be concluded that

among the nitro containing amide and sulfonamide derivatives

(N1eN9), the effective compound must have nitro group at the 4-

position of the phenyl ring, must be ethyl ether derivative and must

connect the two phenyl rings with sulfonamide bridge to show

good anti-inﬂammatory effect. Among the nitro-free and eF,

eOCH₃, eCONH₂containing amide and sulfonamide derivatives

(N10eN17), while isopropoxy bearing 4-ﬂuorobenzamide com-

pound (N10) showed good anti-inﬂammatory effects, compounds

N14 (sulfonamide derivative), N16 (ethoxy derivative) and N17

(phenoxy derivative) did not show meaningful COX-2 inhibitory

activity. The positive effect of groups (-OCH₃, eCONH₂) except

ﬂuorine at 4- position of phenyl ring was not determined.

moderate cytotoxic effects. Also, it was observed that there was no

correlation between COX inhibition and anticancer results due to

compounds showing high COX-2 inhibitory activity-N8 and N10

were not the most active antitumor agents. However, it can be said

that compound N10 decreased the viability of colon cancer cells to

some extent (49.6%).

3. Conclusions

In this study, a series of new amide/sulfonamide NDs (N1eN17)

were synthesized, characterized, and evaluated for their ability to

inhibit COX enzymes/several pro-inﬂammatory factors and for

their antiproliferative effects on different cancer cell lines.

Accordingly, among seventeen compounds, N8 as a nitro containing

sulfonamide and N10 as a nitro-free amide derivative demon-

strated a highly potent and relatively selective inhibitory effect on

COX-2 enzyme with anti-inﬂammatory potential as evidenced by

cell-free and cell-based in vitro studies. At the same time, N8 and

N10 were not cytotoxic neither in three different cancer cell lines

nor in normal endothelial cells at their active concentrations,

therefore, they could be further optimized and explored as poten-

tially safe lead molecules. For cytotoxicity evaluations, among all

When anticancer results of compounds N1eN17 were evalu-

ated,

ﬂuorobenzamide (N17) was found to be most active anti-

proliferative compound on HT-29 colon cancer cells (IC₅₀: 9.24 M)

and on MCF-7 breast cancer cells (IC₅₀: 11.35 M) with minimal

N-(3-phenoxy-4-(methylsulfonamido)phenyl)-4-

m

effect on healthy cells. In addition, compounds N2, N11 and N13 for

colon cancer, compounds N8, N11, N12 and N15 for prostate cancer,

compounds N5, N9, N12, N13 and N16 for breast cancer showed

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European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 9. 2D and 3D ligand interactions diagrams, interaction fractions and contact map of N17 at the binding pocket residues of BCL-2.

Table 5

action studies, as well as docking and MD simulations provided

partial evidence for the apoptotic cell death and possible involve-

ment of BAX/BCL-2directed pathway. Besides, the results of docking

and MD simulation studies elucidated the reasons for the selec-

tivity of the synthesized compounds against COX enzymes. Overall,

all synthesized compounds obey the druggability rules and can be

promising drug candidates for future developments due to their

acceptable ADME proﬁles and drug-likeness properties. Based on

the ﬁndings in this study, it could be hypothesized that amide and

sulfonamide derivatives of nimesulide may confer a new approach

to obtain more potent and safer candidates for NSAIDs.

Average MM/GBSA free energy values of the studied compounds at the human COX-

1, COX-2 and BCL-2 protein targets during the MD simulations.

Target

Compound

Average MM/GBSA score (kcal/mol)

COX -1

COX-1

COX-2

BCL-2

N2

ꢀ71.02

ꢀ52.84

ꢀ58.59

ꢀ59.05

ꢀ60.27

ꢀ66.88

ꢀ58.65

ꢀ65.33

ꢀ69.22

ꢀ46.58

ꢀ72.83

ꢀ67.96

ꢀ74.37

ꢀ48.07

ꢀ50.24

ꢀ68.43

ꢀ66.53

N10

N13

N16

N2

N6

N7

N8

N9

N10

N13

N15

N17

N8

N10

N13

N17

4. Experimental

4.1. Chemistry

All chemicals were supplied from commercial sources and used

directly unless otherwise stated. Melting points were recorded on a

X-4 melting-point apparatus and uncorrected. Thin-layer chroma-

tography was performed using precoated Kieselgel 60GF₂₅₄

(Merck) plates. The visualization of the spots was done by 254 nm

UV light. Column chromatography technique was applied for pu-

riﬁcation of products using silica gel 60 (230e400 mesh, Merck).

PerkinElmer Spectrum 100 FTIR spectrophotometer was used to

record infrared spectra with ATR apparatus. ¹H and ¹³C spectra were

tested compounds only N17 exhibited a good potency, which is

closer to widely used chemotherapeutic agent doxorubicin, in colon

and breast cancer cells with no observed toxicity in healthy cells at

the highest tested concentration (200

mM). The mechanism of

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European Journal of Medicinal Chemistry 221 (2021) 113566

Table 6

Average MM/GBSA free energy values of the reference molecules against COX-1, COX-2, and BCL-2 targets during the MD simulations.

MM/GBSA Scores of Reference Molecules (kcal/mol)

COX-1

COX-2

Celecoxib

ꢀ72.58

ꢀ65.56

ꢀ53.62

ꢀ81.85

ꢀ36.39

Rofecoxib

Nimesulide

Venetoclax

Nimesulide

BCL-2

Table 7

doublet; t, triplet; q, quartet; sept, septet; m, multiplet; dd, double

doublet; dt, double triplet; td, triple doublet and ddd, doublet of

doublet of doublets. The molecular weights of products were

detected by Shimadzu LC-MS/MS 8040 Liquid Chromatograph Mass

Spectrometer with an ESI source. The purity of compounds (>95%)

was conﬁrmed by the Shimadzu Prominence LC-20A Semi-Pre-

parative HPLC system with a PDA detector and Shim-pack ODS(H)

Therapeutic activity predictions of studied compounds by the

Inﬂammation-QSAR models.

Compound

Inﬂammation-QSAR^a

N1

N2

N3

N4

N5

N6

N7

N8

0.38

0.40

0.46

0.41

0.30

0.34

0.40

0.39

0.66

0.40

0.58

0.54

0.67

0.45

0.68

0.65

250 ꢂ 4.6 mm, 5

mm C18 column. The gradient program was set 20%

H₂O (B) in methanol (A) was used from 0 to 5 min, 20e60% B from 5

to 30 min, 60% B from 30 to 35 min, 60-20% B from 35 to 40 min and

20% B from 40 to 45 min with a ﬂow rate 1.0 mL/min. Literature

search of compounds was done using Reaxys database.

N9

N10

N11

N12

N13

N14

N15

N16

N17

4.1.1. General procedure a for the synthesis of N1eN4, N6eN11 and

N14eN17

N-(4-Amino-2-alkoxyphenyl)methanesulfonamide derivative

was dissolved in enough chloroform. Triethylamine (Et₃N) and

substituted benzoyl chloride or substituted benzenesulfonyl chlo-

ride were added dropwise to the solution in an ice bath. The

mixture was stirred at room temperature for various reaction times.

After completion of the reaction detected by TLC (hexane:ethyl

acetate), the mixture was poured into ice-water and extracted with

CHCl₃(3 times). The combined organic layer was washed with 10%

HCl and water, respectively, dried over anhydrous Na₂SO₄and

concentrated in vacuo. The crude product was puriﬁed using

crystallization or column chromatography techniques.

a

Potential anti-inﬂammatory activity. Cutoff is 0.5. Values

higher than 0.5 indicate potentially active compounds. Training

set consists of approved drugs. Model description: Training set

N ¼ 598, Test set N ¼ 93, Sensitivity ¼ 0.86, Speciﬁcity ¼ 0.84,

Accuracy ¼ 0.85, MCC ¼ 0.69. Reference: Clarivate Analytics.

Table 8

Therapeutic activity predictions of studied compounds by

the Cancer- QSAR models.

Compound

Cancer-QSAR^a

4.1.1.1. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-2-

nitrobenzamide (N1). The reaction was performed under general

procedure A conditions using N-(4-amino-2-ethoxyphenyl)meth-

anesulfonamide (0.217 mmol; 50 mg), 2-nitrobenzoyl chloride

N1

N2

N3

N4

N5

N6

N7

N8

0.36

0.32

0.45

0.46

0.54

0.56

0.44

0.43

0.51

0.56

0.62

0.60

0.29

0.45

(0.26 mmol; 48.3 mg; 34.4

mL; 1.2 equiv.) and Et₃N (0.26 mmol;

26.4 mg; 36.3 L; 1.2 equiv.) with 2.5 h reaction time. The crude

m

product was puriﬁed by crystallization with ethyl alcohol to give

compound N1. Cream-colored solid; 54 mg; 65% yield;

m.p.149e151 ^ꢁC; R_f¼ 0.28 (hexane:ethyl acetate, 1:1); IR (ATR) w

3308, 3258, 3113, 3080, 3024, 2986, 2937, 2879, 1655, 1608, 1509,

N9

N10

N11

N12

N13

N14

N15

N16

N17

1475, 1420, 1331, 1315, 1257, 1147, 964 cm^ꢀ1

;

¹H NMR (400 MHz,

10.67 (s, 1H), 8.79 (s, 1H), 8.11 (dd, J ¼ 8.5 and

DMSO‑d₆, ppm):

d

1.3 Hz, 1H), 7.84 (td, J ¼ 7.8 and 1.0 Hz, 1H), 7.72e7.74 (m, 2H), 7.45

(d, J ¼ 2.0 Hz, 1H), 7.11e7.17 (dd and t, J ¼ 8.6, 2.6 and 8.6 Hz, 2H),

4.01 (q, J ¼ 6.9 Hz, 2H), 2.88 (s, 3H), 1.35 (t, J ¼ 6.9 Hz, 3H); ¹³C NMR

(100 MHz, DMSO‑d₆, ppm): 164.6, 153.1, 146.9, 138.6, 134.6, 133.1,

131.6, 129.8, 128.2, 124.8, 121.8, 111.9, 104.6, 64.3, 40.6, 15.1; LC/MS

(ESI) m/z: 378.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 3.47 min, 99.66%

purity.

a

Potential activity against cancer. Cutoff is 0.5. Values

higher than 0.5 indicate potentially active compounds.

Training set consists of approved drugs. Model descrip-

tion: Training set

N

¼

886, Test set

N

¼

167,

Sensitivity ¼ 0.89, Speciﬁcity ¼ 0.83, Accuracy ¼ 0.86,

MCC ¼ 0.72. Reference: Clarivate Analytics.

4.1.1.2. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-3-

nitrobenzamide (N2). The reaction was performed under general

procedure A conditions using N-(4-amino-2-ethoxyphenyl) meth-

anesulfonamide (0.217 mmol; 50 mg), 3-nitrobenzoyl chloride

(0.26 mmol; 48.3 mg; 1.2 equiv.) and Et₃N (0.26 mmol; 26.4 mg;

collected on 400/100 MHz Jeol and 600/150 MHz Agilent High-

Performance Digital FT-NMR spectrometers using DMSO‑d₆as

solvent. Splitting patterns are reported as follows: s, singlet; d,

36.3 mL; 1.2 equiv.) with 3 h reaction time. The crude product was

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European Journal of Medicinal Chemistry 221 (2021) 113566

Fig. 10. An overview of biological data of potent nimesulide derivatives.

puriﬁed by crystallization with ethyl alcohol to give compound N2.

White solid; 54 mg; 65% yield; m.p.177e179 ^ꢁC; R_f¼ 0.43 (hex-

ane:ethyl acetate, 1:1); IR (ATR) w 3349, 3243, 3088, 2983, 2939,

compound N4. Yellow-green solid; 59 mg; 73% yield;

m.p.214e217 ^ꢁC; R_f¼ 0.63 (hexane:ethyl acetate, 1:1); IR (ATR) w

3394, 3277, 3108, 3082, 3060, 3016, 2979, 2931, 2864, 1680, 1602,

1658, 1610, 1533, 1475, 1335, 1320, 1272, 1156, 972 cm^ꢀ1

;

¹H NMR

1510, 1416, 1400, 1346, 1323, 1264, 1155, 979 cm^ꢀ1

;

¹H NMR

(400 MHz, DMSO‑d₆, ppm):

d

10.56 (s, 1H), 8.81 (s, 1H), 8.75 (t,

(600 MHz, DMSO‑d₆, ppm): d 10.52 (s, 1H), 8.69 (s, 1H), 8.35 (d,

J ¼ 1.9 Hz, 1H), 8.41 (ddd, J ¼ 8.2, 2.2 and 0.8 Hz, 1H), 8.36 (dt, J ¼ 7.9

and 1.4 Hz, 1H), 7.81 (t, J ¼ 7.9 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz, 1H), 7.32

(dd, J ¼ 8.5 and 2.1 Hz, 1H), 7.18 (d, J ¼ 8.5 Hz, 1H), 4.1 (q, J ¼ 7.0 Hz,

2H), 2.89 (s, 3H), 1.36 (t, J ¼ 6.9 Hz, 3H); ¹³C NMR (100 MHz,

DMSO‑d₆, ppm): 163.8, 152.9, 148.3, 138.3, 136.7, 134.7, 130.8, 127.8,

126.8, 122.9, 122.0, 112.8, 105.6, 64.4, 40.7, 15.1; LC/MS (ESI) m/z:

378.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 3.62 min, 98.48% purity.

J ¼ 7.6 Hz, 2H), 8.16 (d, J ¼ 7.2 Hz, 2H), 7.58 (s, 1H), 7.31 (d, J ¼ 8.1 Hz,

1H), 7.20 (d, J ¼ 7.6 Hz, 1H), 4.57 (m, 1H), 2.91 (s, 3H), 1.33 (s, 6H);

¹³C NMR (150 MHz, DMSO‑d₆, ppm): 164.3, 151.5, 149.6, 141.0, 138.1,

129.6, 127.7, 124.0, 122.5, 112.5, 106.4, 70.6, 40.6, 22.1; LC/MS (ESI)

m/z: 392.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 26.54 min, 98.15% purity.

4.1.1.5. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-2-

nitrobenzenesulfonamide (N6). The reaction was performed under

general procedure A conditions using N-(4-amino-2-ethoxyphenyl)

methanesulfonamide (0.217 mmol; 50 mg), 2-nitrobenzenesulfonyl

chloride (0.26 mmol; 57.7 mg; 1.2 equiv.) and Et₃N (0.26 mmol;

4.1.1.3. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-4-

nitrobenzamide (N3). The reaction was performed under general

procedure A conditions using N-(4-amino-2-ethoxyphenyl)meth-

anesulfonamide (0.217 mmol; 50 mg), 4-nitrobenzoyl chloride

(0.26 mmol; 48.3 mg; 1.2 equiv.) and Et₃N (0.26 mmol; 26.4 mg;

26.4 mg; 36.3 mL; 1.2 equiv.) with 12 h reaction time. The crude

product was puriﬁed by column chromatography (silica gel) with

hexane:ethyl acetate (2:1) to give compound N6. White solid;

67 mg; 75% yield; m.p.174e178 ^ꢁC; R_f¼ 0.43 (hexane:ethyl acetate,

1:1); IR (ATR) w 3264, 3106, 3023, 2982, 2938, 2887, 1611, 1539,

36.3 mL; 1.2 equiv.) with 2 h reaction time. The crude product was

puriﬁed by washing with hot ethyl alcohol to give compound N3.

Yellow solid; 58 mg; 71% yield; m.p.242e244 ^ꢁC; R_f¼ 0.50 (hex-

ane:ethyl acetate, 1:1); IR (ATR) w 3393, 3267, 3111, 3068, 3017,

2973e2862, 1682, 1605, 1557, 1515, 1478, 1413, 1388, 1325, 1258,

1483, 1409, 1360, 1333, 1154, 1122, 916 cm^ꢀ1

;

¹H NMR (400 MHz,

10.71 (s, 1H), 8.76 (s, 1H), 7.92e7.96 (m, 2H),

DMSO‑d₆, ppm):

d

1155, 975 cm^ꢀ1; ¹H NMR (600 MHz, DMSO‑d₆, ppm):

d

10.55 (s, 1H),

7.76e7.83 (m, 2H), 7.05 (d, J ¼ 8.5 Hz, 1H), 6.75 (d, J ¼ 2.3 Hz, 1H),

6.59 (dd, J ¼ 8.5 and 2.3 Hz, 1H), 3.92 (q, J ¼ 6.9 Hz, 2H), 2.83 (s, 3H),

1.28 (t, J ¼ 6.9 Hz, 3H); ¹³C NMR (100 MHz, DMSO‑d₆, ppm): 153.1,

148.5, 136.0, 135.3, 133.1, 131.7, 130.5, 128.1, 125.1, 122.8, 112.7, 105.4,

64.3, 40.8, 14.8; LC/MS (ESI) m/z: 414.0 [M ꢀ H]^-; HPLC analysis,

t_R¼ 3.33 min, 98.07% purity.

8.79 (s, 1H), 8.36 (d, J ¼ 8.5 Hz, 2H), 8.16 (d, J ¼ 8.3 Hz, 2H), 7.58 (s,

1H), 7.32 (d, J ¼ 8.2 Hz,1H), 7.20 (d, J ¼ 8.4 Hz,1H), 4.06 (q, 2H), 2.92

(s, 3H), 1.39 (t, 3H); ¹³C NMR (150 MHz, DMSO‑d₆, ppm): 164.3,

152.7, 149.6, 141.0, 138.2, 129.6, 127.6, 124.0, 122.0, 112.7, 105.5, 64.3,

40.6, 15.0; LC/MS (ESI) m/z: 378.0 [M ꢀ H]^-; HPLC analysis,

t_R¼ 7.83 min, 99.56% purity.

4.1.1.6. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-3-

nitrobenzenesulfonamide (N7). The reaction was performed under

general procedure A conditions using N-(4-amino-2-ethoxyphenyl)

methanesulfonamide (0.217 mmol; 50 mg), 3-nitrobenzenesulfonyl

chloride (0.26 mmol; 57.7 mg; 1.2 equiv.) and Et₃N (0.26 mmol;

4.1.1.4. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

nitrobenzamide (N4). The reaction was performed under general

procedure A conditions using N-(4-amino-2-isopropoxyphenyl)

methanesulfonamide (0.205 mmol; 50 mg), 4-nitrobenzoyl chlo-

ride (0.246 mmol; 45.6 mg; 1.2 equiv.) and Et₃N (0.246 mmol;

26.4 mg; 36.3 mL; 1.2 equiv.) with 12 h reaction time. The crude

24.9 mg; 34.3

m

L; 1.2 equiv.) with 2.5 h reaction time. The crude

product was puriﬁed by crystallization with ethyl alcohol to give

product was puriﬁed by washing with cold diethyl ether to give

compound N7. White solid; 70 mg; 78% yield; m.p.181e184 ^ꢁC;

15

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European Journal of Medicinal Chemistry 221 (2021) 113566

R_f¼ 0.43 (hexane:ethyl acetate, 1:1); IR (ATR) w 3225, 3106, 3079,

3033, 2981e2878, 1612, 1526, 1487, 1405, 1351, 1343, 1297, 1151,

127.8, 122.0, 115.9 and 115.7 (d, J_CF¼ 21.79 Hz), 112.4, 106.2, 70.6,

40.6, 22.1; LC/MS (ESI) m/z: 367.0 [M þ H]^þ; HPLC analysis,

t_R¼ 4.29 min, 99.54% purity.

915 cm^ꢀ1; ¹H NMR (400 MHz, DMSO‑d₆, ppm):

d 10.53 (s, 1H), 8.75

(s,1H), 8.45 (t, J ¼ 2.0 Hz,1H), 8.415 (dd, J ¼ 7.9 and 1.51 Hz,1H), 8.12

(dt, J ¼ 7.9 and 1.14 Hz,1H), 7.83 (t, J ¼ 8.0 Hz,1H), 7.04 (d, J ¼ 8.5 Hz,

1H), 6.75 (d, J ¼ 2.3 Hz, 1H), 6.57 (dd, J ¼ 8.5 and 2.3 Hz, 1H), 3.92 (q,

J ¼ 6.9 Hz, 2H), 2.83 (s, 3H), 1.27 (t, J ¼ 6.9 Hz, 3H); ¹³C NMR

(100 MHz, DMSO‑d₆, ppm): 153.1, 148.4, 141.3, 136.1, 133.2, 132.0,

128.2, 128.0, 123.0, 122.0, 113.0, 105.8, 64.4, 40.7, 14.8; LC/MS (ESI)

m/z: 414.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 22.80 min, 99.76% purity.

4.1.1.10. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

methoxybenzamide (N11). The reaction was performed under gen-

eral

isopropoxyphenyl)methanesulfonamide (0.205 mmol; 50 mg), 4-

methoxybenzoyl chloride (0.245 mmol; 41.9 mg; 33.3 L; 1.2

equiv.) and Et₃N (0.245 mmol; 24.8 mg; 34.2 L; 1.2 equiv.) with 2 h

procedure

A

conditions

using

N-(4-amino-2-

m

reaction time. The crude product was puriﬁed by washing with cold

diethyl ether to give compound N11. Cream-colored solid; 58 mg;

75% yield; m.p.182e185 ^ꢁC; R_f¼ 0.53 (hexane:ethyl acetate, 1:1); IR

(ATR) w 3345, 3242, 3057, 3024, 3006, 2981e2838,1648,1605,1506,

4.1.1.7. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-4-

nitrobenzenesulfonamide (N8). The reaction was performed under

general procedure A conditions using N-(4-amino-2-ethoxyphenyl)

methanesulfonamide (0.217 mmol; 50 mg), 4-nitrobenzenesulfonyl

chloride (0.26 mmol; 57.7 mg; 1.2 equiv.) and Et₃N (0.26 mmol;

1432, 1410, 1383, 1327, 1251, 1122, 1158 cm^ꢀ1

DMSO‑d₆, ppm):

;

¹H NMR (600 MHz,

10.05 (s, 1H), 8.64 (s, 1H), 7.94 (d, J ¼ 7.2 Hz, 2H),

d

26.4 mg; 36.3

mL; 1.2 equiv.) with 12 h reaction time. The crude

7.59 (s, 1H), 7.29 (d, J ¼ 8.2 Hz, 1H), 7.16 (d, J ¼ 8.0 Hz, 1H), 7.04 (d,

J ¼ 7.2 Hz, 2H), 4.57 (m, 1H), 3.83 (s, 3H), 2.90 (s, 3H), 1.33 (s, 6H);

¹³C NMR (150 MHz, DMSO‑d₆, ppm): 165.3,162.4,151.5,138.9,130.0,

127.9, 127.3, 121.7, 114.1, 112.3, 106.1, 70.5, 55.9, 40.6, 22.1; LC/MS

(ESI) m/z: 377.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 25.04 min, 96.45%

purity.

product was puriﬁed by column chromatography (silica gel) with

hexane:ethyl acetate (3:1) to give compound N8. White solid;

63 mg; 70% yield; m.p.199e200 ^ꢁC; R_f¼ 0.50 (hexane:ethyl acetate,

1:1); IR (ATR) w 3244, 3196, 3114, 3082, 3022, 2989, 2936, 2878,

1610, 1533, 1470, 1403, 1343, 1318, 1287, 1157, 982 cm^ꢀ1

;

¹H NMR

(400 MHz, DMSO‑d₆, ppm):

d 10.59 (s, 1H), 8.75 (s, 1H), 8.34 (dt,

J ¼ 8.9 and 2.2 Hz, 2H), 7.97 (dt, J ¼ 8.8 and 2.2 Hz, 2H), 7.03 (d,

4.1.1.11. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

ﬂuorobenzenesulfonamide (N14). The reaction was performed un-

J ¼ 8.5 Hz, 1H), 6.74 (d, J ¼ 2.3 Hz, 1H), 6.57 (dd, J ¼ 8.5 and 2.3 Hz,

1H), 3.92 (q, J ¼ 6.9 Hz, 2H), 2.84 (s, 3H), 1.28 (t, J ¼ 6.9 Hz, 3H); ¹³

C

der general procedure

A

conditions using N-(4-amino-2-

NMR (100 MHz, DMSO‑d₆, ppm): 153.1, 150.4, 145.3, 136.3, 128.9,

128.1, 125.2, 122.8, 112.7, 105.5, 64.4, 40.8, 14.8; LC/MS (ESI) m/z:

414.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 3.53 min, 99.63% purity.

isopropoxyphenyl)methanesulfonamide (0.205 mmol; 50 mg), 4-

ﬂuorobenzenesulfonyl chloride (0.245 mmol; 47.8 mg; 1.2 equiv.)

and Et₃N (0.245 mmol; 24.8 mg; 34.2 mL; 1.2 equiv.) with 12 h re-

action time. The crude product was puriﬁed by column chroma-

tography (silica gel) with hexane:ethyl acetate (6:1) to give

compound N14. White solid; 58 mg; 70% yield; m.p.172e174 ^ꢁC;

R_f¼ 0.65 (hexane:ethyl acetate, 1:1); IR (ATR) w 3244, 3212, 3107,

3082, 2982, 2937, 2897, 1610, 1592, 1493, 1408, 1336, 1239, 1153,

4.1.1.8. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

nitrobenzenesulfonamide (N9). The reaction was performed under

general

procedure

A

conditions

using

N-(4-amino-2-

isopropoxyphenyl)methanesulfonamide (0.41 mmol; 0.1 g), 4-

nitrobenzenesulfonyl chloride (0.62 mmol; 0.14 g; 1.5 equiv.) and

1119 cm^ꢀ1; ¹H NMR (600 MHz, DMSO‑d₆, ppm):

d 10.25 (s, 1H), 8.63

Et₃N (0.62 mmol; 62.0 mg; 86.0

m

L; 1.5 equiv.) with 24 h reaction

(s,1H), 7.79 (q, J ¼ 5.1 and 3.6 Hz, 2H), 7.39 (t, J ¼ 8.8 Hz, 2H), 7.05 (d,

J ¼ 8.5 Hz, 1H), 6.71 (d, J ¼ 2.1 Hz, 1H), 6.57 (dd, J ¼ 8.5 and 2.2 Hz,

1H), 4.41 (sept, J ¼ 6.0 Hz, 1H), 2.84 (s, 3H), 1.20 (d, J ¼ 6.0 Hz, 6H);

¹³C NMR (150 MHz, DMSO‑d₆, ppm): 165.6 and 163.9 (d,

J_CF¼ 251.6 Hz), 151.7, 136.6, 136.18 and 136.16 (d, J_CF¼ 2.95 Hz),

130.23 and 130.16 (d, J_CF¼ 9.57 Hz), 128.1, 123.0, 117.0 and 116.9 (d,

J_CF¼ 22.9 Hz), 112.6, 106.4, 70.6, 40.7, 21.8; LC/MS (ESI) m/z: 401.0

[M ꢀ H]^-; HPLC analysis, t_R¼ 23.06 min, 99.80% purity.

time. The crude product was puriﬁed by column chromatography

(silica gel) with hexane:ethyl acetate (2:1) to give compound N9.

Cream-colored solid; 96 mg; 55% yield; m.p.204e207 ^ꢁC; R_f¼ 0.63

(hexane:ethyl acetate, 1:1); IR (ATR) w 3262, 3220, 3117, 3082, 2980,

2932, 2876, 1608, 1536, 1464, 1396, 1337, 1314, 1286, 1156,

858 cm^ꢀ1; ¹H NMR (600 MHz, DMSO‑d₆, ppm):

d 10.58 (s, 1H), 8.70

(s, 1H), 8.37 (d, J ¼ 8.4 Hz, 2H), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.07 (d,

J ¼ 8.5 Hz, 1H), 6.73 (s, 1H), 6.58 (d, J ¼ 8.5 Hz, 1H), 4.43 (m,

J ¼ 6.0 Hz, 1H), 2.84 (s, 3H), 1.21 (d, J ¼ 6.0 Hz, 6H); ¹³C NMR

(150 MHz, DMSO‑d₆, ppm): 151.7, 150.3, 145.2, 135.9, 128.8, 128.1,

125.2, 123.4, 112.8, 106.6, 70.6, 40.7, 21.8; LC/MS (ESI) m/z: 428.0

[M ꢀ H]^-; HPLC analysis, t_R¼ 8.83 min, 99.49% purity.

4.1.1.12. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

methoxybenzenesulfonamide (N15). The reaction was performed

under general procedure

A conditions using N-(4-amino-2-

isopropoxyphenyl)methanesulfonamide (0.205 mmol; 50 mg), 4-

methoxybenzenesulfonyl chloride (0.245 mmol; 50.6 mg; 1.2

4.1.1.9. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-

ﬂuorobenzamide (N10). The reaction was performed under general

procedure A conditions using N-(4-amino-2-isopropoxyphenyl)

methanesulfonamide (0.205 mmol; 50 mg), 4-ﬂuorobenzoyl chlo-

equiv.) and Et₃N (0.245 mmol; 24.8 mg; 34.2 mL; 1.2 equiv.) with

12 h reaction time. The crude product was puriﬁed by washing with

cold diethyl ether to give compound N15. Cream-colored solid;

43 mg; 51% yield; m.p.182e184 ^ꢁC; R_f¼ 0.45 (hexane:ethyl acetate,

1:1); IR (ATR) w 3237, 3114, 3009, 2974e2849, 1610, 1595, 1498,

ride (0.245 mmol; 38.9 mg; 29.0

mL; 1.2 equiv.) and Et₃N

(0.245 mmol; 24.8 mg; 34.2 L; 1.2 equiv.) with 2 h reaction time.

m

1472, 1328, 1262, 1121, 1153 cm^ꢀ1

ppm):

3H), 6.73 (s, 1H), 6.56 (d, J ¼ 8.2 Hz, 1H), 4.40 (m, 1H), 3.77 (s, 3H),

2.84 (s, 3H), 1.21 (s, 6H); ¹³C NMR (150 MHz, DMSO‑d₆, ppm): 162.9,

151.7, 137.2, 131.5, 129.3, 128.2, 122.5, 114.9, 112.1, 105.9, 70.6, 56.1,

40.7, 21.8; LC/MS (ESI) m/z: 413.0 [M ꢀ H]^-; HPLC analysis,

t_R¼ 22.15 min, 98.48% purity.

;

¹H NMR (600 MHz, DMSO‑d₆,

10.09 (s, 1H), 8.60 (s, 1H), 7.67 (d, J ¼ 7.5 Hz, 2H), 7.04 (m,

The crude product was puriﬁed by washing with cold diethyl ether

to give compound N10. White solid; 60 mg; 81% yield;

m.p.175e177 ^ꢁC; R_f¼ 0.70 (hexane:ethyl acetate, 1:1); IR (ATR) w

3338, 3247, 3081, 2986, 2936, 2910, 1651, 1603, 1505, 1420, 1385,

d

1328, 1273, 1156, 1110 cm^ꢀ^{1 1}H NMR (600 MHz, DMSO‑d₆, ppm):

;

d

10.22 (s, 1H), 8.66 (s, 1H), 8.01 (t, broad, 2H), 7.57 (s, 1H), 7.36 (t,

J ¼ 7.7 Hz, 2H), 7.28 (d, J ¼ 8.2 Hz, 1H), 7.17 (d, J ¼ 7.7 Hz, 1H), 4.565

(m, 1H), 2.90 (s, 3H), 1.33 (s, 6H); ¹³C NMR (150 MHz, DMSO‑d₆,

ppm): 165.4 and 163.7 (d, J_CF¼ 249.11 Hz), 164.8,151.5, 138.6, 131.73

and 131.72 (d, J_CF¼ 2.64 Hz), 130.82 and 130.76 (d, J_CF¼ 9.02 Hz),

4.1.1.13. N-(3-Ethoxy-4-(methylsulfonamido)phenyl)-4-

ﬂuorobenzamide (N16). The reaction was performed under general

procedure

A

conditions using N-(4-amino-2-ethoxyphenyl)

16

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

methanesulfonamide (0.51 mmol; 0.12 g), 4-ﬂuorobenzoyl chloride

(0.61 mmol; 97.0 mg; 1.2 equiv.) and Et₃N (0.61 mmol; 61.9 mg;

85.3 mL; 1.2 equiv.) with 3 h reaction time. The crude product was

ice-bath for 30 min and room temperature for 1 h. Then, Et₃N (3.0

equiv.) and N-(4-amino-2-alkoxyphenyl)methanesulfonamide de-

rivative (1.0 equiv.) were added, respectively. The mixture was

heated at 100e120 ^ꢁC for 24 h (comp. N12) and 43 h (comp. N13).

After completion of the reaction detected by TLC (hexane:ethyl

acetate), the mixture was cooled to room temperature and poured

into ice-water. If there was precipitation, the solid was ﬁltered and

dried, if not, extraction with ethyl acetate (3 times) was performed.

The combined organic layer was washed with NaHCO₃solution and

water, respectively, dried over anhydrous Na₂SO₄and concentrated

in vacuo. The crude products of N12 and N13 were puriﬁed by

washing with cold diethyl ether and column chromatography (sil-

ica gel, hexane:ethyl acetate, 2:3) techniques, respectively.

puriﬁed by washing with cold diethyl ether to give compound N16.

Bright brown solid; 0.149 g; 83% yield; m.p.198e199 ^ꢁC; R_f¼ 0.53

(hexane:ethyl acetate, 1:1); IR (ATR) w 3336, 3259, 3106, 3066,

2995, 2983, 2944, 2905, 1645, 1603, 1509, 1413, 1333, 1235, 1155,

1123 cm^ꢀ1; ¹H NMR (400 MHz, DMSO‑d₆, ppm):

d 10.24 (s, 1H), 8.77

(s, 1H), 8.00 (m, 2H), 7.57 (d, J ¼ 2.1 Hz, 1H), 7.35 (t, J ¼ 8.9 Hz, 2H),

7.29 (dd, J ¼ 8.6 and 1.9 Hz, 1H), 7.16 (d, J ¼ 8.6 Hz, 1H), 4.04 (q,

J ¼ 6.9 Hz, 2H), 2.89 (s, 3H), 1.36 (t, J ¼ 6.9 Hz, 3H); ¹³C NMR

(100 MHz, DMSO‑d₆, ppm): 165.9 and 163.4 (d, J_CF¼ 249.04 Hz),

164.9, 152.9, 138.8, 131.8, 130.9 and 130.8 (d, J_CF¼ 8.92 Hz), 127.9,

121.6, 116.1 and 115.8 (d, J_CF¼ 28.13 Hz), 112.1, 105.4, 64.3, 40.7, 15.1;

LC/MS (ESI) m/z: 353.0 [M þ H]^þ; HPLC analysis, t_R¼ 24.78 min,

98.78% purity.

4.1.3.1. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)tereph-

thalamide (N12). The reaction was performed under general pro-

cedure B conditions as described above. Light brown solid; 24%

yield; m.p.238e241 ^ꢁC; R_f¼ 0.20 (hexane:ethyl acetate, 2:3); IR

(ATR) w 3447, 3396,3346, 3263, 3185, 2982, 2936, 2901, 1649, 1607,

4.1.1.14. N-(3-Phenoxy-4-(methylsulfonamido)phenyl)-4-

ﬂuorobenzamide (N17). The reaction was performed under general

procedure

A

conditions using N-(4-amino-2-phenoxyphenyl)

1527, 1507, 1408, 1327, 1280, 1160 cm^ꢀ1

DMSO‑d₆, ppm):

;

¹H NMR (600 MHz,

d 10.33 (s, 1H), 8.72 (s, 1H), 8.12 (s, 1H), 7.99 (s, 4H),

methanesulfonamide (0.43 mmol; 0.12 g), 4-ﬂuorobenzoyl chloride

(0.52 mmol; 82.5 mg; 1.2 equiv.) and Et₃N (0.52 mmol; 52.6 mg;

72.5 mL; 1.2 equiv.) with 3 h reaction time. The crude product was

7.56 (d, 2H), 7.32 (d, J ¼ 8.6 Hz, 1H), 7.18 (d, J ¼ 8.6 Hz, 1H), 4.57 (m,

J ¼ 6.0 Hz, 1H), 2.89 (s, 3H), 1.33 (d, J ¼ 6.0 Hz, 6H); ¹³C NMR

(150 MHz, DMSO‑d₆, ppm): 167.5, 165.3, 151.5, 138.5, 137.5, 137.3,

128.0, 127.9, 122.1, 112.3, 106.1, 70.5, 40.6, 22.1; LC/MS (ESI) m/z:

390.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 20.72 min, 98.96% purity.

puriﬁed by washing with cold diethyl ether to give compound N17.

Cream-colored solid; 0.136 g; 79% yield; m.p.159e162 ^ꢁC; R_f¼ 0.73

(hexane:ethyl acetate, 1:1); IR (ATR) w 3336, 3240, 3083, 3061,

3029, 3012, 2934, 1651, 1603, 1588, 1506, 1488, 1399, 1324, 1200,

1157, 1115 cm^ꢀ1; ¹H NMR (400 MHz, DMSO‑d₆, ppm):

d

10.25 (s,1H),

4.1.3.2. N-(3-Phenoxy-4-(methylsulfonamido)phenyl)tereph-

thalamide (N13). The reaction was performed under general pro-

cedure B conditions as described above. Cream-colored solid; 25%

yield; m.p.228e231 ^ꢁC (dec.); R_f¼ 0.23 (hexane:ethyl acetate, 2:3);

IR (ATR) w 3453, 3340, 3232, 3060, 3023, 2935, 2870, 2839, 1673,

1654, 1606, 1544, 1509, 1492, 1406, 1389, 1320, 1277, 1160 cm^ꢀ1; ¹H

9.23 (s, 1H), 7.93 (m, 2H), 7.52 (dd, J ¼ 8.7 and 1.4 Hz, 1H), 7.42e7.28

(m, 3Hþ3H), 7.16 (m, 1H), 7.07 (m, 2H), 2.94 (s, 3H); ¹³C NMR

(100 MHz, DMSO‑d₆, ppm): 165.9 and 163.4 (d, J_CF¼ 249.3 Hz),

164.9, 156.6, 151.3, 138.6, 131.6, 131.0 and 130.9 (d, J_CF¼ 9.05 Hz),

130.6, 128.0, 124.4, 123.7, 119.7, 115.9 and 115.73 (d, J_CF¼ 22.0 Hz),

115.69, 110.5, 40.9; LC/MS (ESI) m/z: 401.0 [M þ H]^þ; HPLC analysis,

t_R¼ 27.93 min, 99.66% purity.

NMR (600 MHz, DMSO‑d₆, ppm): d 10.36 (s, 1H), 9.28 (s, 1H), 8.10 (s,

1H), 7.94 (m, J ¼ 8.5 and 3.4 Hz, 4H), 7.55 (m, 2H), 7.42 (m, 3H), 7.36

(d, J ¼ 8.8 Hz, 1H), 7.17 (t, J ¼ 7.3 Hz,1H), 7.08 (d, J ¼ 8.3 Hz, 2H), 2.95

(s, 3H); ¹³C NMR (150 MHz, DMSO‑d₆, ppm): 167.6, 165.3, 156.4,

151.2, 138.4, 137.28, 137.27, 130.5, 128.0, 127.9, 127.8, 124.4, 123.7,

119.7, 115.6, 110.4, 40.8; LC/MS (ESI) m/z: 424.0 [M ꢀ H]^-; HPLC

analysis, t_R¼ 18.47 min, 98.30% purity.

4.1.2. N-(3-Isopropoxy-4-(methylsulfonamido)phenyl)-4-methoxy-

2-nitrobenzamide (N5)

N-(4-Amino-2-isopropoxyphenyl)methanesulfonamide

(0.204 mmol; 50 mg) was dissolved in 3 mL DCM. 2-Nitro-4-

methoxybenzoic acid (0.204 mmol; 40.2 mg), DCC (0.225 mmol;

46.4 mg; 1.1 equiv.) and DMAP (5.0 mg; 0.2 equiv.) were added to

the solution in an ice bath. The mixture was reﬂuxed for 16 h. After

completion of the reaction detected by TLC (hexane:ethyl acetate),

the mixture was poured into ice-water (10 mL) and extracted with

ethyl acetate (3 ꢂ 10 mL). The combined organic layer was washed

with water (20 mL), dried over anhydrous Na₂SO₄and concentrated

in vacuo. The crude product was puriﬁed by column chromatog-

raphy (silica gel, hexane:ethyl acetate, 3:2). Light yellow solid;

60 mg; 69% yield; m.p.166e169 ^ꢁC; R_f¼ 0.33 (hexane:ethyl acetate,

1:1); IR (ATR) w 3319, 3246, 3154, 3114, 3068, 2980, 2931, 2850,

4.2. Biological evaluation

4.2.1. Screening of cyclooxygenase Inhibitory

The inhibitory activity of the new compounds on human re-

combinant COX-1 and COX-2 were examined by following the in-

structions given for the COX (human) Inhibitor Screening Assay Kit

(Cayman Chemical). The assay kit provides the quantiﬁcation of

prostanoid products as a yield of the COX reaction. Brieﬂy, the

puriﬁed COX-2 enzyme was pre-incubated with the compounds at

1

m

M for 10 min at 37 ^ꢁC. The COX reaction was initiated by adding

arachidonic acid as a substrate at 100 M and 30 s later stannous

1657, 1613, 1506, 1421, 1342, 1318, 1275, 1162, 1115, 972 cm^ꢀ1

;

¹H

m

NMR (600 MHz, DMSO‑d₆, ppm): 10.59 (s, 1H), 8.72 (s, 1H), 7.71 (d,

d

chloride solution was used to stop enzyme catalysis. The com-

pounds showing an inhibitory effect for the COX-2 enzyme were

J ¼ 8.5 Hz, 1H), 7.60 (s, 1H), 7.47 (s, 1H), 7.38 (d, J ¼ 9.9 Hz, 1H), 7.17

(d, J ¼ 8.5 Hz, 1H), 7.13 (d, J ¼ 8.6 Hz, 1H), 4.54 (m, J ¼ 5.9 Hz, 1H),

3.89 (s, 3H), 2.89 (s, 3H), 1.31 (d, J ¼ 5.9 Hz, 6H); ¹³C NMR (150 MHz,

DMSO‑d₆, ppm): 164.1, 161.0, 157.0, 151.7, 148.9, 138.5, 131.1, 128.2,

124.8, 122.1, 119.2, 111.6, 109.9, 105.4, 70.6, 56.8, 40.6, 22.0; LC/MS

(ESI) m/z: 422.0 [M ꢀ H]^-; HPLC analysis, t_R¼ 6.92 min, 99.91%

purity.

also screened on COX-1 at 10

mM with the same experimental

process. Inhibitory activity of each compound was determined in

two independent experiments, in triplicate. As positive controls,

nimesulide and celecoxib were used in this assay.

4.2.2. Cell culture

For the inﬂammation studies, human monocyte THP-1 cells

were cultured in the RPMI-1640 medium supplemented with 100

4.1.3. General procedure B for the synthesis of N12 and N13

Terephthalamic acid (1.0 equiv.) was dissolved in enough DMF.

EDCI.HCl (1.0 equiv.) and HOBt (1.0 equiv.) were added dropwise to

the solution in an ice bath, respectively. The mixture was stirred in

U/mL penicillin, 100

mg/mL streptomycin, and 10% fetal bovine

serum (FBS). When the concentration of suspension culture

reached enough point, the cells were plated at a density of

17

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T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

1 ꢂ 10⁶cells/mL in 6 well plates and treated with 10 ng/mL of PMA

to differentiate the monocyte into macrophage for 24 h. For the

resting stage, cells were maintained in PMA-free fresh medium for

another 24 h under the same culturing condition before treatment.

RAW264.7 was maintained in high glucose Dulbecco's modiﬁed

Eagle medium (DMEM) containing 10% FBS, 100 U/mL penicillin,

wells to solubilize the protein-bound dye on the gyratory shaker for

10 min. The absorbance was determined by using a microplate

reader (Tecan Inﬁnite® 200 PRO, Switzerland) at 565 nm. IC₅₀

values of compounds for each cell line were evaluated by GraphPad

Prism 5 software. In both assays, doxorubicin was used as a positive

control.

and 100 mg/mL streptomycin. The cell lines HT-29 (human colon

cancer cells), PC-3 (human prostate cancer cells), MCF-7 (human

breast cancer cells), and HUVEC (human healthy umbilical cells)

were routinely propagated in the identical medium of the murine

macrophage cell line for the screening of anticancer effects of

compounds. All cells were incubated in a humidiﬁed atmosphere at

37 ^ꢁC and 5% CO₂. For all the treatment processes, compounds were

dissolved in DMSO and maximum DMSO content in wells did not

exceed 1.0%.

4.2.2.4. Gene expression analyses by quantitative PCR. The mRNA

expression levels of COX-1, COX-2, iNOS, TNF-a, IL-1b and as

housekeeping gene -Actin were quantiﬁed by using speciﬁc Taq-

b

Man® probes (Thermo Scientiﬁc, Rockford, IL, USA). The total RNA

content was isolated from THP-1 cells for the analysis of COX genes;

and RAW 264.7 cell lines for other pro-inﬂammatory genes. Isola-

tion was taken place by using a commercially available Pure Link®

RNA mini kit with slight modiﬁcation and a high-capacity cDNA

reverse transcription kit (Applied Biosystems, Foster City, CA) was

utilized for cDNA synthesis as described in previous studies [1 7,45].

In the presence of cDNA samples and TaqMan® probes, real-time

PCR ampliﬁcations were recorded by Applied Biosystems 7500

Real Time PCR Systems (Applied Biosystems, Thermo Fisher Sci-

entiﬁc). The comparative DDCt method was implemented to

determine the change in the mRNA levels.

4.2.2.1. Determination of LPS-Induced PGE₂release. The effect of

compounds on the release of PGE₂in medium were investigated

according to the manufacturer's instructions of Thermo Fisher

invitrogen PGE₂ELISA Kit (EHPGE2). Differentiated THP-1 cells

were treated with compounds at 50

mM doses and simultaneously

stimulated with 1 g/mL of LPS. After 24 h incubation, supernatants

m

were collected for the ELISA process and the total protein content of

viable cells was quantiﬁed with Thermo Fisher Pierce BCA Protein

Assay Kit for normalization.

4.2.3. Immunoblotting assay

The effects of N17 on protein expression levels of BAX and BCL-2

were assigned by western blotting as previously described [45].

Brieﬂy, MCF-7 cells were seeded at 6-well plates with a density

1 ꢂ 10⁶cell/mL. The cells were incubated with the presence of test

compounds for 24 h and total protein samples were isolated from

cells by using RIPA buffer. The concentration of total protein lysates

was determined by the BCA assay. Equally loaded samples were run

throughout the discontinuous buffer system including 4% stacking

and 12% separating gel [47]. By gel electrophoresis, proteins in the

sample were forced to separate under an electrical current based on

their sizes. The gel was subjected to Western blotting to be labelled

4.2.2.2. Quantiﬁcation of NO release. The concentration of released

NO in media was quantiﬁed by using the Griess reaction method as

previously described [45]. Basically, RAW264.7 macrophage was

plated to a 24-well plate at 4 ꢂ 10⁵cells/mL density in phenol-free

media and left for 24 h incubation. Then, cells were treated with

compounds at 20 and 50

mM doses and simultaneously stimulated

with LPS (1 g/mL) for 24 h. At the end of the incubation, super-

m

natants were collected for the quantiﬁcation of NO concentration

and the total protein content of viable cells were quantiﬁed with

BCA assay for normalization. 20

itive control.

m

M of 1400 W was used as a pos-

with BAX, BCL-2, and b-actin primary antibodies (Santa Cruz

Biotechnology, Santa Cruz, CA) at 4 ^ꢁC. The non-binding primary

antibody was removed by washing steps with TBST and the

membrane was subjected to 2 h incubation with the recommended

dilution of horseradish peroxidase (HRP) -conjugated secondary

antibody. The substrate of HRP was supplied by the reagent named

SuperSignal™ West Pico PLUS Chemiluminescent. The intensity of

the chemiluminescence signal was scanned by the C-DiGit® Blot

Scanner. The densitometric analysis was taken place by using Image

Studio Digits Software 5.0 and the results were given as relative

change.

4.2.2.3. MTT & sulforhodamine B (SRB) cell cytotoxicity assays.

The cytotoxicity of NDs was determined by MTT assay. Brieﬂy,

cancer and healthy cell lines, in the exponential growth phase, were

plated in 96-well plate at 5 ꢂ 10⁴cells/mL (100

mL per well). After

24 h of incubation at 37 ^ꢁC, cells were treated with compounds at

100 mM doses for 48 h. At the end of the incubation period, MTT

solution was added to each well at 1 mg/mL ﬁnal concentration for

2 h. Cell medium was aspirated from each well and precipitated

formazan crystal was dissolved in dimethyl sulfoxide and the

absorbance was read at 570 and for background subtraction at

620 nm by using a microplate reader (Tecan Inﬁnite® 200 PRO,

Switzerland).

4.2.4. Clonogenic assay

MCF-7 cells were seeded in 6-well plates at a concentration of

5 ꢂ 10²(1 mL/well) and left to attach for 24 h. After attachment, the

medium was replaced, and the cells were treated with increasing

concentrations of N17 (25e100) in every 2 days for a total of 14

days. Next, cells were ﬁxed in 1 mL of ﬁxation solution (75%

methanol and 25% acetic acid) for 30 min at room temperature. The

ﬁxation solution was taken away and wells were rinsed with dH₂O.

The cells were stained with 1 mL of 0.5% crystal violet solution for

30 min at room temperature. Finally, wells were rinsed with dH₂O,

photographed and colonies were counted using ImageJ Studio

software.

To determine the IC₅₀value of compounds, a slightly modiﬁed

SRB assay, which is addressed to Vanicha&Kanyawim, was per-

formed in each cell line [46]. Brieﬂy, cells were seeded in 96-well

plate at 5 ꢂ 10⁴cells/mL (100

mL per well) for SRB assay. The cells

were left at 37 ^ꢁC for 24 h incubation in their exponential growth

phase then treated with compounds (0e200 mM) for 48 h. After the

treatment period, supernatant from each well was aspirated care-

fully and cells were ﬁxed with 10% (m/V) trichloroacetic acid (TCA)

solution at 4 ^ꢁC for 1 h. At the end of the ﬁxation step, TCA solution

was removed and washed with distilled water ﬁve times. Then the

plate was left for air-drying at room temperature. When plates

4.2.5. SEM analyses for observation of apoptotic ﬁgures

were completely dried,100

to each well for 30 min at room temperature. To remove the un-

bound dye, the plate was rinsed in 1% (V/V) acetic acid solution four

m

L of 0.04 (m/V) SRB solution was added

The ultrastructural changes in colon cancer cells were detected

by SEM analyses. Brieﬂy, the cells were plated on coverslips in a 24-

well culture plate and overnight incubated. Then, cells were treated

times. 200

mL of 10 mM of Tris base solution (pH:10.5) was added to

with N17 at 25 mM doses for 48 h. At the end of incubation, the cells

18

€

T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

were washed with a PIPES buffer. The cell ﬁxation was achieved in a

2.5% glutaraldehyde solution for 1 h at 4 ^ꢁC. The ﬁxed cells were

maintained and dehydrated by a series of incubations in the

ethanol bath (50-70-80-90-100-100%) for 7 min each. For the

complete drying process, coverslips were subjected to HMDS for

overnight incubation. In the last step, the slides were mounted on a

metal stub and coated with conductive metal for the observing

under FESEM (Zeiss Supra 40 VP).

4.6. Binary QSAR models

The MetaCore/MetaDrug platform of Clarivate Analytics was

used for investigating the ADME features, toxicity predictions and

therapeutic activities of the molecules. All molecules in the study

were screened in 25 common diseases and 26 different toxicity

QSAR models. The quality of binary QSAR models was validated

using the sensitivity, speciﬁcity, accuracy and Matthews correlation

coefﬁcient (MCC) parameters.

4.3. Preparation of ligands N1eN17

4.7. Statistical analysis

The molecules (N1eN17) were sketched in Maestro [48] 2D

Sketcher. 2D molecules were converted to 3D structures and pre-

pared for docking simulations by LigPrep module of the Maestro

[49] with OPLS3e force ﬁeld [50] was used in the restrain mini-

mization with heavy atom convergence of 0.3 Å. Epik was used in

the determination of the protonation states at neutral pH for all

molecules [51]. After ligand preparation, atomic ESP charges for all

the ligand molecules were determined by Semi-empirical NDDO

Module of Maestro [52]. PM3 method was used for semiempirical

calculations. Partial charges obtained from PM3 optimization were

used in docking.

Data are obtained by at least three independent biological rep-

licates and expressed as standard error of the mean. The differ-

ences between treatment groups were assessed by the statistical

technique called one-way ANOVA with Dunnett's post hoc test

(*p < 0.02, **p < 0.005, ***p < 0.001).

Author contributions

The project was designed and supervised by T.B.T., M.A. and T.G.

The studied compounds were designed by T.B.T., M.A. and T.G,

synthesized and characterized by T.G and M.A. The bioactivity

studies, data analysis, and construction of ﬁgures of the related

parts were performed by A.O, Y.B.Y, and T.B.T. The draft of their

relative sections was written by T.B.T, T.G and A.O. All the in silico

simulations and molecular modeling studies were conducted by

S.D and P.S. and corresponding sections were drafted by these au-

thors. All authors contributed to the writing of article.

4.4. Preparation of human COX proteins

Crystal structure of the human COX-1 (PDB ID: 6Y3C) and COX-2

structures (PDB ID: 5KIR) downloaded from Protein Data Bank

[53e55]. The initial structures were simpliﬁed by removing ‘B', C⁰,

and ‘D' chains. Of the ligands present on the ‘A' chain of the human

COX-2 structure, only the co-crystallized ligand (Rofecoxib) was

kept, and other ligands were removed. The used human COX-1

protein is crystallized in apo form. Therefore, another COX-1 pro-

tein crystallized from Ovis aries (PDB ID: 3KK6) [56] which includes

the co-crystallized ligand (Celecoxib) at the binding pocket, was

aligned with the 6Y3C-coded structure and ligand was merged to

the binding pocket of the 6Y3C. As a result, human COX-1 protein

containing the Celecoxib ligand, and human COX-2 protein con-

taining the Rofecoxib ligand were prepared for docking studies.

Protein preparations was performed by using Schrodinger's

Maestro Molecular modeling package [48]. OPLS3e force ﬁeld [50]

was used in the restrain minimization with heavy atom conver-

gence of 0.3 Å. Disulﬁde bonds were formed and missing side

chains were ﬁxed using Prime module [57]. Using PROPKA, pro-

tonation states were calculated according to physiological pH 7.4.

BCL-2 protein, which was prepared as published in our previous

study, was used [55].

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgement

This study was ﬁnancially supported by a research grant from

Scientiﬁc and Technological Research Council of Turkey (TUBITAK;

Grant No. 117Z398) and partially Çanakkale Onsekiz Mart Univer-

sity (Scientiﬁc Research Projects, ID: FYL-2019-3123). Authors

ꢀ

thank to Dr. Tugba Tas¸ kın Tok for contributions at the beginning of

the TUBITAK project.

Appendix A. Supplementary data

4.5. Molecular docking and molecular dynamic simulations

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113566.

All molecular docking studies were accomplished by using the

€

Glide module of the Schrodinger Maestro 2018 software [39].

Abbreviations Used

Standard precision was used. The top-docking poses of protein-

ligand complex structures were located in simulation boxes and

solvated with TIP3P water models. Partial charges were used.

Counter ions and 0.15 M NaCl solution were added in order to

neutralize the simulation systems. MD simulations were performed

at Desmond [44]. TIP3P water models were used and OPLS3e force

ﬁeld was applied. Simulations conducted for 100 ns and 2000

frames with equal intervals during the simulations were recorded

for each simulation. All simulations were performed at constant

physiological temperature (310 K) and constant pressure

(1.01325 bar) by creating isothermal-isobaric ensemble system

with NoseeHoover thermostat [58] and MartynaeTobiaseKlein

barostat [59]. The average MM/GBSA binding free energy of the

compounds were calculated using Prime.

TLC

UV

m.p.

FT-IR

ATR

Thin-layer chromatography

Ultraviolet

Melting point

Fourier-transform infrared spectroscopy

Attenuated total reﬂection

¹H NMR Proton nuclear magnetic resonance

¹³C NMR Carbon nuclear magnetic resonance

TMS

MS

DMSO

DMF

DCM

SAR

Tetramethylsilane

Mass spectroscopy

Dimethyl sulfoxide

Dimethylformamide

Dichloromethane

Structure-activity relationship

19

€

T. Güngor, A. Ozleyen, Y.B. Yılmaz et al.

European Journal of Medicinal Chemistry 221 (2021) 113566

M. Ay, Biological evaluation and molecular docking studies of nitro benzamide

derivatives with respect to in vitro anti-in ﬂ ammatory activity, Int. Immuno-

pharm. 43 (2017) 129e139.

NSAIDs

PGs

COX

PGE₂

NO

NOS

eNOS

nNOS

iNOS

Nonsteroidal anti-inﬂammatory drugs

Prostaglandins

Cyclooxygenase

Prostaglandin E2

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Nitric oxide synthases

Endothelial nitric oxide synthases

Neuronal nitric oxide synthases

Inducible nitric oxide synthase

IL-1

b

Interleukin-1

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NF-

LPS

MTT

k

B

Nuclear Factor kappa

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

b

bromide

SRB

FDA

DOX

CEL

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New nimesulide derivatives with amide/sulfonamide moieties: Selective COX-2 inhibition and antitumor effects

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