Tailoring the substitution pattern of Pyrrolidine-2,5-dione for discovery of new structural template for dual COX/LOX inhibition

Bioorganic Chemistry 112 (2021) 104969

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Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Tailoring the substitution pattern of Pyrrolidine-2,5-dione for discovery of

new structural template for dual COX/LOX inhibition

,

Abdul Sadiq^{a *}, Mater H. Mahnashi^b, Bandar A. Alyami^b, Yahya S. Alqahtani^b, Ali O. Alqarni^b,

,

**

Umer Rashid^c

^aDepartment of Pharmacy, Faculty of Biological Sciences, University of Malakand, Chakdara, 18000 Dir (L), KP, Pakistan

^bDepartment of Pharmaceutical Chemistry, College of Pharmacy, Najran University, Najran, Saudi Arabia

^cDepartment of Chemistry, COMSATS University Islamabad, Abbottabad Campus, 22060 Abbottabad, Pakistan

A R T I C L E I N F O

A B S T R A C T

Keywords:

Dual inhibition of the enzymatic pathways of cyclooxygenases (COX-1/COX-2) and lipoxygenase (LOX) is a

rational approach for developing more efﬁcient and safe anti-inﬂammatory agents. Herein, dual inhibitors of

COX and LOX for the management of inﬂammation are reported. The structural modiﬁcations of starting

pyrrolidine-2,5-dione aldehyde derivatives resulted in two structurally diverse families (Family A & B). Syn-

thesized derivatives from both Families displayed preferential COX-2 afﬁnity in submicromolar to nanomolar

ranges. Disubstitution pattern of the most active series of compounds having N-(benzyl(4-methoxyphenyl)amino

moiety presents a new template that is mimic to the diaryl pattern of traditional COX-2 inhibitors. Compound 78

NN-disubstituted pyrrolidine-2,5-dione

Cyclooxygenases

Lipoxygenase

Leukotriene

Carbonic anhydrase

with IC₅₀value of 0.051 0.001 μM emerged as the most active compound. Highly potent COX-2/5-LOX in-

hibitors have also demonstrated appreciable in-vivo anti-inﬂammatory activity through carrageenan induced paw

edema test. Moreover, the involvement of histamine, bradykinin, prostaglandin, and leukotriene mediators to

adjust the inﬂammatory response were also studied. Apart from COX inhibition, sulfonamide is considered an

important template for carbonic anhydrase inhibition. Hence, we also evaluated six sulfonamide derivatives for

off-target in-vitro bovine carbonic anhydrase-II inhibition. Biological results were ﬁnally rationalized by docking

simulations. Typically, most active COX-2 inhibitors interact with the amino acid residues responsible for the

COX-2 selectivity.

1. Introduction

acid bound covalently as ester in the cell membranes of most body cells.

During the inﬂammatory process, AA is released and metabolized by

From ancient Egyptian and Greek civilizations, plant remedies such

as extracts from the bark of trees and dried myrtle leaves have been used

to alleviate pain, inﬂammation, and fever. In 1763, the analgesic and

antipyretic effects of willow bark were published for the ﬁrst time.

Salicin was identiﬁed as its active component. Salicylic acid, the phar-

macologically active form of salicin, was later synthesized from phenol

in 1860 by Kolbe and Lautemann. Later, a more palatable drug ace-

tylsalicylic acid, commonly known as aspirin, was developed, and

marketed by Bayer in 1899. This development was followed by some

drug discoveries, grouped as Nonsteroidal anti-inﬂammatory drugs

(NSAID), with analgesic, anti-inﬂammatory, and antipyretic properties

[1–6].

cyclooxygenases (COX) and lipoxygenase (LOX) which lead to the for-

mation of signaling mediators eicosanoids. Proinﬂammatory prosta-

glandins (PGs) produced through the COX pathway, and leukotrienes

(LTs, non-cyclized eicosanoids) produced via the LOX pathway are

associated with potent inﬂammatory properties [7–9].

The major biochemical pathway of traditional NSAIDS involves the

inhibition of the cyclooxygenase (COX) enzyme which is involved in the

catalysis of the rate-limiting step of the oxidation of AA into prosta-

glandins (PGs) and thromboxane A2 (TXA2) [10–13]. Among two

identiﬁed isoforms, cyclooxygenase-2 (COX-2) is mainly responsible for

inﬂammation and is rapidly induced by various inﬂammatory stimuli.

Cyclooxygenase-1 (COX-1) is involved in the housekeeping function of

various tissues, including, kidney, gastrointestinal tract and, platelets

Arachidonic acid (AA) is the most abundant polyunsaturated fatty

* Corresponding author.

** Corresponding author.

E-mail addresses: sadiquom@yahoo.com (A. Sadiq), umerrashid@cuiatd.edu.pk (U. Rashid).

https://doi.org/10.1016/j.bioorg.2021.104969

Received 5 February 2021; Received in revised form 24 April 2021; Accepted 3 May 2021

Available online 7 May 2021

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

[14–19]. The development of selective COX-2 inhibitors “COXIBs”

intended to have anti-inﬂammatory properties. COXIBs have provided

relief to the patients suffering from inﬂammatory disease, the problem

of gastrointestinal lesions, and discomforts. Some selective COX-2 in-

hibitors are characterized by 1,2-diarylheterocycles (Celecoxib, rofe-

coxib, Valdecoxib and DuP696) and sulfanilamide (nimesulide).

However, the cardiovascular (CV) risk associated with COXIBs led to the

withdrawal of rofecoxib, and valdecoxib from the market [20–22].

There is the number of strategies reported to decrease NSAID-

associated gastrointestinal lesions and discomforts. These include the

introduction of novel therapeutics and dual inhibitions of COX and LOX.

It has been reported that the use of NSAIDs (COX-1/COX-2 inhibitors)

lead to decreased accumulation of leukotrienes (LT) via the lipoxygenase

(LOX) pathway. Hence, dual inhibitions of COX/LOX is a new approach

to retain anti-inﬂammatory effects of NSAIDs with reduced serious GI

toxicity associated with them [23–25]. Carboxylic acid-containing

diphenylpyrrole analog Licofelone (1, Fig. 1a) is an example of a dual

COX/LOX inhibitor [26–27].

Family A). However, synthesis of comparatively less hydrophobic and

ﬂexible acetic acid/sulfonamides (–CH₂-) and propanoic acid/ sulfon-

– –

C H) were also planned to synthesize (Family B). The

amides (–CH₃

derivatives with the best in-vitro afﬁnity and selectivity proﬁle were

further investigated for in-vivo anti-inﬂammatory activity. Biological

results were ﬁnally rationalized by docking simulations.

2. Results and discussion

2.1. Drug design

The development of drugs that are selective for COX-2 inhibition

over COX-1 is a challenge as both isozymes share 60% homology. The

active site of the COX enzyme is a long hydrophobic channel that ex-

tends to the lobby (membrane-binding domain, MBD). The selectivity of

the selective COX-2 inhibitors is associated with their structural fea-

tures. The existence of a spacious selectivity pocket (about 20%) due to

less bulky Val523 allows access to the bulky groups. In the case of COX-

1, access to this pocket is restricted. Esters, amides, and bulky scaffolds

can be accommodated beyond the constriction into the lobby. Tradi-

tional selective COX-2 inhibitors like celecoxib (5, Fig. 3a) possess

vicinal diaryl-substitution on central 5- or 6-membered hetero/carbo-

cyclic ring system bearing a speciﬁc pharmacophoric group (sulfonyl or

sulfonamide) to favor the COX-2 selectivity. Based on the above-

mentioned structural features of selectivity pocket of COX-2 isozyme,

we designed suitable building blocks that should meet two criteria: (i)

The template or center of disubstitution should be mimic the diaryl

pattern of traditional COX-2 inhibitors; (ii) the inhibitor should have a

pharmacophore to facilitate its binding into selectivity pocket. Based on

these two criteria, we selected 1-(disubstituted amino)pyrrolidine-2,5-

dione motif as template and center of disubstitution. We designed a

comprehensive structure–activity relationship (SAR) to explore the ef-

fect of disubstitution on nitrogen atom. Moreover, promising results are

shown by sulfonamide derivatives of marketed NSAIDs reported by

Elkady et al., (e.g. Lonazolac, 6, Fig. 3b) inspired us for COX/LOX in-

hibitor design [39]. Replacement of aldehyde and carboxylic acid to

bulky aryl sulfonamide (Ar-SO₂NH₂) was envisaged as a probable

pharmacophore. We anticipated that this replacement may facilitate the

binding into the selectivity pocket. Alkyl groups are placed between

template and pharmacophore to control the hydrophobicity of the

compounds.

Structurally, NSAIDs are classiﬁed into three main groups: (i) Car-

boxylic aids and/or their eaters (acetic, propionic, salicylic and fenamic

acids); (ii) phenazones (oxicams), and (iii) non-acidic compounds. The

selectivity of the COX-2 inhibitors is associated with their structural

features. The reported selective COX-2 inhibitors have shown wide di-

versity in their chemical structures. Tricyclic class of inhibitors possesses

vicinal diaryl-substitution on central 5- or 6-membered hetero/carbo-

cyclic ring system bearing a speciﬁc pharmacophoric group (sulfonyl or

sulfonamide) to favor the COX-2 selectivity. The non-tricyclic class of

the inhibitors lacked a central cyclic ring and possess a central acyclic

2–3 membered template. Although the two isozymes have nearly iden-

tical structures, however, their selectivity is governed by steric and

hydrophobic interactions. The templates with low polarity were found

to be important for inhibition. Similarly, bulky groups present on the

central core pushed the molecule into the selectivity pocket of COX-2

[28–31]. In 2003, a structurally diverse highly selective (500-fold

selectivity) carboxylic acid COX-2 inhibitor Lumiracoxib (2) (aryl-

alkanoic acid class, Fig. 1) was developed by the structure modiﬁcation

of non-selective diclofenac [32–34]. Similarly, mofezolac (3, carboxylic

acid-containing diarylisoxazole derivative) is a selective inhibitor of

COX-1 (COX-1 IC₅₀= 0.0079 μM, COX-2 IC₅₀= 0.44 μM) [35].

The key role of COX/LOX in the inﬂammatory process and the lim-

itations of the current therapeutics demonstrate the need for new

structural templates. In a previous study, we demonstrated the impor-

tance of substituted succinimides (pyrrolidine-2,5-dione derivatives) as

multitarget anti-inﬂammatory agents. Pyrrolidine-2,5-dione derivative

2.2. Chemistry

(4, Fig. 1) emerged as the potent and selective inhibitor (IC₅₀= 0.98

SI = 31.5) inhibitor of COX-2 isozyme [36].

μM,

The syntheses of maleimide derivatives 14–16 and 31–37 (Scheme

1) have been carried out by the condensation of substituted hydrazine

derivatives with maleic anhydride to form N-maleamic acid in-

termediates (11–13 and 24–30) [36]. The condensation step was fol-

lowed by dehydration of the intermediates that were precipitated in the

solvent. Here, the choice of the solvent matters. N-substituted mal-

eimides from cyclic hydrazine derivatives and maleic anhydride gave

good yield in chloroform. While the synthesis of maleimide derivatives

was carried out in diethyl ether. Synthesized intermediates were

Our research group has already synthesized aldehyde and ketone

derivatives of succinimides via Michael addition reactions using a self-

assembled three-component organocatalyst [36–38]. The structural

modiﬁcation of intermediate pyrrolidine-2,5-dione aldehydes led us to

synthesize and biologically evaluate the two Families (A-B, Fig. 2) with

vast structural diversity. The starting point of our research is the syn-

thesis of hydrophobic 2-methyl propanal succinimides (dimethyl group,

Fig. 1. Chemical structures of Licofelone (1), Lumiracoxib (2), mofezolac (3) and our previously reported most active pyrrolidine-2,5-dione based compound (4).

2

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

Fig. 2. The structural modiﬁcation of starting pyrrolidine-2,5-dione aldehyde derivatives (46–59 and 60–64) resulted in two structurally diverse Family A (66–71)

and Family B (76–79).

Fig. 3. (a) Structural features of celecoxib (5, blue part) and modiﬁed Lonazolac (6, pink part) that combines to design the COX/LOX inhibitors of current research

(b) Design strategy. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

subsequently cyclized without any further puriﬁcation in acetic anhy-

dride in the presence of sodium acetate to give target products 14–16

and 31–37.

benzyl chloride (38) and benzene sulfonyl chloride (39–40) to obtain N-

disubstituted products 41–42 and 43–44 respectively (Scheme 2). First,

we tried potassium carbonate (K₂CO₃) as base in DMF and triethylamine

in DCM. The isolated yield of the target compounds was<40%.

Mono-substituted maleimides 33–34 were further reacted with

3

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

Scheme 1. Synthesis of maleimides 14–16 and 31–37.

Scheme 2. Synthesis of N-disubstituted maleimides 41–44.

However, the reaction was carried put in a 1:2 (v/v) mixture of anhy-

drous DMF and DCM. While 60% sodium hydride (NaH, as mineral oil

suspension) was used as base. The target products were obtained in 4–5

h stirring at 0 ^◦C in 68–72% yield. The reaction was carried out at room

temperature.

natural products and in some drug candidates. Substituted succinimides

can be prepared directly by conjugate addition of carbon nucleophiles.

The carbon nucleophiles can be produced by the deprotonation of acidic

hydrogen containing pro-nucleophiles by using asymmetric organo-

catalyst [40–41]. Among complex organocatalyst systems reported for

these types of transformations, use of self-assembled organocatalyst

system with a single amino acid is revived since last decade. Our

research group has already reported a self-assembled bifunctional

Maleimides are the building blocks for the synthesis of target com-

pounds. Among these target compounds, pyrolidine-2,5-diones (succi-

nimides) are one of the important widespread motifs that are present in

4

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

OtBu-_L-threonine catalyst for the activation of carbonyl compound and

electrophile in Michael addition reactions [37,42–43]. We applied here

OtBu- _L-threonine catalyst for the synthesis of target succinimides. The

addition of isobutyraldehyde (45) to maleimide type Michael acceptors

14–16, 31–37, and 41–44 were carried out by using our previously

reported procedure. Isobutyraldehyde (45) was stirred at room tem-

perature with maleimides in the presence of OtBu- _L-threonine and 1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU) in chloroform to yield 46–59 as

outlined in Scheme 3.

2.3. In-vitro COX inhibition

The COX-1/COX-2 isozyme inhibitory activities of the target com-

pounds (Families A-B, Schemes 1-6) were carried by using celecoxib was

used as a standard drug (Tables 1-4). In-vitro assay results are expressed

as IC₅₀in micromole (μM). Selectivity towards COX-2 isozyme is pre-

sented as the ratio of IC₅₀values of compounds toward COX-1 and IC₅₀

towards COX-2. In the attempt to determine the combined effects of

hydrophobicity, ﬂexibility, and larger functional groups on COX-1/COX-

2 inhibitory potential, we introduced different substituents at the suc-

cinimide core.

Oxidation of aldehyde to corresponding carboxylic acid is commonly

carried out using hazardous heavy metal-based oxidizing agents

(KMnO₄or K₂Cr₂O₇) in acidic or basic media. Borhan et al., used oxone

(Potassium peroxymonosulfate) in dimethylformamide (DMF) for the

oxidation of aromatic aldehydes [44]. Recently, Grunenfelder et al.,

reported oxidation of aldehyde moiety on succinimide by using oxone in

DMF [45]. Here, performed oxidation of aldehyde moieties present in

46–59 using procedure reported by Grunenfelder et al. Oxone (Potas-

sium peroxymonosulfate) was used as an oxidizing agent. The reaction

was carried out at room temperature in DMF under nitrogen. The

scheme was outlined in Scheme 4. Subsequently, we carried out

coupling of synthesized carboxylic acid derivatives with 4-amino-ben-

zene sulfonamide (65) by using the method reported by Grunenfelder

et al. In situ 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-

chloride (EDC.HCl), 1-hydroxybenzo- triazole (HOBt)-activated car-

boxylic acid derivatives of succinimides (60–64) and 4-amino-benzene

sulfonamide (65) were reacted to give amides 66–70. The coupling re-

action was carried out in dry dichloromethane, and N,N-Diisopropyle-

thylamine (Hunig’s base) was used as a base (Scheme 4). Subsequently,

we synthesized the ester derivative (71) by the reaction of the most

active COX-2 inhibitor (61) in ethanol using few drops of sulfuric acid

(Scheme 5).

Table 1 and 2 enlists the IC₅₀values of COX for the Family A and B

aldehydic analogs. Heterocyclic analogues (Family A) are more potent

inhibitors of COX-1 (IC₅₀= 15.21–19.55 μM) than of COX-2 (IC₅₀=

57.24–88.4 μM). In compounds 49–59 (Family B aldehydes), the inﬂu-

ence of N,N-disubstitution was observed in COX-1/COX-2 selectivity.

Monosubstituted products (51–55) emerged as the most potent in-

hibitors of COX-1 than COX-2. While the disubstituted compounds,

except 49, showed selectivity for COX-2 isozyme.

The structural modiﬁcations of Family B disubstituted derivatives

were carried out. We chose potent and selective COX-2 inhibitors 50,

56–59 for further modiﬁcations. Hence, the ﬁrst modiﬁcation we carried

out was the oxidation of selected aldehydes to carboxylic acids. This

transformation led to 2-methylpropanoic acid (60–64). Remarkably, the

synthesized carboxylic acid derivatives proved to be more potent and

selective towards COX-2 isozyme than their aldehyde counterparts.

Benzyl derivative (61) with an IC₅₀value of 0.19 μM (SI = 387) was

found to be the most active compound toward COX-2. In an attempt to

improve the activity of the most active carboxylic acid derivative 61, the

introduction of the ethyl ester group resulted in a considerable decrease

in COX-2 inhibitory activity as well as selectivity (IC₅₀= 0.41 μM (SI =

A synthetic strategy in hand allowed us to explore the SAR of the

most active compound 67. Started with propionaldehyde and acetal-

dehyde, we synthesized sulfamoylphenyl)propenamide (–CH₃-CH-, 78)

and sulfamoylphenyl-acetamide (–CH₂-, 79) derivatives. This SAR also

resulted in the synthesis of two carboxylic acid derivatives (76–77). The

syntheses of the target compounds 76–79 are in Scheme 7. Acetaldehyde

(72) and propionaldehyde (73) was stirred at room temperature with

maleimide (38) in the presence of of OtBu-_L-threonine and 1,8-diazabi-

cyclo[5.4.0]undec-7-ene (DBU) in chloroform to yield 74–75. Carbox-

ylic acid derivatives 76–77 were synthesized by the oxidation of

aldehyde moieties of 74–75 by using oxone as oxidizing agent in DMF at

room temperature under nitrogen. Finally, the synthesized carboxylic

acid derivatives 78–79 were coupled with 76 in the presence of EDC.

HCl, HOBt in dry DCM and DIPEA was used as base (Scheme 6).

113).

There is a well-known aspect that the introduction of sulfonyl

(-SO₂CH₃) or sulfonamide (-SO₂NH₂) substituents on the aromatic ring

are preferred for COX-2 selectivity due to the presence of a larger hy-

drophobic channel. In other words, the increase in the bulkiness would

result in more efﬁcient interactions with COX-2 selectivity pocket resi-

dues. Based on these facts, we decided to introduce sulfonamide moiety

to obtain sulfamoylphenyl)propenamide derivatives (66–70). Apart

from their range of activity and selectivity against the COX-2, the

compounds of this family were not active against COX-1 isoform. The

nature of N-disubstitution on 1-aminopyrrolidine-2,5-dione inﬂuenced

the COX-2 inhibitory potential of these compounds. Here, again 1-

benzyl(4-methoxypheny)amino group in compound 67 (COX-2 IC₅₀

0.06 M) was found to be most potent than diphenylamino derivative

(66, COX-2 IC₅₀= 0.37 M), 1-(benzyl(phenyl)amino (68, COX-2 IC₅₀

= 0.17 M) and N-phenylbenzenesulfonamides (69–70, COX-2 IC₅₀

0.10 and 0.74 M respectively). The presence of sulfonamide group

=

μ

=

μ

Scheme 3. Synthesis of 2,5-dioxopyrrolidin-3-yl-2-methylpropanal derivatives (46–59, Family A).

5

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Bioorganic Chemistry 112 (2021) 104969

Scheme 4. Carboxylic acid and sulfonamide derivatives of some 2,5-dioxopyrrolidin-3-yl-2-methylpropanal derivatives.

Scheme 5. Synthesis of ethyl 2-(1-(benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-2-methylpropanoate (71).

made a considerable increase in the activity of the compounds.

2.4. In-vitro 5-LOX inhibition

Our research ains to optimize the potency and selectivity of COX-2

isozyme versus COX-1 isozyme as well as drug-like properties, particu-

larly lipophilicity. We started with our research with more lipophilic 1-

substituted aminopyrrolidine-2,5-dione derivatives of 2-methylpropa-

nal (dimethyl group). A synthetic strategy in hand allowed us to

explore the SAR of the most active compound 67. The synthetic strategy

involved is the use of propionaldehyde and acetaldehyde as starting

materials. Keeping 1-benzyl(4-methoxyphenyl)amino and sulfonamide

group in compound 67 unchanged, sulfamoylphenylacetamide, (–CH₂-,

78) and sulfamoylphenyl)propenamide (–CH₃-CH-, 79) derivatives were

synthesized. This SAR also resulted in the synthesis of a small set of

carboxylic acid derivatives (76–77). All four synthesized compounds

were evaluated for COX inhibition potential. Despite of the improved

inhibition potential of methylene (–CH₂-) carboxylic acid derivative 76

A dual inhibition of COX and LOX enzymatic pathways presents a

rational approach for the design of more effective and safe anti-

inﬂammatory agents. In literature, a number of derivatives containing

thiophene, pyrrolidine, hydrazine, aryl pyrazoline scaffolds are recog-

nized a dual COX/LOX inhibitor. Zileuton, a benzothiophene N-hydroxy

urea derivative, is the only 5-LOX inhibitor in the market. It inhibits 5-

LOX by chelating with Fe present in the active site.

Licofelone (2, Fig. 1), a dihydropyrrolizine acetic acid derivative, is a

dual COX/LOX inhibitor and is under development for the treatment of

osteoarthritis. From the structure features of Lonazolac (6, Fig. 3)., we

envisioned that diverse N-substituted pyrrolidine-2,5-dione could prove

effective 5-LOX inhibitor. The results of the 5-LOX inhibition of the

compounds are presented in Table 5. The synthesized compounds were

screened for their 5-LOX inhibitory potentials. N,N-disubstituted alde-

hyde group-containing derivatives 56–59 showed inhibition in the low

(IC₅₀= 0.17 μM), the selectivity was decreased from 176 (compound

61) to 148. However, no change in COX-2 inhibition potential for

compound 77 versus 61 was observed. On the other hand, the modiﬁ-

cation compound 78 to sulfamoylphenylacetamide derivative 88 (IC₅₀

micromolar range (1.18–3.63

μM). Substitution of aldehyde by the

carboxylic group was effective but depends mainly upon the structure of

the substituent at N-atom of succinimide. Most active carboxylic acid-

based COX-2 inhibitors 61, 86–87 showed good 5-LOX inhibition. The

= 0.05 μM) resulted in a 1.2-fold increase in COX-2 inhibition potency.

While for compound 79, the COX-2 inhibition potential was decreased

3.6-fold versus 67.

IC₅₀values of these compounds are 4.01, 8.41, and 2.14 μM, respec-

tively. However, compound 63 (bearing the N-sulfone group) with IC₅₀

value of 0.94 M emerged as a potent compound of this family.

μ

6

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

Scheme 6. Synthesis of Family B carboxylic acid (76–77) and sulfonamide derivatives (78–79) from 2-(1-(benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-

yl)acet- (72) and propan- (73) aldehydes.

Tested 2-methyl-sulfamoylphenyl) propenamide derivatives 66–73,

sulfamoylphenyl acetamide derivative 78, and sulfamoylphenyl acet-

amide and propenamide analog 79 revealed excellent 5-LOX inhibitory

potential. We also tested the in-vivo anti-inﬂammatory response at

lower doses for all three compounds, i.e. 10 & 5 mg/kg of compounds

61, 67, 78. Compound 61, drop the anti-inﬂammatory activity when the

dose was reduced to 10 and then to 5 mg/kg. The maximum observed

activity at the 3rd hour for compound 61 was 51.64 and 37.26% at 10

and 5 mg/kg respectively. There is not a big difference in the activity

proﬁle of compound 61 at 20 and 10 mg/kg. However, a clear reduction

in the activity can be noted when used 5 mg/kg concentration. In can be

concluded from the observed results, that the optimum dose for our

compound can be considered 10 mg/kg.

potential in submicromolar range (IC₅₀= 0.61–0.97 μM) greater than

standard Zileuton.

2.5. In-vivo anti-inﬂammatory activity

For in-vivo studies, we have selected compounds 61, 67, and 78. The

anti-inﬂammatory activity of the compounds was assessed by the

carrageenan-induced rat paw edema method. Carrageenan induced paw

edema model is a COX-2–dependent model of inﬂammation. Therefore,

2.5.3. Anti-inﬂammatory mechanism of the synthesized compounds

To check the possible role of proinﬂammatory mediator for mecha-

nistic studies, we choose compounds 61, 67 and 78.

we selected two compounds 67 (IC₅₀= 0.064 μM) and 78 (IC₅₀= 0.051

μ

M) having high COX-2 inhibition potential (high selectivity index).

While we also selected the most active COX-2 inhibitor 61 of the car-

2.5.3.1. Effect of compounds on paw edema induced by histamine. With

the administration of 1 mg/kg (bw) of chlorpheniramine maleate, the

histamine-induced inﬂammation was observed to be altered at ﬁrst hour

(72.64%) and remained signiﬁcant till the 3rd hour. Similarly, the

observed percent inhibition with compound 61 was 45.45% at 1st hour

at a concentration of 10 mg/kg and remained signiﬁcant till 3rd hour

(48.07%). Moreover, the compounds 67 and 77 at concentrations of 10

mg/kg displayed moderate anti-inﬂammatory effect at the ﬁrst hour

(23.55% for 78, 26.01% for 77) in the histamine-induced model of

inﬂammation (Fig. 5a).

boxylic series (IC₅₀= 0.19 μM).

2.5.1. Acute toxicity results

The experimental animals showed no mortalities with receiving a

single concentration of 1000 mg/kg of the compounds 61, 67, or 78

(Table 6). There were no obvious signs and symptoms on experimental

animals even with maximum concentration of the compound. The safety

deﬁned by the Organization for Economic Cooperation and Develop-

ment is that an LD₅₀concentration > 300 to 2000 is classiﬁed as cate-

gory 4 safe drug.

2.5.3.2. Effect of compounds on paw edema induced by bradykinin. In

2.5.2. Carrageenan induced paw edema test for 61, 67 and 78

proinﬂammatory method of bradykinin (20 μg/ml), the change in paw

In the carrageenan induced model of inﬂammation, we observed that

compound 61 produced a practical activity proﬁle as compared to 67

and 78 as shown in Fig. 4. The compound 61 with a carboxylic acid

group was more dominant in in-vivo anti-inﬂammatory activity

compared to the compounds with sulfanilamide group (67, 78). The

compound 61 showed 46.18% anti-inﬂammatory potential at 1st hour

which gradually increased up to the 3rd hour, i.e. 52.77% at the highest

concentrations of mg/kg. The activity of compound 61 was in parallel

comparison to the standard drug aspirin which was 48.54 to 53.64%.

The activity pattern of compounds 67 and 78 were also encouraging.

There was a dose-dependent response in the in-vivo anti-inﬂammatory

volume was observed and the data was recorded between 1 and 5 h. In

comparison to the positive control, the synthesized compounds showed

lower percent inhibition in bradykinin-induced inﬂammation. The

percent inhibition of 61 was 37% at 3rd h of bradykinin injection.

Similarly, compounds 67 and 78 showed 19.00 and 26.00% inhibitions

respectively at the 2nd hour which was very less effective in comparison

to the positive control HOE 140 as shown in Fig. 5b.

2.5.3.3. Effect of compounds on paw edema induced by prostaglandin E₂.

The administration of 0.01 μg/ml of prostaglandin E2 was correlated

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A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

Table 1

In-vitro assays on substituted 2-(1-amino-2,5-dioxopyrrolidin-3-yl)-2-methylpropanal.

Comp No.

R¹

R²

X

IC₅₀

(μ

M) ± SEM^[a]

SI ^[b]

COX-1

COX-2

46

47

48

49

50

–

CH₂

O

17.56 ± 1.34

15.21 ± 0.98

19.55 ± 0.65

6.3 ± 0.97

88.4 ± 1.46

60.91 ± 1.37

57.24 ± 1.66

> 100

0.20

0.25

0.34

> 0.26

47

–

N-CH₃

CH₃

–

74.22 ± 1.29

1.58 ± 0.03

–

51

52

53

54

H

11.27 ± 0.06

10.02 ± 0.01

17.36 ± 0.09

7.59 ± 0.01

14.35 ± 0.01

13.61 ± 0.01

21.03 ± 1.26

25.34 ± 0.11

0.78

0.74

0.82

0.30

–

55

H

0.97 ± 0.01

7.18 ± 0.01

0.13

–

56

57

58

39.2 ± 1.32

44.8 ± 2.01

79.2 ± 1.97

0.76 ± 0.01

2.54 ± 0.12

1.23 ± 0.01

52

18

64

–

59

3.4 ± 2.10

1.09 ± 0.03

86

Celecoxib

14.05

0.05 0.01

281

[a]

Data shown are the mean ± (SEM (n = 3). ^[b]Selectivity index: ratio of [IC₅₀(COX-1)]/IC₅₀(COX-2)].

Table 2

In-vitro COX1/COX-2 inhibition assays on carboxylic (60–64) and ester (75) derivatives of substituted 2-(1-amino-2,5-dioxopyrrolidin-3-yl)-2-methylpropanal.

R¹

R²

R³

IC₅₀

(

μM) ± SEM

SI

COX-1

COX-2

60

61

OH

63.4 ± 2.56

33.4 ± 1.41

0.99 ± 0.11

64

0.19 ± 0.02

0.43 ± 0.01

0.88 ± 0.06

176

62

63

OH

38.0 ± 1.33

66.3 ± 2.03

88

75

64

71

OH

82.6 ± 1.68

71.9 ± 1.53

0.73 ± 0.10

0.41 ± 0.01

137

113

OC₂H₅

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Bioorganic Chemistry 112 (2021) 104969

Table 3

Table 5

In-vitro assays on sulfonamide derivatives of substituted 2-(1-amino-2,5-diox-

In-vitro human 5-LOX assays on substituted 2-(1-amino-2,5-dioxopyrrolidin-3-

yl)-2-methylpropanal.

opyrrolidin-3-yl)-2-methylpropanal (66–70).

Compound No.

IC₅₀

(

μM) ± SEM

Compound No.

IC₅₀(μM) ± SEM

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

32.6 ± 1.23

ND

61

4.01 ± 0.06

ND

62

19.4 ± 1.05

59.7 ± 2.09

6.4 ± 1.04

ND

63

0.94 0.02

1.06 ± 0.01

0.83 ± 0.08

0.94 ± 0.08

0.97 ± 0.12

0.80 ± 0.11

0.61 0.10

ND

64

66

R¹

R²

COX-1 (%

COX-2

IC₅₀

SI

67

inhibition @

27.9 ± 1.13

31.8 ± 1.09

ND

68

10

μM

(μM) ±

69

SEM

70

ND

71

66

67

n.a*.

17%

0.37 ±

–

3.63 ± 0.11

1.18 ± 0.09

3.51 ± 0.14

1.24 ± 0.01

1.49 ± 0.01

76

8.41 ± 0.29

2.14 ± 0.10

0.99 0.10

0.69 0.10

0.64 ± 0.06

0.05

77

0.064

±

78

79

0.001

0.17 ±

0.01

Zileuton

68

69

23%

n.a.

–

0.10 ±

Table 6

0.01

Animal group speciﬁcation and quantity of drug administered for acute toxicity

studies with compounds 61, 67 and 78.

70

n.a.

0.74 ±

–

Groups

Animals

Compounds (61, 67 and 78) mg/kg body weight

0.04

Male

Female

* n.a. = no activity found at 10 μM

1

8

5

2

25

3

50

4

100

200

300

400

500

1000

2000

Table 4

5

In-vitro assays on carboxylic acid and sulfonamide derivatives of 2-(1-(benzyl(4-

methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)acet- (86, 88) and propan-

(87, 89) aldehydes 2-(1-(benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-

3-yl)acetaldehyde.

6

7

8

9

10

procedure as shown in Fig. 5c.

2.5.3.4. Effect of compounds on paw edema induced by leukotriene. In

leukotriene-induced model of inﬂammation, our selected compounds

61, 67 and 78 showed concentration-dependent anti-inﬂammatory re-

R¹

R²

R³

COX-1

COX-2

IC₅₀

SI

IC₅₀(μM) ±

sponses at 10 mg/kg body weight (Fig. 5d). The leukotriene (10 μg/ml)

SEM

(μM) ±

induced edema was inhibited by our selected compounds in a dose-

dependent manner. The compound 61 exhibited maximum inhibition

of 70.11% at 3rd hour which was comparatively much closer in potency

to that of standard drug. The encouraging values produced by 67 and 78

were also observed to be 49.08 and 57.00% at 3rd hour after the

administration of leukotriene. Montelukast used as positive control

exhibiting a 77.40% inhibition of paw inﬂammation at 3rd hour.

In the in-vivo studies, our compounds have enhanced activities in

prostaglandins E₂and leukotrienes pathways. Moreover, our com-

pounds have moderate activities in histamine pathways and very low

percent inhibitions when tested on bradykinin pathways. Overall, it

shows that our compounds act both on prostaglandins E₂and leuko-

triene pathways. Unlike NSAIDs, our compounds inhibit the major

pathways in the production of arachidonic acid. Bradykinin is the

vasoactive peptides produced by the action kallikreins on the plasma

kininogen. It mainly produces the dilation of small blood vessels, in-

creases vascular permeability, and induction of pain which are present

in the inﬂammatory processes. Moreover, the compounds working on

the major pathways have very poor activity on bradykinin B1 and B2

receptors. Consequently, our compounds also proved to be very on the

bradykinin pathway validating the actions of our compounds on both

cyclooxygenase and lipoxygenase pathways. Bradykinin stimulated the

arachidonic acid release and prostaglandin synthesis in a variety of

tissues. This will also increase the inositol phosphate formation via

activating the phosphatidylinositol-speciﬁc phospholipase C. It has been

(% inhibition

SEM

@ 10 μM)

76

77

78

H

OH

25.1 ± 1.11

37.0 ± 1.44

(19%)

0.175 ±

0.01

148

195

–

CH₃

H

0.196 ±

0.03

0.051

0.001

79

H

CH₃

(15%)

0.18 ±

–

0.02

with increase in paw volume. The observed changes in connection with

PGE2 inﬂammation were signiﬁcantly modiﬁed by our selected com-

pounds 61, 67 and 78 (each 10 mg/kg) and standard Celecoxib (50 mg/

kg). The compound 61 signiﬁcantly reduced the PGE2-induced paw

edema with 64.55% (at 1st hour) reaching to a maximal level of 81.08%

(4th hour) and continued to be signiﬁcant till the 5th hour. Similarly, 67

and 78 displayed promising results (51.38–59.20% and 55.88–65.25%

respectively) till the 3rd hour of observations. The maximum observed

percent inhibition of standard drug Celecoxib was 87.14% in the same

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Bioorganic Chemistry 112 (2021) 104969

Fig. 4. Percent inhibition produced by compounds 61, 67 and 78 (5, 10 and 20 mg/kg) in the carrageenan induced inﬂammation. Each percent point shown mean ±

SEM. Values are presented as mean ± SEM, n = 08. Data were analyzed by Two-way ANOVA followed by Bonferroni posttest. ### Shows difference between Vehicle

and Standard drug group while *** shows signiﬁcant values in standard drug and tested compounds groups. *p < 0.01, **p < 0.001, ***p < 0.0001 and n.s;

not signiﬁcant.

Fig 5. (a) Percent inhibitions produced by the compounds 61, 67 and 78 (10 mg/kg) in histamine induced paw edema; (b) Percent inhibitions produced by the

compounds 61, 67 and 78 (10 mg/kg) in bradykinin induced paw edema model of inﬂammation; (c) Percent inhibitions produced by the compounds 61, 78 and 88

(10 mg/kg) in prostaglandin E2 induced paw edema assay; (d) Percent inhibitions produced by the compounds 61, 78 and 88 (10 mg/kg) in leukotriene induced paw

edema model in mice. Each percent point represents the mean for group of 08 mice. Data was analyzed by two-way ANOVA posttest. *P < 0.05, **P < 0.01, ***P <

0.001, n.s; indicates non-signiﬁcant.

Table 7

In-vitro bovine carbonic anhydrase assay on sulfonamide derivatives of substituted 2-(1-amino-2,5-dioxopyrrolidin-3-yl)-2-methylpropanal (77–81).

Compound No.

bCA-II IC₅₀(μM) ± SEM

66

0.43 ± 0.11

0.51 ± 0.13

0.41 ± 0.08

0.76 ± 0.10

0.59 ± 0.06

0.48 ± 0.02

0.13 ± 0.01

0.17 ± 0.01

67

68

69

70

78

79

Acetazolamide (Standard)

suggested that free arachidonate may be released by the action of

phosphatidylinositol-speciﬁc phospholipase C followed by diglyceride

or monoglyceride lipases. Bradykinin receptors coupled to a G protein

and activate Phospholipase A2 to release arachidonate for PG synthesis.

2.6. Off-target carbonic anhydrase inhibition

The presence of the primary sulfonamide moiety is a key structural

feature of our synthesized compounds 66–70 and 78–79. Apart from

COX inhibition, sulfonamide is considered an important template for

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Bioorganic Chemistry 112 (2021) 104969

various pharmacological activities. It has been reported that the primary

sulfonamide COX-2 inhibitors celecoxib and valdecoxib also have the

potential to inhibit a number of isozymes of carbonic anhydrase (CA, EC

4.2.1.1) in nanomolar afﬁnity [46–48]. Interestingly, rofecoxib (a

methyl sulfone analog) was not able to show signiﬁcant human CA-II

inhibition. These results encouraged us to test our sulfonamide de-

rivatives against bovine carbonic anhydrase (bCA-II). The results in

terms of IC₅₀values are presented in Table 7. Acetazolamide was used as

a standard drug. All the tested compounds showed bCA-II inhibition in

the submicromolar range. sulfamoylphenyl)propenamide derivative 79

2-methylpropoic acid ester (71) and sulfonamides (67, 78, 79) de-

rivatives of 1-(benzyl(4-methoxyphenyl)amino)-1H-pyrrole-2,5-dione

(38) into the binding site of COX-2 isozyme (PDB = 1CX2). The choice of

these represented compounds is to highlight the required features

essential for COX-2 selectivity. It is important to note that we have

explored the predictive power of docking simulations and performed

docking studies were carried out on R- and S-isomers.

As illustrated in Fig. 6, carboxylic acid derivatives fully insert into

the selectivity pocket of COX-2. Fig. 7 showed the orientations of the

carboxylic acid derivatives 61, 76–77, ester 71, and compares them

with the binding orientation of native SC-558. As shown in Fig. 7a, 1-

(benzyl(4-methoxyphenyl)amino moiety of 2-methylpropoic acid de-

rivative (61) is well accommodated in the selectivity pocket of COX-2.

and forms hydrogen bond interactions with Arg513 and His90.

Methoxy ring is directed towards the constriction site and forms hy-

drophobic interactions with Tyr355. Carboxylic acid group oriented it-

self towards the small hydrophobic pocket and forms hydrogen bond

with Gly526. Dimethyl group forms hydrophobic interactions (not

shown in Figure due to clarity) with Trp387, Phe518, and Ala527.

Propanoic acid derivative (76) forms hydrogen bond interactions with

(IC₅₀= 0.13 μM) emerged as the most potent inhibitor than the standard

acetazolamide.

Inhibition of CA-II is a signiﬁcant ﬁnding of the current study. The

synthesized dual hybrids compounds can interact simultaneously with

two different pathways i.e. COX-2 and CA-II. Evidence from the litera-

ture revealed that the use of dual COX-2 and CA inhibitors could be used

for adjuvant therapy of different cancers or CA related diseases.

Furthermore, dual inhibitors are less prone to drug resistance and

toxicity [47]. Our ﬁndings on the dual COX/CA inhibition highlighted

the promising future of synthesized sulfonamide derivatives for devel-

oping improved and nontoxic inhibitors on both targets.

Arg513 and Ser353. His90 displayed

π

-alkyl interactions with a methyl

group. While benzyl group ring also forms

π

-alkyl interactions with

Leu352 (Fig. 7b). In contrast, acetic acid derivative (77), binds in the

COX-2 active site in an inverted binding orientation with carboxylic acid

moiety is embedded deep into the selectivity pocket and forms hydrogen

bond interactions with Arg513 and His90. While hydroxyl group also

interacts with Leu352 via hydrogen bond. Methoxy group of the com-

pound 87 directed towards the top of the COX-2 active site and forms

2.7. Docking studies

The active site of the COX enzyme is a long hydrophobic channel that

extends to the lobby (membrane-binding domain, MBD). The selectivity

of the selective COX-2 inhibitors is associated with their structural fea-

tures. This is mainly due to the structural difference in the two isozymes.

The existence of a spacious selectivity pocket (about 20%) due to less

bulky Val523 allows access to the bulky groups. The selectivity pocket is

present above the Arg120, Tyr355, and Glu524 constriction site which

play important role in the binding of inhibitors especially carboxylic

acids. Arg513 is located at the base of the selectivity pocket and forms

hydrogen bonds with ligands. Other residues important for COX-2

selectivity are His90, Gln192, Leu352, and Ser353.

hydrogen bond interactions with –OH of Tyr385. Met 522 forms

π-sulfur

interactions with the benzyl ring (Fig. 7c). Ester derivative 71 hydrogen

bond interactions with His90. Ser353 established π-σ interactions. While

–

π-alkyl interactions, C H types

Arg120, Phe518, and Val523 form weak

of interactions.

Target compounds 68, 78, 79 each containing bulky sulfonamide

group were also studied to highlight the required features essential for

COX-2 selectivity. Fig. 8 illustrated the binding orientation of the sul-

fonamides. Bulky sulfonamide accommodated well into the selectivity

pocket of COX-2. Within this family, we studied the binding orientation

of R- and S-isomers of 2-methylpropanamide (78). S-67 positioned with

their sulfonamide moiety deeply embedded into the selectivity pocket

and aromatic functional groups projecting towards constriction-site and

forms a hydrogen bond with Guandidinium-NH₂of Arg120. Similarly,

guanidine-NH₂of Arg513 forms a hydrogen bond interactions with

sulfonyl oxygen. Sulfonamide-NH forms hydrogen bond with Gln192,

To rationalize the results obtained via in-vitro COX-2 inhibition

assay, we performed docking simulations by using Molecular Operating

Environment (MOE 2016.0208) software. Crystal structure of COX-2 in

complex with SC-558 was retrieved from Protein Data Bank (PDB code

1CX2). First, the validation of the docking procedure was carried out by

the comparison of binding orientation and position of re-docked native

ligand (SC-558) with the experimentally determined X-ray crystal

structure. The lowest energy conformation computed by the MOE pro-

gram was found very close to crystal structure bound conformation. The

computed root-mean-square deviation (RMSD) between the re-docked

pose and crystal structure bound conformation was 0.74 Å indicating

that MOE can reproduce the correct binding pose.

and Leu352. Sulfur atom displayed

Benzene sulfonamide ring forms

functional groups in R-67 projecting towards the top of the active site

and forms T-shaped interactions with Trp387 and -lone pair type of

interaction with Ser530. It is important to note that highly selective

π

-sulfur interactions with His90.

π-σ interactions. In contrast, aromatic

π-

π

After validation, we docked most active 2-methylpropoic (61,

– –

C H) acetic (77 –CH₂-) acids,

dimethyl group), propanoic (76, –CH₃

Fig. 6. Ribbon model of the superimposed binding poses of carboxylic acid derivatives 61, 76 and 77, ester derivative 75 and native SC-558. into the binding site of

human COX-2 (1CX2).

11

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Bioorganic Chemistry 112 (2021) 104969

Fig. 7. Close-up depiction of the lowest-energy three-dimensional (3-D) docking poses of carboxylic acid derivatives (a) 61 (b) 76 (c) 77 and (d) ester 71 into the

binding site of human COX-2 (1CX2).

Fig. 8. Ribbon model of the superimposed binding poses of sulfonamide derivatives S-67, R-67, 78, 79 into the binding site of human COX-2 (1CX2). The secondary

pocket residues (His90, Gln192, Leu352, Ser353, Arg513 and Val523 are shown as turquoise spheres (left). While, on right side these amino acids are shown in

individual color for clarity.

Fig. 9. Close-up depiction of the lowest-energy three-dimensional (3-D) docking poses of sulfonamide derivatives (a) S-67, (a) R-67 (c) 78, (d) 79 into the binding

site of human COX-2 (1CX2).

12

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

lumiracoxib and diclofenac also form hydrogen bond interactions with

Ser530, and Trp385.¹Sulfonamide group in R-67 is oriented toward

selectivity pocket and establish hydrogen bond interactions with His90,

Gln192, Leu352 and Ser353. His90 also interacts with S-atom via

value of 0.54 (Hexane/Ethyl acetate 6:4). Isolated yield = 74%. ¹H NMR

(CDCl₃, 400 MHz): δ 6.75 (s, 2H), 3.78 (t, J = 3.98 Hz, 4H), 2.95 (t, J =

4.63 Hz, 4H). ¹³C NMR (CDCl₃, 100 MHz): δ 171.2, 133.6, 65.3, 54.8.

π

-sulfur interactions. Binding-poses and interaction plots of prop-

4.2.3. 1-(4-Methylpiperazin-1-yl)-1H-pyrrole-2,5-dione (16)

The compound appeared as Yellowish-White solid with an observed

R_fvalue of 0.51 (Hexane/Ethyl acetate 6:4). Isolated yield = 70%. ¹H

NMR (CDCl₃, 400 MHz): δ 6.75 (s, 2H), 2.84 (t, J = 8.01 Hz, 4H), 2.49

(t, J = 7.34 Hz, 4H), 2.27 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 171.2,

135.2, 158.3, 153.5, 145.3.

anamide (78) and acetamide (79) are shown in Fig. 9a-c.

3. Conclusions

In an effort to synthesize better and selective COX-2 inhibitors than

traditional marketed inhibitors, new analogs of pyrrolidine-2,5-dione

were designed, wherein the benzene sulfonamide pharmacophore re-

ported in our previous study is replaced by N-(benzyl(4-methoxyphenyl)

amino-pyrrolidine-2,5-dione. In-vitro enzyme inhibition results against

COX /LOX revealed that this new template is crucial for selectivity and

potency. The carboxylic acid group was substituted for aldehyde moiety

in the lead compounds. Further substitution of the carboxylic group with

sulfonamide generated the most potent and selective-COX-2 inhibitors.

The IC₅₀values of compounds 67 (64 nM) and 78 (51 nM) were found to

be very close to with IC₅₀value close to the standard drug celecoxib (50

nM). Compounds 61, 67 and 78 were further investigated for anti-

inﬂammatory activity via carrageenan-induced paw edema test. The

multiple mechanistic approaches applied for compounds 61, 67, and 78

indicates a reduction in the level of the prostaglandins and leukotrienes.

This study provide the scientiﬁc support for the use of investigated

compounds for the treatment of inﬂammatory diseases. Another signif-

icant ﬁnding of the current study is the off-target inhibition of bovine

carbonic anhydrase-II (bCA-II). All the tested compounds showed bCA-II

inhibition in the submicromolar range. Evidence from the literature

revealed that the use of dual COX-2 and CA inhibitors could be used for

adjuvant therapy of different cancers. Furthermore, dual inhibitors are

less prone to drug resistance and drug-drug interactions. Our ﬁndings on

the dual COX/CA inhibition highlighted the promising future of syn-

thesized sulfonamide derivatives for developing improved and nontoxic

inhibitors on both targets. Biological results were ﬁnally rationalized by

docking simulations. Typically, the compounds interact with deeply

located Arg513 via hydrogen bond. Moreover, most active COX-2 in-

hibitors interact with the amino acid residues important for selectivity.

Overall, the current study would be interesting to take highly potent

compounds to the next level of studies.

4.2.4. 1-(Dimethylamino)-1H-pyrrole-2,5-dione (31)

The compound appeared in reddish color with an observed R_fvalue

of 0.56 (Hexane/Ethyl acetate 6:4). Isolated yield = 75%. ¹H NMR

(CDCl₃, 400 MHz): δ 6.78 (s, 2H), 2.71 (s, 6H). ¹³C NMR (CDCl₃, 100

MHz): δ 174.1, 134.9, 47.3.

4.2.5. 1-(Diphenylamino)-1H-pyrrole-2,5-dione (32)

The compound appeared as dark Brown oily with an observed R_f

value of 0.50 (Hexane/Ethyl acetate 6:4). Isolated yield = 73%. ¹H NMR

(CDCl₃, 400 MHz): δ 7.45–7.41 (m, 4H), 7.36–7.26 (m, 2H), 7.23–7.15

(m, 3H), 7.13–7.10 (m, 1H), 6.79 (s, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ

171.2, 142.6, 138.1, 131.2, 124.3, 122.0.

4.2.6. 1-(Phenylamino)-1H-pyrrole-2,5-dione (33)

The compound appeared in dark color semisolid with an observed R_f

value of 0.50 (Hexane/Ethyl acetate 6:4). Isolated yield = 76%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.69 (bs, 1H), 7.39–7.25 (m, 5H), 6.75 (s, 2H).

¹³C NMR (CDCl₃, 100 MHz): δ 172.3, 146.1. 135.6, 132.5, 130.5, 124.3,

119.3.

4.2.7. 1-((4-Methoxyphenyl)amino)-1H-pyrrole-2,5-dione (34)

The compound appeared as dark color semisolid with an observed R_f

value of 0.58 (Hexane/Ethyl acetate 6:4). Isolated yield = 73%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.63 (bs, 1H), 6.99 (d, J = 7.15 Hz, 2H), 6.84 (d,

J = 7.21 Hz, 2H), 6.77 (s, 2H), 3.80 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz):

δ 171.3, 154.0, 143.1, 132.6, 120.6, 118.4, 57.3.

4.2.8. 1-(p-Tolylamino)-1H-pyrrole-2,5-dione (35)

The compound appeared as dark color oily/semisolid with an

observed R_fvalue of 0.61 (Hexane/Ethyl acetate 6:4). Isolated yield =

77%. ¹H NMR (CDCl₃, 400 MHz): δ 10.51 (s, 1H), 7.05 (d, J = 8.02 Hz,

2H), 6.98 (d, J = 7.89 Hz, 2H), 6.77 (s, 2H), 2.34 (s, 3H). 13C NMR

(CDCl3, 100 MHz): δ 172.4, 141.5, 133.6, 130.2, 128.6, 119.8, 23.4.

4. Materials and methods

4.1. General

4.2.9. N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide (36)

The compound appeared as dark color solid with an observed R_f

value of 0.44 (Hexane/Ethyl acetate 6:4). Isolated yield = 79%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.88 (s, 1H), 7.94–7.62 (m, 5H), 6.73 (s, 2H). 13C

NMR (CDCl3, 100 MHz): δ 169.3, 137.6, 133.7, 131.6, 129.8, 128.4.

Purchase of chemicals and characterization methods are presented in

Supporting Information

4.2. Synthesis of N-Monosubstituted amino-1H-pyrrole-2,5-diones

(14–16, 31–37)

4.2.10. N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-

Different hydrazine derivatives were reacted with maleic anhydride

which produced substituted maleanic acid intermediates. The corre-

sponding intermediates were then cyclized with acetic anhydride at high

temperature in the presence of sodium acetate to produce the respective

maleimide (1–10).

methylbenzenesulfonamide (37)

The compound appeared as Brown solid with an observed R_fvalue of

0.48 (Hexane/Ethyl acetate 6:4). Isolated yield = 77%. ¹H NMR (CDCl₃,

400 MHz): δ 10.63 (bs, 1H), 8.03 (d, J = 5.80 Hz, 2H), 7.73 (d, J = 6.13

Hz, 2H), 6.78 (s, 2H), 2.39 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 170.8,

139.6, 135.2, 133.7, 130.8, 128.7, 24.1.

4.2.1. 1-(Piperidin-1-yl)-1H-pyrrole-2,5-dione (14)

The compound appeared as White solid with an observed R_fvalue of

0.53 (Hexane/Ethyl acetate 6:4). Isolated yield = 71%. ¹H NMR (CDCl₃,

400 MHz): δ 6.77 (s, 2H), 2.79–2.69 (m, 4H), 1.45–1.31 (m, 2H),

1.26–1.11 (m, 4H). ¹³C NMR (CDCl3, 100 MHz): δ 170.3, 131.8, 52.1,

22.4, 19.8.

4.3. Synthesis of disubstituted maleimides

Under nitrogen atmosphere, sodium hydride (60% in mineral oil,

10.6 mmol, 700 mg) was dispersed in a 1:2 mixture of anhydrous DMF

and DCM. (10 mL) at 0 ^◦C. A solution of maleimides (33–34) in 1:2

mixture of anhydrous DMF and DCM. (10 mL) was gently added and

continued stirring at 0 ^◦C 45–50 min. Afterwards, chlorobenzyl chloride

4.2.2. 1-Morpholino-1H-pyrrole-2,5-dione (15)

The compound appeared as White color solid with an observed R_f

13

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

(38, 1.5 equiv) / sulfonyl chlorides (39–40) in solvent mixture was

added dropwise. The resulting mixture was further stirred at room

temperature for 4–5 hr. After completion of reaction 20 mL brine was

added. The aqueous phase was extracted with 3 × 30 mL of ethyl ace-

tate. The combined organic layers were dried over MgSO₄, ﬁltered, and

evaporated in vacuo. The crude maleimide was puriﬁed by silica gel

column chromatography (heptane/EtOAc 2:1)

4.4.2. 2-Methyl-2-(1-morpholino-2,5-dioxopyrrolidin-3-yl)propanal (47)

The compound appeared as solid with an observed R_fvalue of 0.37

(Hexane/Ethyl acetate 6:4). Isolated yield = 73%. ¹H NMR (CDCl₃, 400

MHz): δ 9.51 (s, 1H), 3.73–3.58 (m, 4H), 3.31 (dd, J = 5.80, 9.21 Hz,

1H) 3.04 (dd, J = 9.35, 18.37 Hz, 1H), 2.93–2.74 (m, 4H), 2.59 (dd, J =

5.82, 18.42 Hz, 1H), 1.35 (s, 3H), 1.26 (s, 3H). ¹³C NMR (CDCl₃, 100

MHz): δ 203.6, 179.8, 176.7, 69.4, 68.8, 54.1, 53.6, 52.1, 41.4, 31.1,

20.0, 18.7.

4.3.1. 1-(Benzyl(4-methoxyphenyl)amino)-1H-pyrrole-2,5-dione (41)

The compound appeared as light Reddish color semisolid with an

observed R_fvalue of 0.46 (Hexane/Ethyl acetate 6:4). Isolated yield =

4.4.3. 2-Methyl-2-(1-(4-methylpiperazin-1-yl)-2,5-dioxopyrrolidin-3-yl)

propanal (48)

1

72%. H NMR (CDCl₃, 400 MHz): δ 7.40–7.27 (m, 5H), 7.01 (d, J =

The compound appeared as solid with an observed R_fvalue of 0.36

(Hexane/Ethyl acetate 6:4). Isolated yield = 75%. ¹H NMR (CDCl₃, 400

MHz): δ 9.55 (s, 1H), 3.23 (dd, J = 5.20, 9.57 Hz, 1H) 2.83 (dd, J = 9.21,

18.41 Hz, 1H), 2.69–2.55 (m, 5H), 2.42–2.33 (m, 4H), 2.28 (s, 3H), 1.30

(s, 3H), 1.25 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 203.8, 178.0, 175.4,

55.8, 54.7, 53.3, 52.1, 51.5, 42.6, 31.4, 19.5, 18.7.

9.05 Hz, 2H), 6.85 (d, J = 8.73 Hz, 2H), 6.75 (s, 2H), 4.63 (s, 2H), 3.83

(s, 3H). 13C NMR (CDCl3, 100 MHz): δ 170.5, 156.3, 140.6, 138.2,

134.6, 130.4, 129.6, 129.1, 127.8, 118.4, 117.2, 67.5, 54.8.

4.3.2. 1-(Benzyl(phenyl)amino)-1H-pyrrole-2,5-dione (42)

The compound appeared as light dark semisolid with an observed R_f

value of 0.58 (Hexane/Ethyl acetate 6:4). Isolated yield = 71%. ¹H NMR

(CDCl₃, 400 MHz): δ 7.39–7.25 (m, 5H), 7.13–6.79 (m, 5H), 6.78 (s,

2H), 4.65 (s, 2H). 13C NMR (CDCl3, 100 MHz): δ 172.5, 152.1, 144.3,

134.6, 130.5, 129.8, 129.0, 128.5, 128.1, 124.3, 116.3, 65.8.

4.4.4. 2-(1-(Dimethylamino)-2,5-dioxopyrrolidin-3-yl)-2-methylpropanal

(49)

The compound appeared as light color solid with an observed R_f

value of 0.35 (Hexane/Ethyl acetate 6:4). Isolated yield = 61%. ¹H NMR

(CDCl₃, 400 MHz): δ 9.51 (s, 1H), 3.18 (dd, J = 5.6, 9.6 Hz, 1H) 3.01

(dd, J = 9.6, 18.4 Hz, 1H) 2.87 (s, 6H), 2.67 (dd, J = 5.6, 18.4 Hz, 1H),

1.29 (s, 3H), 1.25 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 205.3, 177.4,

177.4, 50.1, 43.7, 42.4, 42.4, 31.2, 20.0, 19.0.

4.3.3. N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-

phenylbenzenesulfonamide (43)

The compound appeared as light color solid with an observed R_f

value of 0.48 (Hexane/Ethyl acetate 6:4). Isolated yield = 69%. ¹H NMR

(CDCl₃, 400 MHz): δ 7.69–7.57 (m, 3H), 7.45–7.35 (m, 4H), 7.30–7.26

(m, 3H), 6.75 (s, 2H). 13C NMR (CDCl3, 100 MHz): δ 173.2, 145.3,

144.2, 134.5, 131.2, 130.5, 129.8, 128.3, 127.3, 124.6 116.5.

4.4.5. 2-(1-(Diphenylamino)-2,5-dioxopyrrolidin-3-yl)-2-methylpropanal

(50)

The compound appeared as dark sticky semisolid with an observed R_f

value of 0.28 (Hexane/Ethyl acetate 6:4). Isolated yield = 68%. ¹H NMR

(CDCl₃, 400 MHz): δ 9.57 (s, 1H), 7.54–7.43 (m, 6H), 7.23–7.19 (m,

2H), 7.15–7.12 (m, 2H), 3.34 (dd, J = 4.6, 9.3 Hz, 1H) 3.06 (dd, J = 9.4,

18.9 Hz, 1H), 2.60 (dd, J = 4.6, 18.9 Hz, 1H), 1.37 (s, 3H), 1.33 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 205.7, 179.8, 175.9, 147.8, 147.6,

130.8, 130.3, 128.8, 128.7, 128.1, 128.0, 126.2, 124.0, 123.8, 52.3,

42.8, 32.0, 19.4, 18.0.

4.3.4. N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-methyl-N-

phenylbenzenesulfonamide (44)

The compound appeared as light-Yellow semisolid with an observed

R_fvalue of 0.50 (Hexane/Ethyl acetate 6:4). Isolated yield = 68%. ¹H

NMR (CDCl₃, 400 MHz): δ 7.73 (d, J = 9.63 Hz 2H), 7.52 (d, J = 8.69

Hz, 2H), 7.42–7.27 (m, 5H), 6.75 (s, 2H), 2.37 (s, 3H). 13C NMR (CDCl3,

100 MHz): δ 173.6, 145.2, 139.5, 135.3, 133.4, 130.8, 129.3, 128.9,

125.4, 116.3, 24.3.

4.4.6. 2-(2,5-Dioxo-1-(phenylamino)pyrrolidin-3-yl)-2-methylpropanal

(51)

4.4. Synthesis of disubstituted amin-2,5-dioxopyrrolidin-3-yl)-2-

The compound appeared as dark semisolid with an observed R_fvalue

of 0.28 (Hexane/Ethyl acetate 6:4). Isolated yield = 73%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.50 (bs, 1H), 9.55 (s, 1H), 7.52–7.45 (m, 2H),

7.42–7.30 (m, 3H), 3.23 (dd, J = 5.9, 9.5 Hz, 1H) 2.88 (dd, J = 9.5, 18.2

methylpropanal derivatives (46–59, 74–75)

The isobutyraldehyde (45)/acetaldehyde (82)/ propionaldehyde

(83) in excess amount (1.5 equiv) was mixed with O-tert-^L-threonine and

potassium hydroxide (each 5 mol%) to produce the nucleophilic

enamine. The maleimides (31–37, 41–44) (1.0 equiv) were added and

continued stirring at normal laboratory temperature. The conversion of

staring materials into product was monitored by TLC. At complete

conversion (for each reaction), water was added to quench the catalyst

activity. The crude product was extracted with chloroform three times.

The organic layers were combined and dried. The crude succinimide

product (in each reaction) was puriﬁed by column chromatography

using n-hexane and ethyl acetate as solvent system.

Hz, 1H) 2.69 (dd, J = 5.9, 18.2 Hz, 1H), 1.30 (s, 3H), 1.25 (s, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 204.2, 178.7, 176.2, 132.1, 129.2, 128.7,

125.8, 53.7, 43.8, 32.6, 20.7, 19.3.

4.4.7. 2-(1-((4-Methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-2-

methylpropanal (52)

The compound appeared as semisolid with an observed R_fvalue of

0.33 (Hexane/Ethyl acetate 6:4). Isolated yield = 71%. ¹H NMR (CDCl₃,

400 MHz): δ 10.45 (bs, 1H), 9.53 (s, 1H), 7.22 (d, J = 6.8 Hz, 2H), 7.14

(d, J = 7.2 Hz, 2H), 3.86 (s, 3H), 3.27 (dd, J = 5.9, 9.6 Hz, 1H), 2.86 (dd,

J = 9.6, 18.3 Hz, 1H), 2.58 (dd, J = 5.9, 18.3 Hz, 1H), 1.28 (s, 3H), 1.21

(s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 205.2, 177.2, 175.2, 150.3,

141.9, 122.7, 118.1, 58.178, 49.1, 40.3, 29.8, 18.7, 18.0.

4.4.1. 2-(2,5-Dioxo-1-(piperidin-1-yl)pyrrolidin-3-yl)-2-methylpropanal

(46)

The compound appeared as light color powder with an observed R_f

value of 0.38 (Hexane/Ethyl acetate 6:4). Isolated yield = 79%. ¹H NMR

(CDCl₃, 400 MHz): δ 9.54 (s, 1H), 3.30 (dd, J = 4.8, 9.2 Hz, 1H) 3.00

(dd, J = 9.2, 18.8 Hz, 1H) 2.90–2.70 (m, 4H), 2.63 (dd, J = 4.8, 18.8 Hz,

1H), 1.63–1.48 (m, 6H), 1.29 (s, 3H), 1.22 (s, 3H). ¹³C NMR (CDCl₃,

100 MHz): δ 204.4, 180.0, 176.4, 53.6, 52.1, 51.7, 45.0, 30.8, 27.8,

26.2, 25.4, 19.0, 18.4.

4.4.8. 2-(2,5-Dioxo-1-(p-tolylamino)pyrrolidin-3-yl)-2-methylpropanal

(53)

The compound appeared as Half White solid with an observed R_f

value of 0.34 (Hexane/Ethyl acetate 6:4). Isolated yield = 69%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.43 (bs, 1H), 9.55 (s, 1H), 7.14 (d, J = 8.12 Hz,

2H), 7.08 (d, J = 7.14 Hz, 2H), 3.20 (dd, J = 5.24, 9.51 Hz, 1H), 3.01

(dd, J = 9.51, 18.44 Hz, 1H), 2.61 (dd, J = 5.24, 18.45 Hz, 1H), 2.37 (s,

3H), 1.25 (s, 3H), 1.21 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 204.2,

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A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

177.6, 174.8, 147.4, 140.3, 123.3, 119.8, 51.9, 42.7, 31.2, 23.5, 20.4,

18.2.

122.6, 120.1, 118.2, 51.9, 42.1, 31.5, 24.9, 20.7, 19.3.

4.4.15. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)

acetaldehyde (74)

4.4.9. N-(3-(2-Methyl-1-oxopropan-2-yl)-2,5-dioxopyrrolidin-1-yl)

benzenesulfonamide (54)

In the synthesis of compound 84, acetaldehyde was used as Michael

donor. The compound appeared as solid with an observed Rf value of

0.49 (hexane/ethyl acetate 6:4). Isolated yield = 50%. ¹H NMR (CDCl₃,

400 MHz): δ 9.59 (s, 1H), 7.50–7.44 (m, 2H), 7.41–7.37 (m, 1H),

7.29–7.26 (m, 2H), 7.06 (d, J = 8.87 Hz, 2H), 6.78 (d, J = 9.14 Hz, 2H),

4.66 (d, J = 6.79 Hz, 2H), 3.83 (s, 3H), 3.42–3.27 (m, 2H), 2.97 (dd, J =

9.62, 18.41 Hz, 1H), 2.88–2.80 (m, 1H), 2.69 (dd, J = 5.88, 18.44 Hz,

1H). ¹³C NMR (CDCl₃, 100 MHz): δ 202.9, 177.6, 175.2, 154.6, 135.5,

133.1, 128.8, 128.7, 128.7, 128.0, 122.8, 118.2, 116.8, 54.3, 50.5, 44.2,

35.1, 30.9.

The compound appeared as dark semisolid with an observed R_fvalue

of 0.24 (Hexane/Ethyl acetate 6:4). Isolated yield = 79%. ¹H NMR

(CDCl₃, 400 MHz): δ 10.48 (bs, 1H), 9.55 (s, 1H), 8.06–7.97 (m, 2H),

7.73–7.58 (m, 3H), 3.45 (dd, J = 5.59, 9.66 Hz, 1H) 2.99 (dd, J = 9.71,

18.60 Hz, 1H), 2.68 (dd, J = 5.65, 18.57 Hz, 1H), 1.31 (s, 3H), 1.27 (s,

3H). ¹³C NMR (CDCl₃, 100 MHz): δ 205.3, 179.3, 175.8, 133.9, 129.3,

127.2, 123.3, 51.9, 42.7, 31.3, 20.0, 18.6.

4.4.10. 4-Methyl-N-(3-(2-methyl-1-oxopropan-2-yl)-2,5-dioxopyrrolidin-

1-yl)benzene- sulfonamide (55)

The compound appeared as solid with an observed R_fvalue of 0.25

(hexane/ethyl acetate 6:4). Isolated yield = 76%. ¹H NMR (CDCl₃, 400

MHz): δ 10.45 (bs, 1H), 9.55 (s, 1H), 8.08–8.04 (m, 2H), 7.51–7.48 (m,

2H), 3.46 (dd, J = 5.63, 9.64 Hz, 1H) 3.02 (dd, J = 9.65, 18.56 Hz, 1H),

2.69 (dd, J = 5.63, 18.57 Hz, 1H), 2.37 (s, 3H), 1.32 (s, 3H), 1.25 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 205.0, 180.4, 174.8, 133.9, 129.3,

129.0, 124.8, 50.8, 43.5, 29.8, 23.5, 20.7, 18.3.

4.4.16. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)

propanal (75)

The propionaldehyde was used for the synthesis of compound 85.

The compound appeared as Yellowish solid with an observed R_fvalue of

0.44 (Hexane/Ethyl acetate 6:4). Isolated yield = 53%. ¹H NMR (CDCl₃,

400 MHz): δ 9.59 (s, 1H), 7.38–7.25 (m, 5H), 7.03 (d, J = 9.37 Hz, 2H),

6.80 (d, J = 9.31 Hz, 2H), 4.65 (d, J = 3.86 Hz, 2H), 3.85 (s, 3H),

3.33–3.24 (m, 1H), 3.20–3.05 (m, 1H), 2.83 (dd, J = 9.33, 18.41 Hz,

4.4.11. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-

2-methylpropanal (56)

1H), 2.40 (dd, J = 5.11, 18.41 Hz, 1H), 1.29 (d, J = 8.22 Hz, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 203.1, 177.2, 175.5, 153.4, 139.1, 135.7,

128.8, 128.7, 128.7, 128.6, 127.9, 119.2, 116.0, 57.5, 51.9, 42.4, 42.1,

31.9, 15.8.

The compound appeared as semisolid with an observed R_fvalue of

0.43 (Hexane/Ethyl acetate 6:4). Isolated yield = 53%. ¹H NMR (CDCl₃,

400 MHz): δ 9.52 (s, 1H), 7.40–7.35 (m, 2H), 7.33–7.25 (m, 3H), 7.11

(d, J = 9.05 Hz, 2H), 6.85 (d, J = 8.87 Hz, 2H), 4.76 (d, J = Hz, 2H), 3.87

(s, 3H), 3.24 (dd, J = 5.00, 9.47 Hz, 1H) 2.82 (dd, J = 9.38, 18.30 Hz,

4.5. Synthesis of carboxylic acid derivatives (60–64, 76–77)

1H), 2.50 (dd, J = 5.04, 18.51 Hz, 1H), 1.28 (s, 3H), 1.27 (s, 3H). ¹³

C

The aldehyde product (compounds 50, 56–59, 1 equiv) was diluted

in 10 mL anhydrous dimethylformamide followed by the addition of 1.1

equivalent of potassium peroxymonosulfate. The mixture was stirred at

normal laboratory temperature. After 3 h of stirring, the reaction was

quenched with aqueous HCl (1 M, 100 mL). The organic layer was

separated three time with ethyl acetate (100 mL each time). The organic

layers were diluted with 1 M aq.HCl and brine (each 100 mL), mixed

with anhydrous sodium sulfate, ﬁltered and then concentrated by rotary

evaporator. The crude oxidized products were then puriﬁed by gravity

column chromatography using ethyl acetate and 0.2% formic acid.

NMR (CDCl₃, 100 MHz): δ 204.3, 177.7, 175.3, 142.5, 141.6, 135.6,

128.8, 128.7, 128.1, 128.1, 123.8, 121.7, 119.6, 52.1, 43.1, 42.6, 31.5,

22.2, 18.5, 17.9.

4.4.12. 2-(1-(Benzyl(phenyl)amino)-2,5-dioxopyrrolidin-3-yl)-2-

methylpropanal (57)

The compound appeared as clear solid with an observed R_fvalue of

0.42 (Hexane/Ethyl acetate 6:4). Isolated yield = 56%. ¹H NMR (CDCl₃,

400 MHz): δ 9.51 (s, 1H), 7.42–7.24 (m, 5H, 3H, 2H), 4.71 (d, J = 6.63

Hz, 2H), 3.23 (dd, J = 5.70, 9.33 Hz, 1H) 2.82 (dd, J = 9.38, 18.30 Hz,

1H), 2.51 (dd, J = 5.79, 18.20 Hz, 1H), 1.25 (s, 3H), 1.24 (s, 3H). ¹³

C

4.5.1. 2-(1-(Diphenylamino)-2,5-dioxopyrrolidin-3-yl)-2-methylpropanoic

acid (60)

NMR (CDCl₃, 100 MHz): δ 203.5, 177.6, 174.5, 139.2, 137.8, 131.9,

129.2, 128.7, 128.0, 126.5, 123.9, 118.9, 116.9, 52.8, 42.6, 39.5, 31.9,

19.1, 18.5.

The compound appeared as solid with an observed R_fvalue of 0.30

(Hexane/Ethyl acetate 1:1). Isolated yield = 61%. ¹H NMR (CDCl₃, 400

MHz): δ 8.96 (bs, 1H), 7.36–7.29 (m, 5H), 7.26–7.22 (m, 3H), 7.18–7.16

(m, 2H), 3.52 (dd, J = 5.02, 9.40 Hz, 1H), 2.96 (dd, J = 9.50, 18.68 Hz,

4.4.13. N-(3-(2-Methyl-1-oxopropan-2-yl)-2,5-dioxopyrrolidin-1-yl)-N-

phenylbenzene- sulfonamide (58)

1H), 2.68 (dd, J = 4.95, 18.58 Hz, 1H), 1.40 (s, 3H), 1.36 (s, 3H). ¹³

C

The compound appeared as semisolid with an observed R_fvalue of

0.40 (Hexane/Ethyl acetate 6:4). Isolated yield = 59%. ¹H NMR (CDCl₃,

400 MHz): δ 9.57 (s, 1H), 7.75–7.60 (m, 6H), 7.29–7.26 (m, 2H),

7.07–7.01 (m, 2H), 3.72 (dd, J = 4.94, 9.30 Hz, 1H) 2.97 (dd, J = 9.45,

18.93 Hz, 1H), 2.57 (dd, J = 4.91, 18.91 Hz, 1H), 1.37 (s, 3H), 1.31 (s,

3H). ¹³C NMR (CDCl₃, 100 MHz): δ 204.2, 178.3, 175.4, 147.7, 146.7,

135.6, 128.7, 128.6, 128.1, 128.0, 123.7, 120.9, 119.6, 51.9, 42.5, 31.5,

20.0, 18.9.

NMR (CDCl₃, 100 MHz): δ 178.3, 175.2, 174.1, 145.3, 145.0, 132.5,

131.9, 130.9, 129.2, 128.8, 128.7, 126.5, 125.9, 125.6, 48.4, 40.4, 32.7,

18.7, 18.0.

4.5.2. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-

2-methylpropanoic acid (61)

The compound appeared as semisolid with an observed R_fvalue of

0.25 (Hexane/Ethyl acetate 1:1). Isolated yield = 59%. ¹H NMR (CDCl₃,

400 MHz): δ 8.94 (bs, 1H), 7.38–7.25 (m, 5H), 7.13 (d, J = 7.64 Hz, 2H),

7.01 (d, J = 7.83 Hz, 2H), 4.65 (d, J = 3.86 Hz, 2H), 3.85 (s, 3H), 3.33

(dd, J = 5.53, 8.76 Hz, 1H), 2.83 (dd, J = 9.33, 18.41 Hz, 1H), 2.53 (dd,

J = 5.71, 18.34 Hz, 1H), 1.30 (s, 3H), 1.26 (s, 3H). ¹³C NMR (CDCl₃,

100 MHz): δ 178.7, 176.2, 175.2, 143.9, 143.1, 135.3, 130.9, 129.2,

128.8, 128.7, 126.5, 125.5, 124.5, 56.8, 42.6, 40.4, 32.7, 23.8, 18.7,

18.0.

4.4.14. 4-Methyl-N-(3-(2-methyl-1-oxopropan-2-yl)-2,5-dioxopyrrolidin-

1-yl)-N-phenylbenzene sulfonamide (59)

The compound appeared as oily with an observed R_fvalue of 0.37

(Hexane/Ethyl acetate 6:4). Isolated yield = 56%. ¹H NMR (CDCl₃, 400

MHz): δ 9.55 (s, 1H), 7.73 (d, J = 8.93 Hz, 2H), 7.54–7.45 (m, 4H),

7.40–7.35 (m, 1H), 7.26–7.22 (m, 2H), 3.72 (dd, J = 5.60, 9.35 Hz, 1H)

3.04 (dd, J = 9.25, 18.37 Hz, 1H), 2.64 (dd, J = 5.58, 18.35 Hz, 1H),

2.37 (s, 3H), 1.38 (s, 3H), 1.35 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

204.8, 180.1, 177.5, 148.5, 147.0, 135.8, 128.7, 128.6, 127.8, 127.9,

15

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Bioorganic Chemistry 112 (2021) 104969

4.5.3. 2-(1-(Benzyl(phenyl)amino)-2,5-dioxopyrrolidin-3-yl)-2-

methylpropanoic acid (62)

4.6.1. Ethyl 2-(1-(benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-

yl)-2-methyl- propanoate (71)

The compound appeared as solid with an observed R_fvalue of 0.29

(Hexane/Ethyl acetate 1:1). Isolated yield = 63%. ¹H NMR (CDCl₃, 400

MHz): δ 8.91 (s, 1H), 7.46–7.21 (m, 5H, 3H, 2H), 4.68 (d, J = 6.89 Hz,

2H), 3.25 (dd, J = 4.96, 9.23 Hz, 1H) 2.85 (dd, J = 9.22, 18.21 Hz, 1H),

2.56 (dd, J = 4.99, 18.20 Hz, 1H), 1.26 (s, 3H), 1.23 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 179.0, 178.1, 176.5, 140.6, 138.2, 132.6, 131.9,

129.1, 129.0, 127.5, 122.5, 117.5, 117.0, 54.5, 43.7, 41.5, 35.6, 18.5,

18.0.

The compound appeared as White powder with an observed R_fvalue

of 0.46 (n-hexane/ethyl acetate 3:2). Isolated yield = 71%. ¹H NMR

(CDCl₃, 400 MHz): δ 7.52–7.44 (m, 2H), 7.43–7.36 (m, 1H), 7.29–7.25

(m, 2H), 7.13 (d, J = 9.17 Hz, 2H), 6.86 (d, J = 8.84 Hz, 2H), 4.73 (d, J

= 6.71 Hz, 2H), 4.04 (q, J = 8.96 Hz, 2H), 3.87 (s, 3H), 3.25 (dd, J =

5.84, 9.53 Hz, 1H), 2.95 (dd, J = 9.59, 18.31 Hz, 1H), 2.59 (dd, J = 5.72,

18.33 Hz, 1H), 1.30 (s, 3H), 1.26 (s, 3H), 1.14 (t, J = 7.70 Hz, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 177.2, 176.2, 174.1, 137.6, 136.7, 133.3,

129.6, 129.5, 129.1, 129.1, 128.3, 127.1, 125.6, 65.9, 55.4, 39.6, 38.6,

31.3, 24.6, 18.3, 16.6, 13.7. LC-MS: m/z = 425.1 [M + H]⁺;

4.5.4. 2-(2,5-Dioxo-1-(N-phenylphenylsulfonamido)pyrrolidin-3-yl)-2-

methylpropanoic acid (63)

The compound appeared as solid with an observed R_fvalue of 0.28

(Hexane/Ethyl acetate 1:1). Isolated yield = 62%. ¹H NMR (CDCl₃, 400

MHz): δ 8.94 (s, 1H), 7.64–7.50 (m, 6H), 7.29–7.28 (m, 2H), 6.99–6.96

(m, 2H), 3.70 (dd, J = 4.87, 8.96 Hz, 1H) 2.99 (dd, J = 8.95, 18.14 Hz,

4.7. Synthesis of sulfonamides (66–70, 78–79)

The oxidized products (compounds 60–64, 1 equiv.) were diluted in

DCM 4 mL using an inert condition of nitrogen gas. Subsequent addi-

tions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(EDC⋅HCl, 1.2 equiv), 1-hydroxy-benzotriazole (HOBt, 1.2 equiv) and

Hunig’s base (2.5 equiv) were performed and the was stirred for 5 min at

normal laboratory temperature. Sulfanilamide (1.1 equiv.) was added to

the reaction and continued stirring at laboratory temperature. After 24

h, the reaction was diluted with ethyl acetate and washed with aq. HCl

(0.5 M, 20 mL). The extraction with ethyl acetate was repeated three

times (50 mL each time). The three organic layers were combined and

was added saturated sodium bicarbonate and brine (each 20 mL). The

mixture was dried, ﬁltered and the organic solvents were removed with

reduced pressure of a rotary evaporator. The crude products (Com-

pounds 66–70) were puriﬁed by gravity column chromatography

eluting with hexane and ethyl acetate solvent system.

1H), 2.60 (dd, J = 4.90, 18.11 Hz, 1H), 1.36 (s, 3H), 1.35 (s, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 179.3, 177.7, 173.5, 145.3, 144.5, 134.5,

128.5, 128.3, 128.2, 127.3, 124.5, 121.5, 119.2, 50.5, 44.5, 31.5, 18.5,

18.0.

4.5.5. 2-Methyl-2-(1-((4-methyl-N-phenylphenyl)sulfonamido)-2,5-

dioxopyrrolidin-3-yl) propanoic acid (64)

The compound appeared as solid with an observed R_fvalue of 0.34

(Hexane/Ethyl acetate 1:1). Isolated yield = 66%. ¹H NMR (CDCl₃, 400

MHz): δ 8.96 (s, 1H), 7.72–7.71 (m, 2H), 7.56–7.48 (m, 4H), 7.43–7.37

(m, 1H), 7.26–7.22 (m, 2H), 3.70 (dd, J = 5.94, 9.87 Hz, 1H) 3.08 (dd, J

= 9.85, 18.48 Hz, 1H), 2.66 (dd, J = 5.98, 18.47 Hz, 1H), 2.35 (s, 3H),

1.36 (s, 3H), 1.34 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 178.5, 178.1,

176.5, 146.4, 145.5, 134.2, 128.6, 128.3, 128.1, 127.7, 124.3, 121.8,

119.2, 54.6, 42.5, 32.5, 22.4, 17.2, 15.2.

4.7.1. 2-(1-(Diphenylamino)-2,5-dioxopyrrolidin-3-yl)-2-methyl-N-(4-

sulfamoylphenyl) propenamide (66)

4.5.6. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)

acetic acid (76)

The compound appeared as dark color oily/semisolid with an

observed R_fvalue of 0.32 (Hexane/Ethyl acetate 6:4). Isolated yield =

71%. ¹H NMR (CDCl₃, 400 MHz): δ 10.31 (s, 1H), 7.69–7.67 (m, 3H),

7.32–7.24 (m, 5H), 7.15–7.13 (m, 3H), 7.11–7.05 (m, 3H), 6.03 (s, 2H),

3.28–3.24 (m, 1H), 2.72 (dd, J = 9.60, 18.23 Hz, 1H), 2.51 (dd, J = 6.40,

18.16 Hz, 1H), 1.35 (s, 3H), 1.33 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

178.1, 176.4, 168.6, 148.0, 135.8, 132.4, 132.0, 130.5, 128.8, 128.8,

128.8, 128.7, 128.1, 128.0, 126.7, 126.7, 124.0, 120.4, 119.4, 51.9,

43.2, 33.8, 17.4, 16.5. LC-MS: m/z = 506.5 [M + H]⁺; Analysis calcu-

lated for C₂₆H₂₆N₄O₅S; C, 61.65; H, 5.17; N, 11.06; O, 15.79; S, 6.33.

Observed: C, 61.52; H, 5.19; N, 11.09; O, 15.79; S, 6.31.

The compound appeared as solid with an observed R_fvalue of 0.25

(Hexane/Ethyl acetate 1:1). Isolated yield = 52%. ¹H NMR (CDCl₃, 400

MHz): δ 8.89 (s, 1H), 7.53–7.37 (m, 3H), 7.26–7.25 (m, 2H), 6.94 (d, J

= 7.62 Hz, 2H), 6.74 (d, J = 7.68 Hz, 2H), 4.69 (d, J = 6.38 Hz, 2H), 3.86

(s, 3H), 3.46–3.41 (m, 2H), 2.99 (dd, J = 9.40, 18.21 Hz, 1H), 2.97–2.86

(m, 1H), 2.67 (dd, J = 5.35, 18.17 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz):

δ 178.7, 176.0, 174.4, 151.4, 134.4, 133.5, 128.9, 128.7, 128.4, 128.2,

124.5, 120.9, 116.8, 54.6, 48.5, 44.6, 34.7, 31.7. LC-MS: m/z = 369.2

[M + H]⁺;

4.5.7. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)

propanoic acid (77)

4.7.2. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-

2-methyl-N-(4-sulfamoylphenyl)propenamide (67)

The compound appeared as white solid with an observed R_fvalue of

0.24 (Hexane/Ethyl acetate 1:1). Isolated yield = 50%. ¹H NMR (CDCl₃,

400 MHz): δ 8.89 (bs, 1H), 7.40–7.26 (m, 5H), 7.05 (d, J = 9.55 Hz, 2H),

6.96 (d, J = 9.61 Hz, 2H), 4.67 (d, J = 4.11 Hz, 2H), 3.85 (s, 3H),

3.32–3.28 (m, 1H), 3.07–2.98 (m, 1H), 2.87 (dd, J = 9.45, 18.74 Hz,

The compound appeared as semisolid with an observed R_fvalue of

0.31 (Hexane/Ethyl acetate 6:4). Isolated yield = 66%. HPLC purity =

98.8% (C₁₈RP, 1% TFA in MeOH / H₂O-92:8), T_R= 8.2 min. ¹H NMR

(CDCl₃, 400 MHz): δ 10.37 (s, 1H), 7.72–7.70 (m, 1H), 7.54–7.37 (m,

4H), 7.29–7.26 (m, 1H), 7.00–6.89 (m, 7H), 5.96 (m, 2H), 4.61–4.53 (m,

2H), 3.88 (s, 3H), 3.31 (dd, J = 5.33, 9.70 Hz, 1H), 2.92 (dd, J = 9.75,

18.33 Hz, 1H), 2.59 (dd, J = 5.38, 18.53 Hz, 1H), 1.25 (s, 6H). ¹³C NMR

(CDCl₃, 100 MHz): δ 177.4, 175.1, 169.6, 149.0, 137.2, 133.3, 132.5,

131.9, 130.9, 129.2, 128.8, 128.7, 127.6, 127.5, 126.5, 123.8, 119.0,

115.9, 56.8, 40.4, 38.6, 32.6, 23.7, 18.7, 18.0. LC-MS: m/z = 550.6 [M

+ H]+; Analysis calculated for C₂₈H₃₀N₄O₆S; C, 61.08; H, 5.49; N,

10.18; O, S, 5.82. Observed, C, 61.16; H, 5.51; N, 10.14; O, 17.43; S,

5.79.

1H), 2.47 (dd, J = 5.80, 18.74 Hz, 1H), 1.29 (d, J = 7.95 Hz, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 180.2, 178.5, 177.3, 150.4, 142.5, 134.5,

129.0, 128.7, 128.6, 128.4, 128.1, 120.5, 118.6, 54.4, 50.3, 45.2, 42.5,

33.3, 15.3. LC-MS: m/z = 383.4 [M + H]⁺;

4.6. Synthesis of ester (71)

The compounds 61 (300 mg) was dissolved ethanol (3 mL) followed

by the addition of sulfuric acid (0.4 mL). The stirring of reaction was

4.7.3. 2-(1-(Benzyl(phenyl)amino)-2,5-dioxopyrrolidin-3-yl)-2-methyl-N-

(4-sulfamoyl- phenyl)propenamide (68)

◦

continued at 60 C for 2 h. The reaction was quenched with water (2

mL). The crude product was extracted with ethyl acetate and was

concentrated under reduce pressure using rotary evaporator. The pure

product was obtained by washing in methanol.

The compound appeared as oily semisolid with an observed R_fvalue

of 0.38 (Hexane/Ethyl acetate 6:4). Isolated yield = 67%. HPLC purity

= 98.8% (C₁₈RP, 1% TFA in MeOH / H₂O-92:8), T_R= 8.2 min. ¹H NMR

16

A. Sadiq et al.

Bioorganic Chemistry 112 (2021) 104969

(CDCl₃, 400 MHz): δ 10.30 (s, 1H), 7.80–7.75 (m, 2H), 7.68–7.58 (m,

4H), 7.35–7.24 (m, 6H), 6.91–6.86 (m, 2H), 5.95 (s, 2H), 4.64 (d, J =

4.96 Hz, 2H), 3.28 (dd, J = 4.90, 9.56 Hz, 1H), 2.89 (dd, J = 9.65, 18.43

5. 4.10–4.11. COX-1 and COX-2 and LOX inhibitions assays

COX-1 (from sheep, Sigma EC Number 1.14.99.1), COX-2 (from

human recombinant, Sigma Catalog Number C0858), 5-LOX (from

Human Recombinant, Sigma Catalog Number 437996), lineolic acid

(Sigma, CAS no. 60–33-3), arachidonic acid (Sigma, Cat No 150384), N,

N,N,N-tetramethyl-p-phenylenediamine dihydrochloride (Sigma, CAS

637–01-4), glutathione (Sigma, CAS 70–18-8), hematin (Sigma, CAS

15489–90-4). The methodology is presented in Supporting information.

Hz, 1H), 2.53 (dd, J = 5.08, 18.53 Hz, 1H), 1.28 (s, 3H), 1.23 (s, 3H). ¹³

C

NMR (CDCl₃, 100 MHz): δ 178.6, 176.5, 168.6, 147.5, 139.4, 134.0,

132.3, 131.0, 130.8, 129.6, 128.6, 128.6, 127.7, 127.6, 126.4, 122.3,

119.7 115.3, 54.6, 42.5, 39.9, 31.0, 19.3, 17.3. LC-MS: m/z = 521.3 [M

+ H]⁺.

4.7.4. 2-(2,5-Dioxo-1-(N-phenylphenylsulfonamido)pyrrolidin-3-yl)-2-

methyl-N-(4-sulfamoyl-phenyl)propenamide (69)

5.1. In-vivo based inﬂammation

The compound appeared as dark oil with an observed R_fvalue of

0.34 (Hexane/Ethyl acetate 6:4). Isolated yield = 75%. HPLC purity =

98.8% (C₁₈RP, 1% TFA in MeOH / H₂O-92:8), T_R= 8.2 min. ¹H NMR

(CDCl₃, 400 MHz): δ 10.41 (s, 1H), 7.79–7.74 (m, 3H), 7.67–7.53 (m,

4H), 7.36–7.26 (m, 5H), 6.89–6.83 (m, 2H), 5.86 (s, 2H), 3.54 (dd, J =

5.02, 9.11 Hz, 1H), 3.07 (dd, J = 9.05, 18.64 Hz, 1H), 2.70 (dd, J = 5.01,

18.63 Hz, 1H), 1.37 (s, 3H), 1.33 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

177.8, 176.1, 169.1, 146.4, 138.9, 132.7, 130.1, 130.0, 129.4, 128.7,

128.6, 128.4, 128.7, 127.1, 126.7, 123.3 120.9 116.4, 51.1, 41.4, 33.8,

18.1, 17.3. LC-MS: m/z = 571.2 [M + H]⁺.

The Swiss albino mice (either sex) with an average weight of 30 to

35 g were used as experimental animals in this study. The animals were

obtained from National Institute of Health Islamabad Pakistan. The

animals were kept as per the standard protocols of light–dark cycle,

food, water and temperature throughout the experimental observations.

These animals were used as per the approval of Ethical Committee of

Department of Pharmacy, FBS, University of Malakand, Pakistan via

letter No. DREC/21. After the pharmacological experiments, the animals

were euthanized using the standard protocols [49]. The detailed meth-

odology is presented in Supporting information

4.7.5. 2-Methyl-2-(1-((4-methyl-N-phenylphenyl)sulfonamido)-2,5-

dioxopyrrolidin-3-yl)-N-(4-sulfamoylphenyl)propenamide (70)

5.2. Evaluations of phlogistic agents/Irritants

The compound appeared as dark oil with an observed R_fvalue of 0.38

(Hexane/Ethyl acetate 6:4). Isolated yield = 73%. HPLC purity = 98.8%

(C₁₈RP, 1% TFA in MeOH / H₂O-92:8), T_R= 8.2 min. ¹H NMR (CDCl₃,

400 MHz): δ 10.44 (s, 1H), 7.83–7.76 (m, 2H), 7.74–7.60 (m, 3H),

7.41–7.28 (m, 4H), 7.09 (d, J = 9.33 Hz, 2H), 6.79 (d, J = 9.31 Hz, 2H),

5.79 (s, 2H), 3.57 (dd, J = 4.67, 8.97 Hz, 1H), 3.02 (dd, J = 9.01, 17.95

Hz, 1H), 2.68 (dd, J = 4.69, 18.03 Hz, 1H), 2.35 (s, 3H), 1.35 (s, 3H),

1.31 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 177.7, 174.6, 168.6, 147.3,

142.3, 131.0, 130.6, 128.7, 128.5, 128.4, 128.1, 128.0, 127.4, 127.2,

127.1, 125.6 122.6 119.3, 53.7, 44.6, 31.3, 25.6, 19.6, 17.9. LC-MS: m/z

= 585.2 [M + H]⁺.

To check the possible anti-inﬂammatory mechanism, different

phlogistic agents like histamine, prostaglandin E2, bradykinin and

leukotriene were used in experimental animals. The detailed method-

ology is presented in Supporting information.

5.3. Docking studies

We have performed docking studies of synthesized compounds by

using Molecular Operating Environment (MOE 2016.08) [50] on the

selected target of COX-2 obtained from protein data bank (PDB) with

accession code 1CX2. The docking protocol used for the current study is

reported in our previous reports [51–54] and also presented in Sup-

porting Information.

4.7.6. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-

N-(4-sulfamoyl- phenyl) acetamide (78)

The compound appeared as semisolid with an observed R_fvalue of

0.38 (Hexane/Ethyl acetate 3:2). Isolated yield = 52%. HPLC purity =

97.9% (C₁₈RP, 1% TFA in MeOH / H₂O-92:8), T_R= 7.1 min. ¹H NMR

(CDCl₃, 400 MHz): δ 10.37 (s, 1H), 7.77–7.63 (m, 4H), 7.54–7.39 (m,

1H), 7.04–6.91 (m, 5H), 6.69–6.67 (m, 2H), 6.58–6.56 (m, 1H), 5.92 (s,

2H), 4.67 (s, 2H), 3.85 (s, 3H), 3.21–3.18 (m, 1H), 3.06–3.03 (m, 1H),

2.95–2.99 (m, 1H), 2.84 (dd, J = 9.38, 18.58 Hz, 1H), 2.48 (dd, J = 5.99,

18.42 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ 178.0, 175.2, 168.1,

152.4, 151.3, 146.8, 135.6, 133.3, 132.1, 130.9, 130.4, 128.7, 128.7,

128.0, 128.0, 125.7, 123.7, 121.5, 119.7, 58.5, 42.5, 40.1, 31.3, 23.8.

LC-MS: m/z = 522.7 [M + H]+; Analysis calculated for C₂₆H₂₆N₄O₆S; C,

59.76; H, 5.01; N, 10.72; S, 6.14. Observed: C, 59.90; H, 5.03; N, 10.68;

O, 18.37; S, 6.11.

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

The authors are thankful to the Najran University, Ministry of Edu-

cation, Kingdom of Saudi Arabia for extending their support in

completion of the project (Project No. NU/MID/17/222). We are also

thankful to the Higher Education Commission (HEC) Pakistan for their

ﬁnancial support via Project No. 10562/KPK/R&D/HEC/2017. UR is

also thankful to HEC for ﬁnancial support for the purchase of MOE li-

cense under HEC-NRPU project 5291/Federal/NRPU/R&D/HEC/2016.

4.7.7. 2-(1-(Benzyl(4-methoxyphenyl)amino)-2,5-dioxopyrrolidin-3-yl)-

N-(4-sulfamoyl- phenyl) propenamide (79)

The compound appeared as semisolid with an observed R_fvalue of

0.34 (Hexane/Ethyl acetate 3:2). Isolated yield = 58%. HPLC purity =

98.8% (C₁₈RP, 1% TFA in MeOH / H₂O-90:10), T_R= 9.4 min. ¹H NMR

(CDCl₃, 400 MHz): δ 10.33 (s, 1H), 7.81–7.64 (m, 2H), 7.57–7.47 (m,

2H), 7.42–7.39 (m, 1H), 7.33–7.26 (m, 2H), 6.78–6.28 (m, 6H), 5.94 (s,

2H), 4.67 (d, J = 4.39 Hz, 2H), 3.86 (s, 3H), 3.47–3.26 (m, 2H), 2.87 (dd,

J = 9.51, 18.21 Hz, 1H), 2.63 (dd, J = 6.13, 18.16 Hz, 1H), 1.27 (d, J =

7.14 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 177.5, 175.8, 169.5,

158.8, 149.1, 137.2, 136.5, 132.5, 128.6, 128.6, 128.2(2), 125.3, 124.2,

122.2, 121.3, 120.6, 117.3, 56.9, 41.1, 39.4, 31.9, 23.8, 16.9. LC-MS: m/

z = 536.6 [M + H]⁺; Analysis calculated for C₂₇H₂₈N₄O₆S: C, 60.44; H,

5.26; N, 10.44; S, 5.98. Observed; C, 60.53; H, 5.28; N, 10.41; S, 5.96.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.104969.

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Tailoring the substitution pattern of Pyrrolidine-2,5-dione for discovery of new structural template for dual COX/LOX inhibition

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