Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents

Journal Pre-proof Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents Muhammad Saeed Jan, Sajjad Ahmad, Fida Hussain, Ashfaq Ahmad, Fawad Mahmood, Umer Rashid, Obaid-ur-Rahman Abid, Farhat Ullah, Muhammad Ayaz, Abdul Sadiq PII:

S0223-5234(19)31015-3

DOI:

https://doi.org/10.1016/j.ejmech.2019.111863

Reference:

EJMECH 111863

To appear in:

European Journal of Medicinal Chemistry

Received Date: 12 June 2019 Revised Date:

5 November 2019

Accepted Date: 6 November 2019

Please cite this article as: M.S. Jan, S. Ahmad, F. Hussain, A. Ahmad, F. Mahmood, U. Rashid, O.u.-R. Abid, F. Ullah, M. Ayaz, A. Sadiq, Design, synthesis, in-vitro, in-vivo and in-silico studies of pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory agents, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111863. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

1 2

Design, Synthesis, In-vitro, In-vivo and In-silico studies of

3

Pyrrolidine-2,5-dione Derivatives as Multitarget Anti-inflammatory

4

Agents

5 6

Muhammad Saeed Jan1, Sajjad Ahmad1, Fida Hussain1,2, Ashfaq Ahmad1, Fawad Mahmood3,

7

Umer Rashid*, 4, Obaid-ur-Rahman Abid 5, Farhat Ullah1, Muhammad Ayaz1,

8

Abdul Sadiq**,1

9 10

1

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

11 2

12 13

3

Department of Pharmacy, University of Swabi, Swabi, KP, Pakistan.

Department of Pharmacy, Sarhad University of Science & Technology, Peshawar, KPK, Pakistan.

14 15

4

Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, 22060 Abbottabad, Pakistan.

16 17

5

Department of Chemistry, Hazara University, Mansehra, Pakistan

18 19

Corresponding author (*): Email: [email protected] (Umer Rashid)

20 21

Corresponding author (**): Email: [email protected], Contact: +92 (0)301-2297 102

22 23 24

1

25

Abstract

26

In recent years, drug discovery paradigm has been shifted from conventional single target

27

inhibition toward multitarget design concept. In current research, we have reported synthesis, in-

28

vitro, in-vivo and acute toxicity determination of N-substituted pyrrolidine-2,5-dione derivatives

29

as multitarget anti-inflammatory agents. We synthesized cycloalkyl, alkyl and aryl carbonyl

30

derivatives by the Michael addition of ketones to N-substituted maleimides using self-assembled

31

three component system as an organocatalyst. Anti-inflammatory potential of the compounds

32

was determined by using different in-vitro assays, like cyclooxygenase-1, cyclooxygenase-2 and

33

5-lipoxygenase, albumin denaturation and anti-protease assays. Amongst the synthesized

34

compounds, 13a-e series of compounds showed inhibition in low micromolar to submicromolar

35

ranges. These compounds also demonstrated COX-2 selectivity. Compound 13e with IC50 value

36

0.98 µM and SI of 31.5 emerged as the most potent inhibitor of COX-2. Based on in-vitro

37

results, in-vivo anti-inflammatory investigations were performed on compounds 3b and 13e via

38

carrageenan induced paw edema test. The possible mode of action of compounds 3b and 13e

39

were ascertained with various mediators like histamine, bradykinin, prostaglandin and

40

leukotriene. In-vivo acute toxicity study showed the safety of synthesized compounds up to 1000

41

mg/kg dose. The selectivity of the compounds against cyclooxygenase isoforms was supported

42

by docking simulations. Selective COX-2 inhibitors showed significant interactions with the

43

amino acid residues present in additional secondary COX-2 enzyme pocket. Furthermore, in-

44

silico pharmacokinetic predictions confer the drug-like characteristics.

45 46

Keywords: Michael addition; Succinimides; 5-Lipoxygenase; Cyclooxygenase-1/2; Albumin

47

denaturation; Protease inhibition.

48

2

49

1. Introduction

50

Inflammation is a multifactorial disorder frequently associated with pain. It involves raise of

51

vascular permeability, membrane alteration and protein denaturation [1]. The normal cells or

52

tissues upon exposure to microbes, chemical or physical agents can get inflamed which may lead

53

to injury [2]. The intensity of the loss of function depends on the extent and site of the injury.

54

Inflammation is the body defensive mechanism that is triggered by various stimuli including

55

radiation, heat, microbial infections and is frequently associated with tissue damages [3-4].

56

Arachidonic acid metabolism play a vital role in the mechanism of inflammation [5].

57

Arachidonic acid metabolized to thromboxane A2 and prostaglandins by the cyclooxygenase-2

58

(COX-2) cascade, or by the 5-lipoxygenase (5-LOX) pathway upon suitable stimulus of

59

neutrophils. The arachidonic acid is break down from phospholipids membrane and converted to

60

prostaglandins and leukotrienes through COX-2 or 5-LOX cascades [6]. Inhibitions of COX-2

61

and 5-LOX may direct to reduce the production of prostaglandins and leukotrienes. Hence, the

62

drug having capability to inhibit these enzymes have the potential to give anti-inflammatory and

63

analgesic effects with a decrease in the gastro-intestinal side effects [7]. Membrane stabilization

64

is a process of maintaining the integrity of biological membranes [8]. Denaturation of proteins is

65

a process in which proteins lose their secondary and tertiary structures due to the appliance of the

66

external stress such as a concentrated inorganic salts, strong acids or bases and organic solvents

67

or heat [9]. When the biological proteins are denatured, they lose their functions. Proteins

68

denaturation is a well-known reason of inflammation [10]. Proteases, also called proteinases or

69

peptidases, are group of enzymes that perform proteolysis, i.e. hydrolysis of peptide bonds which

70

link the amino acids jointly in the polypeptide chain forming the protein [11]. Proteases

71

constitute one of the largest functional group of proteins involved in many normal and 3

72

pathological processes. Proteases inhibitions may help in control of several diseases including

73

inflammation [12-14].

74

For the treatment of multifactorial pathologies such as Alzheimer’s disease (AD) and

75

inflammation, the drug discovery paradigm has been shifted from conventional single target

76

inhibition towards multitarget design concept. In anti-inflammatory drug discovery, the

77

development of licofelone as dual cyclooxygenase/lipoxygenase inhibitor is a success of the later

78

strategy. Aspirin, a nonsteroidal anti-inflammatory drug (NSAID), is the oldest clinically

79

accepted example of multitarget anti-inflammatory drug [15].

80

Organic compounds with cyclic amide group have been reported to possess great

81

pharmacological importance [16]. Substituted succinimides (pyrrolidine-2,5-diones) are

82

important compounds in medicinal chemistry [17-18]. Due to the presence of amide group, they

83

showed excellent in-vivo activity because it can easily cross the biological membrane [19]. A

84

diversity of biological activities and pharmaceutical uses has been attributed to them [20-31].

85

Based on the literature evidences, it is obvious that specific functional groups are responsible for

86

biological activities, and may specifically be effective in the management of inflammation [32-

87

33]. Some of the ketoester derivatives of succinimides showed strong anti-inflammatory activity

88

[34].

89

The organocatalytic asymmetric Michael addition is a strong reaction for making the C-C bond

90

formation [35-36]. The asymmetric Michael addition of ketones to maleimides has been explored

91

by a limited number of researchers. Among them, none has explored the biological importance of

92

the synthesized compounds. Our synthetic strategy relied on Michael addition of ketones to N-

93

substituted maleimides using self-assembled three component system as an organocatalyst. We

4

94

have previously synthesized aldehyde and ketoester derivatives of succinimides [37-38]. We

95

noticed that the synthesis of some of the designed compounds have previously investigated using

96

different catalysis systems [39-42]. However, to the best of our literature search, we have noticed

97

that these compounds have not been previously evaluated for any biological activity including

98

anti-inflammatory activity. Herein, we report synthesis, in-vitro, in-vivo and molecular docking

99

studies of N-substituted pyrrolidine-2,5-dione derivatives as multitarget anti-inflammatory

100

agents.

101

2. Results and discussion

102

2.1 Design strategy

103

General structure of selective COX-2 inhibitor includes two vicinal aryl rings on central 5- or 6-

104

memebered heterocyclic/acyclic scaffold bearing a small lipophilic moiety or sulfonamide

105

(responsible for prooxidant activity). COX-2 selectivity of celecoxib is associated with these

106

structural features (Figure 1a). In the current study, we have constructed pyrrolidine-2,5-dione

107

based derivatives as shown in Figure 1b. Our design strategy aims to synthesize N-substituted

108

pyrrolidine-2,5-dione connected at position-3 with cycloalkyl, alkyl and aryl carbonyl

109

substituents (Figure 1b-c). Cycloalkyl carbonyl compounds are planned to connect with N-

110

substituted pyrrolidine-2,5-dione ring directly (Figure 1b). While, aryl carbonyl derivatives relate

111

to a flexible methylene linker (Figure 1c). Considering the importance of rational development of

112

drug to tame multifactorial inflammation, we decided to design “single-drug-multitarget”

113

strategy. The investigated multiple targets are represented in Figure 1d.

5

114 115

Figure 1: (a) General structure of selective COX-2 inhibitor; (b-c) Design strategy of current research;

116

(d) Multiple anti-inflammatory targets evaluated in current study.

6

117

2.2 Asymmetric ketone derivatives of pyrrolidine-2,5-dione

118

We synthesized ketone derivatives (3a-l) of pyrrolidine-2,5-dione by adding various cyclic alkyl

119

/ alkyl ketones to N-substituted maleimides. Except 3i-j, all the reactions are simple single step

120

and completed in short time with good isolated yields. We used a tricomponent non-covalent

121

organocatalyst system for these reactions for the first time as shown in Scheme 1. The synthesis

122

of 3i-j was carried out in two-step. In first step, N-substituted sulfonamide derivative 7 was

123

synthesized by the reaction of maleic anhydride with sulfanilamide in diethyl ether to obtain N-

124

sufamoyl-phenylmaleanic

125

intermediate. The synthesized intermediate was then cyclized in acetic anhydride in the presence

126

of sodium acetate to give sulfanilamide derivative 7 (Scheme 2). Cyclohexanone and

127

cycloheptanone reacted with the N-sulfonamide derivative 7 to yield 3i-j.

acid

([(4‐sulfamoylphenyl)carbamoyl]prop‐2‐enoic

acid,

O O

R1 N O

O R

H3C

O

L-isoleucine, thiourea and KOH (20 mol% each)

1a-e

2a-c, 7

1f-g

O

N R1 O

Chloroform (1.0 M), rt

R'

O

O R H3C R'

3a-j

R1 =

O S NH2 O

Br

128 Scheme 1: Synthesis of the pyrrolidine-2,5-dione derivatives 3a-j. O

O O S NH2 O

O + H 2N O

131

O

3k-l

O

CH3

Cyclic alkyl

130

N R1

R,R' = H, H (1f, 3k); CH3, CH3 (1g, 3l)

=

129

6)

4

5

20 oC CHCl3

O O S NH2 O

HN O HO

6

80-100 oC Ac2O, NaOAc

O S NH2 O

N O

7

Scheme 2: Synthesis of the N-benzene sulfonamide derivative (7) of pyrrolidine-2,5-dione

7

132

The addition of aromatic ketones to maleimide type Michael acceptors is very rare in the

133

literature. There is only a recent example of aromatic ketones additions to N-phenylmaleimide

134

[42]. However, to the best of our literature survey, there is no single example of aromatic or any

135

other ketones to the maleimide Michael acceptors that we used in our anti-inflammatory study.

136

In the next Scheme, relevant 4-substituted acetophenones (aryl ketones) were stirred at room

137

temperature with 7 in the presence of OtBu-L-threonine and 1,8-diazabicyclo[5.4.0]undec-7-ene

138

(DBU) in chloroform to obtain 13a-e as outlined in Scheme 3. O

O

7

X

+

CH3

O

OtBu-L-threonine, DBU X

8-12

O S NH2 O

O

CHCl3, rt, 51.3-63.2%

R

N

R

13a-e

X = N (13a), X = CH (13b-e) R = -H (13a-b), -OCH3 (13c), -Cl (13d), -CH3 (13e)

139 140

Scheme 3: Synthesis of the pyrrolidine-2,5-dione derivatives 13a-e.

141

The individual yields and other details of all the compounds are available in experimental section

142

and the 1H and

143

screened compounds are > 95 % pure as determined by HPLC.

144

2.3 In-vitro enzyme inhibition assays

145

2.3.1 Cyclooxygenase (COX-1/COX-2) and 5-LOX inhibition assay

146

The COX-1 / COX-2 inhibitory activity of the synthesized compounds (3a-j and 13a-c) were

147

carried out using COX-1 and COX-2 screening assay kits. Celecoxib, indomethacin, diclofenac

148

sodium and Zileuton was used as corresponding positive controls. The in-vitro enzymes

149

inhibition results are presented as IC50 values as means of three acquired determinations and

13

C NMR spectra are provided in the supporting information. Biologically

8

150

presented in Table 1. The selectivity index (SI) was calculated as IC50 (COX-1) / IC50 (COX-2)

151

(Table 1). It is evident from the in-vitro results that overall cyclic ketones (3a-h) and alkyl

152

ketones (3i-j) exhibited poor to moderate inhibition of both COX isoforms. N-benzyl derivatives

153

3b and 3e showed selectivity towards COX-2. They showed selectivity index of 3.13 and 2.35

154

respectively. We noticed that presence of bulkier cycloalkyl carbonyl derivatives improve the

155

COX-2

156

(cycloheptanone) and 3h (cyclopentanone) was 1.45, 1.66 and 0.58 respectively. N-sulfonamide-

157

cyclohexanone/ cycloheptanone derivatives (3i and 3j respectively) showed moderate COX-2

158

inhibition with selectivity index of 2.04 and 2.36 respectively.

159

It is evident from Table 1 that in-vitro results of aryl ketone / N-sulfonamide derivatives (13a-e)

160

demonstrated marked inhibitions and selectivity against COX-2 isozyme. Compounds of this

161

series showed IC50 values in submicromolar to low micromolar range. Compound 13e with IC50

162

value of 0.98 µM and SI 31.5 emerged as the most potent inhibitor of COX-2. The SI for

163

compounds 13a-d is 4.88, 11.5, 18.7 and 10.9 respectively.

164

The synthesized compounds were also screened for their 5-LOX inhibitory potentials.

165

Compounds of series 1 (3a-l) demonstrated moderate to poor 5-LOX inhibition. Sulfonamide-

166

cycloheptanone derivative 3j exhibited IC50 value of 16.15 µM. Aryl ketone derivatives 13a-e

167

showed good to excellent inhibition of human lipoxygenase (5-LOX). Among all the tested

168

compounds of this series, 13a and 13e exhibited highest inhibitions attaining IC50 values of 0.81

169

and 0.86 µM respectively, while the standard drug zileuton exhibited IC50 of 0.63 µM (Table 1).

170

Concentrations of the synthesized compounds at which 50% of inhibition is observed (IC50) were

171

calculated among the inhibition percentages against the tested concentrations using the MS-

172

Excel program. All the assays were performed in triplicate and values were expressed as means ±

selectivity.

The

selectivity index

for

compounds

3a

(cyclohexanone),

3g

9

173

Standard error means (SEM). Statistical analysis was performed by Two-Way analysis of

174

difference (ANOVA), followed by Bonferroni tests. The difference was measured to be

175

statistically significant when the p value ˂ 0.05.

176

Table 1: In-vitro cyclooxygenase-1 / 2 and 5-LOX inhibition activity of the compounds

No.

IC50 (µM)

Structures COX-1

COX-2

SI

5-LOX

65.56 + 1.34

45.08 + 1.29

1.45

78.3 + 1.87

62.57 + 1.12

19.98 + 0.07

3.13

36.0 + 0.92

186.35 + 3.16

113.44 + 2.22

1.64

130.5 + 1.64

53.02 + 1.82

56.49 + 1.41

0.93

69.18 + 2.02

51.52 + 1.31

21.86 + 0.62

2.35

56.2 + 1.00

48.92 + 1.16

56.58 + 0.98

0.86

26.83 + 0.32

65.64 + 2.38

39.45 + 1.49

1.66

18.44 + 0.09

O O

3a

N O

O O

3b

N O O

O

3c

N

Br

O

O O

N

3d O

O O

N

3e O

O O

3f

N O

O

O O

3g

N O

10

O O

3h

30.26 + 0.82

51.35 + 1.55

0.58

98.55 + 2.09

43.39 + 1.19

21.26 + 1.07

2.04

21.31 + 1.28

49.10 + 1.81

20.73 + 0.82

2.36

16.15 + 0.09

33.31 + 1.63

63.88 + 1.93

0.52

62.38 + 1.66

39.25 + 1.23

53.47 + 1.41

0.73

53.45 + 1.39

43.70 + 2.01

8.94 + 0.06

4.88

0.81 + 0.02

48.66 + 1.89

4.23 + 0.19

11.5

5.29 + 0.26

O S NH2 O

39.21 + 1.13

2.10 + 0.03

18.7

4.74 + 0.02

O S NH2 O

68.79 + 2.15

6.29 + 0.01

10.9

12.59 + 0.42

O S NH2 O

30.87 + 1.53

0.98 + 0.01

31.5

0.86 + 0.01

72.52 + 1.32

0.28 + 0.02

269

-

N O O

O

3i

O S NH2 O

N O O O

O S NH2 O

N

3j

O

O O

3k

N O

O O

3l

N O O O

13a

O S NH2 O

N

N O

O O

13b

O S NH2 O

N O

O O

N

13c

O O O O

13d

N O

Cl O O

13e

N O

H 3C

177 Celecoxib

11

Diclofenac

0.48 + 0.01

10.05 + 1.02

0.05

-

Indomethacin

0.25 + 0.01

0.07 + 0.01

3.57

-

-

-

-

0.63 + 0.03

Zileuton

178

SI= IC50 (COX-1) / IC50 (COX-2)

179 180

2.3.2. Inhibition of Albumin denaturation and protease

181

Albumin denaturation and protease inhibitory assays were also performed for the synthesized

182

compounds as shown in Table 2. Among our tested compounds of series-A, compound 3b

183

showed good albumin denaturation and protease inhibition potentials with IC50 values of 24.53

184

and 17.52 µM respectively. Compound 13a showed excellent inhibition with IC50 values of 5.36

185

(Albumin denaturation) and 13.39 µM (protease inhibition). Most active COX-2 and 5-LOX

186

inhibitor 13e exhibited IC50 values of 16.89 and 15.29 µM respectively.

187

Table 2: In-vitro albumin denaturation and protease inhibition activities of the compounds. Compound

Albumin denaturation IC50 (µM)a

Protease inhibition IC50 (µM)

3a

40.54

66.34

3b

24.53

17.52

3c

157.04

99.94

3d

59.58

77.10

3e

53.44

63.47

3f

50.57

117.10

3g

94.62

63.08

3h

97.17

85.51

3i

ND

b

ND

3j

ND

ND

3k

77.84

151.35

3l

65.56

88.70

13a

5.36

c

13.39

13b

16.11

45.65

13c

24.85

14.91

12

188 189 190 191 192 193 194

13d

19.66

21.48

13e

16.89

15.29

Diclofenac sodium

30.52

42.72

a

The calculated IC50 value for was µM. Data is represented as mean ± S.E.M; n = 3; b ND = not determined; c bold value = most potent compound

2.4. In-vivo anti-inflammatory activity

195

Anti-inflammatory activity of the compounds was assessed by carrageenan-induced paw edema

196

method of the experimental animals. Carrageenan induced paw edema model is a COX-2–

197

dependent model of inflammation. Therefore, we selected two compounds having high COX-2

198

inhibitory potentials (high selectivity index) from the synthesized two series of derivatives

199

(compound 3b from series 1 and 13e from series 2).

200

2.4.1. Acute toxicity

201

The dose chosen for in-vivo testing were based upon the results of preliminary range finding

202

tests from LD0 to LD100 and were ranged from 5-2000 mg/kg body weight consisting of 8 doses.

203

Three replicates were tested for each of eight doses with positive and negative control. The

204

detailed dosing regimens and animal specifications for the tested compound such as 3b and 13e

205

were given in above Table 3.

206

In the acute toxicity study of both the tested compounds (3b and 13e), no related mortalities were

207

recorded in animals treated with a single dose of 1000 mg/kg body weight. Therefore, the

208

approximate lethal dose (LD50) of both compounds in the experimental mice was 1000 mg/kg.

209

There were no clinical signs in the, eyes and mucus membrane (nasal), skin and fur, respiratory

210

rate, autonomic effects (salivation, piloerection, perspiration, defecation and urinary

211

incontinence) circulatory signs and central nervous system (ptosis, gait, drowsiness, convulsion

212

and tremors) among mice administered 1000 mg/kg body weight of both tested compounds (3b 13

213

& 13e). According to organization for economic cooperation and development (OECD)

214

guidelines for acute oral toxicity, an LD50 dose of > 300 – 2000 is categorized as category 4 and

215

hence the drug is found to be safe.

216

Table 3: Animal group specification and quantity of drug administered for acute toxicity

217

studies with compound 3b and 13e. Group 1 2 3 4 5 6 7 8 9 10

Animals Male 8 8 8 8 8 8 8 8 8 8

Female 8 8 8 8 8 8 8 8 8 8

Tested drug 3b + 13e (mg/kg b.wt) 5 25 50 100 200 300 400 500 1000 2000

218

219

2.4.2 Carrageenan induced paw edema test

220

In preliminary screening test of compound 3b and 13e for anti-inflammatory activity, it has been

221

observed from Table 3 that the tested compound 3b and 13e revealed better anti-inflammatory

222

properties. The anti-inflammatory activity of the tested compound 3b was 34.54 % (P˂0.001) at

223

1st h and remained significant till 5th h (39.49%, P˂0.001) at highest dose (100mg/kg)

224

comparable to that of standard drug aspirin (47.54-57.64%). In addition, the tested compound

225

13e displayed excellent activity (55.17-68.22%) than that of positive control at 100 mg/kg body

226

weight.

227

2.4.3 Anti-inflammatory mechanism of the synthesized compounds

14

228

For the investigation of possible anti-inflammatory mechanism of tested compounds 3b and 13e

229

we used various mediators in in-vivo animal model.

230

2.4.3.1. Effect of compounds on paw edema induced by Histamine

231

Histamine induced inflammation was significantly changed when treated with the tested dose of

232

positive control i.e. chlorpheniramine maleate at 1mg/kg body weight at 1st h (71.80%) and

233

remained significant till 5th h.

234

inhibitory potential (32.40%) at 1st h at the dose of 100 mg/kg and was remained significant till

235

5th h. Likewise, the tested compound 3b at dose of 100 mg/kg did not displayed good anti-

236

inflammatory effect (17.10%) till 4th h of the administration of histamine induced inflammation

237

(Figure 2a).

238

2.4.3.2. Effect of compounds on paw edema induced by Bradykinin

239

Mean changes in paw edema volume of mouse pretreated with the test compounds at 100 mg/kg

240

body weight were measured at 1, 2, 3, 4 and 5 h after administration of bradykinin (20 µg/ml).

241

Both the tested compounds exhibited less activity in bradykinin induced inflammation as

242

compared to the positive control. The tested compound 3b displayed 12.40% inhibition at 3rd h

243

of bradykinin injection while 13e showed 27.13% inhibition which was not comparable to that of

244

positive control HOE 140. The results are shown in Figure 2b.

245

2.4.3.3. Effect of compounds on paw edema induced by Prostaglandin

246

Administration of prostaglandin E2 (0.01 µg/ml) was associated with an increase in paw edema.

247

The prostaglandin E2 inflammatory changes were significantly modified by treatment with tested

248

compounds 3b, 13e (100 mg/kg) and Celecoxib (50 mg/kg). The tested compound 3b

249

significantly reduced the PGE2 induced paw edema with 50.50 % at 1st h which reached to

250

maximal level at 4th h (64.60%) and remained significant till 5th h. Similarly, 13e displayed

Similarly, tested compound 13e demonstrated significant

15

251

promising result (61.60-81.80%) till 5th h of the experimental procedure. Celecoxib

252

comparatively displayed maximum percent inhibition of paw inflammation (65.90-87.10%)

253

(Figure 2c).

254 255

2.4.3.4. Effect of compounds on paw edema induced by leukotriene

256

In leukotriene induced inflammatory effect, the tested compounds 3b and 13e demonstrated a

257

concentration dependent anti-inflammatory activity at 100 mg/kg body weight (Figure 2d). The

258

edema produced by leukotriene injection (10 µg/ml) was inhibited by the tested compound in

259

dose dependent manner. The compound 3b exhibited maximum inhibition (60.10 %) at 2nd h

260

while 13e displayed marked anti-inflammatory activity with 74.40 % at 3rd h after administration

261

of leukotriene which was comparatively much closer to that of standard drug. Montelukast used

262

as positive control displayed 77.40% percent inhibition of paw inflammation at 3rd h.

263 264

Table 4: Anti-inflammatory activity of the tested compound using carrageenan induced paw edema in mice. Compound

Dose

Percentage inhibition of Edema (Mean + SEM) 1h

Vehicle

265 266 267

10ml/kg

2h

7.66±3.753 ***

3h

7.39±2.475 ***

4h

11.41±3.069 ***

5h

14.45±1.568 ***

7.31±3.204

Aspirin

100mg/kg

47.54±4.12

54.63±1.47

53.26±1.36

56.92±2.95

57.64±1.55***

3b

25 mg/kg 50 mg/kg 100mg/kg

24.33±3.28*** 34.54±4.59*** 44.68±4.79***

28.33±1.20*** 37.43±2.12*** 49.63±2.15***

23.33±1.20* 41.14±2.19*** 51.64±1.81***

27.81±2.79** 44.51±3.43*** 49.68±3.28***

23.37±2.16*** 47.84±2.56*** 55.07±2.33***

13e

25 mg/kg 50 mg/kg 100mg/kg

45.63±1.47*** 51.52±2.82*** 55.17±3.09***

53.03±1.93*** 57.33±2.97*** 63.84±2.94***

51.54±4.12*** 54.24±5.62*** 61.65±2.69***

54.17±2.42*** 57.26±1.36*** 66.07±2.33***

56.62±3.24*** 59.33±2.97*** 68.22±3.98***

Data expressed as mean percent inhibition ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test. ns; non significance, *P<0.05, **P<0.01, ***P<0.001, compared to vehicle control. n=8 mice per group.

16

268 269

Figure 2: (a) Percent inhibition produced by tested compound 3b and 13e (100 mg/kg) in histamine

270

induced paw edema model in mice. (b) Percent inhibition produced by tested compound 3b and 13e (100

271

mg/kg) in bradykinin induced paw edema model in mice; (c) Percent inhibition produced by tested

272

compound 3b and 13e (100 mg/kg) in prostaglandin E2 induced paw edema model in mice; (d) Percent

273

inhibition produced by tested compound 3b and 13e (100 mg/kg) in leukotriene induced paw edema

274

model in mice. Each percent point represents the mean for group of 08 mice. Data was analyzed by two-

275

way ANOVA post test. *P < 0.05, **P < 0.01, ***P < 0.001, n.s; indicates non-significant;

276

2.5. Docking studies

277

Docking studies on the targets was carried out using Molecular Operating Environment (MOE

278

2016.08) software. Crystal structure of COX-2 in complex with SC-558 was retrieved from

279

Protein Data Bank (PDB code 1CX2). In protein data bank repository, two forms of 5-LOX are

280

available. Crystal structure human 5-LOX with no co-crystallized ligand was obtained from PDB

281

(accession No. 3O8Y). While, the crystal structure of another human 5-LOX with co-crystalized

282

substrate, arachidonic acid, is also available (PDB code = 3V99). We preferred to carry out 17

283

docking studies on 3O8Y due to mutation (S663D) and absence of many amino acids in 3V99

284

[43-44].

285

2.5.1. Docking studies on COX-2 enzyme

286

Docking studies on the target enzymes were carried out to support our in-vitro experimental

287

results and to analyze the binding orientation and ligand-enzyme interactions. We have explored

288

the predictive power of docking simulations and performed docking studies were carried out on

289

all possible enantiomers. All the synthesized compounds of two series (3a-j and 13a-d) were

290

docked into the binding pockets of COX-1 and COX-2 isoforms. Although, the active sites of

291

both isoforms are very similar, however in COX-2, an additional secondary side pocket is

292

present above the Arg120/Tyr355. This additional pocket is bordered by small Val523

293

(isoleucine in COX-1). Furthermore, COX-2 also contains conserved Arg513 (replaced with

294

His513 in COX-1). Other residues important for COX-2 selectivity are His90, Gln192, Leu352

295

and Ser353. To investigate the selectivity, we only performed docking simulations on COX-2

296

isozyme. Docking simulations on the reference drugs celecoxib and indomethacin were also

297

performed. Two-dimensional (2-D) binding interaction pattern of both drugs is shown in

298

Supporting Information (Figure S-1). Analysis of the 2-D interaction plot of celecoxib, a

299

selective COX-2 inhibitor, reveals that it interacts amino acid residues (His90, Leu352, Ser353

300

and Arg513) present in additional pocket COX-2 pocket (Figure S1-b in Supporting

301

Information). Similarly, indomethacin displayed interactions with Ser353 and Tyr355.

302

Compounds of series 1 (3a-j) were docked into the binding site of COX-2. It is interesting to

303

note that (S, S)-isomer, with few exceptions, have more negative binding affinity values than (R,

304

R)-isomer, (S, R)-isomer and (R, S)-isomer. Interestingly, the most active COX-2 inhibitor 3b has

305

shown high negative value in (R, R)-form. The computed binding affinity for (R, R)-isomer of

18

306

most active COX-2 inhibitor 3b is -6.95 kcal/mol. While, for (S, S)- 3b, (S, R)- 3b and (R, S)- 3b,

307

it was found to be -6.81, -6.71 and -6.60 kcal/mol. Binding interaction pattern shown in Figure

308

3a revealed that in (S, S)- 3b, cyclohexanone ring oriented towards the secondary pocket and

309

forms hydrogen bond interactions with His90. While, the (R, R)-3b forms hydrogen bond

310

interactions with His90 and π-H interactions between Tyr355. Hence, we can predict here that

311

(R, R)-2 may have the more contribution towards bioactivity against COX-2.

312 313

Figure 3: Two-dimensional (2-D) binding interaction pattern of; (a) (S, S)-3b; (b) (R, R)-3b in the

314

binding site of 1CX2.

315

Analysis of docked poses of aryl ketone / N-sulfonamide derivatives (13a-e) showed that all the

316

compounds adopted similar conformations to the native ligand SC-558 (highly selective COX-2

317

inhibitor). Figure 4a represents the superposed binding poses of R-isomers (13a-e) and native

318

SC-558. The secondary pocket residues are shown in red spheres. While, the orientations of all

319

the ligands in S-isomeric form are shown in Figure 4b. The two-dimensional binding

320

interactions of R-sulfonamides (13a-b and 13d) are shown in Supporting Information (Figure

321

S-2).

19

322 323

Figure 4: (a) Ribbon model of the superimposed binding poses of compounds 13a-e (R-isomer) into the

324

binding site of human COX-2 (1CX2). The secondary pocket residues (His90, Gln192, Leu352,

325

Ser353 Arg513 and Val523 are shown as red spheres. While, some other important residues are shown

326

as yellow spheres; (b) Binding orientation of all compound 13a-e (S-isomer).

327 328

Figure 5: Two-dimensional (2-D) binding interaction pattern of; (a) R-13c; (b) R-13e in the binding site

329

of 1CX2.

330 331 332 333

20

334

2.5.2. Docking studies on 5-LOX enzyme

335

All the synthesized compounds were also docked into the binding site of human 5-LOX (PDB

336

accession code 3O8Y). Human 5-LOX consist of a hydrophobic cavity having Leu367, Leu362,

337

Ile406, Ala410, Val604, Leu607. The iron atom, liganded with His367, His372, His550 and

338

Asn554, is present at the center of the active site. Phe177 is also an important residue and

339

participates in the complex stabilization. Docking simulations on the reference drug Zileuton was

340

also performed. Two-dimensional (2-D) binding interaction pattern of Zileuton in the binding

341

site of 3O8Y is shown in Supporting Information (Figure S-3). Analysis of the 2-D interaction

342

plot of Zileuton reveals that it forms hydrogen bond interactions with Asn554, Gln607 and Ala

343

672. While, Leu607 and Asn180 forms arene-H interactions.

344

We docked our both series (3a-j and 13a-e) into the binding site of 3O8Y. All the possible

345

isomers were included, and their binding affinities were also analyzed. To explore the binding

346

mode of most active series of the compounds (13a-e). The 2-D interaction plot of R- and S-13e

347

into the active site of 5-LOX is shown in Figure 6. S-13e forms hydrogen bond interactions with

348

Asn180, His550, Pro668 and Ala672. While, R-13e established hydrogen bond interactions with

349

Asn180, Leu607 and Gln611. Oxygen atom of sulfonamide coordinated with Fe to stabilize the

350

ligand-enzyme complex. The computed binding affinity for S-13e and R-13e is -8.3684 and -

351

8.9405 kcal/mol respectively. Complete docking analysis of the most active compound of 3g

352

from first series of the compounds (3a-j) is presented in Supporting Information (Figure S-4

353

and S-5).

354 355 356

21

357 358

Figure 6: Two-dimensional (2-D) binding interaction pattern of; (a) S-13e; (b) R-13e in the binding site

359

of 5-LOX (3O8Y).

360

2.6. Preliminary In-silico pharmacokinetic studies

361

The aim of this study is to predict the human intestinal absorption, blood brain barrier

362

penetration and toxicities of the synthesized compounds. These in-silico

363

properties were predicted by using online AdmetSAR (http://lmmd.ecust.edu.cn/admetsar1)

364

server. These properties with probability output are tabulated in Table 5. All the compounds are

365

predicted to absorbed in intestine. The blood brain barrier (BBB) penetration was also predicted.

366

Inflammation has played a critical role in Alzheimer’s disease (AD) and other neurodegenerative

367

disorders (NDs). Cyclooxygenases (COX-1 and COX-2) and lipoxygenases (LOXs) affects the

368

progression of neurodegenerative diseases [45]. Lipoxygenase-5 (LOX-5) enzyme is widely

369

distributed in central nervous system (CNS) and in NDs its expression level increases [46].

370

Therefore, LOX-5 is considered as important target for neuroinflammation (NI) [47-48].

371

Similarly, COX-2 inhibition is also important to treat NDs [49]. The BBB penetration data in

372

Table 5 showed that all compounds are permeable and hence CNS active to treat the NDs like

pharmacokinetic

22

373

AD. Moreover, AMES toxicity and carcinogenicity was also computed. All the compounds were

374

found to be non-AMES toxic and non-carcinogens.

375

Table 5: In-silico pharmacokinetic descriptors for compounds 3a-j and 13a-e and reference

376

Drugs. HIA

BBB penetration

AMES Toxicity

Carcinogens

3a

+ (1.000a)

+ (0.9964)

Non-AMES toxic (0.8625)

Non-carcinogens (0.8819)

3b

+ (1.000)

+ (0.9968)

Non-AMES toxic (0. 0.8142)

Non-carcinogens (0.9068)

3c

+ (0.9973)

+ (0.9954)

Non-AMES toxic (0.8191)

Non-carcinogens (0.8458)

3d

+ (1.000)

+ (0. 9923)

3e

+ (1.000)

+ (0.9940)

3f

+ (0.9944)

+ (0.9877)

3g

+ (09961)

+ (0.9963)

3h

+ (0.9973)

+ (0.9966)

3i

+ (0.9958)

+ (0.9781)

3j

+ (0.9925)

+ (0.9798)

3k

+ (1.000)

+ (0.9971)

3l

+ (1.000)

+ (0.9799)

Non-carcinogens (0.8748) Non-carcinogens (0. 0.8976) Non-carcinogens (0.9180) Non-carcinogens (0.9003) Non-carcinogens (0.88143) Non-carcinogens (0.7634) Non-carcinogens (0.7984) Non-carcinogens (0.7982) Non-carcinogens (0.7029)

13a

+ (0.9912)

+ (0.9213)

13b

+ (0.9936)

+ (0.9785)

13c

+ (0.9964)

+ (0.9674)

13d

+ (0.9950)

+ (0.9482)

Non-AMES toxic (0.8376) Non-AMES toxic (0.8245) Non-AMES toxic (0.7192) Non-AMES toxic (0.8601) Non-AMES toxic (0.8720) Non-AMES toxic (0.7037) Non-AMES toxic (0.6862) Non-AMES toxic (0.8768) Non-AMES toxic (0.8774) Non-AMES toxic (0.8774) Non-AMES toxic (0.8774) Non-AMES toxic (0.8774) Non-AMES toxic (0.8774)

Compound

Non-carcinogens (0.7029) Non-carcinogens (0.7029) Non-carcinogens (0.7029) Non-carcinogens (0.7029)

23

13e

+ (0.9963)

+ (0.9717)

Celecoxibb

+ (1.000)

+ (0.9713)

Indomethacinb + (0.9509) 377

a

+ (0.9381)

Non-AMES toxic (0.8774) Non-AMES toxic (0.7185) Non-AMES toxic (0.9133)

Non-carcinogens (0.7029) Non-carcinogens (0.7905) Non-carcinogens (0.8728)

Probability output is given in parenthesis; b Reference drugs

378 379

3. Conclusions

380

Inflammation has been described and investigated by multiple researchers due to its association

381

with numerous diseased conditions. The use of various types of drugs for the management of

382

inflammation has been reported with different molecular targets and peculiar mechanisms. The

383

exploitation of each target against inflammation has been provided with myriads of applications

384

and drawbacks. The way a specific drug bind with a receptor, and functional groups of a specific

385

drug candidate carry a lot of information regarding its fate and association with adverse drug

386

reactions. Worldwide, various groups of researchers are continuously exploring novel molecular

387

targets and novel drug candidates for various diseases including inflammation. Our current

388

investigation describes the synthesis of N-substituted pyrrolidine-2,5-dione connected at

389

position-3 with cycloalkyl, alkyl and aryl carbonyl substituents. The synthesized compounds

390

were evaluated for their anti-inflammatory potentials by using different in-vitro assays like

391

cyclooxygenase-1, cyclooxygenase-2, 5-lipoxygenase, albumin denaturation and anti-protease

392

assays. It is evident from the in-vitro results that overall cyclic ketones (3a-h) and alkyl ketones

393

(3i-j) exhibited poor to moderate inhibitions of both COX isoforms. N-benzyl derivatives 3b and

394

3e showed selectivity towards COX-2. Compound 3b demonstrated IC50 value of 19.98 µM and

395

selectivity index (SI) of 3.13. Amongst the synthesized compounds, 13a-e series of compounds

396

showed inhibitions in low micromolar to submicromolar ranges. These compounds also 24

397

demonstrated COX-2 selectivity. Compound 13e with IC50 value 0.98 µM and SI of 31.5

398

emerged as the most potent inhibitor of COX-2. Compounds 3b and 13e were further

399

investigated for in-vivo anti-inflammatory activity via carrageenan induced paw edema test. The

400

possible mode of action of compounds 3b and 13e were ascertained with various mediators like

401

histamine, bradykinin, prostaglandin and leukotriene. In-vivo acute toxicity study showed the

402

safety of synthesized compounds up to 1000 mg/kg dose. Extensive docking analysis were

403

carried out considering all the possible isomers of the compounds. Unfortunately, we were

404

unable to separate and identify the ratio of isomers in our compounds. However, by exploiting

405

the predictive power of computational tools, binding affinity data and binding orientation of all

406

the possible isomers were investigated. The selectivity of the compounds against cyclooxygenase

407

isoforms was supported by docking simulations. Selective COX-2 inhibitors showed significant

408

interactions with the amino acid residues His90, Gln192, Leu352 and Ser353 present in

409

additional secondary COX-2 enzyme pocket. Preliminary in-silico pharmacokinetic studies have

410

shown that all compounds are CNS active and thus these compounds can also be used to treat

411

neuroinflammation.

412

The focus of the current research is to identify ligands that can act on multiple anti-inflammatory

413

targets. This study represents a unique example of asymmetric N-substituted pyrrolidine-2,5-

414

dione derivatives targeting anti-inflammatory enzymes. The findings of the research in terms of

415

asymmetric catalysis, scaffold structure, in-vitro/in-vivo screening results, in-silico docking on

416

various targets and preliminary pharmacokinetic predictions are encouraging. Further

417

investigations are required to explore the type and position of substituents. Current study

418

described substitution at position-3 of N-substituted pyrrolidine-2,5-dione, however, the effect of

419

substitution at position-4 (vicinal substituents) of N-substituted pyrrolidine-2,5-dione will be

25

420

taken into consideration. For COX-2 selectivity, cycloalkyl / alkyl groups were not favorable

421

substituents. In conclusion, pyrrolidine-2,5-dione is still an important scaffold and further

422

structural modification may lead to design and synthesize of potent anti-inflammatory drugs.

423

4. Materials and methods

424

4.1. General

425

All the reagents and solvents were purchased from standard commercial vendors and were used

426

without any further purification. N-substituted pyrrolidine-2,5-dione, sulfanilamide, cycloalkyl,

427

alkyl and aryl carbonyl reagents were purchased from Sigma Aldrich. 1H and 13C-NMR spectra

428

were recorded in deuterated solvents on a Bruker spectrometer at 400 and 100 MHz respectively

429

using tetramethyl silane (TMS) as internal reference. Chemical shifts are given in δ scale (ppm).

430

The progress of all the reactions was monitored by TLC on 2.0 x 5.0 cm aluminum sheets pre-

431

coated with silica gel 60F254 with a layer thickness of 0.25 mm (Merck). LC-MS spectra were

432

obtained using Agilent technologies 1200 series high performance liquid chromatography

433

comprising of G1315 DAD (diode array detector) and ion trap LCMS G2445D SL. Final

434

products were analyzed for their purity on Schimadzu system using C18 reversed phase column

435

and isocratic solvent system of water/methanol (10:90) at room temperature. Biologically

436

screened compounds are > 95 % pure as determined by HPLC. Elemental analyses were

437

conducted using Elemental Vario EI III CHN analyzer (for Series 1) and LECO-932 CHNS

438

Analyzer (LECO Corporation, USA) (for Series 2).

439

4.2. General information of the Synthesized compounds (3a-l)

440

All the compounds were synthesized by the addition of different ketones to maleimides. The

441

respective ketone (2.0 equiv) was added to maleimide (1.0 equiv). Organocatalyst assembly

442

consisting of L-isoleucine, thiourea and KOH (0.2 equiv each) were added to chloroform (1M). 26

443

All the reactions were completed at room temperature. Thin layer chromatography (TLC) was

444

used for monitoring of reactions. The finishing of the starting material on TLC plate was

445

considered as completion of each reaction. The reaction was diluted with chloroform (15 ml) and

446

transferred to a separating funnel. Then added 15 ml of water, shake well and allowed to separate

447

the two layers. The organic layer containing crude compound was separated from the water

448

layer. Repeat the same extraction three times. Concentrate the crude compound by using rotary

449

evaporator and absorbed on silica gel surface. The solid form of the crude compound was loaded

450

to column chromatography for purification. In column chromatography, n-hexane and ethyl

451

acetate were used as eluting solvents. The yield of the final product was calculated from the

452

obtained pure compound. The products' structures were confirmed by 1H &

453

data was also compared with the reported literature.

454

4.2.1. 3-(2-oxocyclohexyl)-1-phenylpyrrolidine-2,5-dione (3a) [39- 41]

455

White solid. Yield = 71%. Rf = n-hexane-ethyl acetate (3:1) = 0.41. 1H NMR (400 MHz, CDCl3)

456

(ppm): 1.51-1.82 (m, 3H), 1.96-2.04 (m, 1H), 2.07-2.22 (m, 2H), 2.31-2.48 (m, 2H), 2.52-2.67

457

(m, 1H), 2.82-2.90 (m, 1H), 3.02-3.12 (m, 1H), 3.19-3.33 (m, 1H), 7.24-7.33 (m, 2H), 7.35-7.40

458

(m, 1H), 7.44-7.50 (m, 2H).

459

32.6, 38.5, 40.4, 41.7, 50.1, 52.3, 126.5, 126.5, 128.7, 129.2, 131.9, 175.1, 177.4, 210.2. HPLC

460

purity = 95.2 %, TR = 10.0 min. LC-MS found for C16H17NO3 (m/z) = 272.2 [M+H].

461

calcd (%): C, 70.83; H, 6.32; N, 5.16. Found (%): C, 71.03; H, 6.29; N, 5.21.

462

4.2.2. 1-benzyl-3-(2-oxocyclohexyl)pyrrolidine-2,5-dione (3b) [39-41]

463

Off-white solid. Yield = 69 %. Rf = n-hexane-ethyl acetate (3:1) = 0.45. The Rf value in n-

464

hexane and ethyl acetate (4:1) was calculated as 0.45. 1H NMR (400 MHz, CDCl3) (ppm): 1.49-

465

1.79 (m, 3H), 1.83-2.01 (m, 2H), 2.12-2.26 (m, 1H), 2.33-2.63 (m, 3H), 2.80-2.99 (m, 2H), 3.02-

13

13

C NMR and the

C NMR (100 MHz, CDCl3) (ppm): 22.1, 25.4, 29.3, 30.1, 31.4,

Analysis

27

466

3.16 (m, 1H), 4.65 (d, J = 7.38 Hz, 2H), 7.26-7.38 (m, 5H). 13C NMR (100 MHz, CDCl3) (ppm):

467

24.0, 26.2, 27.9, 32.0, 32.9, 39.5, 40.2, 40.9, 41.8, 49.9, 51.5, 126.9, 127.6, 128.3, 129.1, 129.8,

468

133.9, 175.2, 178.3, 210.2. HPLC purity = 96.4 %, TR = 10.9 min. LC-MS found for C17H19NO3

469

(m/z) = 286.2 [M+H]. Analysis calcd (%): C, 71.56; H, 6.71; N, 4.91; Found (%): C, 71.43; H,

470

6.73; N, 4.94.

471

4.2.3. 1-(4-bromophenyl)-3-(2-oxocyclohexyl)pyrrolidine-2,5-dione (3c) [40, 41]

472

Yellow solid. Yield = 75% %. Rf = n-hexane-ethyl acetate (4:1) = 0.43. 1H NMR (400 MHz,

473

CDCl3) (ppm): 1.64-1.77 (m, 3H), 2.06-2.21 (m, 4H), 2.47-2.52 (m, 3H), 1.96-3.02 (m, 2H),

474

7.24-7.28 (m, 2H), 7.59-7.64 (m, 2H).

475

28.1, 30.7, 31.5, 32.5, 33.8, 41.8, 42.5, 43.6, 54.1, 54.8, 124.1, 129.2, 131.0, 133.6, 175.6, 177.8,

476

211.6. HPLC purity = 98.1 %, TR = 13.9 min. LC-MS found for C16H16BrNO3 (m/z) = 350.1

477

[M+H]. Analysis calcd (%): C, 54.87; H, 4.61; N, 4.00 %. Found (%): C, 54.97; H, 4.60; N, 4.03.

478

4.2.4. 3-(5-methyl-2-oxocyclohexyl)-1-phenylpyrrolidine-2,5-dione (3d)

479

White solid. Yield = 79 % %. Rf = n-hexane-ethyl acetate (4:1) = 0.49. 1H NMR (400 MHz,

480

CDCl3) (ppm): 0.95-0.96 (m, 1H), 1.14-1.20 (m, 3H), 1.26-1.44 (m, 1H), 1.61-1.83 (m, 1H),

481

1.87-2.03 (m, 2H), 2.16-2.36 (m, 2H), 2.43-2.81 (m, 2H) 2.94-2.97 (m, 1H), 3.03-3.15 (m, 1H),

482

7.19-7.27 (m, 2H), 7.31-7.33 (m, 1), 7.38-7.41 (m, 2H). 13C NMR (100 MHz, CDCl3) (ppm): 17.

483

7, 17.7, 21.3, 21.4, 26.9, 26.9, 32.0, 32.4, 33.4, 33.6, 34.9, 35.2, 35.7, 37.1, 37.2, 37.4, 38.2, 41.0,

484

41.2, 41.3, 46.1, 47.5, 51.3, 126.8, 126.8, 126.9, 128.7, 129.3, 132.2, 132.5, 175.8, 175.9, 178.6,

485

178.7, 210.4, 210.8. HPLC purity = 96.5 %, TR = 11.2 min. LC-MS found for C17H19NO3 (m/z)

486

= 286.5 [M+H]. Analysis calcd (%): C, 71.56; H, 6.69; N, 4.91. Found (%): C, 71.76; H, 6.71; N,

487

4.93.

488

4.2.5. 1-benzyl-3-(5-methyl-2-oxocyclohexyl)pyrrolidine-2,5-dione (3e)

13

C NMR (100 MHz, CDCl3) (ppm): 23.1, 24.3, 27.4,

28

489

Yellow solid. Yield = 63% % yield. Rf = n-hexane-ethyl acetate (4:1) = 0.46. 1H NMR (400

490

MHz, CDCl3) (ppm): 1.00 (d, J = 6.58 Hz, 3H), 1.16-1.26 (m, 1H), 1.32-1.45 (m, 2H), 1.83-2.02

491

(m, 4H), 2.24-2.36 (m, 3H), 2.63-3.08 (m, 1H), 4.62-4.73 (m, 2H), 7.23-7.39 (m, 5H). 13C NMR

492

(100 MHz, CDCl3) (ppm): 17.8, 17.8, 21.2, 21.3, 26.8, 26.9, 31.8, 32.3, 32.7, 32.9, 34.9, 35.2,

493

37.2, 37.4, 39.9, 41.0, 41.2, 41.4, 42.6, 45.5, 46.7, 50.5, 9.54, 100.1, 127.8, 128.0, 128.6, 128.6,

494

128.7, 128.8, 136.1, 176.4, 176.5, 179.4, 210.2, 212.5. HPLC purity = 95.7 %, TR = 12.1 min.

495

LC-MS found for C18H21NO3 (m/z) = 300.2 [M+H]. Analysis calcd (%): C, 72.22; H, 7.07; N,

496

4.68. Found (%): C, 72.01; H, 7.09; N, 4.71.

497

4.2.6. 3-(4-oxotetrahydro-2H-pyran-3-yl)-1-phenylpyrrolidine-2,5-dione (3f) [41]

498

Off-white solid. Yield = 73 %. Rf = CHCl3-MeOH (6:1) = 0.44. 1H NMR (400 MHz, CDCl3)

499

(ppm): 2.33-2.41 (m, 1H), 2.71-3.19 (m, 5H), 3.52-3.78 (m, 2H), 4.26-4.75 (m, 2H), 7.25-7.48

500

(m, 5H). 13C NMR (100 MHz, CDCl3) (ppm): 31.47 32.3, 36.9, 41.6, 43.1, 51.2, 53.1, 66.6, 69.0,

501

70.2, 126.3, 128.7, 129.2, 129.8, 174.9, 177.6, 204.0. HPLC purity = 98.4 %, TR = 6.3 min. LC-

502

MS found for C15H15NO4 (m/z) = 274.1 [M+H]. Analysis calcd (%): C, 65.92; H, 5.53; N, 5.13.

503

Found (%): C, 65.73; H, 5.52; N, 5.15.

504

4.2.7. 3-(2-oxocycloheptyl)-1-phenylpyrrolidine-2,5-dione (3g) [39]

505

White solid. Yield = 78 %. Rf = n-hexane-ethyl acetate (4:1) = 0.53. 1H NMR (400 MHz, CDCl3)

506

(ppm): 1.23-1.99 (m, 6H), 2.00-2.27 (m, 2H), 2.61-2.75 (m, 2H), 2.75-3.01 (m, 2H), 3.22-3.38

507

(m, 1), 3.42-3.55 (m, 1H), 7.24-7.27 (m, 1H), 7.32-7.35 (m, 1H), 7.37-7.41 (m, 1H), 7.45-7.50

508

(m, 2H). 13C NMR (100 MHz, CDCl3) (ppm): 25.2, 28.9, 30.3, 30.5, 30.6, 31.0, 31.7, 33.3, 33.5,

509

34.1, 38.6, 53.1, 54.1, 127.0, 127.3, 128.2, 129.7, 130.1, 133.6, 134.6, 174.9, 175.1, 177.2, 213.8,

510

214.0. HPLC purity = 97.1 %, TR = 13.5 min. LC-MS found for C17H19NO3 (m/z) = 286.1

511

[M+H]. Analysis calcd (%): C, 71.56; H, 6.71; N, 4.91. Found (%): C, 71.70; H, 6.72; N, 4.88.

29

512 513 514 515

4.2.8. 3-(2-oxocyclopentyl)-1-phenylpyrrolidine-2,5-dione (3h) [40]

516

Yellow solid. Yield = 74% %. Rf = n-hexane-ethyl acetate (2:1) = 0.40. The reaction was

517

completed in 20 h and the color of the obtained product was yellowish with 80% isolated yield.

518

The Rf value in n-hexane and ethyl acetate (4:1) was calculated as 0.55.

519

1

520

2.36-2.45 (m, 1H), 2.95 (dd, J = 5.26 and 18.39 Hz, 1H), 2.82-2.89 (m, 1H), 2.99 (dd, J = 9.66

521

and 18.39 Hz, 1H), 3.45 (ddd, J = 8.43, 5.26 and 3.17 Hz, 1H), 7.23-7.27 (m, 2H), 7.36-7.41 (m,

522

1H), 7.44-7.49 (m, 2H). 13C NMR (100 MHz, CDCl3) (ppm): 23.6, 25.5, 30.6, 37.9, 40.0, 51.0,

523

127.0, 128.9, 129.81, 132.7, 176.8, 179.8, 216.1. HPLC purity = 97.3 %, TR = 9.1 min. LC-MS

524

found for C15H15NO3 (m/z) = 258.1 [M+H]. Analysis calcd (%): C, 70.02; H, 5.88; N, 5.44.

525

Found (%): C, 70.21; H, 5.86; N, 5.47.

526

4.2.9. 4-(2,5-dioxo-3-(2-oxocyclohexyl)pyrrolidin-1-yl)benzenesulfonamide (3i)

527

The synthesis of 3i was synthesized in a 2-step procedure. The synthesis of intermediate 4-(2,5-

528

dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide (7) was carried out as described in

529

Section 4.3 (Step-1). White solid. Yield = 61%. Rf = CHCl3-MeOH (4:1) = 0.47. 1H NMR (400

530

MHz, CDCl3) (ppm): 1.62-1.68 (m, 2H), 1.69-1.77 (m, 1H), 1.79-1.97 (m, 1H), 2.03-2.19 (m,

531

1H), 2.29-2.36 (m, 1H), 2.44-2.48 (m, 1H), 2.73-2.86 (m, 1H), 2.93-3.14 (m, 1H), 3.17-3.39 (m,

532

3H), 5.93 (s, 2H), 7.64-7.74 (m, 2H), 7.82-7.94 (m, 2H).

533

26.6, 27.6, 28.8, 30.1, 32.6, 40.4, 44.5, 126.4, 128.6, 129.1, 131.8, 175.0, 177.4, 213.7. HPLC

H NMR (400 MHz, CDCl3) (ppm): 1.82-1.95 (m, 2H), 2.06-2.17 (m, 2H), 2.18-2.26 (m, 2H),

13

C NMR (100 MHz, CDCl3) (ppm):

30

534

purity = 95.1 %, TR = 7.5 min. LC-MS found for C16H18N2O5S (m/z) = 351.1 [M+H]. Analysis

535

calcd (%): C, 54.85; H, 5.18; N, 7.99. Found (%): C, 54.73; H, 5.20; N, 7.96.

536 537 538

4.2.10. 4-(2,5-dioxo-3-(2-oxocycloheptyl)pyrrolidin-1-yl)benzenesulfonamide (3j)

539

The synthesis of 3j was synthesized in a 2-step procedure. The synthesis of intermediate 4-(2,5-

540

dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide (7) was carried out as described in

541

Section 4.3 (Step-1). White solid. Yield = 67 %. Rf = n-hexane-ethyl acetate (2:1) = 0.55. 1H

542

NMR (400 MHz, CDCl3) (ppm): 1.53-1.59 (m, 2H), 1.62-1.72 (m, 2H), 1.73-1.86 (m, 3H), 1.87-

543

1.97-2.19 (m, 1H), 2.33-2.48 (m, 3H), 2.53-2.59 (m, 1H), 2.62-2.80 (m, 1H), 2.81-2.99 (m, 1H),

544

5.88 (s, 2H), 7.79 (d, J = 5.8 Hz, 2H), 7.89 (m, J = 5.7 Hz, 2H).

545

(ppm): 25.7, 26.0, 27.1, 28.7, 31.9, 37.5, 39.3, 45.0, 48.4, 125.3, 126.1, 128.2, 128.5, 134.4,

546

136.5, 175.3, 178.0, 214.6. HPLC purity = 96.3 %, TR = 8.3 min. LC-MS found for C17H20N2O5S

547

(m/z) = 365.3 [M+H]. Analysis calcd (%): C, 56.03; H, 5.53; N, 7.69. Found (%): C, 56.18; H,

548

5.52; N, 7.73.

549

4.2.11. 3-(2-oxopropyl)-1-phenylpyrrolidine-2,5-dione (3k) [40]

550

Yellow solid. Yield = 76 %. Rf = n-hexane-ethyl acetate (2:1) = 0.43.

551

CDCl3) (ppm): 2.18 (s, 3H), 2.52-2.58 (m, 1H), 2.99-2.21 (m, 4H), 2.26-7.49 (m, 5H). 13C NMR

552

(100 MHz, CDCl3) (ppm): 29.9, 35.1, 36.4, 41.9, 126.3, 128.2, 129.0, 131.2, 175.1, 178.3, 205.5.

553

HPLC purity = 98.1 %, TR = 6.2 min.

554

4.2.12. 3-(2-methyl-3-oxobutan-2-yl)-1-phenylpyrrolidine-2,5-dione (3l) [39]

555

Yellow solid. Yield = 59 %. Rf = n-hexane-ethyl acetate (4:1) = 0.51. The Rf value in n-hexane

556

and ethyl acetate (4:1) was calculated as 0.45. 1H NMR (400 MHz, CDCl3) (ppm): 1.12 (s, 3H),

13

C NMR (100 MHz, CDCl3)

1

H NMR (400 MHz,

31

557

1.35 (s, 3H), 2.15 (s, 3H), 2.52 (dd, J = 5.40 and 9.39Hz, 1H), 2.94 (dd, J = 9.39 and 18.31 Hz,

558

1H), 3.2 (dd, J = 5.40 and 18.31 Hz, 1H), 7.25-7.28 (m, 2H), 7.29-7.33 (m, 1H), 7.34-7.39 (m,

559

2H).

560

133.0, 175.2, 176.9, 212.5. HPLC purity = 98.5 %, TR = 6.2 min.

13

C NMR (100 MHz, CDCl3) (ppm): 22.2, 24.0, 25.6, 44.3, 50.6, 128.0, 128.6, 128.7,

561 562

4.3. General information of the Synthesized compounds (13a-c)

563

Synthesis of the target compounds were carried out in two steps. In first step, 4-(2,5-dioxo-2,5-

564

dihydro-1H-pyrrol-1-yl)benzenesulfonamide (7) was synthesized by the following procedure.

565

Step-1

566

To a suspension of 20 mmol of sulfanilamide in chloroform, a solution of maleic anhydride (20

567

mmol) in chloroform was added drop-wise. The resulting viscous suspension was stirred at room

568

temperature for 2 h and then left overnight at room temperature. The resulting N-sufamoyl-

569

phenylmaleanic acid ([(4‐sulfamoylphenyl)carbamoyl]prop‐2‐enoic acid, 6) was obtained by

570

filtration. The crude compound was dried and added to a flask already contained anhydrous

571

sodium acetate (10 mmol) in 10 mL of acetic anhydride. The reaction mixture was stirred over a

572

steam bath for 1h. The reaction mixture after cooling to room temperature was poured into ice

573

water. The resulting precipitates were filtered and dried and recrystallize from ethanol.

574

Yield = 63%; 1H NMR (400 MHz, CDCl3) (ppm): 5.84 (s, 2H, NH2), 6.81 (s, 2H, CH=CH), 7.41

575

(d, 2H, J = 7.60 Hz, Ar-H), 7.55 (d, 2H, J = 7.60 Hz, Ar-H).

576

Step-2

577

To a stirred reaction mixture of aromatic ketone (1.5 equiv), OtBu-L-threonine (0.1 equiv), 1,8-

578

diazabicyclo[5.4.0]undec-7-ene (DBU) (0.1 equiv) in chloroform (1M) was added 4-(2,5-dioxo-

579

2,5-dihydro-1H-pyrrol-1-yl)benzenesulfonamide (7, 1.0 equiv). The reaction was stirred at room

32

580

temperature and monitored by TLC. After complete conversion of the limiting reagent, the

581

reaction was diluted with H2O. The chloroform layer was extracted three times and all the three

582

layers were then combined. The crude product was concentrated by rotary evaporator and

583

subjected to column chromatography for purification.

584 585

4.3.1. 4-(2,5-dioxo-3-(2-oxo-2-(pyridin-2-yl)ethyl)pyrrolidin-1-yl)benzenesulfonamide (13a)

586

Off-white solid. Yield = 67 %. Rf = CHCl3-MeOH (3:1) = 0.52.

587

(ppm): 2.58 (dd, J = 18.30 and 5.40 Hz, 1H), 3.17 (dd, J = 18.30 and 9.30 Hz, 1H), 3.25-3.34 (m,

588

1H), 3.49-3.63 (m, 2H), 5.88 (s, 2H), 7.15 (d, J = 7.80 Hz, 2H), 7.21 (d, J = 7.80 Hz, 2H), 7.44-

589

7.48 (m, 1H), 7.81-7.85 (m, 1H), 8.07-8.09 (m, 1H), 8.70-8.72 (m, 1H).

590

CDCl3) (ppm): 39.4, 40.9, 45.0, 122.0, 127.7, 127.7, 127.9, 129.1, 137.2, 139.4, 149.1, 152.5,

591

176.6, 179.5, 198.7. HPLC purity = 98.1 %, TR = 5.3 min. LC-MS found for C17H15N3O5S (m/z)

592

= 374.2 [M+H]. Analysis calcd (%): C, 54.68; H, 4.05; N, 11.25; S, 8.59. Found (%): C, 54.75;

593

H, 4.06; N, 11.23; S, 8.56.

594

4.3.2. 4-(2,5-dioxo-3-(2-oxo-2-phenylethyl)pyrrolidin-1-yl)benzenesulfonamide (13b) [42]

595

Off-white solid. Yield = 60 % %. Rf = CHCl3-MeOH (6:1) = 0.45. 1H NMR (400 MHz, CDCl3)

596

(ppm): 2.50 (dd, J = 18.10 and 6.10 Hz, 1H), 3.00 (dd, J = 18.10 and 8.60 Hz, 1H), 3.31-3.38

597

(m, 1H), 3.61-3.72 (m, 2H), 5.87 (bs, 2H), 7.12-7.16 (m, 2H), 7.25-7.40 (m, 3H), 7.70-7.78 (m,

598

2H), 7.79-7.90 (m, 2H).

599

128.0, 128.7, 128.7, 128.8, 130.5, 132.0, 132.4, 175.4, 178.0, 197.0. HPLC purity = 99.3 %, TR =

600

8.1 min. LC-MS found for C18H16N2O5S (m/z) = 372.1 [M+H]. Analysis calcd (%): C, 58.05; H,

601

4.33; N, 7.52; S, 8.61. Found (%): C, 58.17; H, 4.32; N, 7.50; S, 8.58.

1

H NMR (400 MHz, CDCl3)

13

C NMR (100 MHz,

13

C NMR (100 MHz, CDCl3) (ppm): 38.6, 39.8, 40.4, 126.6, 126.7,

33

602

4.3.3. 4-(3-(2-(4-methoxyphenyl)-2-oxoethyl)-2,5-dioxopyrrolidin-1-yl)benzenesulfonamide

603

(13c)

604

Off-white solid. Yield = 66 %. Rf = CHCl3-MeOH (6:1) = 0.57. 1H NMR (400 MHz, CDCl3)

605

(ppm): 2.63 (dd, J = 17.80 and 6.30 Hz, 1H), 3.10 (dd, J = 17.80 and 9.10 Hz, 1H), 3.30-3.37 (m,

606

1H), 3.54-3.62 (m, 2H), 3.88 (s, 3H), 5.90 (bs, 2H), 7.19-7.23 (m, 2H), 7.34-7.45 (m, 2H), 7.50-

607

7.58 (m, 3H), 7.93-7.97 (m, 1H).

608

113.5, 121.7, 123.2, 123.9, 124.1, 125.8, 125.8, 127.8, 163.4, 176.6, 178.6, 199.9. HPLC purity

609

= 98.7 %, TR = 7.9 min. LC-MS found for C19H18N2O6S (m/z) = 403.5 [M+H]. Analysis calcd

610

(%): C, 56.71; H, 4.51; N, 6.96; S, 7.97. Found (%): C, 56.84; H, 4.49; N, 6.94; S, 7.99.

611

4.3.4.

612

(13d)

613

Off-white solid. Yield = 61 %. Rf = CHCl3-MeOH (6:1) = 0.54. The reaction was completed in

614

24 h with 51.3 % isolated yield. The Rf value in n-hexane and ethyl acetate (4:1) was calculated

615

as 0.36. 1H NMR (400 MHz, CDCl3) (ppm): 2.61 (dd, J = 18.20 and 6.10 Hz, 1H), 3.11 (dd, J =

616

18.20 and 9.20 Hz, 1H), 3.22-3.29 (m, 1H), 3.54-3.74 (m, 2H), 5.92 (bs, 2H), 7.28-7.32 (m, 1H),

617

7.37-7.41 (m, 1H), 7.45-7.50 (m, 1H), 7.80-7.97 (m, 4H).

618

36.3, 37.0, 38.7, 121.6, 123.1, 123.8, 124.0, 125.6, 125.7, 127.7, 128.4, 129.3, 174.9, 176.6,

619

195.8. HPLC purity = 98.6 %, TR = 8.8 min. LC-MS found for C18H15ClN2O5S (m/z) = 407.1

620

[M+H]. Analysis calcd (%): C, 53.14; H, 3.72; N, 6.89; S, 7.88. Found (%): C, 53.05; H, 3.70; N,

621

6.91; S, 7.90.

622

4.3.5. 4-(2,5-dioxo-3-(2-oxo-2-(p-tolyl)ethyl)pyrrolidin-1-yl)benzenesulfonamide (13e)

623

Off-white solid. Yield = 68 %. Rf = CHCl3-MeOH (6:1) = 0.59. 1H NMR (400 MHz, CDCl3)

624

(ppm): 2.40 (s, 3H), 2.58 (dd, J = 17.90 and 5.90 Hz, 1H), 3.14 (dd, J = 17.90 and 8.90 Hz, 1H),

13

C NMR (100 MHz, CDCl3) (ppm): 36.4, 37.2, 39.7, 54.7,

4-(3-(2-(4-chlorophenyl)-2-oxoethyl)-2,5-dioxopyrrolidin-1-yl)benzenesulfonamide

13

C NMR (100 MHz, CDCl3) (ppm):

34

625

3.33-3.43 (m, 1H), 3.54-3.63 (m, 2H), 5.92 (bs, 2H), 7.25-7.37 (m, 3H), 7.43-7.53 (m, 1H), 7.56-

626

7.71 (m, 4H).

627

129.1, 130.8, 130.9, 131.9, 132.4, 175.0, 177.4, 196.8. HPLC purity = 99.1 %, TR = 8.1 min. LC-

628

MS found for C19H18N2O5S (m/z) = 387.1 [M+H]. Analysis calcd (%): C, 59.06; H, 4.70; N,

629

7.25; S, 8.30. Found (%): C, 59.13; H, 4.69; N, 7.23; S, 8.28.

13

C NMR (100 MHz, CDCl3) (ppm): 22.0, 37.6, 38.7, 40.4, 126.5, 128.6, 128.8,

630 631

4.4. In-vitro anti-inflammatory assessments of the compounds

632

4.4.1. Anti-Cyclooxygenase-2 assay (COX-1 / COX-2)

633

Cyclooxygenase-1

634

Cyclooxygenase (COX-2) from human recombinant (Catalog Number C0858) SIGMA

635

ALDRICH and lipoxygenase (5-LOX) from Human Recombinant (Catalog Number 437996)

636

from SIGMA ALDRICH. Enzyme substrate arachidonic acid (CAT No 150384) and lineolic

637

acid (CAS no. 60-33-3) SIGMA ALDRICH. Indicator and co-factor substances, glutathione

638

(CAS 70-18-8), N, N_, N_-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) CAS 637-

639

01-4 and hematin (CAS 15489-90-4) from SIGMA ALDRICH. Linoleic acid substrate in 5-lox

640

(CAS 60-33-3) Cayman chemical USA.

641

Experiments were carried out according to Glassman and White, with some modification [50].

642

Briefly, the enzymes COX-1 (10 µL, 0-.7-0.8 µg) and COX-2 (300 units/ml) were activated on

643

ice for 5 min with the addition of 50 µl co-factor solution containing 0.9 mM glutathione, 01 mM

644

hematin and 0.24 mM TMPD (N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride) in 0.1

645

M Tris HCl buffer with pH 8.0 for the activation. After that the 60 µl of enzyme solution and 20

646

µl of test samples having various concentrations ranging from 31.25 to 1000µg/ml were kept at

647

room temperature for five minutes. Similarly, the reaction was started by the addition of

(cox-1)

from

sheep

(EC

Number 1.14.99.1)

SIGMA

ALDRICH,

35

648

arachidonic acid (30 mM, 20 µl). Incubate the reaction mixture for 5 min and then absorbance

649

was measured at 570 nm using UV-visible spectrophotometer. The percent inhibition of COX-1

650

and COX-2 was calculated from absorbance value per unit time. The IC50 values (in µM) were

651

determined by plotting the inhibition against the sample solution concentrations. In the current

652

study indomethacin and celecoxib were used as positive controls for COX-1 and COX-2

653

respectively.

654

4.4.2. 5-Lipoxygenase (5-LOX) inhibitory assay

655

The 5-lipoxygenase inhibitory activity was performed by following the previously reported

656

procedure. Briefly, different concentrations of the synthesized compounds were prepared ranging

657

from 31.25 to 1000 µg/ml. Secondly, the enzyme 5-lipoxygenase (10,000 U/ml) solution was

658

prepared. The linoleic acid (80 mM) was used as substrate. Similarly, 50 mM phosphate buffer

659

was prepared with 6.3 pH. Different concentrations of the synthetic compounds were dissolved

660

in 0.25 ml of phosphate buffer and 0.25 ml of lipoxidase enzyme solution were added and

661

incubated for 5 min at 25 oC. Afterwards, 1.0 ml of lenoleic acid solution (0.6 mM) was added,

662

mixed well and absorbance were measured at 234 nm. The experiment was performed three

663

times. Zileuton was used as standard drug [51]. The percent inhibition was calculated from the

664

following equation; % Inhibition =

Abs of control − Abs of control × 100 Abs of control

665

The IC50 values (µM) were determined by plotting the inhibition against the sample solution

666

concentrations.

667

4.4.3. Inhibition of albumin denaturation

668

The anti-inflammatory activity of the synthesized compounds was determined using inhibition of

669

albumin denaturation technique. Briefly, 0.05 mL of various concentrations ranging from 31.25 36

670

to 1000 µg/ml of the synthesized compounds and 0.5 ml of 5% aqueous solution of albumin were

671

mixed. The pH of the reaction mixture was adjusted to 6.3 by adding small amount of 1N HCl.

672

The reaction mixture was then incubated at 37 oC for 20 min and heated to 51 oC for further 3

673

min. The reaction mixture was allowed to cool. After cooling the reaction mixture, 2.5 ml of the

674

phosphate buffer saline was added, and the turbidity was measured at 660 nm at UV visible

675

Spectrophotometer. Diclofenac sodium was used as a standard [52]. The experiment was

676

performed three times. The percent inhibition of protein denaturation was calculated by the given

677

formula; % Inhibition =

Abs of control − Abs of sample × 100 Abs of control

678

The IC50 values (µM) were determined by plotting the inhibition against the sample solution

679

concentrations.

680

4.4.4. Protease inhibitory assay

681

The reaction mixture (2 ml) consists of trypsin 0.06 mg/ml, 1 ml 20 mM Tris HCl buffer (pH

682

7.4) and 1 ml of the compound with various concentrations (31.25-1000 µg/ml). The mixture

683

was incubated at 37 oC for 5 min and then 1 ml of 0.8% (w/v) casein was added. The mixture

684

was incubated for additional 20 min and 2 ml of 70% perchloric acid was added. Cloudy

685

suspension was centrifuged at 3000 rpm for 4 to 5 min and the absorbance of the supernatant was

686

measured at 217 nm against buffer was used as blank. The experiment was performed three

687

times. The percent inhibition of protease was calculated using the given formula and diclofenac

688

sodium was used as standard drug [4]. % Inhibition =

Abs of control − Abs of sample × 100 Abs of control

37

689

The IC50 values (µM) were determined by plotting the inhibition against the sample solution

690

concentrations.

691

4.5. In-vivo Anti-inflammatory activity

692

4.5.1 Experimental animals

693

In this study Swiss albino mice of either sex (30-35 g) were used. Animals were purchased from

694

the Pharmacology section of the National Institute of Health, Islamabad, Pakistan. Animals were

695

kept in appropriate cages under controlled laboratory circumstances of 22-25 °C with 12 h

696

light/dark cycle and had a free admittance to water and food throughout acclimatization period.

697

The experimental protocols were approved by the ethical committee of the Department of

698

Pharmacy University of Malakand, KPK, Pakistan.

699

4.5.2 Acute toxicity

700

For the determination of possible toxicity of the synthesized compounds, acute toxicity was

701

performed. Mice were randomly divided into four groups of either sex (n = 8) and were treated

702

with 50, 100, 250 and 500 mg/kg, i.p. The control group received 10 mL/kg normal saline. All

703

the animals were observed randomly for any allergic or abnormal behavioral effect during first 4

704

h and then the numbers of dead animals were counted after 24 h. According to organization for

705

economic cooperation and development (OECD) guidelines for acute oral toxicity, an LD50 dose

706

of > 300 – 2000 is categorized as category 4 and hence the drug is found to be safe [53].

707

4.5.3 Carrageenan induced inflammation

708

The preliminary in-vivo anti-inflammatory potential of the synthesized compounds was assessed

709

on mice of both sexes (30-35 g). Forty (40) mice were randomly divided in five (05) groups

710

(Groups 1-5) each group having eight (08) mice. Group 1 served as a negative control, received

711

10 % (v/v) DMSO, 10ml/kg p.o. along with 150 µL Phosphate buffer saline as vehicle [54-55], 38

712

group 2 served as positive control and was administered Aspirin 100 mg/kg in 0.9 % normal

713

saline, while group 3, 4 and 5 given 25, 50 and 100 mg/kg of tested compounds in DMSO,

714

Tween-80 and normal saline in a ratio of 5:1:94. Rests of the chemicals were dissolved in 0.9

715

% normal saline solution respectively. After 30 minutes, freshly prepared 0.05 ml of 1 % w/v

716

saline suspension of carrageenan was administered subcutaneously in the sub planter surface of

717

right hind paw of each mouse. The paw edema volume was instantly measured via

718

paleothermometer (LE 7500 Plan Lab S.L) after the injection of the carrageenan (irritant) at 1-5

719

h interval. Paw volume of the tested drug and positive control were measured at various intervals

720

and were compared with that of vehicle. Percent inhibition of inflammation was measured via

721

the following formula; Percent inhibition =

−

× 100

722

Where “C” is the average inflammation of control and “T” is the paw volume of tested group.

723

4.5.4 Possible anti-inflammatory mechanism of the synthesized compounds

724

The anti-inflammatory mechanism was evaluated using the histamine, bradykinin, prostaglandin

725

E2 and leukotriene induced paw edema assays. BALB/c mice (30–35 g) of either sex were

726

administered intraperitoneally (i.p.) injection of 10 % DMSO or montelukast (lipoxygenase

727

inhibitor) or chlorpheniramine maleate 25 mg/kg (antihistaminic) 100 mg/kg or HOE 140

728

(Bradykinin inhibitor) 1mg/kg or Celecoxib (cyclooxygenase inhibitor) 50 mg/kg or tested

729

compound (100 mg/kg). After 1 h, paw edema was induced by sub planter injection of 10 µg/ml

730

leukotriene or 0.1 ml of histamine (1 mg/ml) or bradykinin (20 µg/ml) or prostaglandin E2 (0.01

731

µg/ml). Paw volume of each mouse was immediately measured before and after the sub planter

732

administration of different irritants (inflammatory agents) at 1, 2, 3, 4 and 5 h.

733

4.6. Docking studies 39

734

Docking studies were carried out by using Molecular Operating Environment (MOE 2016.08)

735

[56]. Crystal structure of COX-2 in complex with SC-558 was retrieved from Protein Data Bank

736

(PDB code 1CX2). In protein data bank repository, two forms of 5-LOX are available. Crystal

737

structure human 5-LOX with no co-crystallized ligand was obtained from PDB (accession No.

738

3O8Y). While, the crystal structure of another human 5-LOX with co-crystalized substrate,

739

arachidonic acid, is also available (PDB code = 3V99). For COX-2, the docking procedure was

740

validated by re-docking of the native ligands. For 5-LOX, we superposed the mutated LOX

741

(3V99) and human 5-LOX and docking studies were carried out by using arachidonic acid as

742

reference [57]. In another method, we identified binding site at 10 Å to Fe.

743

Preparation of ligands and downloaded enzymes (3D protonation, energy minimization and

744

determination of binding site was carried out by our previously reported methods [58-62]. All the

745

ligand structures were drawn using Builder option in MOE. A data base of compounds was built

746

as ligand.mdb. The compounds were then energy minimized upto 0.01 Gradient using

747

MMFF94X forcefield. The enzyme structure was opened in MOE window. The water molecules

748

(if present) were removed. The 3D protonation was done for all atoms in implicit solvated

749

environment at pH = 7, temperature = 300 K and salt concentration of 0.1. The complete

750

structure was energy minimized using MMFF94X forcefield. Finally, all the compounds were

751

docked into the binding sites of the prepared enzymes. Default docking parameters were set, and

752

ten different conformations were generated for each compound. Lowest binding energy ligand

753

enzyme complexes were analyzed by MOE ligand interaction module. While, for 3-D interaction

754

plot, discovery studio visualizer was used [63].

755

Acknowledgement

40

756

Dr. Umer Rashid is thankful to Higher Education Commission for financial support for the

757

purchase of MOE license under HEC-NRPU project 5291/Federal/NRPU/R&D/HEC/2016. We

758

are thankful to the Higher Education Commission of Pakistan for providing research funding to

759

complete the project under the project No. 22-1/HEC/R&D/PPCR/2018.

760 761 762 763 764 765

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49

Highlights •

Cycloalkyl, alkyl and aryl carbonyl derivatives by the Michael addition of ketones to Nsubstituted maleimides

•

Anti-inflammatory potential of the compounds was determined by using in-vitro and invivo assays.

•

In-vivo acute toxicity study showed the safety of the tested compounds 3b and 13e.

•

Molecular docking studies on COX-2 and 5-LOX were carried out.

•

In-silico pharmacokinetic predictions were also performed

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: