Pyrrolidin-2-one linked benzofused heterocycles as novel small molecule monoacylglycerol lipase inhibitors and antinociceptive agents

DR FAREEDA ATHAR (Orcid ID : 0000-0002-6566-7573)

DR OBAID AFZAL (Orcid ID : 0000-0002-4188-5592)

Article type : Research Article

Pyrrolidin-2-one Linked Benzofused Heterocycles as Novel Small Molecule

Monoacylglycerol Lipase Inhibitors and Antinociceptive Agents

Running title: Pyrrolidin-2-one derivatives as MAGL inhibitors.

1

2

3

4

Abdulmalik Saleh Alfawaz Altamimi , Sandhya Bawa , Fareeda Athar , Md Quamrul Hassan ,

1

1*

Yassine Riadi , Obaid Afzal

1

Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University, AlKharj-

1942, Saudi Arabia.

1

2

Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New

Delhi-110062, India.

3

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India.

4

Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi-

1

10062, India.

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

1

0.1111/CBDD.13751

*

Corresponding author

Obaid Afzal (Ph.D.)

Assistant Professor

Department of Pharmaceutical Chemistry

College of Pharmacy

Prince Sattam Bin Abdulaziz University

AlKharj-11942, Saudi Arabia.

Tel.: +966115886094, Mob.: +966544322364

E-mail: obaid263@gmail.com; o.akram@psau.edu.sa

Abstract

Eighteen pyrrolidin-2-one linked benzothiazole, and benzimidazole derivatives (10-27) were

designed and synthesized. The structure of the compounds was confirmed by elemental and

1

spectral (IR, H-NMR, and MS) data analysis. All the compounds were screened by human

monoacylglycerol lipase (hMAGL) inhibition assay. Three benzimidazole compounds, 22 (4-Cl

phenyl), 23 (3-Cl,4-F phenyl), and 25 (4-methoxy phenyl) were found to be the most potent,

having an IC value of 8.6 nM, 8.0 nM and 9.4 nM, respectively. Among them, the halogen-

5

0

substituted phenyl derivatives, compound 22 (4-Cl phenyl) and compound 23 (3-Cl,4-F phenyl),

showed micromolar potency against fatty acid amide hydrolase (FAAH), having an IC value of

5

0

3

5 µM and 24 µM, respectively. Benzimidazole derivative having 4-methoxyphenyl substitution

(

compound 25) was found to be a selective MAGL inhibitor (IC = 9.4 nM), with an IC value

5

0

50

above 50 µM against FAAH. In the formalin-induced nociception test, compound 25 showed a

dose-dependent reduction of pain response in both acute and late phases. At 30 mg/kg dose, it

significantly reduced the pain response and showed greater potency than the reference drug

gabapentin (GBP).

Keywords

Pyrrolidin-2-one; Benzothiazole; Benzimidazole; MAGL inhibitors; Pain

1

. Introduction

2

-Arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA; Anandamide) are

bioactive lipids (major endocannabinoids), released upon cell membrane activation. Their

functions are mediated by the stimulation of two G-protein-coupled receptors, CB and CB

1

2

(cannabinoid receptors). Moreover, 2-AG and AEA have also been identified to act on several

other receptors like transient receptor potential vanilloid (TRPV1) and peroxisome proliferator

activated receptors (PPARs) [1-4]. 2-AG (major endocnnabinoid) and AEA are hydrolysed by the

enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively

[5]. MAGL is an α/β-hydrolase enzyme that catalyses 2-AG into free fatty acid (FFA) and glycerol

via a catalytic triad (Ser122, Asp239 and His269) [6]. MAGL has been suggested to be the main

α/β-hydrolase that terminates 2-AG actions in the central nervous system (CNS), contributing

~85% of total 2-AG hydrolysis. Residual ~15% of 2-AG is stated to be hydrolysed by α/β-

hydrolase domain (ABHD)-containing proteins (ABHD6 and ABHD12) [7].

The hydrolysis of 2-AG generates arachidonic acid (AA), which acts as the precursor for the

production of inflammatory prostaglandins (PGE and PGD ) by COX-dependent mechanism. In

2

the brain, 2-AG hydrolysis was shown to provide the principal source of the neuro-inflammatory

prostaglandins. The inhibition of MAGL has exerted substantial anti-inflammatory and

neuroprotective properties in animal models of Alzheimer’s and Parkinson’s disease [8,9]. MAGL

inhibitors are reported to increase 2-AG levels in the CNS and consequently caused weakened

mechanical and cold allodynia, by the action of 2-AG on CB receptors, founding their ability to

1

reduce neuropathic pain [10,11]. The role of 2-AG in neuropathic pain has been comprehensively

documented. It has been suggested that 2-AG exhibits antinociception by acting both in the CNS

and the periphery [12-15]. JJKK-048 (MAGL, IC 363 pM) is a potent and selective MAGL

5

0

inhibitor and appeared to be extremely potent in vivo. JJKK-048 causes a considerable surge of 2-

AG levels without affecting AEA levels in mouse brain. It has been shown that JJKK-048 caused

significant analgesia in a writhing test and tail immersion test, without any notable

cannabimimetic side effects [16]. Thus, MAGL inhibitors are potentially useful for the treatment

of pain, inflammation, and CNS disorders. The interesting findings advocated that MAGL

inhibition convene significant therapeutic benefits in neurological disorders and neuropathic pain

[17-19].

SAR629 and congeners (a triazolo carboxamide) were discovered as potent dual MAGL-FAAH

inhibitors. The crystal structure of the MAGL inbound with the inhibitor SAR629 was also

reported [20]. ML30 has been reported to be a potent (IC 0.54 nM) human MAGL inhibitor with

5

0

a 1,000-fold selectivity versus FAAH [21]. In agreement, Aaltonen et al. observed a similar

potency for ML30 (IC₅₀1.5 nM) toward hMAGL. Both compounds, JJKK-048 and KML29

exhibited outstanding MAGL selectivity over FAAH (>10,000-fold) [22,23]. The comprehensive

description of the active site of MAGL was revealed by X-ray diffraction method (Protein Data

Bank, PDB ID: 3HJU), at 2.2 Å resolution [24]. Another X-ray crystal structure of hMAGL bound

with the inhibitor ZYH was determined recently with the use of molecular replacement method to

a resolution of 1.35 Å (PDB ID: 3PE6) [25]. A detailed explanation of the crystal structure of

MAGL and its inhibitors was published recently [26]. Based on the promising therapeutic benefits

of MAGL inhibitors, Abide Therapeutics (California, United States) announces the clinical study

of ABX-1431, which is currently in clinical trials for neurological disorders [27]. Moreover, PF-

6795071 (a triﬂuoromethyl glycol carbamate derivative) was discovered by Pfizer

Massachusetts, United States), as a potent and selective covalent MAGL inhibitor (IC = 3 nM)

0

(

5

0

for the treatment of neuro-inﬂammation [28]. Takeda Pharmaceuticals Co. Ltd. (Kanagawa, Japan)

identified a potent and reversible MAGL inhibitor (IC = 3.6 nM), comprising of piperazinyl

5

0

linked pyrrolidin-2-one moiety (Compound 23, Figure 1). The coordinates of the crystal structures

of MAGL in complex with a piperazinyl pyrrolidin-2-one derivative (PDB ID: 5ZUN) have also

been reported [29]. A benzoylpiperidine derivative (compound 23, IC = 80 nM) is reported as

5

0

potent and selective reversible MAGL inhibitor [30]. A benzisothiazolinone (BTZ) derivative

Compound 13, Figure-1) has been revealed recently as MAGL inhibitor (IC₅₀= 34.1 nM),

(

targeting allosteric regulatory cysteine residues (Cys201 and Cys208) [31]. Our group have

identified two novel human MAGL inhibitors, ZINC24092691 (IC₅₀= 10 nM) and

ZINC12863377 (IC₅₀= 39 nM) by docking based virtual screening of ZINC database.

Furthermore, the structure-activity relationship study of ZINC24092691 lead to a derivative

having an IC value of 6.5 nM [32,33]. The structures of potent MAGL inhibitors, along with

5

0

their IC values, are presented in Figure 1.

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0

The MAGL active site comprises three parts; viz. (a) a hydrophobic void for bulky fatty acid chain

of 2-AG, (b) nucleophilic elbow/oxyanion hole, consisting critical amino acid residues, Ser122,

Ala51, and Met123, where the formation of H-bonds or covalent bonds in the catalytic site of the

enzyme takes place and (c) a small amphiphilic pouch/space for the exiting group glycerol.

Bearing in mind, the possibility of structural modification in the MAGL native substrate (2-AG)

and the binding configuration of MAGL inhibitors, ZINC12863377 (IC₅₀= 39 nM) and

ZINC24092691 (IC = 10 nM), novel small molecules MAGL inhibitors (10-27) were designed

5

0

(Figure-2). All the designed and synthesized compounds were assayed for their human MAGL

inhibitory potential. To find selective inhibitors, the most potent compounds were further assayed

against human FAAH. The most potent and selective MAGL inhibitor was then evaluated for their

antinociceptive activity on formalin-induced hyperalgesia.

2

. Results and discussion

The binding pattern of ZINC24092691 (IC = 10 nM) and ZINC12863377 (IC = 39 nM), at the

5

0

50

active site of MAGL protein, was used to design novel small molecule MAGL inhibitors (10-27).

Their design and route of synthesis are depicted in Figure 2 and Figure 3, respectively.

2

.1. Chemistry

1

-(substituted phenyl)-5-oxopyrrolidine-3-carboxylic acids (1-9) were synthesized by direct

condensation of appropriate anilines and itaconic acid (methylene succinic acid) at their fusion

point, as reported in the literature [34]. The condensation of the terminal carboxylic group in

compounds 1-9 with 2-aminobenzenethiol or 1,2-diaminobenzene was carried out with some

modification in the procedure reported earlier [35]. The condensation was driven by

polyphosphoric acid, which gives us better yield and purity of benzothiazole (10-18) and

benzimidazole derivatives (19-27). Based on the generated elemental and spectral (infra-red

spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry) data, the

structure of the compounds was confirmed.

In the IR spectrum of compound 1 (1-Phenyl-5-oxopyrrolidine-3-carboxylic acid), the

−

1

−1,

−1

characteristic absorption bands at 3252 cm , 1734 cm and 1692 cm were attributed to the O-H

1

of carboxylic acid, C=O of pyrrolidine ring and C=O of carboxylic acid, respectively. The H-

NMR spectrum of compound 1 exhibited a characteristic singlet at δ 11.79 due to the presence of

–

COOH proton, which is D O exchangeable. The doublet appearing at δ 7.65-7.62 (J = 9 Hz) was

2

assigned to two ortho proton of the phenyl ring, while two triplets at δ 7.39-7.34 (J = 7.5 Hz) and

δ 7.16-7.11 (J = 7.2 Hz), were attributed to two meta and one para proton of the phenyl ring,

respectively. The multiplet resonating at δ 4.08-3.93 was assigned to two NCH and one CH

2

proton of the pyrrolidin-2-one ring, while the other two COCH protons of the pyrrolidin-2-one

2

ring appeared as a multiplet at δ 2.80-2.65. The spectral data of compound 10 (benzothiazole

−

1

−1

derivative having unsubstituted phenyl) exhibits specific IR bands at 1691 cm and 1602 cm ,

due to the presence of C=O bond of the pyrrolidin-2-one ring and C=N function of the

1

benzothiazole ring, respectively. In the H-NMR spectrum of compound 10, all the nine aromatic

protons of benzothiazole and phenyl ring appeared resonating at δ 8.17-7.15 as multiplet. The

multiplet resonating at δ 4.39-4.17 was assigned to two NCH and one CH proton of the

2

pyrrolidin-2-one ring, while the other two COCH protons of the pyrrolidin-2-one ring appeared as

2

a multiplet at δ 3.19-2.97. Moreover, the mass spectrum of compound 10 showed the molecular

+

ion peak, i.e., m/z (M ) at 294.10, which confirmed its successful synthesis. The IR spectrum of

compound 19 (benzimidazole derivative having unsubstituted phenyl) showed stretching bands at

−

1

−1

3

387 cm , 1695 cm , and 1592 cm , due to the characteristic N-H of benzimidazole, C=O of

1

pyrrolidine, and C=N of benzimidazole, respectively. In the H NMR spectrum of compound 19,

the broad singlet appearing at δ 12.48 showed the presence of D O exchangeable N-H proton. Two

2

protons of benzimidazole (H-4 and H-7) appeared as a doublet at δ 7.69-7.67 (J = 8.1 Hz).

Remaining seven aromatic protons of benzimidazole and phenyl ring appeared as a multiplet at δ

7

3

.52-7.36 and δ 7.17-7.12. Three multiplets, resonating between δ 4.32-4.19, δ 4.05-3.95, and δ

.10-2.93, were assigned to two NCH , one CH and two COCH protons of the pyrrolidin-2-one

2

+

ring. The mass spectrum of the compound 19, having m/z 277.15 (M ), further confirmed its

successful synthesis.

2

.2. In vitro hMAGL enzyme inhibition assay

The hMAGL enzyme inhibition assay was done with the method reported previously using

Cayman’s assay kit [33,36]. All the eighteen compounds (10-27) were screened for their ability to

inhibit MAGL protein. Overall, the benzimidazole linked pyrrolidin-2-one derivatives (19-27)

showed better activity than their benzothiazole analogs (10-18). Three compounds (22, 23, and 25)

were found to decrease the MAGL activity below 50 % at 10 nM concentration. Among them, the

halogen-substituted phenyl derivatives, compound 22 (4-Cl phenyl) and compound 23 (3-Cl,4-F

phenyl) were found to be most potent, having an IC value of 8.6 nM and 8.0 nM, respectively.

5

0

Also, compound 25 (4-methoxy phenyl) was almost equipotent as their halogen-substituted

counterparts, with an IC₅₀value of 9.4 nM. The structure-activity relationship (SAR) of

benzimidazoles derivatives showed their MAGL inhibitory potential as follows: 3-Cl,4-F > 4-Cl >

4

-OCH > 2-CH > 4-CH > H > 4-NO > 4-OH > 4-SO NH . The IC of standard control, a

3 3 3 2 2 2 50

selective MAGL inhibitor, CAY10499 (IC = 415 nM) was found to be comparable as reported in

5

0

literature (IC = 350 nM) [36]. The results of the hMAGL inhibition assay are provided in Table

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0

1

.

2

.3. In vitro hFAAH enzyme inhibition assay

MAGL inhibitors having an IC value less than 10 nM (compound 22, 23, and 25) were selected

5

0

for the inhibition assay against a closely related hydrolase, FAAH [37]. Among them, the halogen-

substituted phenyl derivatives, compound 22 (4-Cl phenyl) and compound 23 (3-Cl,4-F phenyl)

showed micromolar potency against FAAH, having an IC₅₀value of 35 µM and 24 µM,

respectively. Surprisingly, the benzimidazoles derivative having 4-methoxyphenyl substitution

(

compound 25) was found to be a selective MAGL inhibitor (IC = 9.4 nM), with FAAH, IC

50 50

value more than 50 µM. The IC of a selective FAAH inhibitor, URB597 (standard control), was

5

0

found to be 5 nM, comparable as reported in literature (IC = 4.6 nM) [38]. The results of the

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0

hFAAH inhibition assay are provided in Table 1.

2

.4. Computational studies

.4.1. Molecular docking

To gain insights into the binding pattern of compound 25, it was docked into the active site of

MAGL protein (PDB ID: 5ZUN, Resolution-1.35 Å), by Glide XP docking using Maestro GUI of

Schrodinger. The binding pose of compound 25 showed that the carbonyl function of pyrrolidin-2-

one is positioned into the oxyanion hole, forming three prominent hydrogen bonds (~ 2Å) with

critical amino acid residues; Ala51, Met123, and catalytic Ser122. The benzimidazole moiety is

projected into the amphiphilic pocket, forming π-π stacking interaction with Tyr194. However, the

4

-methoxy phenyl part of the ligand is involved in close van der Waals interactions in the

hydrophobic atmosphere of Leu148, Leu213, and Leu241. 3D and 2D representation of the

binding pose of compound 25 into the active site of MAGL is presented in Figure-4.

2

.4.2. Physicochemical and pharmacokinetic properties

To develop a potent, selective, and orally active MAGL inhibitor, that can efficiently cross the

blood-brain barrier (BBB), we analysed thirty-five physicochemical and pharmacokinetic

properties of the three potent compounds having MAGL IC value less than 10 nM (compounds

5

0

2

2, 23 and 25) with the QikProp module of Schrodinger. Ghose et al. derived a guideline for the

selection and optimization of CNS compounds, through the analysis of thirty-five physicochemical

and pharmacokinetic properties of 317 CNS and 626 non-CNS oral drugs [40]. The guidelines for

2

designing high-quality CNS drugs are: (i) TPSA <76 Å (25-60 Å ), (ii) no. of N atoms = 1 to 2,

including one aliphatic amine), (iii) 2 to 4 linear chains outside rings, (iv) no. of polar H atoms <3

3

2

(

0-1), (v) molecular volume = 740-970 Å , (vi) SASA = 460-580 Å , and (vii) a positive QikProp

CNS activity parameter. Interestingly, the physicochemical and pharmacokinetic profile of

compounds 22, 23, and 25, was found to be within the limits as proposed and presented in the

table in supplementary material.

2

.5. Antinociceptive activity

.5.1. Formalin-induced nociception test

The formalin-induced test is considered a widely accepted animal pain model. The behavioural

responses to formalin consist of two distinctive phases I and II. Phase-I is an acute pain of intense

licking and biting of injected paw, which lasts up to 5 minutes of formalin injection, and reflects

the activity of C-fiber afferent nociceptors. Phase II reflects the state of central sensitization of the

spinal dorsal horn neurons and involves periods of licking and biting, which peaks from 10-30

minutes of formalin injection, but lasts up to 60 minutes [41,42]. Compound 25 was administered

orally, 4 hours before formalin injection, at concentrations of 5, 10, 30, and 50 mg/kg, suspended

in 0.5% methylcellulose. Gabapentin (GBP), a drug used in neuropathic pain, was used as a

reference drug (100 mg/kg, i.p, dissolved in 0.9% normal saline). The duration of paw licking and

biting during phases I and II are presented as mean±SEM in Figure 5. GBP showed only a limited

effect on acute nociceptive responses (phase I), as compared to control. However, it showed a

significant suppression of paw licking during phase II, confirming its central effect. Compound 25

showed a dose-dependent reduction of pain response in both acute and late phases, indicating its

peripheral as well as central effect. At 30 mg/kg dose, it significantly reduced the pain response

and showed greater potency than the reference drug, gabapentin (Figure 5).

3

. Summary and conclusion

Pyrrolidin-2-one linked benzothiazole and benzimidazole derivatives (10-27) were designed as per

the structural requirements of MAGL inhibitors and based on previously identified compounds

reported by our group [32, 33]. All the designed compounds were successfully synthesized and

characterized. Compounds (10-27) were screened for their potential to inhibit hMAGL. Three

benzimidazole compounds, 22 (4-Cl phenyl), 23 (3-Cl,4-F phenyl), and 25 (4-methoxy phenyl)

were found to be the most potent, having an IC₅₀value of 8.6 nM, 8.0 nM and 9.4 nM,

respectively. Among them, halogen-substituted phenyl derivatives, compound 22 (4-Cl phenyl)

and compound 23 (3-Cl,4-F phenyl), showed micromolar potency against FAAH, having an IC₅₀

value of 35 µM and 24 µM respectively. The benzimidazoles derivative having 4-methoxyphenyl

substitution (compound 25) was found to be a selective MAGL inhibitor (IC = 9.4 nM), with an

5

0

IC value of more than 50 µM against FAAH. In molecular docking analysis, the binding pattern

5

0

of compound 25 showed that the carbonyl function of pyrrolidin-2-one is positioned into the

oxyanion hole, forming three prominent hydrogen bonds (~ 2Å) with critical amino acid residues:

Ala51, Met123, and catalytic Ser122. The benzimidazole moiety is projected into the amphiphilic

pocket, forming π-π stacking interaction with Tyr194. However, the 4-methoxy phenyl part of the

ligand is involved in close van der Waals interactions in the hydrophobic atmosphere of Leu148,

Leu213, and Leu241. This pattern of binding is in full agreement with the inhibitor bound crystal

structures reported earlier [20, 24, 26, 29]. In the formalin-induced nociception test, compound 25

showed a dose-dependent reduction of pain response in both acute and late phases. At 30 mg/kg

dose, it significantly reduced the pain response and showed higher potency than the reference

drug, gabapentin (GBP). Recently, MAGL inhibitors (ABX-1431, Abide Therapeutics, USA, and

PF-06795071, Pfizer, USA) were discovered and are currently under clinical trials for neurological

disorders and neuropathic pain [27, 28]. Moreover, compound 25, is a potential lead compound for

further exploitation at position 1 and 4 of the pyrrolidin-2-one moiety for the development of more

potent MAGL inhibitors. In conclusion, the present study confirmed that pharmacological

inhibition of enzyme MAGL might convene significant therapeutic benefits in neurological

disorders and neuropathic pain.

4

. Experimental

4

.1. Chemistry

Chemicals and reagents were purchased from Sigma Aldrich (New Delhi, India), Merck Ltd.,

(New Delhi, India) and S. D. Fine Chemicals Ltd. (New Delhi, India). Thin-layer chromatography

(TLC) was used to assess the progress of the reaction and purity of the compounds. Melting points

(M.P.) of the synthesized compounds were obtained and uncorrected by using open capillary tubes

on a melting point apparatus. Elemental analysis was performed on the CHNOS-Elemental

analyzer (Vario EL III) using sulphanilic acid as standard and tungsten (VI) oxide as combusting

agent. Sample for analysis was prepared in Tin boats of dimension 6×6×12 mm. Infra-red (IR)

-1

spectrum was recorded by Shimadzu FT-IR spectrometer from 4000-400 cm range, by using KBr

(potassium bromide) pellets of the synthesized compounds. Bruker 300 MHz NMR instrument is

1

used for the recording of the H-NMR spectrum of compounds in solvent CDCl or DMSO-d .

3

6

UPLC–MS (Q-TOF-ESI) (Waters Corp., USA) with an ESI technique was used for molecular

mass detection (m/z) of the synthesized compounds.

4

.1.1. General procedure for the synthesis of 1-(substitutedphenyl)-5-oxopyrrolidine-3-carboxylic

acids (1-9)

To substituted aniline (20 mmol), an equimolar amount of itaconic acid (20 mmol, 2.6 g) was

added and the temperature of the reaction mixture was raised till their fusion point. After 30

minutes of stirring, mixture was cooled and chilled in an ice bath. The obtained solid was

dissolved in 10% aqueous NaOH solution, treated with activated charcoal and filtered. The filtrate

was acidified with dil. HCl. The resulting precipitate was filtered, washed with water and

recrystallized from methanol.

4

.1.1.1. 1-Phenyl-5-oxopyrrolidine-3-carboxylic acids (1)

-1

White solid; Yield: 87 %; M.P.: 190-191 ºC; IR (KBr, cm ): 3252 (COO-H), 3045 (Ar C-H), 1734

1

(

C=O), 1692 (C=OOH), 1522 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.79 (s, 1H,

6

COOH, D O exchangeable), 7.65-7.62 (d, 2H, H-2 & H-6, J = 9 Hz), 7.39-7.34 (t, 2H, H-3 & H-5,

2

J = 7.5 Hz), 7.16-7.11 (t, 1H, H-4, J = 7.2 Hz), 4.08-3.93 (m, 3H, NCH & CH ), 2.80-2.65 (m,

2

pyrr

+

2

6

4

H, COCH ); ESI-MS (m/z): 205.11 (M ); Anal. calcd. For C H NO : C, 64.38; H, 5.40; N,

2

11 11

3

.83. Found: C, 64.44; H, 5.47; N, 6.91.

.1.1.2. 1-(2-Methylphenyl)-5oxopyrrolidine-3-carboxylic acids (2)

-1

White solid; Yield: 63 %; M.P.: 153-154 ºC; IR (KBr, cm ): 3243 (COO-H), 3048 (Ar C-H), 1741

1

(

C=O), 1711 (C=OOH), 1605 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.18 (s, 1H,

6

COOH, D O exchangeable), 7.55-7.18 (m, 3H, H-3, H-4 & H-5), 7.10-7.05 (d, 1H, H-6, J = 7.2

2

+

Hz), 4.06-3.91 (m, 3H, NCH & CH ), 2.82-2.64 (m, 2H, COCH ); ESI-MS (m/z): 219.12 (M );

2

pyrr

2

Anal. calcd. For C H NO : C, 65.74; H, 5.98; N, 6.39. Found: C, 65.82; H, 6.00; N, 6.47.

1

2

13

3

4

.1.1.3. 1-(4-Methylphenyl)-5oxopyrrolidine-3-carboxylic acids (3)

-1

White solid; Yield: 85 %; M.P.: 186-187 ºC; IR (KBr, cm ): 3261 (COO-H), 3056 (Ar C-H), 1726

1

(

C=O), 1695 (C=OOH), 1592 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.23 (s, 1H,

6

COOH, D O exchangeable), 7.52-7.49 (d, 2H, H-3 & H-5, J = 8.1 Hz), 7.18-7.15 (d, 2H, H-2 &

2

H-6, J = 8.1 Hz), 4.05-3.90 (m, 3H, NCH & CH ), 2.81-2.62 (m, 2H, COCH ), 2.27 (s, 3H,

2

pyrr

2

+

CH ); ESI-MS (m/z): 219.12 (M ); Anal. calcd. For C H NO : C, 65.74; H, 5.98; N, 6.39.

3

12 13

3

Found: C, 65.83; H, 6.02; N, 6.45.

4

.1.1.4. 1-(4-Chlorophenyl)-5oxopyrrolidine-3-carboxylic acids (4)

-1

White solid; Yield: 85 %; M.P.: 151-152 ºC; IR (KBr, cm ): 3245 (COO-H), 3037 (Ar C-H), 1743

1

(

C=O), 1698 (C=OOH), 1577 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.95 (s, 1H,

6

COOH, D O exchangeable), 7.70-7.67 (d, 2H, H-3 & H-5, J = 9 Hz), 7.43-7.40 (d, 2H, H-2 & H-

2

6

, J = 8.7 Hz), 4.08-3.93 (m, 3H, NCH & CH ), 2.84-2.66 (m, 2H, COCH ); ESI-MS (m/z):

2 pyrr 2

+

2

39.06 (M ), 241.06 (M + 2); Anal. calcd. For C H ClNO : C, 55.13; H, 4.21; N, 5.84. Found:

11 10 3

C, 55.19; H, 4.29; N, 5.92.

4

.1.1.5. 1-(3-Chloro-4-fluorophenyl)-5oxopyrrolidine-3-carboxylic acids (5)

-1

White solid; Yield: 82 %; M.P.: 143-144 ºC; IR (KBr, cm ): 3289 (COO-H), 3081 (Ar C-H), 1737

1

(

C=O), 1702 (C=OOH), 1561 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.57 (s, 1H,

6

COOH, D O exchangeable), 7.96-7.93 (q, 1H, H-5), 7.61-7.56 (m, 1H, H-2), 7.44-7.37 (t, 1H, H-

2

6

, J = 9 Hz), 4.14-3.93 (m, 3H, NCH & CH ), 2.84-2.66 (m, 2H, COCH ); ESI-MS (m/z):

2 pyrr 2

+

2

57.07 (M ), 259.07 (M + H); Anal. calcd. For C H ClFNO : C, 51.28; H, 3.52; N, 5.44. Found:

11 9 3

C, 51.33; H, 3.57; N, 5.51.

4

.1.1.6. 1-(4-Hydroxyphenyl)-5oxopyrrolidine-3-carboxylic acids (6)

-1

White solid; Yield: 75 %; M.P.: 200-201 ºC; IR (KBr, cm ): 3267 (COO-H), 3064 (Ar C-H), 1751

1

(

C=O), 1713 (C=OOH), 1537 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.61 (s, 1H,

6

COOH, D O exchangeable), 7.35-7.30 (d, 2H, H-2 & H-6, J = 7.5 Hz), 7.07-7.04 (d, 2H, H-3 &

2

H-5, J = 9 Hz), 5.43 (s, 1H, OH), 4.11-3.92 (m, 3H, NCH & CH ), 2.80-2.62 (m, 2H, COCH );

2

pyrr

2

+

ESI-MS (m/z): 221.11 (M ); Anal. calcd. For C H NO : C, 59.73; H, 5.01; N, 6.33. Found: C,

1

11

4

5

9.82; H, 5.08; N, 6.41.

.1.1.7. 1-(4-Methoxyphenyl)-5oxopyrrolidine-3-carboxylic acids (7)

4

-1

White solid; Yield: 85 %; M.P.: 173-174 ºC; IR (KBr, cm ): 3254 (COO-H), 3051 (Ar C-H), 1739

1

(

C=O), 1710 (C=OOH), 1571 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.73 (s, 1H,

6

COOH, D O exchangeable), 7.53-7.50 (d, 2H, H-2 & H-6, J = 9 Hz), 6.94-6.91 (d, 2H, H-3 & H-

2

5

2

6

4

, J = 9 Hz), 4.04-3.89 (m, 3H, NCH & CH ), 2.80-2.61 (m, 2H, COCH ); ESI-MS (m/z):

2 pyrr 2

+

35.13 (M ); Anal. calcd. For C H NO : C, 61.27; H, 5.57; N, 5.95. Found: C, 61.30; H, 5.63; N,

1

2

13

4

.02.

.1.1.8. 1-(4-Nitrophenyl)-5oxopyrrolidine-3-carboxylic acids (8)

-1

Pale yellow solid; Yield: 33 %; M.P.: 175-177 ºC; IR (KBr, cm ): 3296 (COO-H), 3067 (Ar C-H),

1

758 (C=O), 1722 (C=OOH), 1605 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.64 (s, 1H,

6

COOH, D O exchangeable), 7.92-7.87 (d, 2H, H-3 & H-5, J = 7.5 Hz), 7.06-7.01 (d, 2H, H-2 &

2

H-6, J = 7.2 Hz), 4.13-3.95 (m, 3H, NCH & CH ), 2.84-2.67 (m, 2H, COCH ); ESI-MS (m/z):

2

pyrr

2

+

2

50.11 (M ); Anal. calcd. For C H N O : C, 52.80; H, 4.03; N, 11.20. Found: C, 52.91; H, 4.07;

11 10 2 5

N, 11.31.

4

.1.1.9. 1-(4-Sulfamoylphenyl)-5oxopyrrolidine-3-carboxylic acids (9)

-1

White solid; Yield: 72 %; M.P.: 211-213 ºC; IR (KBr, cm ): 3278 (COO-H), 3068 (Ar C-H), 1760

1

(

C=O), 1716 (C=OOH), 1591 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 11.65 (s, 1H,

6

COOH, D O exchangeable), 7.86-7.83 (d, 2H, H-3 & H-5, J = 9.3 Hz), 7.28-7.25 (d, 2H, H-2 &

2

H-6, J = 8.1 Hz), 4.19-3.99 (m, 3H, NCH & CH ), 3.56 (s, 2H, NH ), 2.85-2.70 (m, 2H,

2

pyrr

2

+

COCH ); ESI-MS (m/z): 284.10 (M ); Anal. calcd. For C H N O S: C, 46.47; H, 4.25; N, 9.85.

2

11 12

2

5

Found: C, 46.62; H, 4.37; N, 9.95.

4

.1.2. General procedure for the synthesis of 4-(benzothiazolyl/benzimidazolyl)-1-

(substitutedphenyl)pyrrolidin-2-one derivatives (10-27)

To an equimolar amount of appropriate 1-(substitutedphenyl)-5-oxopyrrolidine-3-carboxylic acids

10 mmol) and 2-aminobenzenethiol (10 mmol, 1.25 g) or 1,2-diaminobenzene (10 mmol, 1.08 g)

(

in a round bottom flask, polyphosphoric acid (10 g) was added. The reaction mixture was stirred at

o

1

50-160 C for 2-3 hours. The reaction mixture was then cooled to room temperature, 5% sodium

carbonate solution (15 ml) was added, refluxed for 10 min and left to cool. The content of the flask

was poured in a beaker containing 50 ml of water and stirred for 10 minutes. The crude product

was filtered, washed with water and recrystallized from ethanol. The compounds were further

purified through column chromatography by hexane:ethylacetate (4:1) as eluent.

4.1.2.1. 4-(Benzo[d]thiazol-2-yl)-1-phenylpyrrolidin-2-one (10)

-1

Pale yellow solid; Yield: 60 %; M.P.: 148-149 ºC; IR (KBr, cm ): 3062 (Ar C-H), 1691 (C=O),

1

4

2

4

606 (C=N), 1575 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.17-7.15 (m, 9H, Ar-H),

6

+

.39-4.17 (m, 3H, NCH & CH ), 3.19-2.87 (m, 2H, COCH ); ESI-MS (m/z): 294.10 (M ),

2

pyrr

2

+

95.10 (M +H); Anal. calcd. For C H N OS: C, 69.36; H, 4.79; N, 9.52. Found: C, 69.43; H,

1

7

14

2

.82; N, 9.61.

.1.2.2. 4-(Benzo[d]thiazol-2-yl)-1-(2-methylphenyl)pyrrolidin-2-one (11)

-1

Pale yellow solid; Yield: 63 %; M.P.: 155-156 ºC; IR (KBr, cm ): 3069 (Ar C-H), 1661 (C=O),

1

597 (C=N), 1567 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.11-8.08 (d, 1H, H-

6

4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 8.00-7.97 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.1 Hz), 7.53-7.35 (m, 4H, H-

5_b_e_n_z_o_t_h_i_a_z_o_l_e, H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, H-3_p_h_e_n_y_l& H-4_p_h_e_n_y_l), 7.20-7.17 (m, 2H, H-5_p_h_e_n_y_l& H-6_p_h_e_n_y_l), 4.38-4.25

(

m, 2H, NCH ), 4.17-4.12 (m, 1H, CH ), 3.18-2.88 (m, 2H, COCH ), 2.17 (s, 3H, CH ); ESI-MS

2 pyrr 2 3

+

(

m/z): 308.13 (M ), 309.13 (M +H); Anal. calcd. For C H N OS: C, 70.10; H, 5.23; N, 9.08.

18 16 2

Found: C, 70.27; H, 5.31; N, 9.17.

4

.1.2.3. 4-(Benzo[d]thiazol-2-yl)-1-(4-methylphenyl)pyrrolidin-2-one (12)

-1

Pale yellow solid; Yield: 65 %; M.P.: 152-153 ºC; IR (KBr, cm ): 3071 (Ar C-H), 1657 (C=O),

1

591 (C=N), 1560 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.11-8.08 (d, 1H, H-

6

4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 8.00-7.97 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.1 Hz), 7.57-7.41 (m, 4H, H-

5_b_e_n_z_o_t_h_i_a_z_o_l_e, H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, H-3_p_h_e_n_y_l& H-5_p_h_e_n_y_l), 7.20-7.17 (d, 2H, H-2_p_h_e_n_y_l& H-6_p_h_e_n_y_l, J = 8.4

Hz), 4.38-4.23 (m, 2H, NCH ), 4.16-4.12 (m, 1H, CH ), 3.17-2.87 (m, 2H, COCH ), 2.28 (s, 3H,

2

pyrr

2

+

CH ); ESI-MS (m/z): 308.13 (M ), 309.13 (M +H); Anal. calcd. For C H N OS: C, 70.10; H,

3

18 16

2

5

.23; N, 9.08. Found: C, 70.22; H, 5.33; N, 9.20.

4

.1.2.4. 4-(Benzo[d]thiazol-2-yl)-1-(4-chlorophenyl)pyrrolidin-2-one (13)

-1

Pale yellow solid; Yield: 63 %; M.P.: 174-175 ºC; IR (KBr, cm ): 3066 (Ar C-H), 1689 (C=O),

1

610 (C=N), 1581 (C=C), 762 (C-Cl); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.10-8.08 (d, 1H,

6

H-4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 7.99-7.97 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 7.74-7.71 (t, 2H, H-

5_b_e_n_z_o_t_h_i_a_z_o_l_e& H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.7 Hz), 7.59-7.42 (m, 4H, Ar-H_p_h_e_n_y_l), 4.40-4.22 (m, 2H, NCH₂),

+

4

.19-4.14 (m, 1H, CH ), 3.19-2.87 (m, 2H, COCH ); ESI-MS (m/z): 328.08 (M ), 330.08

pyrr

2

+

(

M +2); Anal. calcd. For C H ClN OS: C, 62.10; H, 3.98; N, 8.52. Found: C, 62.21; H, 4.05; N,

17 13 2

8

4

.63.

.1.2.5. 4-(Benzo[d]thiazol-2-yl)-1-(3-chloro-4-fluorophenyl)pyrrolidin-2-one (14)

-1

Pale yellow solid; Yield: 66 %; M.P.: 195-196 ºC; IR (KBr, cm ): 3112 (Ar C-H), 1680 (C=O),

1

602 (C=N), 1535 (C=C), 771 (C-Cl); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.12-8.10 (d, 1H,

6

H-4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 8.01-7.99 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 7.59-7.42 (m, 5H, Ar-H),

4

.40-4.26 (m, 2H, NCH ), 4.20-4.14 (m, 1H, CH ), 3.17-2.92 (m, 2H, COCH ); ESI-MS (m/z):

2

pyrr

2

+

3

46.05 (M ), 348.05 (M +2); Anal. calcd. For C H ClFN OS: C, 58.88; H, 3.49; N, 8.08. Found:

1

7

12

2

C, 58.97; H, 3.55; N, 8.21.

4

.1.2.6. 4-(Benzo[d]thiazol-2-yl)-1-(4-hydroxyphenyl)pyrrolidin-2-one (15)

-1

Pale yellow solid; Yield: 52 %; M.P.: 169-170 ºC; IR (KBr, cm ): 3433 (O-H), 3096 (Ar C-H),

1

680 (C=O), 1593 (C=N), 1525 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.11-8.09 (d,

6

1

H, H-4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 7.97-7.95 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 7.70-7.67 (t, 2H, H-

5_b_e_n_z_o_t_h_i_a_z_o_l_e& H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.7 Hz), 7.40-7.31 (m, 4H, Ar-H_p_h_e_n_y_l), 5.65 (bs, 1H, OH, D O

2

exchangeable), 4.38-4.23 (m, 2H, NCH ), 4.15-4.11 (m, 1H, CH ), 3.17-2.91 (m, 2H, COCH );

2

pyrr

2

+

ESI-MS (m/z): 310.10 (M ), 311.10 (M +H); Anal. calcd. For C H N O S: C, 65.79; H, 4.55; N,

1

7

14

2

9

.03. Found: C, 65.91; H, 4.67; N, 9.18.

.1.2.7. 4-(Benzo[d]thiazol-2-yl)-1-(4-methoxyphenyl)pyrrolidin-2-one (16)

4

-1

Pale yellow solid; Yield: 58 %; M.P.: 164-165 ºC; IR (KBr, cm ): 3091 (Ar C-H), 1685 (C=O),

1

600 (C=N), 1547 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.12-8.09 (d, 1H, H-

6

4_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 7.8 Hz), 8.01-7.98 (d, 1H, H-7_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.1 Hz), 7.59-7.42 (m, 4H, H-

5_b_e_n_z_o_t_h_i_a_z_o_l_e, H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, H-3_p_h_e_n_y_l& H-5_p_h_e_n_y_l), 7.18-7.15 (d, 2H, H-2_p_h_e_n_y_l& H-6_p_h_e_n_y_l, J = 8.4

Hz), 4.40-4.22 (m, 2H, NCH ), 4.15-4.09 (m, 1H, CH ), 3.69 (s, 3H, OCH ), 3.15-2.86 (m, 2H,

2

pyrr

3

+

COCH ); ESI-MS (m/z): 324.12 (M ), 325.12 (M +H); Anal. calcd. For C H N O S: C, 66.65;

2

18 16

2

H, 4.97; N, 8.64. Found: C, 66.74; H, 5.08; N, 8.78.

4

.1.2.8. 4-(Benzo[d]thiazol-2-yl)-1-(4-nitrophenyl)pyrrolidin-2-one (17)

-1

Yellow solid; Yield: 55 %; M.P.: 209-210 ºC; IR (KBr, cm ): 3082 (Ar C-H), 1665 (C=O), 1598

1

(

C=N), 1537 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.28-8.11 (m, 4H, H-4_b_e_n_z_o_t_h_i_a_z_o_l_e, H-

6

7_b_e_n_z_o_t_h_i_a_z_o_l_e, H-3_p_h_e_n_y_l& H-5_p_h_e_n_y_l), 7.52-7.38 (t, 2H, H-5_b_e_n_z_o_t_h_i_a_z_o_l_e, H-6_b_e_n_z_o_t_h_i_a_z_o_l_e, J = 8.7 Hz), 7.02-

.99 (d, 2H, H-2_p_h_e_n_y_l& H-6_p_h_e_n_y_l, J = 8.4 Hz), 4.37-4.18 (m, 2H, NCH ), 4.13-4.10 (m, 1H,

6

2

+

CH ), 3.12-2.85 (m, 2H, COCH ); ESI-MS (m/z): 339.10 (M ), 340.10 (M +H); Anal. calcd. For

pyrr

2

C H N O S: C, 60.17; H, 3.86; N, 12.38. Found: C, 60.25; H, 3.97; N, 12.51.

1

7

13

3

4

.1.2.9. 4-(Benzo[d]thiazol-2-yl)-1-(4-sulfamoylphenyl)pyrrolidin-2-one (18)

-1

Pale yellow solid; Yield: 52 %; M.P.: 188-189 ºC; IR (KBr, cm ): 3093 (Ar C-H), 1674 (C=O),

1

7

4

604 (C=N), 1534 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 8.17-8.05 (m, 4H, Ar-H),

6

.55-7.42 (m, 4H, Ar-H), 5.71 (s, 2H, SO NH , D O exchangeable) 4.37-4.22 (m, 2H, NCH ),

2

+

.19-4.14 (m, 1H, CH ), 3.19-2.87 (m, 2H, COCH ); ESI-MS (m/z): 373.09 (M ), 374.09

pyrr

2

+

(

M +H); Anal. calcd. For C H N O S : C, 54.68; H, 4.05; N, 11.25. Found: C, 54.82; H, 4.16; N,

17 15 3 3 2

1

4

1.31.

.1.2.10. 4-(1H-benzo[d]imidazol-2-yl)-1-phenylpyrrolidin-2-one (19)

-1

Off white solid; Yield: 75 %; M.P.: 215-216 ºC; IR (KBr, cm ): 3387 (N-H), 3161 (Ar C-H), 1695

1

(

C=O), 1592 (C=N), 1568 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.48 (bs, 1H, NH,

6

D O exchangeable), 7.69-7.67 (d, 2H, H-4

2

and H-7_b_e_n_z_i_m_i_d_a_z_o_l_e, J = 8.1 Hz), 7.52-7.36 (m,

benzimidazole

4

2

H, ArH), 7.17-7.12 (m, 3H, ArH), 4.32-4.19 (m, 2H, NCH ), 4.05-3.95 (m, 1H, CH ), 3.10-

2 pyrr

+

.93 (m, 2H, COCH ); ESI-MS (m/z): 277.15 (M ), 278.15 (M +H); Anal. calcd. For C H N O:

2

17 15

3

C, 73.63; H, 5.45; N, 15.15. Found: C, 73.70; H, 5.51; N, 15.22.

.1.2.11. 4-(1H-benzo[d]imidazol-2-yl)-1-(2-methylphenyl)pyrrolidin-2-one (20)

4

-1

Off white solid; Yield: 72 %; M.P.: 220-221 ºC; IR (KBr, cm ): 3415 (N-H), 3189 (Ar C-H), 1695

1

(

C=O), 1605 (C=N), 1576 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.45 (bs, 1H, NH,

6

D O exchangeable), 7.69-7.51 (m, 4H, ArH), 7.47-7.31 (m, 4H, ArH), 4.32-4.20 (m, 2H, NCH ),

2

4

.07-3.97 (m, 1H, CH ), 3.10-2.89 (m, 2H, COCH ), 2.22 (s, 3H, CH ); ESI-MS (m/z): 291.17

pyrr 2 3

+

(

M ), 292.17 (M +H); Anal. calcd. For C H N O: C, 74.20; H, 5.88; N, 14.42. Found: C, 74.32;

18 17 3

H, 5.96; N, 14.55.

4

.1.2.12. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-methylphenyl)pyrrolidin-2-one (21)

-1

Off white solid; Yield: 75 %; M.P.: 218-219 ºC; IR (KBr, cm ): 3395 (N-H), 3177 (Ar C-H), 1670

1

(

C=O), 1587 (C=N), 1572 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.19 (bs, 1H, NH,

6

D O exchangeable), 7.57-7.51 (m, 4H, ArH), 7.20-7.15 (m, 4H, ArH), 4.29-4.16 (m, 2H, NCH ),

2

4

.06-3.95 (m, 1H, CH ), 3.08-2.92 (m, 2H, COCH ), 2.28 (s, 3H, CH ); ESI-MS (m/z): 291.17

pyrr 2 3

+

(

M ), 292.17 (M +H); Anal. calcd. For C H N O: C, 74.20; H, 5.88; N, 14.42. Found: C, 74.35;

18 17 3

H, 5.95; N, 14.57.

4

.1.2.13. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-chlorophenyl)pyrrolidin-2-one (22)

-1

Off white solid; Yield: 72 %; M.P.: 212-213 ºC; IR (KBr, cm ): 3405 (N-H), 3117 (Ar C-H), 1683

1

(

C=O), 1588 (C=N), 1552 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.38 (bs, 1H, NH,

6

D O exchangeable), 7.72-7.69 (d, 2H, H-4

2

and H-7_b_e_n_z_i_m_i_d_a_z_o_l_e, J = 9 Hz), 7.52-7.34 (m,

benzimidazole

4

2

H, ArH), 7.16-7.13 (m, 2H, ArH), 4.30-4.16 (m, 2H, NCH ), 4.05-3.95 (m, 1H, CH ), 3.09-

2 pyrr

+

.93 (m, 2H, COCH ); ESI-MS (m/z): 311.12 (M ), 313.12 (M +2); Anal. calcd. For

2

C H ClN O: C, 65.49; H, 4.53; N, 13.48. Found: C, 65.58; H, 4.65; N, 13.56.

1

7

14

3

4

.1.2.14. 4-(1H-benzo[d]imidazol-2-yl)-1-(3-chloro-4-fluorophenyl)pyrrolidin-2-one (23)

-1

Off white solid; Yield: 74 %; M.P.: 253-254 ºC; IR (KBr, cm ): 3380 (N-H), 3121 (Ar C-H), 1678

1

(

C=O), 1590 (C=N), 1542 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.49 (bs, 1H, NH,

6

D O exchangeable), 8.01-7.98 (m, 1H, ArH), 7.66-7.41 (m, 4H, ArH), 7.17-7.14 (m, 2H, ArH),

2

4

.32-4.19 (m, 2H, NCH ), 4.05-3.95 (m, 1H, CH ), 3.11-2.94 (m, 2H, COCH ); ESI-MS (m/z):

2

pyrr

2

+

3

29.10 (M ), 331.10 (M +2); Anal. calcd. For C H ClFN O: C, 61.92; H, 3.97; N, 12.74. Found:

1

7

13

3

C, 62.18; H, 4.09; N, 12.85.

4

.1.2.15. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-hydroxyphenyl)pyrrolidin-2-one (24)

-1

Off white solid; Yield: 68 %; M.P.: 228-229 ºC; IR (KBr, cm ): 3421(O-H), 3106 (Ar C-H), 1679

1

(

C=O), 1595 (C=N), 1525 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.37 (bs, 1H, NH,

6

D O exchangeable), 7.73-7.58 (m, 4H, ArH), 7.44-7.15 (m, 4H, ArH), 5.63 (bs, 1H, OH, D O

2

exchangeable), 4.33-4.20 (m, 2H, NCH ), 4.06-3.97 (m, 1H, CH ), 3.10-2.89 (m, 2H, COCH );

2

pyrr

2

+

ESI-MS (m/z): 293.14 (M ), 294.14 (M +H); Anal. calcd. For C H N O : C, 69.61; H, 5.15; N,

1

7

15

3

2

1

4.33. Found: C, 69.80; H, 5.22; N, 14.49.

.1.2.16. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphenyl)pyrrolidin-2-one (25)

4

-1

Off white solid; Yield: 70 %; M.P.: 224-225 ºC; IR (KBr, cm ): 3380 (N-H), 3172 (Ar C-H), 1692

1

(

C=O), 1603 (C=N), 1575 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.40 (bs, 1H, NH,

6

D O exchangeable), 7.77-7.54 (m, 4H, ArH), 7.43-7.13 (m, 4H, ArH), 4.31-4.17 (m, 2H, NCH ),

2

4

.07-3.98 (m, 1H, CH ), 3.72 (s, 3H, OCH ), 3.08-2.92 (m, 2H, COCH ); ESI-MS (m/z): 307.16

pyrr 3 2

+

(

M ), 308.17 (M +H); Anal. calcd. For C H N O : C, 70.34; H, 5.58; N, 13.67. Found: C, 70.42;

18 17 3 2

H, 5.65; N, 13.74.

4

.1.2.17. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-nitrophenyl)pyrrolidin-2-one (26)

-1

Yellow solid; Yield: 60 %; M.P.: 271-272 ºC; IR (KBr, cm ): 3120 (Ar C-H), 1670 (C=O), 1587

1

(

C=N), 1531 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.35 (bs, 1H, NH, D O

6

2

exchangeable), 7.81-7.55 (m, 4H, Ar-H), 7.41-7.13 (m, 4H, ArH), 4.31-4.17 (m, 2H, NCH ), 4.06-

2

+

3

.96 (m, 1H, CH ), 3.10-2.93 (m, 2H, COCH ); ESI-MS (m/z): 322.13 (M ), 323.13 (M +H);

pyrr 2

Anal. calcd. For C H N O : C, 63.35; H, 4.38; N, 17.38. Found: C, 63.47; H, 4.55; N, 17.51.

1

7

14

4

3

4

.1.2.18. 4-(1H-benzo[d]imidazol-2-yl)-1-(4-sulfamoylphenyl)pyrrolidin-2-one (27)

-1

Pale yellow solid; Yield: 62 %; M.P.: 233-234 ºC; IR (KBr, cm ): 3122 (Ar C-H), 1690 (C=O),

1

598 (C=N), 1541 (C=C); H NMR (DMSO-d , 300 MHz) δ (ppm): 12.41 (bs, 1H, NH, D O

6

2

exchangeable), 7.61-7.49 (m, 4H, Ar-H), 7.34-7.19 (m, 4H, ArH), 5.78 (s, 2H, SO NH , D O

2

exchangeable), 4.37-4.22 (m, 2H, NCH ), 4.19-3.14 (m, 1H, CH ), 3.19-2.87 (m, 2H, COCH );

2

pyrr

2

+

ESI-MS (m/z): 356.10 (M ), 357.11 (M +H); Anal. calcd. For C H N O S: C, 57.29; H, 4.53; N,

1

7

16

4

3

1

5.72. Found: C, 57.38; H, 4.64; N, 15.88.

4

.2. In vitro hMAGL enzyme inhibition assay

The hMAGL enzyme inhibition assay was done by the method reported previously and as per the

manufacturer's instructions supplied with Cayman’s assay kit, USA [33,36]. In short, six

concentration of test compounds (10-27) and standard (CAY10499) in DMSO as solvent, ranging

1

00 μM to 1 nM, were prepared by serial dilution (tenfold) method. The samples were tested for

their potential to inhibit MAGL in triplicate, using 180 μl, in 96-well plates, as provided with the

assay kit. Tris-HCl (10 mM, pH 7.2), containing EDTA (1mM) and BSA (0.1 mg/ml) was used as

assay buffer and for the dilution of hMAGL protein. 4-nitrophenylacetate (4-NPA) was used as

MAGL substrate, which gives yellow coloured product (4-nitrophenol) upon MAGL action.

Average absorbance of background well containing DMSO and buffer (BW), 100% initial activity

wells (IA), test samples and standard sample were recorded at 405 nm using microplate reader

(PerkinElmer®). The average absorbance of BW was then subtracted from the average absorbance

of IA, test samples and standard samples. The percentage MAGL activity for the test and standard

samples were determined by the formula: % MAGL activity = Test / IA × 100. GraphPad Prism

(

ver. 6.01) was used for the calculation of IC values (Table 1).

50

4

.3. In vitro hFAAH enzyme inhibition assay

The hFAAH enzyme inhibition assay was done by the method reported previously and as per the

manufacturer's instructions supplied with Cayman’s assay kit, USA [37]. Six concentrations of test

compounds (22, 23 and 25) and standard (URB597) in DMSO as solvent, ranging 100 μM to 1

nM, were prepared by serial dilution (tenfold) method. The samples were tested for their potential

to inhibit FAAH in triplicate, using 180 μl, in 96-well plates, as provided with the assay kit. Tris-

HCl (125 mM, pH 9), containing EDTA (1mM) and BSA (0.1 mg/ml) was used as assay buffer

and for the dilution of hFAAH protein. AMC arachidonoylamide (AMCAA) was used as FAAH

substrate. Average fluorescence of BW (background well containing DMSO and buffer), 100%

initial activity wells (IA), test samples and standard sample were recorded at 340 nm (excitation

wavelength) and 460 nm (emission wavelength) using microplate reader (PerkinElmer®). The

average fluorescence of BW was then subtracted from the average absorbance of IA, test samples

and standard samples. The percentage FAAH activity for the test and standard samples were

determined by the formula: % FAAH activity = Test / IA × 100. GraphPad Prism (ver. 6.01) was

used for the calculation of IC values (Table 1).

5

0

4

.4. Computational study

Computations were performed on an Intel Core i7, 4.90 GHz processor with 16 GB memory and 2

GB graphics running with Windows 10 operating system. Maestro10.1 graphical user interface

(GUI) of Schrödinger was utilized for computations.

4

.4.1. Molecular docking

Glide 5.9 implemented in Maestro 9.4 (GUI of Schrodinger) was used for extra precision (XP)

docking of potential ligand (compound 25). The X-ray crystal structure of hMAGL (PDB ID:

5

ZUN, Resolution-1.35 Å) was retrieved from Protein Data Bank (PDB) and utilized for

molecular docking study [29]. Protein Preparation Wizard in Maestro was used for the protein

structure preparation like deletion of water molecules, assignment of bond orders, the inclusion of

hydrogen atoms, and treatment of formal charges. An exhaustive sampling option was utilized for

the optimization of the hydrogen bonding network. The energy of the protein structure was

minimized to an RMSD of 0.3 Å by using the Impref module (Impact 5.9) with the OPLS_2005

force field. Glide scoring grids (docking grid box of 20×20×20 Å) were generated by defining the

active binding site residues in the protein structure. LigPrep 2.6 with Epik 2.4, were used to

expand protonation, and tautomeric states of ligand (compound 25) at pH 7.0 ± 2.0 and then

energy was minimized using the OPLS_2005 force field. The docking simulation of the prepared

ligand was performed by Glide XP docking [39].

4

.4.2. Physicochemical and pharmacokinetic properties

The QikProp 3.6 of Schrodinger is used for the calculation of physicochemical and

pharmacokinetic properties of the selected compounds (compound 22, 23 and 25). Structures of

the compounds were neutralised prior to the generation of QikProp (QP) properties. Thirty-five

physicochemical and pharmacokinetic properties were predicted and compared with the qualifying

limits as proposed for orally active CNS drug discovery [40].

4

.5. Antinociceptive activity

Animals were obtained from Central Animal House Facility, Jamia Hamdard (Hamdard

University), New Delhi-110062 (Registration Number: 173/CPCSEA; 28 Jan., 2000). All the

experimental procedures were carried out with the permission of Institutional Animal Ethics

Committee (Proposal No. 1048). The rats were housed under standard laboratory conditions

(Temperature: 22±3ºC; RH: 60±5% and 12/12 hours light-dark cycle) in polypropylene cages.

Animals were provided free access to standard diet pellets (Amrut rat feed, Mfd. by Nav

Maharastra Chakan Oil Mills Ltd., New Delhi, India) and water ad libitum. The animals were

selected randomly, marked and housed in their cages for 7 days prior to dosing to allow for

acclimatisation to the laboratory conditions.

4

.5.1. Formalin-induced nociception test

Formalin test was performed by the method reported in literature [41,42]. Male Wistar rats (180-

00 g, at the time of dosing) were taken in groups of ten animals each. Animals were administered

vehicle (0.5% methylcellulose) or test compound 25 (5, 10, 30 and 50 mg/kg; p.o; suspended in

.5% methylcellulose), 4 hours before an intraplantar injection of formalin (2.5%, 50 µl) into the

2

0

dorsal surface of the right hind paw. The reference drug, GBP (100 mg/kg, i.p, dissolved in 0.9%

normal saline), was injected 30 minutes before formalin injection. The duration of time (in

seconds) spent licking and biting the injected paw, was recorded in the time interval of 0-5

minutes (phase-I, acute chemical pain consist of intense licking and biting) and 10-30 minutes

(

phase-II, state of central sensitization, which involves periods of licking and biting), after

formalin injection.

.6. Statistical analysis

4

GraphPad Prism (ver. 6.01) was used for statistical analysis of data. Treatment effects were

compared with the control by using analysis of variance (ANOVA) followed by Dunnett’s test.

Results are expressed as mean±SEM.

Conflict of Interest

The authors have declared no conflict of interest.

Acknowledgments

The authors would like to thank the Deanship of Scientific Research at Prince Sattam Bin

Abdulaziz University for providing financial support for this project (grant no. 2019/03/11064).

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding

author.

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Table 1: In vitro hMAGL and hFAAH inhibition assay of the synthesized compounds (10-27).

Compound

X

S

R

hMAGL IC50 hFAAH IC50

1

0

1

2

H

>100 μM

88 μM

ND

2-CH

4-CH

3

>100 μM

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

S

4-Cl

60 μM

48 μM

>100 μM

72 μM

>100 μM

680 nM

255 nM

290 nM

8.6 nM

8.0 nM

22 μM

9.4 nM

760 nM

45 μM

415 nM

--

ND

S

3-Cl,4-F

4-OH

ND

S

ND

S

4-OCH

4-NO

3

ND

S

2

ND

S

4-SO

H

2

NH

2

ND

NH

ND

NH 2-CH

NH 4-CH

NH 4-Cl

3

ND

3

ND

35 μM

24 μM

ND

NH 3-Cl,4-F

NH 4-OH

NH 4-OCH

NH 4-NO

NH 4-SO

3

52 μM

ND

2

NH

2

ND

CAY10499 --

URB597 --

--

5 nM

ND: Not Determined; CAY10499 and URB597 (standard control); IC50 values were calculated from GraphPad Prism

ver. 6.01).

(

Figure 1: MAGL substrate (2-AG) and representative potent MAGL inhibitors.

Figure 2: Design of novel MAGL inhibitors. (A) MAGL Substrate, 2-AG; (B) Binding pattern of

MAGL inhibitors (ZINC12863377 AND ZINC24092691); (C) Designed compounds (10-27).

Figure 3: (Scheme 1): Synthesis of the intermediates (1-9) and target compounds (10-27).

Figure 4: 3D and 2D representation of MAGL-compound 25 binding pattern, obtained after glide

XP docking.

Figure 5: Formalin induced nociception test; a single dose of the test compounds (5, 10, 30 and 50

mg/kg, p.o, suspended in 0.5% methylcellulose) was administered 4 hours before formalin

injection (50 µl, 2.5%). Reference drug, gabapentin (100 mg/kg, i.p, dissolved in 0.9 % normal

saline) was injected 30 minutes before formalin injection. Total paw licking and biting duration,

Phase-I (white bar, 0-5 min.) and Phase-II (black bar, 10-30 min.) was recorded as a measure of

pain behaviour. Data is represented as mean±SEM from a group of 10 animals. *p<0.05,

*

*p<0.01, p>0.05 (NS, non-significant) vs. control (vehicle). GBP: gabapentin; T5, T10, T30, T50

are the dose concentration of tested compound-25.

Article Doi

Pyrrolidin-2-one linked benzofused heterocycles as novel small molecule monoacylglycerol lipase inhibitors and antinociceptive agents

DOI: 10.1111/cbdd.13751

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Article abstract of DOI:10.1111/cbdd.13751

Full text of DOI:10.1111/cbdd.13751

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