Analgesic and anticancer activity of benzoxazole clubbed 2-pyrrolidinones as novel inhibitors of monoacylglycerol lipase

Article abstract of DOI:10.3390/molecules26082389

Ten benzoxazole clubbed 2-pyrrolidinones (11–20) as human monoacylglycerol lipase inhibitors were designed on the criteria fulfilling the structural requirements and on the basis of previously reported inhibitors. The designed, synthesized, and characterized compounds (11–20) were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Compounds 19 (4-NO₂ derivative) and 20 (4-SO₂ NH₂ derivative), with an IC₅₀ value of 8.4 and 7.6 nM, were found most active, respectively. Both of them showed micromolar potency (IC₅₀ value above 50 μM) against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of 2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three hydrogen bonds (~2 ?) with Ala51, Met123, and Ser122, the amino acid residues responsible for the catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test, compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They significantly demonstrated the reduction in pain response, having better potency than the positive control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA, for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth inhibition (% GI), respectively.

Full text of DOI:10.3390/molecules26082389

molecules
Article
Analgesic and Anticancer Activity of Benzoxazole Clubbed
2-Pyrrolidinones as Novel Inhibitors of
Monoacylglycerol Lipase
Obaid Afzal 1,
*
, Abdulmalik Saleh Alfawaz Altamimi 1,*, Mir Mohammad Shahroz 2, Hemant Kumar Sharma 2,
Yassine Riadi 1 and Md Quamrul Hassan 3
1
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
Al Kharj 11942, Saudi Arabia; y.riadi@psau.edu.sa
2
Department of Pharmaceutical Chemistry, College of Pharmacy, Sri Satya Sai University of Technology and
Medical Sciences, Sehore 466001, Madhya Pradesh, India; mirshahroz@gmail.com (M.M.S.);
hkspharma@rediffmail.com (H.K.S.)
Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard,
New Delhi 110062, India; quamrulhassan309@gmail.com
3
*
Correspondence: o.akram@psau.edu.sa (O.A.); as.altamimi@psau.edu.sa (A.S.A.A.);
Tel.: +96-611-588-6094 (O.A.); +96-611-588-6072 (A.S.A.A.)
Abstract: Ten benzoxazole clubbed 2-pyrrolidinones (11–20) as human monoacylglycerol lipase
inhibitors were designed on the criteria fulfilling the structural requirements and on the basis of
previously reported inhibitors. The designed, synthesized, and characterized compounds (11–20
were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Com-
pounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative), with an IC50 value of 8.4 and 7.6 nM,
were found most active, respectively. Both of them showed micromolar potency (IC50 value above
)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Afzal, O.; Altamimi, A.S.A.;
Shahroz, M.M.; Sharma, H.K.; Riadi,
Y.; Hassan, M.Q. Analgesic and
Anticancer Activity of Benzoxazole
Clubbed 2-Pyrrolidinones as Novel
Inhibitors of Monoacylglycerol
Lipase. Molecules 2021, 26, 2389.
https://doi.org/10.3390/
50 µM) against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective
inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of
2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three
hydrogen bonds (~2 Å) with Ala51, Met123, and Ser122, the amino acid residues responsible for the
catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties
of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per
the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test,
compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They
significantly demonstrated the reduction in pain response, having better potency than the positive
control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA,
for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to
have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth
inhibition (% GI), respectively.
molecules26082389
Academic Editor: Laura Micheli
Received: 20 March 2021
Accepted: 14 April 2021
Published: 20 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: analgesic; anticancer; pyrrolidin-2-one; benzoxazole; MAGL inhibitors; molecular docking
1. Introduction
Endocannabinoids (endogenous ligands), cannabinoid (CB) receptors, and proteins
for their biological synthesis and degradation constitute the endocannabinoid system
(ECS) [1]. Endocannabinoids are biosynthesized from the membrane phospholipids [2].
Endocannabinoid, N-arachidonoyl ethanolamine (AEA, Anandamide) functions as partial
agonist on CB1 and CB2 receptors. It has low affinity for CB2 and moderate affinity for CB1.
Endocannabinoids, 2-arachidonoylglycerol (2-AG) function as full agonist and have mod-
erate affinity for both the receptors. Interestingly, 2-AG is the major endocannabinoid and
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
is found to be approximately 170-fold higher in concentration than AEA, in the brain [
3].
AEA and 2-AG hydrolysis and degradation are facilitated by fatty acid amide hydrolase
Molecules 2021, 26, 2389. https://doi.org/10.3390/molecules26082389
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2389
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(FAAH) and monoacylglycerol lipase (MAGL) enzymes, correspondingly [
1]. MAGL (an
α
/β
-hydrolase) hydrolyzes 2-AG into glycerol and free fatty acid (FFA), by the action of
the catalytic triad organized with Ser122, Asp239 and His269 amino acids in the active
site of the enzyme [ ]. MAGL hydrolyzes approximately 85% of total 2-AG in the CNS [
and generates arachidonic acid (AA), giving rise to neuro-inflammatory PGE2 and PGD2
prostaglandins [ ]. MAGL inhibitors ameliorates neuropathic pain by increasing 2-AG and
decreasing neuro-inflammatory prostaglandins in the CNS (Figure 1) ]. In addition, 2-AG
demonstrated analgesic activity by acting on CB1 receptors in the CNS and periphery [ 11].
4
5]
6
[7
8
–
It is reported that JJKK-048 (MAGL, IC50 363 pM) exhibited analgesic activity in tail immer-
sion and writhing test [12]. MAGL inhibition has shown significant neuroprotective and
anti-inflammatory potential in Parkinson’s and Alzheimer’s disease [13,14]. PF-06795071
(IC50 3 nM), a MAGL inhibitor, is reported to have considerable anti-neuroinflammatory
potential [15]. Another MAGL inhibitor, ABX-1431 (IC50 14 nM) is under clinical trials
for broad range of CNS disorders like Tourette syndrome [16]. Many more research find-
ings encouraged that the inhibitors of MAGL has therapeutic potentials in pain and CNS
disorders [17–20].
Figure 1. Role of MAGL inhibitors in the alleviation of pain and cancer. PG: prostaglandins; PA: phosphatidic acid; LPA:
lysophosphatidic acid; and S1P: sphingosine-1-phosphate.
The governing importance of MAGL in abnormal lipolysis in cancer has been demon-
strated [21]. MAGL was originally known for its lipolytic action on monoacylglycerols from
stored triacylglycerols into glycerol and free fatty acids (FFA) [22]. Cancer cells utilizes this
lipolytic pathways for their hastened proliferation [23]. The cancer-supporting action of
MAGL is due to elevated FFA levels. This MAGL-FFA pathway promotes in vivo tumor
growth by increasing FFA-derived oncogenic signaling lipids (PA, LPA, S1P, and PGE2) [18].
These protumorigenic lipid mediators encourage tumor growth, angiogenesis, and metas-
tasis in cancer (Figure 1) [24]. MAGL is reported to expressed vastly in aggressive type of
cancer cells and is associated with pathogenesis, proliferation, and in vivo tumor growth.
MAGL inhibition disrupts cancer cell proliferation, growth and metastasis [25
–27]. The
anticancer effect of MAGL inhibition in prostate cancer was totally abolished by cotreat-

Molecules 2021, 26, 2389
3 of 18
ment with SR141716 (rimonabant; CB1 receptor antagonist) and fatty acids, signifying that
amplified endocannabinoid action and reduced stock of FFA from MAGL inhibition is the
reason behind antitumor effect [26].
MAGL inhibitors identified till date includes URB602 (IC50 28
µM) [28], CAY10499
(IC50 144 nM) [29], SPB01403 (IC50 31 M) [30], JZL184 (IC50 8 nM) [31], SAR629 (IC50
µ
1.1 nM) [32], KML29 (IC50 3.6 nM) [33], ML30 (IC50 0.54 nM) [34], JJKK-048 (IC50 363 pM) [35],
ABX1431 (IC50 14 nM) [16], R(3t) (IC50 3.6 nM) [36], PF-06795071 (IC50 3 nM) [15], a ben-
zoylpiperidine derivative (IC50 80 nM) [37], and a benzisothiazolinone derivative (IC50
34.1 nM) [38]. JJKK-048 and KML29 both were reported to be highly selective MAGL
inhibitors, and their selectivity is more than 10,000-fold over FAAH. [33
crystal structures of human MAGL enzyme were elucidated by X-ray crystallography, and
are available on protein data bank [32 36 39 40]. A comprehensive description of MAGL
,35]. In addition, 3D
,
, ,
crystal structures and inhibitors were reviewed [41]. Our group has also identified nanomo-
lar MAGL inhibitors, viz. ZINC24092691 (IC50 10 nM), ZINC12863377 (IC50 39 nM), a
ZINC24092691 analogue (IC50 6.5 nM), a thiazole-5-carboxylate derivative (IC50 37 nM), and
a pyrrolidin-2-one linked benzimidazole derivative (compound 25; IC50 9.4 nM) [42–45].
In continuation of our work on MAGL inhibitors, we have designed novel 2-pyrrolidinone
linked benzoxazole derivatives and screened them for analgesic and anticancer effects.
2. Results
The binding pattern of pyrrolidin-2-one derivatives (ZINC12863377, compound 25
and compound R-3t), and the basic structural requirement for MAGL inhibitors was kept
in mind to design novel pyrrolidin-2-one linked benzoxazole derivatives (Figure 2). The
route of synthesis of compounds (11–20) is presented in Figure 3.
2.1. Chemistry
Intermediate compound 1, (1-Benzyl-5-oxopyrrolidine-3-carboxylic acid), was success-
fully prepared by fusion of benzylamine and methylidenesuccinic acid in water, while
compounds 2–10 were synthesized as reported by us in our previous publication [45].
The fusion of synthesized 1-substituted-5-oxopyrrolidine-3-carboxylic acids (1–10) with
2-aminophenol was done by the procedure reported earlier with some minor modifica-
tions [46]. The fusion of acids (1–10) and 2-aminophenol was carried out by the use of
polyphosphoric acid, giving better yields (57–70%) and purity of benzoxazole deriva-
tives (11–20).
The prototype intermediate compound 1, (1-Benzyl-5-oxopyrrolidine-3-carboxylic acid),
revealed typical pea−k1s at 1627 cm−1 (carbonyl of acid), 1734 cm−1 (carbonyl of pyrrolidi-
none), and 3241 cm (O-H of acid) in IR spectrum. In 1H-NMR spectrum of compound
1,
two methyl protons were found resonating as singlet at
at 2.62–2.76 indicated two COCH2 protons, and at 4.29–4.44 they indicated two NCH2
and one CH proton of 2-pyrrolidinone ring. Five aromatic protons of the benzyl ring
were found resonating at 7.20–7.36 as a multiplet. The typical singlet was assigned to
COOH proton at 11.35 and was found to be D2O exchangeable. Final prototype com-
δ 3.62. The multiplets resonating
δ
δ
δ
δ
pound 11 (4-(Benzo[d]oxazol-2-yl)-1-benzylpyrrolidin-2-one) exhibited specific IR bands
at 1621 cm−1 (C=N of benzoxazole) and 1703 cm−1 (C=O of pyrrolidin-2-one). The two
protons of CH2 of benzyl appeared at
the 2-pyrrolidinone ring was assigned at
at 3.13–3.18 and 3.81–3.86 were ascribed to one CH proton and two NCH2 protons of the
2-pyrrolidinone ring, correspondingly. Six aromatic protons were found resonating at
7.38–7.58 as a multiplet. The doublet resonating at 7.73–7.78 was assigned to the aromatic
δ
3.95–4.01 as a multiplet. Two COCH2 protons of
δ
2.60–2.77 as a multiplet. The multiplets located
δ
δ
δ
protons of benzoxazole. The other two protons of benzoxazole were found resonating at
δ
7.94–7.96 as doublet. The M+ (molecular ion) peak of compound 11 was found at 292.14,
validating its successful synthesis.

Molecules 2021, 26, 2389
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Figure 2. Design of novel pyrrolidin-2-one linked benzoxazole MAGL inhibitors. (A) Binding pattern of reported pyrrolidin-
2-one MAGL inhibitors; ZINC12863377 [42], compound 25 [45] and (R)-3t [36]. (B) Designed compounds (11–20).
Figure 3. Scheme for synthesis of the intermediates (
(b) Fusion at 130–140 C (Method 2); (c) 2-aminophenol, polyphosphoric acid, 150–160 C, sodium carbonate.
1
–
10) and target compounds (11
–
20). (
a
) Reflux in water (Method 1);
◦
◦

Molecules 2021, 26, 2389
5 of 18
2.2. Human MAGL Assay
The assay was executed by Cayman’s assay kit by the reported procedure [29]. All the
ten compounds (11 20) were screened for hMAGL inhibitory potential. The substituted
phenyl derivatives (13 20) were established to reduce the MAGL activity at 100 M con-
–
–
µ
centration below 50%. Compound 19 (4-NO2 derivative) and compound 20 (4-SO2NH2
derivative) were the most potent, with an IC50 of 8.4 and 7.6 nM, correspondingly. The
structure–function relationship of benzoxazole derivatives is displayed hMAGL inhibitory
activity as follows: 4-SO2NH2 > 4-NO2 > 3-Cl,4-F > 4-OCH3 > 4-Cl > 4-OH > 4-CH3 > 2-
CH3 > phenyl/benzyl. The IC50 of standard controls, selective MAGL inhibitors, CAY10499
(IC50 = 415 nM), and for JZL184 (IC50 = 10 nM) were comparable to the reported values [29].
The outcomes of the experiments are presented in Table 1.
Table 1. In vitro hMAGL and hFAAH inhibition assay of the synthesized compounds (11–20).
Compound
11
R
hMAGL; IC50
>100 µM
hFAAH; IC50
ND
12
13
>100 µM
ND
ND
85 ± 1.5 µM
14
15
72 ± 2.1 µM
65 ± 3.2 nM
ND
28 ± 1.8 µM
16
34 ± 1.7 nM
25 ± 2.3 µM
17
18
62 ± 2.7 µM
42 ± 1.5 nM
ND
37 ± 2.2 µM
19
20
8.4 ± 1.9 nM
7.6 ± 0.8 nM
55 ± 2.7 µM
68 ± 2.1 µM
CAY10499
JZL184
URB597
–
–
–
415 ± 3.2 nM
10 ± 0.8 nM
–
–
–
5 ± 0.6 nM
ND: Not determined; CAY10499, JZL184 and URB597 (standard control); IC50 values were calculated from
GraphPad Prism (ver. 8.0.2). Results are expressed as mean ± SEM (n = 3).

Molecules 2021, 26, 2389
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2.3. Human FAAH Assay
Derivatives having IC50 in nanomolar range (compound 15
,
16,
18,
19, and 20) were
nominated for further screening against FAAH, an allied hydrolase of MAGL [47]. They
displayed micromolar potency, with an IC50 value ranging from 25 to 68 M against FAAH.
The benzoxazole derivatives having 4-NO2 phenyl (19) and 4-SO2NH2 phenyl (20), with an
FAAH IC50 value greater than 50 M, were considered selective MAGL inhibitors. The IC50
µ
µ
of standard control, URB597 (selective FAAH inhibitor), was 5 nM, equivalent to the value
reported (IC50 = 4.6 nM) [48]. The outcomes of the experiments are presented in Table 1.
2.4. Molecular Docking Study
Most active and selective compounds identified by MAGL and FAAH inhibition
assay (19 and 20), were docked at the catalytic center of MAGL, in order to get an insight
of their binding pattern, with the help of XP Glide docking using Maestro (Schrodinger).
Compounds 19 and 20, showed comparable docking scores of
−
9.87 and
−
9.83, respectively.
The binding of compounds 19 and 20 in the active site of MAGL revealed that the carbonyl
group of pyrrolidinone is located exactly in the oxyanion hole and stabilized by three
hydrogen bonds (~2Å) with alanine 51, serine 122, and methionine 123. Serine 122 is one of
the critical amino acid residues of the catalytic triad of MAGL. The benzoxazole moiety
is found to be positioned in the amphiphilic pouch, having π-π stacking contact with the
amino acid Tyr194. The 4-NO2 (19) and 4-SO2NH2 (20) phenyl ring of the ligands were
involved in hydrophobic (van der Waals) attractions with the amino acids, leucine 148,
213, and 241. In addition, the 3D and 2D binding pattern of compounds 19 and 20 in the
catalytic location of MAGL is depicted in Figure 4.
Figure 4. Glide XP docking; 3D and 2D representation of the binding pattern of compound 19 (A), and 20 (B) in the catalytic
site of MAGL. The picture (2D and 3D) for the docked complexes were obtained from Discovery studio visualizer 2020. The
hydrophobicity was calculated from the default option available.

Molecules 2021, 26, 2389
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2.5. Pharmacokinetic and Physicochemical Characteristics
To investigate the potential of the identified derivatives (19 and 20) to cross the se-
lectively permeable membranes of hematoencephalic barrier (BBB), to develop orally
active CNS drugs, their pharmacokinetic and physicochemical features were computed by
QikProp (ADMET predictor) of Schrodinger. Guidelines, concerning the validation and op-
timization of orally active CNS compounds, were developed by Ghose et. al., by analyzing
35 characteristic features of orally bioavailable 317 CNS and 626 non-CNS drugs [49]. This
guideline states that in order to design high-quality CNS drugs, the molecule must qualify
by the following parameters: TPSA less than 76 Å2 (ideally 25–60 Å2), number of N atoms
between 1–2, comprising 1 aliphatic amine, 2–4 side chains on/outside rings, number of
polar H atoms < 3 (ideally 0–1), SASA 460–580 Å2, molecular volume 740–970 Å3, and must
have +ve QikProp CNS property. Remarkably, most of the properties of compounds 19 and
20, computed by QikProp, were found to be in the qualifying range as per the proposed
guideline (Table 2). The properties of compound 19 were found to be within the qualifying
range except dipole moment. For compound 20, 5 out of 35 properties is just slightly above
the upper qualifying limit. Most importantly, qualifying limits for CNS active drugs in
terms of TPSA is from 3.8 to 109, and the calculated TPSA for compound 20 was found to be
119.08. Therefore, the designing of more potent MAGL inhibitors having physicochemical
and pharmacokinetic properties within the preferred CNS limits is required.
Table 2. Physicochemical and pharmacokinetic properties of compounds 19 and 20, predicted by QikProp, Schrodinger, for
CNS activity.
Range of Properties in CNS Drugs
Property
Description
Compound 19
Compound 20
S. No.
QL
PL
PU
QU
drug likeness penalty; the
higher the value, the less
drug-like the molecule
no. of basic amines
no. of amidines groups
no. of carboxylic acid groups
no. of amides groups
no. of rotatable bonds (without
CX3, alkene, amide, small ring)
a qualitative CNS
1
#stars
0
0
0
3
0
0
2
3
4
5
#amine
#amidine
#acid
0
0
0
0
1
0
0
0
1
0
0
0
2
0
0
1
0
0
0
0
0
0
0
0
#amide
6
#rotor
0
3
6
8
1
2
7
CNS
−2
0
1
2
−2
−2
activity parameter
8
9
dipole
SASA
computed dipole moment
solvent accessible surface area
SASA on saturated carbon and
attached hydrogen
0.67
348
1.1
487
3.9
620
8.9
798
9.47
584.85
10.22
617.51
10
FOSA
16
178
314
464
91.99
91.96
SASA on N, O, and H attached
to heteroatoms
11
12
13
14
FISA
PISA
0
0
0
64
292
0
176
343
167.04
325.81
0
210.06
313.57
1.94
π component of SASA
weakly polar component of the
SASA (halogens, P, and S)
solvent accessible volume (Å3)
estimated no. of hydrogen
bonds that would be donated to
the solvent water
160
0
WPSA
volume
0
126
492
830
1104
1388
1002.52
1065.46
15
donorHB
0
0
1
3
0
2
estimated no. of hydrogen
bonds that would be accepted
from the solvent water
a globularity descriptor
(1 for a sphere)
16
17
accptHB
glob
1
2.8
5.2
8.3
6
9.5
0.77
0.82
0.88
0.93
0.82
0.81
predicted polarizability (Å3)
octanol−water logP
18
19
20
QPpolrz
QPlogPo/w
QPlogS
14
28
2.5
−4.6
38
4.7
−2.5
49
6.0
36.43
2.13
−3.99
38.19
0.90
−3.99
−0.16
solubility in log(moles/liter)
log of
−6.5
−
0.42
21
CIQPlogS
conformation-independent
solubility
−6.3
−4.2
−2.3
0.36
−4.16
−3.77
apparent Caco-2 cell
22
23
QPPCaco
QPlogBB
0
0
810
3269
1.2
258.09
100.92
permeability
brain/blood
partition coefficient
−1.2
−0.06
0.75
−1.12
−1.65

Molecules 2021, 26, 2389
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Table 2. Cont.
Range of Properties in CNS Drugs
Property
Description
Compound 19
Compound 20
S. No.
QL
PL
PU
QU
predicted apparent MDCK cell
permeability (nm/s)
prediction of binding to human
serum albumin
24
QPPMDCK
QPlogKhsa
0
0
634
5899
114.43
42.50
25
26
27
−1
2
0.04
3
0.78
3
1.04
3
−0.11
3
−0.34
3
HumanOralAbsorption
PercentHuman
Human oral absorption
Percent of human
61
95
100
100
82.63
68.11
OralAbsorption
oral absorption
van der Waals surface area of
polar nitrogen and
28
TPSA
3.8
12
54
109
98.65
119.08
oxygen atoms
29
30
#NandO
no. of N and O atoms
no. of violations of Lipinski’s
rule of five
1
0
2
0
4
0
7
1
7
0
7
0
RuleOfFive
no. of violations of Jorgensen’s
rule of three
31
32
33
RuleOfThree
#in34
0
0
5
0
0
0
0
1
0
0
0
0
0
no. of atoms in three- or
four-membered rings
no. of atoms in five- or
six-membered rings
no. of atoms not able to form
conjugation in
#in56
11
17
24
20
20
34
35
#noncon
0
8
0
4
10
30
3
3
nonaromatic rings
no. of non-H atoms
#nonHatm
19
25
24
25
Abbreviations: QL, qualifying lower limit; PL, preferred lower limit; QU, qualifying upper limit; PU, preferred upper limit. # QL, PL, QU
and PU values for CNS drug criteria were obtained from reference [49].
2.6. In Silico Absorption and Toxicity Profile
The selected compounds (19 and 20) were evaluated for their absorption and toxicity
profile by a bioinformatics tool admetSAR [50]. The results suggested that both the com-
pounds have high blood–brain barrier (BBB) penetration properties as well as high chance
of human intestinal absorption. In AMES test, compound 19 was found to be mutagenic,
while 20 was non-mutagenic. Carcinogenicity test revealed that both the compounds were
non-carcinogens. The LD50 values in rat were also evaluated, a compound with high value
is considered as less lethal. The LD50 for compounds 19 and 20 were found to be 2.30 and
2.21 mol/kg, respectively. Overall, compound 20 has better toxicity profile as compared to
compound 19 (Table 3).
Table 3. In-silico absorption and toxicity profile of compounds 19 and 20 obtained from admetSAR server [50].
Rat Acute Toxicity
(LD50, mol/kg)
Compound
BBB
HIA
HOB
AMES test
Carcinogenicity
19
20
Yes
Yes
Yes
Yes
Yes
Yes
Mutagenic
Non-carcinogen
Non-carcinogen
2.30
2.21
Non-Mutagenic
BBB: blood–brain barrier; HIA: human intestinal permeability; HOB: human oral bioavailability; AMES test is to detect a probable mutagen;
carcinogenicity estimates the cancer causing ability of a molecule; LD50: lethal dose which could kill 50% of the population of the organism
(rat) on which it is being tested.
2.7. Analgesic Activity
The formalin-induced analgesic test is an extensively acknowledged animal nocicep-
tion model. In order to evaluate both central and peripheral effects of the compound
(20), formalin-induced nociception model was selected for analgesic activity. The formalin
induced behavioral response comprises two typical phases, stage I and II. Stage I persists
up to five minutes after formalin injection and is characterized by acute pain with vigorous
licking and biting of the injected site. Stage I consists the action formalin on afferent
C-fiber nociceptors. While Stage II starts 10–30 min after formalin injection and persists till
60 min, characterized by periodic licking and biting of the injected site. Stage II imitates

Molecules 2021, 26, 2389
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the action of central sensitization of the spinal dorsal horn neurons [51
,
52]. Compound
20 was selected for analgesic activity due to its higher potency (IC50 7.6 nM). Compound
20 (suspensions prepared with 0.5% CMC) were administered per oral (p.o, in doses of 5,
10, 30, and 50 mg/kg body weight, 4 h prior to the formalin injection. Gabapentin (GBP),
(dissolved in 0.9% normal saline), was chosen as positive control (reference drug) and
administered intraperitoneal (i.p) in 100 mg/kg dose. GBP exhibited little analgesic effects
in Stage I (acute nociception), in comparison to the control (0.5% CMC). Though, in Stage II,
it displayed significant reduction of paw licking and biting, endorsing GBP central effects.
However, compound 20, reduced the pain response significantly both in acute (Stage I)
and late (Stage II) phases, in a dose-dependent manner. They significantly demonstrated
the reduction in pain response, having better potency than the positive control GBP at
30 mg/kg. The duration (in seconds) of paw licking and paw biting throughout Stage I
and II is provided in Figure 5.
Figure 5. Formalin-induced analgesic test; a dose of the test compound 20 (5, 10, 30, and 50 mg/kg,
p.o, suspended in 0.5% CMC) was administered 4 h before formalin injection (50 µL, 2.5%). Reference
drug, gabapentin (100 mg/kg, i.p, dissolved in 0.9% normal saline) was injected 30 min before
formalin injection. Total paw licking and biting duration, Stage I (white bar, 0–5 min) and stage II
(black bar, 10–30 min) was recorded as a measure of pain behavior. Data is represented as mean
±
SEM from a group of 10 animals. * p < 0.05, ** p < 0.01, p > 0.05 (NS, nonsignificant) vs. control
(vehicle). GBP: Gabapentin; D5, D10, D30, andD50 are the dose concentrations of test compound 20
.
2.8. Anticancer Activity
Compounds 19 and 20 were supplied to National Cancer Institute (USA), for sulforho-
damine B (SRB) assay and anticancer screening [53 54]. Single-dose (10 M) assay results
,
µ
for compounds 19 and 20 were provided as a mean of percent growth (% G) and growth
inhibition (% GI) against 60 cell lines of nine types of cancers and are tabulated in Table 4.
Derivatives 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer
activity towards SNB-75 cell line of CNS cancer, having % growth inhibition (% GI) of 35.49
and 31.88, respectively. Compound 20 showed 22.22 and 18.03% GI of HOP-92 and HOP-62
cell lines of non-small cell lung cancer, respectively. Both the compounds 19 and 20 were
also found active on UO-31 renal cancer cell line with % GI of 21.18 and 29.95, respectively.
Compound 19 showed % GI of 19.99 on T-47D, while compound 20 showed % GI of 19.89
on MDA-MB-231/ATCC cell lines of breast cancer.

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Table 4. In vitro anticancer screening of compound 19 and 20, against NCI60 cell lines at 10 µM
concentration.
Compound 19
(NSC: 778839)
Compound 20
(NSC: 778842)
Panel
Cell Line
% G
% GI
% G
% GI
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
93.58
100.09
98.41
93.64
101.28
88.92
6.42
−0.09
1.59
94.48
96.15
98.83
92.47
103.53
93.20
5.52
3.85
1.17
Leukemia
6.36
7.53
−1.28
−3.53
11.08
6.80
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
100.60
85.04
104.72
98.63
93.07
94.18
102.33
−0.60
14.96
−4.72
1.37
6.93
5.82
95.28
81.97
77.78
92.75
92.19
99.10
104.20
4.72
18.03
22.22
7.25
7.81
0.90
Non-Small Cell
Lung Cancer
−2.33
−4.20
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
103.79
102.05
102.14
98.10
99.25
105.43
102.52
−3.79
−2.05
−2.14
1.9
104.05
100.24
95.78
101.16
103.23
101.16
102.95
−4.05
−0.24
−4.22
−1.16
−3.23
−1.16
−2.95
12.57
6.29
12.42
2.98
31.88
4.79
Colon Cancer
CNS Cancer
0.75
−5.43
−2.52
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
91.85
98.21
95.59
99.29
64.51
100.97
8.15
1.79
4.41
0.71
35.49
−0.97
87.43
93.71
87.58
97.02
68.12
95.21
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
89.06
88.53
101.37
95.05
102.31
111.25
98.72
106.92
97.59
10.94
11.47
−1.37
4.95
−2.31
−11.25
1.28
92.99
93.29
98.58
100.55
111.54
101.79
98.82
110.78
92.78
7.01
6.71
1.42
−0.55
−11.54
−1.79
−1.18
−10.78
7.22
Melanoma
−6.92
2.41
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
104.14
98.56
106.27
98.30
−4.14
1.44
101.63
98.53
99.47
92.08
97.49
−1.63
1.47
0.53
7.92
2.51
−6.27
Ovarian
Cancer
1.70
101.95
−1.95
1.63
98.37
88.34
101.45
94.77
−1.45
5.23
SK-OV-3
11.66
786-0
A498
ACHN
CAKI-1
SN12C
TK-10
UO-31
104.06
113.46
91.70
97.19
97.21
110.14
78.82
−4.06
−13.46
8.3
2.81
2.79
98.99
113.94
89.48
92.35
95.50
114.82
70.05
1.01
−13.94
10.52
7.65
Renal Cancer
4.50
−10.14
−14.82
21.18
29.95
PC-3
DU-145
91.14
110.09
8.86
−10.09
88.11
111.62
11.89
−11.62
Prostate
Cancer
MCF7
MDA-MB-
231/ATCC
HS 578T
T-47D
MDA-MB-468
99.19
88.41
0.81
92.32
80.11
7.68
11.59
19.89
Breast Cancer
101.81
80.01
98.03
−1.81
19.99
1.97
104.19
83.24
100.34
−4.19
16.24
−0.34

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3. Discussion
Ten benzoxazole clubbed 2-pyrrolidinone derivatives (11
–
20) as the inhibitors of
monoacylglycerol lipase were designed on the criteria fulfilling the structural requirements
and on the basis of previously reported inhibitors [36 42 45]. The designed, synthesized,
and characterized compounds (11 20) were screened against monoacylglycerol lipase
(MAGL) in order to find potential inhibitors. The substituted phenyl derivatives (13–20)
were established to reduce the MAGL activity at 100 M concentration below 50%. Com-
,
–
–
µ
pound 19 (4-NO2 derivative) and compound 20 (4-SO2NH2 derivative) were the most
potent, with IC50 of 8.4 and 7.6 nM, correspondingly. The benzoxazole derivatives having 4-
NO2 phenyl (19) and 4-SO2NH2 phenyl (20), with an FAAH IC50 value greater than 50
were considered selective MAGL inhibitors. In molecular docking studies, compounds 19
and 20 showed comparable docking scores of 9.87 and 9.83, respectively. The binding
µM,
−
−
of compounds 19 and 20 in the active site of MAGL revealed that the carbonyl group of
pyrrolidinone is located exactly in the oxyanion hole and stabilized by three hydrogen
bonds (~2 Å) with alanine 51, serine 122, and methionine 123. Serine 122 is one of the criti-
cal amino acid residues of the catalytic triad of MAGL. The benzoxazole moiety is found
to be positioned in the amphiphilic pouch, having π-π stacking contacts with the amino
acid tyrosine 194. The 4-NO2 phenyl (19) and 4-SO2NH2 phenyl (20) part of the ligand was
engaged in hydrophobic (Van der Waals) attractions with the amino acids leucine 148, 213,
and 241. The binding patterns of compounds 19 and 20 in the catalytic site of MAGL were
found to be similar as those of the reported inhibitors bound crystal structures [32,36,39,41].
Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and
20 computed by QikProp were found to be in the qualifying range as per the proposed
guideline for good orally bioactive CNS drugs. Moreover, compound 20 showed better
toxicity profile than compound 19, as predicted by admetSAR [50]. In formalin-induced
analgesic test, compound 20 reduced the pain response significantly both in acute (stage
I) and late (stage II) phases in a dose-dependent manner. It significantly demonstrated
the reduction in pain response, having better potency than the positive control GBP, at
the dose of 30 mg/kg. Moreover, in one dose (10 µM), anticancer screening by SRB assay,
compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer
activity towards SNB-75 cell line of CNS cancer, having % growth inhibition (% GI) of
35.49 and 31.88, respectively. Therefore, the present work concluded that compound 20
is the potential lead compounds that can be further manipulated at points 1 and 4 of the
2-pyrrolidinone moiety for the discovery and development of more selective and potent
inhibitors of MAGL for neuropathic pain and CNS disorders including cancers.
4. Experimental
4.1. Chemistry
Reagents and solvents were procured from Merck Ltd. (New Delhi, India) and
Sigma-Aldrich Ltd. (New Delhi, India). Progress and completion of the reactions was
checked by thin-layer chromatography (TLC). Melting points of the derivatives were
determined by open tube capillary method and uncorrected. Elemental analysis data
were obtained from CHNOS elemental analyzer (Vario EL III, Elementar Analysensysteme
GmbH, Langenselbold, Germany). Shimadzu FT-IR spectrometer (Shimadzu Analytical Pvt.
Ltd., New Delhi, India) was used for recording IR spectrum (4000–400 cm−1), by preparing
1
KBr pellets. H-NMR spectrum of the derivatives were obtained from Bruker 300 MHz
NMR instrument (Bruker Avance AV-III type, Billerica, MA, USA) using CDCl3 or DMSO-
1
d6 as solvent. H-NMR spectra of compounds 11
–
20 can be found in the Supplementary
Material. Molecular mass (m/z) of the derivatives were obtained by UPLC-MS (Q-TOF-ESI)
(Waters Corp., Milford, MA, USA).

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4.1.1. Synthesis of 1-(Aryl Substituted)-5-Oxopyrrolidine-3-Carboxylic Acids (1–10)
Method-1 (for compound 1)
Equimolar amount of benzylamine (50 mmol) and itaconic acid (50 mmol, 6.5 g) in
50 mL of tripled distilled water was refluxed for about 45–60 min. The contents were
then chilled, filtered, and washed with cold water. The obtained solid was dissolved
in minimum quantity of aq. NaOH (10%). After treatment with activated charcoal, the
solution was filtered and acidified with dil. HCl in order to obtain the precipitate. The
filtered solid was washed with cold water, dried, and purified by recrystallization from
ethanol/water mixture.
1-Benzyl-5-Oxopyrrolidine-3-Carboxylic Acid (
1518 (C=C), 1627 (C=OOH), 1734 (C=O), 2947 (sp3 C-H), 3045 (Ar C-H), 3241 (COO-H); 1H-
NMR (DMSO-d6) (ppm): 2.63–2.76 (m, 2H, COCH2), 3.62 (s, 2H, CH2), 4.29–4.44 (m, 3H,
1
), white solid; yield: 75%; m.p. 142–145◦ C; IR:
δ
NCH2 and CHpyrr), 7.20–7.36 (m, 5H, Ar-H), 11.35 (s, 1H, COOH, D2O exchangeable);
ESI-MS (m/z): 219.12 [M]+; Anal. calcd. For C12H13NO3: C, 65.74; H, 5.98; N, 6.39. Found:
C, 65.80; H, 5.85; N, 6.50.
Method-1 (for compound 1) Method-2 (for compounds 2–10)
Intermediate compounds (2–10) were synthesized as per the procedure reported in
our previous publication [45].
4.1.2. Synthesis of 4-(Benzoxazolyl)-1-(Aryl Substituted)Pyrrolidin-2-Ones (11–20)
Appropriate 1-(aryl substituted)-5-oxopyrrolidine-3-carboxylic acids (20 mmol), 2-
aminophenol (20 mmol, 2.18 g) and polyphosphoric acid (20 g) in an RBF were heated to
150–160◦ C and stirred for 2–3 h. The content of the RBF (round bottom flask) was cooled
at RT; 5% NaCO3 (25 mL) was added and heated for 10 min. The content was chilled and
transferred in a flask having 100 mL of water, and stirred at RT for 15 min. The filtered solid
was washed three times with water (50 mL), dried, and purified by recrystallization with
ethanol. The derivatives were then purified by chromatography using ethylacetate:hexane
(1:4) as solvent.
4-(Benzo[d]oxazol-2-yl)-1-benzylpyrrolidin-2-one (11), pale-yellow solid; yield: 57%; m.p.
160–162 ◦C; IR: 1370 (C-N), 1555 (C=C), 1621 (C=N), 1703 (C=O), 3077 (Ar C-H); 1H-NMR
(DMSO-d6) δ (ppm): 2.60–2.77 (m, 2H, COCH2), 3.13–3.18 (m, 1H, CHpyrr), 3.81–3.86 (m,
2H, NCH2), 3.95–4.01 (m, 2H, CH2), 7.38–7.58 (m, 6H, Ar-H), 7.73–7.78 (d, 1H, Ar-H,
J = 15.6 Hz), 7.94–7.96 (d, 2H, Ar-H, J = 8.4 Hz); ESI-MS (m/z): 292.14 [M]+, 293.14 [M + H]+;
Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.16; H, 5.70; N, 9.82.
4-(Benzo[d]oxazol-2-yl)-1-phenylpyrrolidin-2-one (12), pale-yellow solid; yield: 67%; m.p.
148–150 ◦C; IR: 1395 (C-N), 1552 (C=C), 1615 (C=N), 1698 (C=O), 3070 (Ar C-H); 1H-NMR
(DMSO-d6)
δ (ppm): 2.86–3.20 (m, 2H, COCH2), 4.16–4.40 (m, 3H, NCH2 and CHpyrr),
7.16–8.18 (m, 9H, Ar-H); ESI-MS (m/z): 278.13 [M]+, 279.13 [M + H]+; Anal. calcd. for
C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.60; H, 5.18; N, 10.33.
4-(Benzo[d]oxazol-2-yl)-1-(o-tolyl)pyrrolidin-2-one (13), pale-yellow solid; yield: 68%; m.p.
162–164 ◦C; IR: 1404 (C-N), 1568 (C=C), 1607 (C=N), 1691 (C=O), 3079 (Ar C-H); 1H-NMR
(DMSO-d6)
δ (ppm): 2.18 (s, 3H, CH3), 2.88–3.17 (m, 2H, COCH2), 4.12–4.18 (m, 1H,
CHpyrr), 4.26–4.39 (m, 2H, NCH2), 7.18–7.21 (m, 2H, H-5phenyl and H-6phenyl), 7.36–7.54 (m,
4H, H-5benzoxazole, H-6benzoxazole, H-3phenyl and H-4phenyl), 7.98–8.01 (d, 1H, H-7benzoxazole
,
J = 8.1 Hz), 8.09–8.12 (d, 1H, H-4benzoxazole, J = 7.8 Hz); ESI-MS (m/z): 292.13 [M]+, 293.13
[M + H]+; Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.21; H, 5.82;
N, 9.77.
4-(Benzo[d]oxazol-2-yl)-1-(p-tolyl)pyrrolidin-2-one (14), pale-yellow solid; yield: 67%; m.p.
164–166 ◦C; IR: 1411 (C-N), 1562 (C=C), 1598 (C=N), 1697 (C=O), 3077 (Ar C-H); 1H-NMR
(DMSO-d6)
δ (ppm): 2.28 (s, 3H, CH3), 2.94–3.13 (m, 2H, COCH2), 4.13–4.35 (m, 3H,
NCH2 and CHpyrr), 7.17–7.20 (d, 2H, H-3phenyl and H-5phenyl, J = 8.4 Hz), 7.34–7.42 (m,

Molecules 2021, 26, 2389
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2H, H-2phenyl and H-6phenyl), 7.54–7.57 (d, 2H, H-4benzoxazole and H-7benzoxazole, J = 8.4
Hz), 7.70–7.74 (m, 2H, H-5benzoxazole and H-6benzoxazole); ESI-MS (m/z): 292.13 [M]+, 293.13
[M + H]+; Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.18; H, 5.78;
N, 9.80.
4-(Benzo[d]oxazol-2-yl)-1-(4-chlorophenyl)pyrrolidin-2-one (15), pale-yellow solid; yield: 70%;
◦
m.p. 174–176 C; IR: 758 (C-Cl), 1396 (C-N), 1568 (C=C), 1612 (C=N), 1689 (C=O), 3086 (Ar
C-H); 1H-NMR (DMSO-d6)
δ
(ppm): 3.01–3.09 (m, 2H, COCH2), 4.20–4.33 (m, 3H, NCH2
and CHpyrr), 7.35–7.43 (m, 4H, Ar-H), 7.68–7.72 (t, 4H, Ar-H, J = 6.3 Hz); ESI-MS (m/z):
312.11 [M]+, 314.10 [M + 2]+; Anal. calcd. for C17H13ClN2O2: C, 65.29; H, 4.19; N, 8.96.
Found: C, 65.52; H, 4.35; N, 9.03.
4-(Benzo[d]oxazol-2-yl)-1-(3-chloro-4-fluorophenyl)pyrrolidin-2-one (16), pale-yellow solid; yield:
68%; m.p. 187–189 ◦C; IR: 765 (C-Cl), 1388 (C-N), 1545 (C=C), 1613 (C=N), 1690 (C=O),
3098 (Ar C-H); 1H-NMR (DMSO-d6)
δ (ppm): 2.92–3.19 (m, 2H, COCH2), 4.13–4.21 (m, 1H,
CHpyrr), 4.25–4.41 (m, 2H, NCH2), 7.41–7.60 (m, 5H, Ar-H), 8.05–8.08 (d, 1H, H-7benzoxazole
,
J = 8.1 Hz), 8.11–8.14 (d, 1H, H-4benzoxazole, J = 8.1 Hz); ESI-MS (m/z): 330.10 [M]+, 332.09
[M + 2]+; Anal. calcd. for C17H12ClFN2O2: C, 61.73; H, 3.66; N, 8.47. Found: C, 62.07; H,
3.85; N, 8.61.
4-(Benzo[d]oxazol-2-yl)-1-(4-hydroxyphenyl)pyrrolidin-2-one (17), pale-yellow solid; yield: 62%;
◦
m.p. 242–245 C; IR: 1407 (C-N), 1535 (C=C), 1597 (C=N), 1698 (C=O), 3092 (Ar C-H),
3410 (O-H); 1H-NMR (DMSO-d6)
δ (ppm): 2.91–3.20 (m, 2H, COCH2), 4.12–4.23 (m, 1H,
CHpyrr), 4.25–4.39 (m, 2H, NCH2), 5.64 (bs, 1H, OH, D2O exchangeable), 7.41–7.60 (m, 4H,
Ar-Hphenyl), 7.72–7.77 (t, 2H, H-5benzoxazole and H-6benzoxazole, J = 7.5 Hz), 7.96–7.99 (d, 1H,
H-7benzoxazole, J = 8.4 Hz), 8.08–8.10 (d, 1H, H-4benzoxazole, J = 8.4 Hz); ESI-MS (m/z): 294.12
[M]+, 295.12 [M + H]+; Anal. calcd. for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C,
69.66; H, 4.97; N, 9.78.
4-(Benzo[d]oxazol-2-yl)-1-(4-methoxyphenyl)pyrrolidin-2-one (18), pale-yellow solid; yield:
66%; m.p. 177–179 ◦C; IR: 1412 (C-N), 1546 (C=C), 1600 (C=N), 1690 (C=O), 3081 (Ar C-H);
1H-NMR (DMSO-d6)
δ (ppm): 2.88–3.18 (m, 2H, COCH2), 3.67 (s, 3H, OCH3), 4.08–4.16 (m,
1H, CHpyrr), 4.22–4.39 (m, 2H, NCH2), 7.16–7.19 (d, 2H, H-2phenyl and H-6phenyl, J = 8.4 Hz),
7.42–7.58 (m, 4H, H-5benzoxazole, H-6benzoxazole, H-3phenyl and H-5phenyl), 7.95–7.98 (d, 1H,
H-7benzoxazole, J = 8.4 Hz), 8.09–8.12 (d, 1H, H-4benzoxazole, J = 8.4 Hz); ESI-MS (m/z): 308.13
[M]+, 309.13 [M + H]+; Anal. calcd. for C18H16N2O3: C, 70.12; H, 5.23; N, 9.09. Found: C,
70.35; H, 5.38; N, 9.18.
4-(Benzo[d]oxazol-2-yl)-1-(4-nitrophenyl)pyrrolidin-2-one (19), yellow solid; yield: 62%; m.p.
276–278 ◦C; IR: 1405 (C-N), 1550 (C=C), 1595 (C=N), 1695 (C=O), 3089 (Ar C-H); 1H-NMR
(DMSO-d6)
δ (ppm): 2.89–3.15 (m, 2H, COCH2), 4.11–4.16 (m, 1H, CHpyrr), 4.24–4.37 (m, 2H,
NCH2), 6.79–6.82 (d, 2H, H-2phenyl and H-6phenyl, J = 9 Hz), 7.43–7.48 (t, 2H, H-5benzoxazole
,
H-6benzoxazole, J = 7.5 Hz), 8.01–8.18 (m, 4H, H-4benzoxazole, H-7benzoxazole, H-3phenyl and
H-5phenyl); ESI-MS (m/z): 323.11 [M]+, 324.11 [M + H]+; Anal. calcd. for C17H13N3O4: C,
63.16; H, 4.05; N, 13.00. Found: C, 63.35; H, 4.27; N, 13.23.
4-(4-(Benzo[d]oxazol-2-yl)-2-oxopyrrolidin-1-yl)benzenesulfonamide (20), yellow solid; yield:
◦
60%; m.p. 227–229 C; IR: 1416 (C-N), 1564 (C=C), 1607 (C=N), 1704 (C=O), 3095 (Ar
C-H); 1H-NMR (DMSO-d6)
δ (ppm): 2.91–3.19 (m, 2H, COCH2), 4.15–4.22 (m, 1H, CHpyrr),
4.25–4.38 (m, 2H, NCH2), 5.67 (s, 2H, SO2NH2, D2O exchangeable), 7.42–7.59 (m, 4H, Ar-H),
8.09–8.16 (m, 4H, Ar-H); ESI-MS (m/z): 357.12 [M]+, 358.12 [M + H]+; Anal. calcd. for
C17H15N3O4S: C, 57.13; H, 4.23; N, 11.76. Found: C, 57.36; H, 4.36; N, 11.95.
4.2. Human MAGL Assay
The screening of the synthesized compounds (11–20) for their capability to reduce
hMAGL activity was performed according to the information leaflet provided with Cay-
man’s assay kit (Cayman Chemical, Michigan, USA) by the reported method [29], as

Molecules 2021, 26, 2389
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discussed in detail in our previous publication [45]. The results were compared with
standard MAGL inhibitors, CAY10499 and JZL184, and are provided in Table 1.
4.3. Human FAAH Assay
The screening of the selected compounds (15, 16, 18, 19 and 20) for their potential to
inhibit hFAAH was performed according to the information leaflet provided with Cayman’s
assay kit (Cayman Chemical, Ann Arbor, Michigan, USA) by the reported method [47],
as discussed in detail in our previous publication [45]. The results were compared with
standard FAAH inhibitor, URB597, and are provided in Table 1.
4.4. Molecular Docking Study
Glide executed on Maestro 9.4 (Schrödinger Inc., New York, NY, USA) was utilized for
Glide XP docking of active compounds (19 and 20). The .pdb file of hMAGL X-ray crystal
structure was downloaded from protein data bank having ID 5ZUN (crystal structure
resolution 1.35 Å) for molecular docking study [36]. The protein structure was refined, op-
timized, and energy-minimized with the help of preparation wizard in Maestro. A docking
grid of 20
lized ligand. Ligand (compounds 19 and 20) structures were prepared with the help of
LigPrep 2.6 with Epik 2.4 at pH 7.0 2.0. The methodology was validated by docking the
×
20
×
20 Å, was created around the catalytic site by defining the cocrystal-
±
cocrystallized ligand with Glide XP docking protocol [55].
4.5. Physicochemical and Pharmacokinetic Characteristics
Guidelines, concerning the validation and optimization of orally active CNS com-
pounds, were developed by Ghose et. al. by analyzing 35 characteristic features of orally
bioavailable 317 CNS and 626 non-CNS drugs [49]. For computations of these properties of
the selected compounds (19 and 20), QikProp 3.6 module of Schrodinger was utilized. The
generated data was then matched with the qualifying range as per the suggested guideline
for good orally bioactive CNS drugs.
4.6. In Silico Absorption and Toxicity Profile
The selected compounds (19 and 20) were evaluated for their absorption and tox-
icity profile by a bioinformatics tool admetSAR [50]. Oral bioavailability, intestinal ab-
sorption, and BBB penetration properties were calculated. AMES test for mutagenicity,
carcinogenicity test, and the calculation of LD50 for both the compounds (19 and 20) were
also evaluated.
4.7. Analgesic Activity
Formalin-induced analgesic test was executed by the procedure described by Coderre
and Laughlin [51,52] as discussed in detail in our previous publication [45]. Male Wistar
rats (180–200 g) were obtained with the permission of IAEC (proposal number 1048) from
Jamia Hamdard, New Delhi, India. The results of test compound 20 and reference drug,
Gabapentin (GBP), were statistically compared with the control group.
4.8. Anticancer Screening: Sulforhodamine B Assay
Compounds 19 and 20 were supplied to National Cancer Institute (Bethesda, Mary-
land, USA), for in vitro sulforhodamine B (SRB) assay, anticancer screening on 60 cell lines
of cancers of leukemia, melanoma, and tumors of the kidney, brain, breast, lung, colon,
ovary, and prostate, as per their standard protocol [53,54]. One dose anticancer results
(NCI, USA) of compounds 19 and 20 are provided in the Supplementary Materials.
4.9. Statistical Analysis
The statistical study of the data was accomplished by GraphPad Prism (version 8.0.2;
GraphPad Software, San Diego, CA, USA). The dose response of the test compounds was

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compared with that of control, in formalin-induced analgesic test, by analysis of variance
(ANOVA) followed by Dunnett’s test. Outcomes are communicated as mean ± SEM.
5. Conclusions
Ten benzoxazole clubbed 2-pyrrolidinone derivatives (11–20) as the inhibitors of
MAGL were designed, synthesized, characterized, and assayed against MAGL and FAAH
enzymes, in order to find potential small molecule selective MAGL inhibitor. Compounds
19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative) were found most potent and selective,
with an IC50 of 8.4 and 7.6 nM, respectively. The binding patterns of compounds 19
and 20 in the catalytic site of MAGL were found as expected and similar as those of the
reported inhibitors.
The physiochemical and pharmacokinetic properties of compounds 19 and 20 were
found to be almost in the qualifying range as per the proposed guideline for good orally
bioactive CNS drugs. Compound 20 significantly demonstrated the reduction in pain
response, having better potency than the positive control GBP, at the dose of 30 mg/kg.
The present work concluded that compound 20 is the potential lead compounds that
can be further studied and optimized at points 1 and 4 of the 2-pyrrolidinone moiety
for the discovery and development of more selective and potent inhibitors of MAGL for
neuropathic pain.
Supplementary Materials: The following are available online, 1H-NMR spectra of compounds
11–20 and one dose anticancer results (NCI, USA) of compounds 19 and 20 is provided in the
supporting information.
Author Contributions: Conceptualization, O.A., A.S.A.A. and H.K.S.; data curation, O.A., M.Q.H.
and M.M.S.; formal analysis, A.S.A.A., Y.R., M.Q.H. and H.K.S.; funding acquisition, A.S.A.A. and
O.A.; investigation, O.A., M.Q.H. and M.M.S.; methodology, O.A. and M.Q.H.; project administration,
A.S.A.A., O.A. and Y.R.; resources, A.S.A.A., O.A., M.Q.H. and H.K.S.; software, O.A., M.Q.H. and
H.K.S.; supervision, O.A., A.S.A.A. and M.Q.H.; validation, A.S.A.A., Y.R., M.Q.H., M.M.S. and
H.K.S.; visualization, Y.R., M.Q.H., M.M.S. and H.K.S.; writing—original draft, O.A.; writing—review
and editing, M.Q.H., Y.R., M.M.S. and H.K.S. All authors have read and agreed to the published
version of the manuscript.
Funding: 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).
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Institutional Animal Ethics Committee (Proposal
number 1048) of Jamia Hamdard (Hamdard University, New Delhi-110062 (Registration Number:
173/CPCSEA; 28 January 2000).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding authors.
Acknowledgments: 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).
Conflicts of Interest: Authors declare that they do not have any conflict of interest.
Sample Availability: The samples of the compounds are available on request from the correspond-
ing author.
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