Analgesic and Anticancer Activity of Benzoxazole Clubbed 2-Pyrrolidinones as Novel Inhibitors of Monoacylglycerol Lipase

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. Compounds 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 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.

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 moderate affinity for both the receptors. Interestingly, 2-AG is the major endocannabinoid and 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 (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 [4]. MAGL hydrolyzes approximately 85% of total 2-AG in the CNS [5] and generates arachidonic acid (AA), giving rise to neuro-inflammatory PGE₂ and PGD₂ prostaglandins [6]. MAGL inhibitors ameliorates neuropathic pain by increasing 2-AG and decreasing neuro-inflammatory prostaglandins in the CNS (Figure 1) [7]. In addition, 2-AG demonstrated analgesic activity by acting on CB₁ receptors in the CNS and periphery [8,9,10,11]. It is reported that JJKK-048 (MAGL, IC₅₀ 363 pM) exhibited analgesic activity in tail immersion 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 findings encouraged that the inhibitors of MAGL has therapeutic potentials in pain and CNS disorders [17,18,19,20].

The governing importance of MAGL in abnormal lipolysis in cancer has been demonstrated [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 metastasis 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 [25,26,27]. The anticancer effect of MAGL inhibition in prostate cancer was totally abolished by cotreatment 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 µM) [30], JZL184 (IC₅₀ 8 nM) [31], SAR629 (IC₅₀ 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 benzoylpiperidine 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. [33,35]. In addition, 3D 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 crystal structures and inhibitors were reviewed [41]. Our group has also identified nanomolar 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) [42,43,44,45]. 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 successfully 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 modifications [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 derivatives (11–20).

The prototype intermediate compound 1, (1-Benzyl-5-oxopyrrolidine-3-carboxylic acid), revealed typical peaks at 1627 cm⁻¹ (carbonyl of acid), 1734 cm⁻¹ (carbonyl of pyrrolidinone), and 3241 cm⁻¹ (O-H of acid) in IR spectrum. In ¹H-NMR spectrum of compound 1, two methyl protons were found resonating as singlet at δ 3.62. The multiplets resonating at δ 2.62–2.76 indicated two COCH₂ protons, and at δ 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 δ 7.20–7.36 as a multiplet. The typical singlet was assigned to COOH proton at δ 11.35 and was found to be D₂O exchangeable. Final prototype compound 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 δ 3.95–4.01 as a multiplet. Two COCH₂ protons of the 2-pyrrolidinone ring was assigned at δ 2.60–2.77 as a multiplet. The multiplets located at δ 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 δ 7.38–7.58 as a multiplet. The doublet resonating at δ 7.73–7.78 was assigned to the aromatic 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.

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 μM concentration 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. In vitro hMAGL and hFAAH inhibition assay of the synthesized compounds (11–20).

Compound	R	hMAGL; IC50	hFAAH; IC50
11		>100 μM	ND
12		>100 μM	ND
13		85 ± 1.5 μM	ND
14		72 ± 2.1 μM	ND
15		65 ± 3.2 nM	28 ± 1.8 μM
16		34 ± 1.7 nM	25 ± 2.3 μM
17		62 ± 2.7 μM	ND
18		42 ± 1.5 nM	37 ± 2.2 μM
19		8.4 ± 1.9 nM	55 ± 2.7 μM
20		7.6 ± 0.8 nM	68 ± 2.1 μM
CAY10499	--	415 ± 3.2 nM	--
JZL184	--	10 ± 0.8 nM	--
URB597	--	--	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).

.

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 µM against FAAH. The benzoxazole derivatives having 4-NO₂ phenyl (19) and 4-SO₂NH₂ phenyl (20), with an FAAH IC₅₀ value greater than 50 µM, were considered selective MAGL inhibitors. The IC₅₀ 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.

2.5. Pharmacokinetic and Physicochemical Characteristics

To investigate the potential of the identified derivatives (19 and 20) to cross the selectively 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 optimization 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 Ų (ideally 25–60 Ų), 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 Ų, molecular volume 740–970 ų, 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. Physicochemical and pharmacokinetic properties of compounds 19 and 20, predicted by QikProp, Schrodinger, for CNS activity.

S. No.	Property	Description	Range of Properties in CNS Drugs				Compound 19	Compound 20
			QL	PL	PU	QU
1	#stars	drug likeness penalty; the higher the value, the less drug-like the molecule	0	0	0	3	0	0
2	#amine	no. of basic amines	0	1	1	2	0	0
3	#amidine	no. of amidines groups	0	0	0	0	0	0
4	#acid	no. of carboxylic acid groups	0	0	0	0	0	0
5	#amide	no. of amides groups	0	0	0	1	0	0
6	#rotor	no. of rotatable bonds (without CX3, alkene, amide, small ring)	0	3	6	8	1	2
7	CNS	a qualitative CNS activity parameter	−2	0	1	2	−2	−2
8	dipole	computed dipole moment	0.67	1.1	3.9	8.9	9.47	10.22
9	SASA	solvent accessible surface area	348	487	620	798	584.85	617.51
10	FOSA	SASA on saturated carbon and attached hydrogen	16	178	314	464	91.99	91.96
11	FISA	SASA on N, O, and H attached to heteroatoms	0	0	64	176	167.04	210.06
12	PISA	π component of SASA	0	160	292	343	325.81	313.57
13	WPSA	weakly polar component of the SASA (halogens, P, and S)	0	0	0	126	0	1.94
14	volume	solvent accessible volume (Å3)	492	830	1104	1388	1002.52	1065.46
15	donorHB	estimated no. of hydrogen bonds that would be donated to the solvent water	0	0	1	3	0	2
16	accptHB	estimated no. of hydrogen bonds that would be accepted from the solvent water	1	2.8	5.2	8.3	6	9.5
17	glob	a globularity descriptor (1 for a sphere)	0.77	0.82	0.88	0.93	0.82	0.81
18	QPpolrz	predicted polarizability (Å3)	14	28	38	49	36.43	38.19
19	QPlogPo/w	octanol−water logP	−0.16	2.5	4.7	6.0	2.13	0.90
20	QPlogS	solubility in log(moles/liter)	−6.5	−4.6	−2.5	−0.42	−3.99	−3.99
21	CIQPlogS	log of conformation-independent solubility	−6.3	−4.2	−2.3	0.36	−4.16	−3.77
22	QPPCaco	apparent Caco-2 cell permeability	0	0	810	3269	258.09	100.92
23	QPlogBB	brain/blood partition coefficient	−1.2	−0.06	0.75	1.2	−1.12	−1.65
24	QPPMDCK	predicted apparent MDCK cell permeability (nm/s)	0	0	634	5899	114.43	42.50
25	QPlogKhsa	prediction of binding to human serum albumin	−1	0.04	0.78	1.04	−0.11	−0.34
26	HumanOralAbsorption	Human oral absorption	2	3	3	3	3	3
27	PercentHuman OralAbsorption	Percent of human oral absorption	61	95	100	100	82.63	68.11
28	TPSA	van der Waals surface area of polar nitrogen and oxygen atoms	3.8	12	54	109	98.65	119.08
29	#NandO	no. of N and O atoms	1	2	4	7	7	7
30	RuleOfFive	no. of violations of Lipinski’s rule of five	0	0	0	1	0	0
31	RuleOfThree	no. of violations of Jorgensen’s rule of three	0	0	0	1	0	0
32	#in34	no. of atoms in three- or four-membered rings	0	0	0	0	0	0
33	#in56	no. of atoms in five- or six-membered rings	5	11	17	24	20	20
34	#noncon	no. of atoms not able to form conjugation in nonaromatic rings	0	0	4	10	3	3
35	#nonHatm	no. of non-H atoms	8	19	25	30	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].

). 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.

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 compounds 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. In-silico absorption and toxicity profile of compounds 19 and 20 obtained from admetSAR server [50].

Compound	BBB	HIA	HOB	AMES test	Carcinogenicity	Rat Acute Toxicity (LD50, mol/kg)
19	Yes	Yes	Yes	Mutagenic	Non-carcinogen	2.30
20	Yes	Yes	Yes	Non-Mutagenic	Non-carcinogen	2.21

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 nociception 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 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.

2.8. Anticancer Activity

Compounds 19 and 20 were supplied to National Cancer Institute (USA), for sulforhodamine B (SRB) assay and anticancer screening [53,54]. Single-dose (10 µM) assay results 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. In vitro anticancer screening of compound 19 and 20, against NCI60 cell lines at 10 μM concentration.

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

. 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.

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 [36,42,43,44,45]. 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 μM concentration below 50%. Compound 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 µM, 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 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 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 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 KBr pellets. ¹H-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-d₆ as solvent. ¹H-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).

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 (1), white solid; yield: 75%; m.p. 142–145° C; IR: 1518 (C=C), 1627 (C=OOH), 1734 (C=O), 2947 (sp₃ C-H), 3045 (Ar C-H), 3241 (COO-H); ¹H-NMR (DMSO-d₆) δ (ppm): 2.63–2.76 (m, 2H, COCH₂), 3.62 (s, 2H, CH₂), 4.29–4.44 (m, 3H, NCH₂ and CH_pyrr), 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-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, CH_pyrr), 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 CH_pyrr), 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, CH_pyrr), 4.26–4.39 (m, 2H, NCH₂), 7.18–7.21 (m, 2H, H-5_phenyl and H-6_phenyl), 7.36–7.54 (m, 4H, H-5_benzoxazole, H-6_benzoxazole, H-3_phenyl and H-4_phenyl), 7.98–8.01 (d, 1H, H-7_benzoxazole, J = 8.1 Hz), 8.09–8.12 (d, 1H, H-4_benzoxazole, 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 CH_pyrr), 7.17–7.20 (d, 2H, H-3_phenyl and H-5_phenyl, J = 8.4 Hz), 7.34–7.42 (m, 2H, H-2_phenyl and H-6_phenyl), 7.54–7.57 (d, 2H, H-4_benzoxazole and H-7_benzoxazole, J = 8.4 Hz), 7.70–7.74 (m, 2H, H-5_benzoxazole and H-6_benzoxazole); 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 CH_pyrr), 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, CH_pyrr), 4.25–4.41 (m, 2H, NCH₂), 7.41–7.60 (m, 5H, Ar-H), 8.05–8.08 (d, 1H, H-7_benzoxazole, J = 8.1 Hz), 8.11–8.14 (d, 1H, H-4_benzoxazole, 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, CH_pyrr), 4.25–4.39 (m, 2H, NCH₂), 5.64 (bs, 1H, OH, D₂O exchangeable), 7.41–7.60 (m, 4H, Ar-H_phenyl), 7.72–7.77 (t, 2H, H-5_benzoxazole and H-6_benzoxazole, J = 7.5 Hz), 7.96–7.99 (d, 1H, H-7_benzoxazole, J = 8.4 Hz), 8.08–8.10 (d, 1H, H-4_benzoxazole, 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, CH_pyrr), 4.22–4.39 (m, 2H, NCH₂), 7.16–7.19 (d, 2H, H-2_phenyl and H-6_phenyl, J = 8.4 Hz), 7.42–7.58 (m, 4H, H-5_benzoxazole, H-6_benzoxazole, H-3_phenyl and H-5_phenyl), 7.95–7.98 (d, 1H, H-7_benzoxazole, J = 8.4 Hz), 8.09–8.12 (d, 1H, H-4_benzoxazole, 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, CH_pyrr), 4.24–4.37 (m, 2H, NCH₂), 6.79–6.82 (d, 2H, H-2_phenyl and H-6_phenyl, J = 9 Hz), 7.43–7.48 (t, 2H, H-5_benzoxazole, H-6_benzoxazole, J = 7.5 Hz), 8.01–8.18 (m, 4H, H-4_benzoxazole, H-7_benzoxazole, H-3_phenyl and H-5_phenyl); 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, CH_pyrr), 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 Cayman’s assay kit (Cayman Chemical, Michigan, USA) by the reported method [29], as 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, optimized, and energy-minimized with the help of preparation wizard in Maestro. A docking grid of 20 × 20 × 20 Å, was created around the catalytic site by defining the cocrystallized 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 cocrystallized ligand with Glide XP docking protocol [55].

4.5. Physicochemical and Pharmacokinetic Characteristics

Guidelines, concerning the validation and optimization 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]. 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 toxicity profile by a bioinformatics tool admetSAR [50]. Oral bioavailability, intestinal absorption, 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, Maryland, 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 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.