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Reversible Monoacylglycerol Lipase Inhibitors: Discovery of a New Class of Benzylpiperidine Derivatives

  • Giulia Bononi
    Giulia Bononi
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
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  • Miriana Di Stefano
    Miriana Di Stefano
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    Department of Life Sciences, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
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    Orcidhttps://orcid.org/0000-0001-6727-5816
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  • Giulio Poli
    Giulio Poli
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
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  • Gabriella Ortore
    Gabriella Ortore
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
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  • Philip Meier
    Philip Meier
    Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland
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  • Francesca Masetto
    Francesca Masetto
    Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, DeBoelelaan 1117, 1081HV Amsterdam, The Netherlands
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  • Isabella Caligiuri
    Isabella Caligiuri
    Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy
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  • Flavio Rizzolio
    Flavio Rizzolio
    Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy
    Department of Molecular Sciences and Nanosystems, Ca’ Foscari University, 30123 Venezia, Italy
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  • Marco Macchia
    Marco Macchia
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
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  • Andrea Chicca
    Andrea Chicca
    Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland
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    Orcidhttps://orcid.org/0000-0001-9593-636X
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  • Amir Avan
    Amir Avan
    Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad 91886-17871, Iran
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  • Elisa Giovannetti
    Elisa Giovannetti
    Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, DeBoelelaan 1117, 1081HV Amsterdam, The Netherlands
    Cancer Pharmacology Lab, Fondazione Pisana per la Scienza, via Giovannini 13, 56017 San Giuliano Terme, Pisa, Italy
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  • Chiara Vagaggini
    Chiara Vagaggini
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
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  • Annalaura Brai
    Annalaura Brai
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
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    Orcidhttps://orcid.org/0000-0001-6395-9348
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  • Elena Dreassi
    Elena Dreassi
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
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  • Massimo Valoti
    Massimo Valoti
    Department of Life Sciences, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
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    Orcidhttps://orcid.org/0000-0002-7240-3576
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  • Filippo Minutolo
    Filippo Minutolo
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    Center for Instrument Sharing of the University of Pisa (CISUP), Lungarno Pacinotti 43, 56126 Pisa, Italy
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  • Carlotta Granchi*
    Carlotta Granchi
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    Center for Instrument Sharing of the University of Pisa (CISUP), Lungarno Pacinotti 43, 56126 Pisa, Italy
    *Email: [email protected]. Phone: +39-0502219705.
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    Orcidhttps://orcid.org/0000-0002-5849-0722
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  • Jürg Gertsch
    Jürg Gertsch
    Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland
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  • , and 
  • Tiziano Tuccinardi
    Tiziano Tuccinardi
    Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    Center for Instrument Sharing of the University of Pisa (CISUP), Lungarno Pacinotti 43, 56126 Pisa, Italy
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    Orcidhttps://orcid.org/0000-0002-6205-4069
Cite this: J. Med. Chem. 2022, 65, 10, 7118–7140
Publication Date (Web):May 6, 2022
https://doi.org/10.1021/acs.jmedchem.1c01806

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Monoacylglycerol lipase (MAGL) is the enzyme responsible for the metabolism of 2-arachidonoylglycerol in the brain and the hydrolysis of peripheral monoacylglycerols. Many studies demonstrated beneficial effects deriving from MAGL inhibition for neurodegenerative diseases, inflammatory pathologies, and cancer. MAGL expression is increased in invasive tumors, furnishing free fatty acids as pro-tumorigenic signals and for tumor cell growth. Here, a new class of benzylpiperidine-based MAGL inhibitors was synthesized, leading to the identification of 13, which showed potent reversible and selective MAGL inhibition. Associated with MAGL overexpression and the prognostic role in pancreatic cancer, derivative 13 showed antiproliferative activity and apoptosis induction, as well as the ability to reduce cell migration in primary pancreatic cancer cultures, and displayed a synergistic interaction with the chemotherapeutic drug gemcitabine. These results suggest that the class of benzylpiperidine-based MAGL inhibitors have potential as a new class of therapeutic agents and MAGL could play a role in pancreatic cancer.

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1. Introduction

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Endocannabinoids (eCBs) are signaling lipophilic molecules that exert their biological actions by interacting with cannabinoid receptors (CB) CB1 and CB2. The most important endocannabinoid neuromodulators are represented by anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which regulate several physiological and pathological processes including neuroinflammation, pain, neurodegeneration, and tumor progression. (1−3) These arachidonic acid-derived lipids are synthesized on demand from membrane phospholipid precursors and are quickly metabolized after their release into the extracellular space and reuptake into the cytoplasm. (4) Anandamide degradation is operated by fatty acid amide hydrolase (FAAH), a serine hydrolase localized in postsynaptic neurons, which metabolizes AEA into arachidonic acid (AA) and ethanolamine. (5) On the other hand, 2-arachidonoylglycerol is hydrolyzed by serine hydrolases monoacylglycerol lipase (MAGL) and α/β hydrolase-6 and -12 (ABHD6 and ABHD12). MAGL is the main contributor to 2-AG degradation by hydrolyzing about 85% of 2-AG into AA and glycerol; the remaining 15% is hydrolyzed by ABHD6 and ABDH12. (6,7) MAGL belongs to the α/β hydrolase superfamily and it is highly expressed in presynaptic neurons, but also in peripheral tissues such as kidney, ovaries, testis, adrenal glands, adipose tissue, and heart. (8) This enzyme plays an essential role in the modulation of eCBs and the eicosanoid signaling pathway, as demonstrated in several pharmacological and genetic studies. (9) Increased levels of AA, determined by MAGL hydrolyzing activity, promote the production of thromboxanes, prostaglandins, and other eicosanoids with pro-inflammatory activity. (10) Furthermore, MAGL is overexpressed in aggressive cancer cells and primary tumors, where it modulates an oncogenic signaling network to generate protumorigenic lipids, which favor cancer invasiveness, migration, and growth. (11) eCBs AEA and 2-AG also act as antinociceptive agents and may participate in the control of pain initiation. (2,12) The level of eCBs increases in CNS under inflammatory and neuropathic pain conditions, and this may be due to the response of an endogenous neuroprotective mechanism to a pathological condition. In particular, 2-AG exerted its analgesic effect by stimulating CB2 receptors; therefore, MAGL modulation may influence pain. (13) MAGL inhibition has been shown to alleviate allodynia in some neuropathic pain models. (14−16) Thus, MAGL represents a feasible and promising therapeutic target for the treatment of neurodegenerative diseases, inflammation, pain, and cancer. (17)
The research field aimed at developing new MAGL inhibitors is increasing in the last years, mainly focusing on reversible inhibitors as potential therapeutic agents. (8) Reversible inhibitors (i.e., those compounds that bind temporarily to MAGL, disabling its catalytic activity for a limited period of time) allow maintaining physiological levels of MAGL after its time-limited inhibition, thus avoiding a chronic MAGL blockade (typical of irreversible inhibitors), which leads to desensitization of CB1 receptors provoked by excessive concentrations of 2-AG. In addition, it was found that genetic deletion of MAGL as well as prolonged MAGL inhibition by small molecules determines an impaired CB1-dependent synaptic plasticity, cross-tolerance to exogenous CB1 agonists, and physical dependence in mice, (18−22) thus strongly limiting the potential clinical development of irreversible inhibitors. Presently, some potent irreversible inhibitors are important milestones in the development history of MAGL inhibitors, for example, carbamate derivatives CAY10499, (23) JZL-184, (9) and ABX-1431. (24) Some of the most representative reversible MAGL inhibitors are reported in Figure 1. The naturally occurring terpenoids pristimerin (Figure 1) and euphol (Figure 1) were discovered in 2009 as reversible MAGL inhibitors, although they are characterized by a scarce selectivity for MAGL. (25) The piperazinyl azetidinyl amide ZYH (Figure 1) was patented by Janssen Pharmaceutica in 2010 and published in 2011 as a reversible MAGL inhibitor. The high-resolution X-ray crystal structure of the complex ZYH–human MAGL was reported, thus elucidating conformational changes of MAGL upon ligand binding. (26) Benzo[d][1,3]dioxol-5-ylmethyl 6-phenylhexanoate 1 (Figure 1) is among the early discovered synthetic reversible MAGL inhibitors: compound 1 proved to be efficacious in an experimental autoimmune encephalomyelitis mouse model by reducing the symptoms of multiple sclerosis and delaying the clinical progression of the disease. (27) Compounds 1,5-diphenylpyrazole-3-carboxamide 2 (Figure 1) and salicylketoxime derivative 3 (Figure 1) were developed by our research group: inhibitor 2 mitigated the neuropathic hypersensitivity induced in vivo by oxaliplatin, (28) and compound 3 showed antiproliferative activity in a series of cancer cells. (29) Piperazinyl pyrrolidin-2-one 4 (Figure 1) discovered by Takeda Pharmaceuticals was effective on the isolated enzyme, with inhibition values in the subnanomolar range, and also in vivo, where it decreased arachidonic acid concentration, thus increasing 2-AG levels in the mouse brain. (30) Since 2014, the class of benzoylpiperidine-based MAGL inhibitors has been developed by our group and a series of hit-to-lead optimization processes have enabled the identification of nanomolar inhibitors 5a–c (Figure 1), (31−35) whose phenolic moiety is deeply located in the glycerol cavity of the enzyme, while their diaryl-sulfide extremity fits in the wide lipophilic channel of the enzyme normally hosting the unsaturated 2-AG chain.

Figure 1

Figure 1. Structures of some representative synthetic reversible MAGL inhibitors.

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2. Results and Discussion

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2.1. Design of Benzylpiperidine Derivatives

Our starting point for the design of benzylpiperidine derivatives was the search for chemical moieties potentially able to fit in the MAGL active site through examination of the structures of different serine hydrolase inhibitors reported in the literature. Among them, our attention was attracted by the 2-(3-(piperidin-4-ylmethyl)phenoxy)-5-(trifluoromethyl)pyridine moiety of FAAH inhibitor PF-3845 (compound 6, Figure 2) discovered in the Cravatt lab in 2009. (36) This moiety could be considered to be somewhat similar to the benzoylpiperidine scaffold of our inhibitors (exemplified by compound 5a, Figures 1 and 2) due to (a) the piperidine ring and (b) the presence of two aromatic rings (two phenyl rings in compound 5a or phenyl and pyridine rings in compound 6) connected by means of a linker (sulfur for compound 5a or oxygen for compound 6). Therefore, we envisioned to create the novel hybrid compound 7 (Figure 2) possessing on one side the 2-(3-(piperidin-4-ylmethyl)phenoxy)-5-(trifluoromethyl)pyridine moiety of compound 6 (in blue, Figure 2) and on the other side the amide phenolic moiety of compound 5a, which proved to be fundamental for the MAGL inhibition activity, thus establishing a strategic hydrogen bond network with two active site residues, E53 and H272. (31)

Figure 2

Figure 2. Design of the new benzylpiperidine derivative 7. The moiety deriving from FAAH inhibitor 6 is highlighted in blue, and the moiety deriving from our MAGL inhibitor 5a is highlighted in red.

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As the second step of chemical exploration, compound 7 was modified to assess the importance of the different portions of this new scaffold. First, the trifluoromethyl group in position 5 of the terminal pyridine ring was removed, thus obtaining compound 8 (Figure 3), or shifted to different positions of the pyridine ring, such as in positions 3, 4, or 6, corresponding to compounds 11a, 11b, and 11c, respectively (Figure 3). A further structural simplification was the conversion of the pyridine to a simple unsubstituted phenyl ring in compound 9 (Figure 3). Additionally, we inserted different halogen atoms on the phenolic ring of compound 7; in particular, we based our decision on previously published halogenated benzoylpiperidine derivatives. (32) Indeed, the presence of halogen atoms in specific positions of the benzoylpiperidine-based MAGL inhibitors published in 2019 allowed reaching IC50 values below 1 μM in enzymatic assays (compounds 11c,d and 13b–d of reference (32). In analogy with those MAGL inhibitors, we inserted: (a) the presence of fluorine or chlorine in the para position to the phenolic hydroxyl group (compound 10a and 10c, respectively, Figure 3); (b) the presence of fluorine or chlorine in the para position to the amide carbonyl group (compound 10b and 10d, respectively, Figure 3); and (c) the presence of a bromine atom in the para position to the amide carbonyl group (compound 10e, Figure 3). The last modification consisted of connecting the pyridine ring to the rest of the molecule by a 1,4-disubstituted phenyl ring as in compound 12 (Figure 3), which replaced the 3-(piperidin-4-ylmethyl)phenoxy portion of compound 7. Finally, compound 13 (Figure 3) simultaneously possesses a trifluoromethyl group in position 4 of the pyridine ring (as compound 11b) and a 2-fluoro-5-hydroxyphenyl amide portion (as compound 10a).

Figure 3

Figure 3. Newly synthesized benzylpiperidine derivatives 8, 9, 10a–e, 11a–c, 12, and 13. The modified moieties compared to parent compound 7 are highlighted in blue, in red, or with a dashed square.

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2.2. Chemistry

The synthesis of compounds 7, 10a–e, 11a–c, and 13 follows a common synthetic procedure starting from the reaction of trifluoromethyl-substituted 2-chloropyridine 14–17 with 3-bromophenol 18 in the presence of potassium carbonate as the base and dry N,N-dimethylformamide (DMF) as the solvent (Scheme 1). Compounds 19–22 were subjected to a two-step reaction, which consisted first of a hydroboration of the alkene moiety of 1-Boc-4-methylenepiperidine by the hydroborating reagent 9-borabicyclo[3.3.1]nonane (9-BBN) followed by a cross coupling reaction with the brominated derivatives 19–22 with NaOH as the base, Pd(PPh3)4 as the catalytic system, and tetrabutylammonium iodide as the phase-transfer catalyst in toluene to assemble the central benzylpiperidine scaffold of these compounds (Scheme 1). N-Boc-protected intermediates 23–26 were deprotected by using a solution of HCl in dioxane. The corresponding piperidine hydrochlorides 27–30 were reacted with the properly substituted benzoic acids, which are 3-methoxybenzoic acid for compounds 31 and 37–39, 2-fluoro-5-methoxybenzoic acid for compounds 32 and 40, 4-fluoro-3-methoxybenzoic acid for compound 33, 2-chloro-5-methoxybenzoic acid for compound 34, 4-chloro-3-methoxybenzoic acid for compound 35, and 4-bromo-5-methoxybenzoic acid for compound 36, in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) as the condensing agent, N,N-diisopropylethylamine (DIPEA) as the base, and dry DMF as the solvent, as previously reported (Scheme 1). (32) Deprotection of the methoxy group by boron tribromide in dichloromethane furnished the final hydroxy-substituted compounds.

Scheme 1

Scheme 1. Synthesis of Compounds 7, 10a–e, 11a–c, and 13a
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aReagents and conditions: (a) anhydrous K2CO3, anhydrous DMF, 110 °C, overnight [77–99%]; (b) i. tert-butyl 4-methylenepiperidine-1-carboxylate, 9-BBN 0.5 M solution in THF, anhydrous toluene, 115 °C, 1 h; ii. aq. 3.2 M NaOH, Pd(PPh3)4, TBAI, anhydrous toluene, 115 °C, 18 h [46–99%]; (c) HCl 4.0 M solution in dioxane, anhydrous MeOH, anhydrous CH2Cl2, RT, 1 h [99%]; (d) properly substituted benzoic acid, HATU, DIPEA, anhydrous DMF, RT, 3–12 h [41–75%]; (e) BBr3 1 M solution in CH2Cl2, anhydrous CH2Cl2, −10 to 0 °C, then RT, 1–3 h [46–66%].

A nearly identical synthetic strategy was adopted for the preparation of compounds 8, 9, and 12 (Scheme 2). The only exception is the formation of compounds 44 and 45 by an “Ullmann-type″ reaction that was a copper-catalyzed nucleophilic aromatic substitution between 2-chloropyridine 41 or bromobenzene 42 and 3-bromophenol 18, in the presence of potassium phosphate as the base and anhydrous DMSO as the solvent. This reaction afforded compounds 44 and 45 in low to moderate yields.

Scheme 2

Scheme 2. Synthesis of Compounds 8, 9, and 12a
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aReagents and conditions: (a) for compounds 41 and 42: K3PO4, CuI, anhydrous DMSO, 130 °C, 24 h [11–32%]; for compound 16: anhydrous K2CO3, anhydrous DMF, 110 °C, overnight [84%]; (b) i. tert-butyl 4-methylenepiperidine-1-carboxylate, 9-BBN 0.5 M solution in THF, anhydrous toluene, 115 °C, 1 h; ii. aq. 3.2 M NaOH, Pd(PPh3)4, TBAI, anhydrous toluene, 115 °C, 18 h [30–78%]; (c) HCl 4.0 M solution in dioxane, anhydrous MeOH, anhydrous CH2Cl2, RT, 1 h [90–99%]; (d) 3-methoxybenzoic acid, HATU, DIPEA, anhydrous DMF, RT, 3–12 h [53–72%]; (e) BBr3 1 M solution in CH2Cl2, anhydrous CH2Cl2, −10 to 0 °C, then RT, 1–3 h [29–62%].

The use of 4-bromophenol 43 instead of 3-bromophenol 18 in the first step (step a, Scheme 2) allowed the formation of the different central scaffold bearing the 1,4-disubstituted phenyl ring of the final compound 12 (Scheme 2).

2.3. Enzymatic Assays

The herein reported compounds were tested for their inhibition activity on human MAGL by adopting a spectrophotometric method, which uses 4-nitrophenylacetate as the substrate (Table 1). (33) All the compounds were also evaluated for their inhibition activity on human FAAH, to determine their selectivity, since they all derive from a structural optimization of a fragment belonging to a FAAH inhibitor (Table 1). (36) The enzymatic method used for FAAH assays was similar to that used for MAGL, differing in the used substrate, which is in this case 7-amino-4-methyl coumarin-arachidonamide. (31) The inhibition potencies of the newly synthesized derivatives were compared to the previously published benzoylpiperidine MAGL inhibitors 5a and 5b. (34)
Table 1. In Vitro Inhibitory Activity on Human MAGL and FAAH (hMAGL and hFAAH, IC50, nM)a of Derivatives 7–9, 10a–e, 11a–c, 12, 13, and 40
a

Enzymatic values are the mean of three or more independent experiments, performed in duplicate.

b

Ref (34).

Compound 7, which represents the first synthesized benzylpiperidine MAGL inhibitor, showed encouraging results, possessing an IC50 value on MAGL of 133.9 nM and a good selectivity over FAAH (IC50 = 5.9 μM). The insertion of chlorine atoms on the phenolic ring, as in compounds 10c and 10d, approximately maintains or slightly improves the inhibition activity (IC50 values of 124.6 and 107.2 nM for 10c and 10d, respectively); likewise, the presence of the bromine in compound 10e did not significantly modify the inhibition potency (IC50 = 109.4 nM). On the contrary, when a fluorine atom was introduced in the phenolic ring (compounds 10a and 10b), the inhibition activity improves 1.6-fold in the case of 10b or 5-fold in the case of 10a, compared to initial compound 7; thus, compound 10a reaches an excellent IC50 value of 26.4 nM while still maintaining a good selectivity over FAAH (IC50 = 8.0 μM). Scarce results were provided by the drastic change of the scaffold: the removal of the trifluoromethyl group in compound 8 leads to a maintenance of the inhibition activity shown by derivative 7 (for compound 8: IC50 = 139.3 nM), the substitution of the 5-trifluoromethylpyirdine with a simple benzene ring in compound 9 leads to a slight increase of the IC50 value (148.7 nM), and the worst result concerns compound 12, which dramatically loses potency, showing an IC50 value of 866.7 nM. The shift of the trifluoromethyl group to position 3 of pyridine was detrimental to the activity since a decrease of potency is evident for compound 11a, with an IC50 value of 175.7 nM. Differently, when the CF3 moiety was moved to position 6, the activity improves: compound 11c displays an IC50 value of 36.0 nM. The best result was achieved when CF3 is in position 4 of the pyridine ring in compound 11b, which determines a 10-fold improvement in the IC50 value (IC50 = 13.1 nM) for MAGL and an enhanced selectivity over FAAH (IC50 greater than 10 μM). The combination of the best structural modifications of compound 7, i.e., a fluorine atom in the para position to the phenolic hydroxyl group (as in compound 10a) and the change of position of the trifluoromethyl group from 5 to 4 of the pyridine ring (as in compound 11b), gave rise to compound 13 in which both the modifications exert a synergistic effect on the inhibition activity: 13 shows an IC50 value of 2.0 nM; therefore, it represents the most active MAGL inhibitor of this class but still was endowed with a high degree of selectivity over FAAH (IC50 > 10 μM). Moreover, 13 overtakes benzoylpiperidine 5b in terms of inhibition potency (IC50 = 30.5 nM), which is used as the reference compound. (34) With the aim of confirming the importance of the phenolic OH moiety for the interaction with MAGL, the methoxylated counterpart of compound 13 (compound 40) was tested, and as expected it proved to be inactive (IC50 greater than 1 μM).
To verify whether these compounds could interact with cysteine residues of MAGL, the activity of 13 was also tested in the presence of the thiol-containing agent 1,4-dithio-dl-threitol (DTT). As shown in Figure 4A, the IC50 values of the compound were not significantly influenced by the presence of DTT, thus excluding any significant interaction of compound 13 with MAGL cysteine residues. Furthermore, to confirm the reversible inhibition mechanism, compounds 13 was also subjected to pre-incubation and dilution assays. As shown in Figure 4B, the test suggests a reversible binding mode, as the compound showed very similar activities at all the three different incubation times. As a second test, we investigated the effect of dilution on the inhibition activity. The inhibition produced by incubation with a concentration of 320 nM compound 13 was compared with the inhibition produced by a 40× dilution, and as shown in Figure 4C, the inhibition produced at a concentration of 320 nM was significantly higher compared with that observed at a 40× dilution, comparable to the effect produced by an 8 nM concentration of the compound, thus clearly supporting a reversible mechanism of inhibition. Reference compound JZL-184 was also subjected to the same experimental assays, and as shown in Figure S44, the results confirmed its irreversible mechanism of action with the absence of interactions with DTT.

Figure 4

Figure 4. Analysis of the mechanism of MAGL inhibition of compound 13. (A) Effect of DTT on MAGL inhibition activity. (B) IC50 (nM) values at different preincubation times with MAGL (0, 30, and 60 min). (C) Dilution assay: the first two columns indicate the inhibition percentage of the compound at concentrations of 320 and 8 nM. The third column indicates the inhibition percentage of the compound after dilution (final concentration = 8 nM).

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The mode of inhibition of compound 13 was then evaluated by measuring the Michaelis–Menten kinetics at various inhibitor concentrations. The datasets were plotted as substrate concentration versus enzyme activity and analyzed by applying the mixed model inhibition fit. Kinetic studies indicate for compound 13 an α value greater than 10,000, thus supporting the competitive behavior for this compound, and a measured Ki value of 1.42 ± 0.16 nM (see Figure S45).

2.4. Selectivity Assays

Beyond enzymatic assays on FAAH (Section 2.3.), compound 13 was also profiled for its selectivity toward CB1 and CB2 and it did not significantly bind to any receptor, resulting in IC50 values greater than 10 μM (see Table S4). Then, with the aim to assess the selectivity of 13 in a broader context of the serine hydrolase family, we performed competitive activity-based protein profiling (ABPP) experiments using mouse brain membrane preparations. ABPP is a functional proteomic technology that exploits chemical probes that react with mechanistically related classes of enzymes. (37) TAMRA-fluorophosphonate (TAMRA-FP) is used to visualize serine hydrolases, which include the major eCBs degrading enzymes. (38) An important advantage of ABPP relative to other approaches is that it can detect changes in the activity of very low-abundance enzymes in highly complex samples and can simultaneously assess the potency and selectivity of an inhibitor toward the entire family of serine hydrolases in a specific tissue.
Mouse brain membranes were pre-incubated with control (DMSO), compound 13, and other known inhibitors of serine hydrolases such as JZL-184 (MAGL inhibitor), (9) URB597 (FAAH inhibitor), (39) WWL70 (ABHD6 inhibitor), (40) THL (ABHD6 and ABHD12 inhibitor), (41) and MAFP (unselective serine hydrolase inhibitor) (42) as controls. The TAMRA-FP signal after SDS-PAGE highlighted that compound 13 at a concentration of 10 μM selectively inhibited MAGL (see the two bands associated with MAGL (43) in Figure 5), without affecting other serine hydrolases such as FAAH, ABHD6, and ABHD12, similar to reference MAGL inhibitor JZL-184. Since TAMRA-FP is a highly potent covalent irreversible probe, in this ABPP assay, a reversible inhibitor cannot completely compete with it, despite the high inhibition potency. On the other hand, the covalent irreversible inhibitor JZL-184 can fully compete with the probe. The serine hydrolase bands associated with other enzymes disappeared according to the tested control inhibitor (Figure 5): the FAAH band for URB597, the ABHD6 band for WWL70 and THL, and the ABHD12 band for THL. In the case of pre-treatment with MAFP, all the bands relative to FAAH, MAGL, ABHD6, and ABHD12 disappeared (Figure 5).

Figure 5

Figure 5. ABPP with fluorescent labeling of serine hydrolases in mouse brain membrane homogenates using a TAMRA-FP serine hydrolase probe and different inhibitors as controls. The mouse brain membranes (4 mg/mL) were pre-incubated for 25 min with either DMSO, 13 (10 μM, MAGL inhibitor), JZL-184 (10 μM, MAGL inhibitor), (9) URB597 (4 μM, FAAH inhibitor), (39) WWL70 (10 μM, ABHD6 inhibitor), (40) THL (30 μM, ABHD6 and ABHD12 inhibitor), (41) or MAFP (5 μM, unselective serine hydrolase inhibitor). (42) After additional incubation with TAMRA-FP (125 nM) for 25 min, the samples were separated in SDS-PAGE. A representative image of the TAMRA-FP signal after SDS-PAGE is shown. The presented results could be observed in three independent experiments.

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2.5. Molecular Modeling Studies

To rationalize how this series of inhibitors could interact with MAGL into its binding site, molecular modeling studies were carried out using compound 11b as a reference. The ligand was docked into the crystal structure of MAGL (PDB code 5ZUN) using a robust protocol based on AUTODOCK4 software. The eight potential MAGL-11b complexes predicted by the protocol were then subjected to 1.05 μs of molecular dynamics (MD) simulations and analyzed in terms of RMSD of the ligand disposition during the MD as well as ligand–protein binding free energy evaluations based on the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method (see the Experimental Section for details). The results highlighted binding pose 3 as the most reliable, being the only one associated with an average ligand RMSD below 2.0 Å (Table S1) and corresponding to an interaction energy (ΔPBSA = −8.9 kcal/mol) at least 3.6 kcal/mol higher than those estimated for all other binding poses (Table S2). Figure 6 shows the energy-minimized average structure of MAGL complexed with compound 11b, in the proposed binding mode, obtained from the last 500 ns of MD simulation. The ligand presents a sort of L-shaped binding conformation bent at the level of the methylene linker connecting the two main structural portions of the molecule, in which the benzoylpiperidine moiety occupies the central region of MAGL catalytic site, while the phenoxypyridine fragment is placed at the entrance of the binding cavity, thus closing its access. Due to the absence of a central carbonyl group in the ligand able to interact with the oxyanion hole residues A51 and M123, as observed in the parent MAGL inhibitor 5b, (35) the disposition of compound 11b is shifted toward the entrance of the binding site (Figure S46). Nevertheless, the ligand still establishes the key H-bond interactions with A51 and M123, maintained for more than 90% of the MD simulation, through its benzoyl oxygen. Moreover, the phenolic OH group forms a strong H-bond with H121 that is observed for the whole simulation and thus contributes to firmly anchoring the ligand to the enzyme binding site. Finally, the phenol moiety shows hydrophobic contacts with A51, L184, and H266, while the adjacent piperidine fragment forms lipophilic interactions with the side chains of L148, I179, L213, and L241. The phenyl ring belonging to the phenoxypyridine moiety of 11b fits well the rather narrow entrance of the MAGL catalytic site delimited by S155, F159, I179, L205, and L241; in fact, the phenyl ring shows extensive hydrophobic interactions with I179, L205, and L241 as well as a partial T-shaped stacking with F159. Finally, the trifluoropyridine moiety of 11b is placed in a solvent-exposed area adjacent to the entrance of the binding pocket and MAGL lid domain. In particular, the trifluoromethyl group is placed in a small amphiphilic pocket delimited by S155, T158, and F159, forming van der Waals interactions with these residues, while the pyridine ring shows hydrophobic contacts predominantly with I179. These interactions significantly stabilize the orientation of the trifluoromethyl fragment and thus the whole binding mode of the inhibitor, also limiting its exposure to the solvent.

Figure 6

Figure 6. Minimized average structure of hMAGL in complex with compound 11b in the predicted binding pose. The protein residues surrounding the ligand are shown. Ligand–protein hydrogen bonds are highlighted with black lines. The inner surface of the protein binding site is shown in gray (PDB code 5ZUN).

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The minimized average structure of MAGL bound to 11b was then used as a reference for predicting the binding mode of the other compounds of the series, namely, 7–9, 10a–e, 11a, 11c, 12, 13, and 40, which were subjected to an analogous docking/MD protocol. In addition, ligand–protein binding free energy evaluations were performed using the MM-PBSA approach based on the results of the MD simulations obtained for each MAGL–ligand complex, looking for a correlation between the binding energies estimated for the ligands and their corresponding enzymatic activity that could confirm the reliability of the computational protocol and help interpret the SAR data derived from the MAGL inhibition assays. Considering the significant level of polarizability of the key phenolic moiety of the analyzed ligands, as well as of MAGL binding site, due to the presence of charged residues such as E53 within the inner portion of the catalytic site, various MM-PBSA protocols differing for the internal dielectric constant (εint) value were tested with the aim of identifying the most suitable one for correlating binding energies with activities. (44)Figure 7 shows the correlation obtained between the compounds activities and the binding energies estimated using the best MM-PBSA protocol evaluated, obtained using εint = 4, which showed a squared correlation coefficient of 0.79 (see also Tables S3 and S4).

Figure 7

Figure 7. Correlation between the compound’s activities expressed as pIC50 values and the binding energies estimated using the best MM-PBSA protocol (εint = 4) expressed in kcal/mol.

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The results obtained confirmed the reliability of the binding modes predicted for the series of ligands and helped us decipher the SAR data obtained from the experimental assays. Compound 7, bearing the trifluoromethyl group in position 5 of the pyridine ring, showed a reduced activity with respect to compound 11b and was associated with a 2.7 kcal/mol lower binding free energy. As shown in Figure S1A, the ligand assumes a binding mode very similar to that predicted for 11b, but the trifluoromethyl group is placed outside the amphiphilic pocket formed by S155, F159, I179, L205, and L241, thus losing the interaction with these residues and remaining fully exposed to the solvent. These features may explain the reduced activity of compound 7 compared to 11b. The same considerations are also valid for compound 8, which does not show any interactions with the residues of the amphiphilic pocket because it lacks the terminal trifluoromethyl group. In fact, the ligand shows a MAGL inhibitory activity comparable to 7 and was also associated with the same binding free energy (Table S4). In line with these considerations, the significant drop of activity with respect to 11b observed in compound 12, for which one of the lowest binding energies was estimated (Table S4), could be determined by the complete loss of interactions of its trifluoromethylpyridine moiety, which is connected differently to the rest of the molecule with respect to the other derivatives. Due to this structural change, the trifluoromethylpyridine fragment of the compound is placed in the middle of the solvent-exposed region of the binding site entrance, completely outside the catalytic pocket (Figure S1B); therefore, the ligand loses not only the interactions of the trifluoromethyl group with the residues of the amphiphilic pocket but also the lipophilic contacts between the pyridine ring and I179 as well as the partial T-shaped stacking formed by the adjacent ligand phenyl ring with F159. The destabilizing effect of these features in the binding mode of compound 12 is highlighted by the RMSD of the ligand during the MD, which shows an average value around 3 Å due to the high mobility of the trifluoromethylpyridine moiety. Finally, the highest binding free energy was correctly estimated for compound 13 (Table S4), bearing a fluorine in the para position to the OH group of the phenol ring, for which a binding mode fully comparable with that of 11b was predicted (Figure S47). The fluorine atom is supposed to increase the polarization of the ligand hydroxyl group, thus boosting the strength of the H-bond formed with H121, similarly to what was observed for the series of benzoylpiperidines to which the parent compound 5b belongs. In agreement with this hypothesis, the increase in binding free energy observed for compound 13 with respect to 11b seems to be due to more favorable polar energetic terms and, in particular, to stronger ligand–protein electrostatic interactions (Table S6). On the contrary, the methylation of the OH group prevents the formation of such H-bonds, thus determining the dramatic drop of activity of compound 40, for which one of the lowest binding energies was calculated.

2.6. Biological Studies in Pancreatic Cancer Cells

2.6.1. MAGL Expression in Pancreatic Cancer Cells

MAGL is overexpressed in different tumor types, including pancreatic adenocarcinoma (PAAD) as demonstrated by the analysis of RNA sequencing expression data of 179 pancreatic tumors and 171 normal pancreatic samples from the TCGA and GTEx projects (45) as reported in Figure 8A. Applying the online genomics and visualization platform R2 (http://r2.amc.nl) on the pancreatic adenocarcinoma TCGA dataset (178-rsem-tcgars), 57 patients were classified with high MAGL mRNA expression and 89 with a low MAGL mRNA expression, while 32 samples were excluded due to missing survival data. In the computed Kaplan–Meier curve (Figure 8B), it is shown that a high MAGL mRNA level is significantly (p = 0.007) correlated with a poor overall survival probability compared to a low expression.

Figure 8

Figure 8. MAGL gene expression levels. (A) MAGL mRNA is more expressed in cancer tissues than in normal tissues (http://gepia.cancer-pku.cn/detail.php?gene=MAGL). Pancreatic cancer tissues are among the tumor tissues with the highest expression levels of MAGL. (B) MAGL mRNA expression is a prognostic factor in pancreatic cancer. The expression cutoff between patients with high versus low expression of MAGL (5132, RNA expression units) was obtained by the “R2: Genomics Analysis and Visualization Platform”. (C) Two primary pancreatic cancer cell cultures (PDAC2 and PDAC3) originating from patients undergoing surgery for pancreatic cancer showed significantly different expression levels of MAGL mRNA.

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Our next-generation RNA sequencing (NGS) data showed that MAGL mRNA is expressed in two primary pancreatic cancer cell cultures, with the highest fragments per kilobase million (FPKM) score in PDAC3 (pancreatic ductal adenocarcinoma) cells (Figure 8C), which originated from the most clinically aggressive tumor. (46) Analyzing our transcriptomic data, we found out that PDAC3 cells have a significantly higher expression of arsenite-resistance protein 2 (ARS2), a zinc finger protein that is essential for early mammalian development. Of note, a recent study in glioblastoma models demonstrated that a number of pro-tumorigenic genes are potentially regulated at the transcriptional level by ARS2 and specifically identified MAGL as a novel target of ARS2. (47) This might at least in part explain MAGL differential expression and its impact on cancer aggressiveness. However, the role of ARS2 in the pathophysiology of pancreatic cancer has still to be clarified.

2.6.2. Antiproliferative Activity and Effects on Induction of Apoptosis

The most active compound of this series of benzylpiperidine derivatives, inhibitor 13, was tested in antiproliferative activity assays on different pancreatic ductal adenocarcinoma cancer cells, including the SUIT-2 immortalized cell line, PDAC2 and PDAC3 primary cell cultures, and immortalized ductal normal cells HPNE, by sulforhodamine-B (SRB) assay. PDAC2 and PDAC3 cell cultures were selected as cellular models in most of the following experiments because they maintain the same metastatic, genetic, and histopathological features of the primary tumor.
PDAC2, PDAC3, and SUIT-2 cells showed different sensitivities to compound 13 (Figure 9A). Both SUIT-2 and PDAC2 cells, despite being immortalized and primary cells, show a similar sensitivity. More specifically, the IC50 values of compound 13 in the antiproliferative activity assays with SUIT-2 and PDAC2 cells were 11.19 and 12.61 μM, respectively. However, PDAC3 cells were slightly more sensitive to MAGL inhibitor 13 with an IC50 value of 7.25 μM, and this difference could be in part explained considering the higher MAGL mRNA overexpression in PDAC3 compared to PDAC2 primary cell culture. Of note, the immortalized pancreatic ductal normal cells were not sensitive to compound 13. Reference MAGL inhibitors JZL-184 and ABX-1431 were effective in reducing PDAC3 proliferation, as demonstrated by cell growth curves (Figure 9B). Previous studies suggested that inhibition of MAGL might increase apoptosis and tumor cell sensitivity to chemotherapy. (35,48) Therefore, we evaluated apoptosis induction by 13 using two different assays and compared the pro-apoptotic effects of 13 with gemcitabine, a drug used for the standard treatment of pancreatic cancer (which has IC50 values in the nanomolar range, as reported in our previous studies (34)), and with the two reference MAGL inhibitors, JZL-184 and ABX-1431. In particular, the Annexin-V staining showed that 13 strongly enhanced apoptosis induction in PDAC3 cells (Figure 9C). Remarkably, this compound was able to significantly increase apoptosis induction compared to untreated cells. Gemcitabine has a similar effect and the combination led to an addictive effect. Similar results were observed in PDAC2 cells (i.e., apoptosis fold induction/change of approximately 4, 5, and 9 after treatment with 13, gemcitabine and their combination). JZL-184 and ABX-1431 and their relative combinations with gemcitabine induced apoptosis in PDAC3 cells with a similar or slightly lower potency compared to that of 13 (Figure 9C). Moreover, we demonstrated that the apoptotic response of both PDAC3 and PDAC2 cells after exposure to compound 13 was associated with the concomitant stimulation of caspase-3 (Figure 9D). Indeed, our immunoassay measured significantly higher levels of active caspase-3 in PDAC3 cells treated with 13 or gemcitabine compared to untreated cells. PDAC2 cells showed similar results with slightly lower levels of active caspase-3 (0.35, 0.88, and 1.12 ng/mL in untreated, gemcitabine-treated, and 13-treated cells, respectively). Even in this case, JZL-184 and ABX-1431 were similarly effective in stimulating caspase-3 in PDAC3 cells (Figure 9D).

Figure 9

Figure 9. Antiproliferative and pro-apoptotic effects of MAGL inhibitor 13. (A) IC50 of compound 13 in different pancreatic cancer models and in the immortalized ductal cells HPNE. (B) Representative curves of PDAC3 cells growth inhibitory effects of 13, JZL-184 and ABX-1431, as control. (C) Induction of apoptosis and (D) levels of active caspase-3 in PDAC3 cells treated with 13, gemcitabine, JZL-184, and ABX-1431 for 72 h, compared to control/untreated cells (value = 1, as illustrated by the dashed line). Measurements were performed in triplicate, and data are presented as means ± SEM. *p < 0.05 versus control; #p < 0.05 versus gemcitabine.

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2.6.3. Cell Migration Assays

It is well known that the early metastatic behavior of PDAC is responsible for the poor prognosis of this tumor. Therefore, new therapeutic agents are needed to overcome PDAC aggressiveness and counteract PDAC metastasis. The effect of compound 13 on cell migration was investigated using the wound-healing assay and compared with two reference MAGL inhibitors, JZL-184 and ABX-1431. In the PDAC3 cells (Figure 10), 59 ± 8% of the scratch area was closed after 20 h when treated with 0.1% DMSO (control). After treatment with JZL-184 or ABX-1431, 43 ± 8% or 45 ± 7% of the scratch was closed, respectively, while 13 induced a significant reduction of migration, with 38 ± 6% of the scratch closed (Figure 10). In PDAC2, the control showed 70 ± 3% gap closure, and in these cells, 13 treatment resulted in a closure with 61 ± 3%.

Figure 10

Figure 10. Antimigratory effects of MAGL inhibitors. Statistical evaluation of the results of the wound-healing/migration assay on the PDAC3 cells 20 h after scratch induction and treatment. The percentages of scratch closure for control, 13-, JZL-184-, or ABX-1431-treated cells were compared with one-way analysis of variance (ANOVA)/t test. *p < 0.05 versus control.

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2.6.4. Synergistic Interaction of Compound 13 with Gemcitabine and Potential Mechanisms Underlying Its Effects on Apoptosis, Migration, and Potentiation of Gemcitabine Activity

The pharmacological interaction of the MAGL inhibitor 13 and gemcitabine was determined on PDAC3 cells (Figure 11A) using fixed concentrations of compound 13 corresponding to its IC25 or IC50 value together with a 0–1.25 μM concentration range of gemcitabine. The PDAC3 cells treated with gemcitabine and compound 13 at IC50 showed synergy, whereas compound 13 at IC25 was additive. The PDAC2 cells showed a slight synergy between gemcitabine and 13 at IC50 (CI, 0.76) and additive interaction with 13 at IC25 (CI, 0.96).

Figure 11

Figure 11. Combination assay and modulation of gene expression. (A) CI values of gemcitabine (GEM) combined with compound 13 at IC50 and IC25. The upper line represents an antagonistic CI > 1.2, the lower bar represents a synergistic CI < 0.8. (B) Combined results of different PCR experiments, evaluating the effect of GEM, 13, and JZL-184 on potential determinants of apoptosis induction, migration, and synergistic interaction with gemcitabine compared to control/untreated cells (value = 1, as illustrated by the dashed line). Measurements were performed in triplicate, and data are presented as means ± SEM.

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To elucidate the previous data on apoptosis and migration and the mechanism of interaction, further studies focused on several potential cellular determinants and effectors of drug activities, such as the anti-apoptotic factor Bcl-2, the key matrix metalloproteinase 9 (MMP9), which promotes cell migration, and the main gemcitabine transporter, the human equilibrative nucleoside transporter 1 (hENT1). As shown in Figure 11B, PCR analyses demonstrated the modulation of several of these important factors by compound 13. In particular, 13 induced a slight reduction of Bcl-2, a significant reduction of MMP9, and an increase of hENT1, which might at least in part explain the induction of apoptosis, the antimigratory, activity and the synergistic interaction with gemcitabine, as described previously. (49)

2.7. In Vitro ADME Assays

In vitro ADME properties were assessed for the best MAGL inhibitor of this series, compound 13, in comparison with the previously published benzoylpiperidine derivative 5c, (35) and the results are reported in Table 2. Permeability was measured by the parallel artificial membrane permeability assay (PAMPA) to evaluate the ability of these compounds to reach MAGL in the cytoplasm. Interestingly, compound 13 displayed an increased membrane permeability (Papp of 3.695 × 10–6 cm/s) and a reduced membrane retention (38.9%) compared to 5c (Papp of 2.067 × 10–6 cm/s and membrane retention of 52.6%). Moreover, compound 13 provided a slightly better value of metabolic stability in human liver microsomes (92.7%, expressed as percentage of unmodified compound) compared to 5c (90.5%). Water solubility and stability tests performed in polar solvents (methanol and PBS) and in human plasma gave similar results for both compounds since both displayed solubility lower than 1 ng/mL and showed to be stable in polar solvents and human plasma for more than 24 h.
Table 2. In Vitro ADME Assays of Compounds 5c and 13
    stability
compoundwater solubility ng/mL (logS)Papp × 10–6 cm/sec (RM %)metabolic stability %MeOH (h)PBS pH 7.4 (h)human plasma (h)
5c<12.06790.5>24>24>24
(< −8.702)(52.6)
13<13.69592.7>24>24>24
(< −8.676)(38.9)

3. Conclusions

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In this work, we designed and synthesized a new class of MAGL inhibitors based on a benzylpiperidine scaffold. Among this series, we identified compound 13 as the most potent benzylpiperidine derivative with an IC50 value of 2.0 nM on isolated enzyme. Compound 13 is characterized by a reversible mechanism of action, a competitive behavior (Ki value of 1.42 nM), and a notable selectivity for MAGL compared to other targets of the endocannabinoid system, such as FAAH, CB1, CB2, ABHD6, and ABHD12.
The binding disposition of this class of compounds into the MAGL active site was suggested by molecular docking studies followed by molecular dynamics simulations and binding free energy evaluations.
After demonstrating that MAGL mRNA is overexpressed in pancreatic ductal adenocarcinoma tissues compared to normal pancreatic tissues and considering that MAGL mRNA overexpression was associated with poor patients’ prognosis, compound 13 was also subjected to preliminary pharmacological assays on pancreatic cancer cells. In these studies, 13 showed a moderate antiproliferative activity on SUIT-2 immortalized cancer cells and on PDAC2 and PDAC3 primary cell cultures (IC50 values ranging from 7.25 to 12.61 μM) compared to normal cells HPNE (IC50 > 20 μM). Moreover, compound 13 not only remarkably enhanced apoptosis induction in PDAC cells, but it also significantly reduced cell migration and exerted a synergistic effect when combined with the chemotherapeutic drug gemcitabine. Considering that the chosen PDAC preclinical model shares the same molecular complexity of the originator tumor, representing an important tool for the experimental testing of anti-cancer agents, all these results support the potential applicability to the clinical setting of this class of inhibitors.
Moreover, the completely new scaffold of compound 13, deriving from the merging of the FAAH inhibitor PF-3845 6 (Figure 2) and the benzoylpiperidine-based MAGL inhibitors (exemplified by compound 5a, Figure 2), paves the way for a new chemical class of MAGL inhibitors.
Further research to confirm cellular MAGL engagement and structural optimization of this new class of MAGL inhibitors is ongoing.

4. Experimental Section

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4.1. Synthesis: General Procedures and Materials

All solvents and chemicals were used as purchased without further purification. Chromatographic separations were performed on silica gel columns by flash chromatography (Kieselgel 40, 0.040–0.063 mm; Merck). Reactions were followed by thin layer chromatography (TLC) on Merck aluminum silica gel (60 F254) sheets that were visualized under a UV lamp. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as the drying agent. Proton (1H) and carbon (13C) NMR spectra were obtained with a Bruker Avance III 400 MHz spectrometer using the indicated deuterated solvents. Chemical shifts are given in parts per million (ppm) (δ relative to residual solvent peak for 1H and 13C). 1H-NMR spectra are reported in this order: multiplicity and number of protons. Standard abbreviation indicating the multiplicity was used as follows: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, t = triplet, tt = triplet of triplets, dt = doublet of triplets, td = triplet of doublets, m = multiplet, bm = broad multiplet and bs = broad singlet. HPLC analysis was used to determine purity: all target compounds (i.e., assessed in biological assays) were ≥95% pure by HPLC, as confirmed via UV detection (λ = 254 nm). Analytical reversed-phase HPLC was conducted using a Kinetex EVO C18 column (5 μm, 150 × 4.6 mm, Phenomenex, Inc.); eluent A, water; eluent B, CH3CN; after 5 min. at 25% B, a gradient was formed from 25% to 75% of B in 5 min and held at 75% of B for 10 min; flow rate was 1 mL/min. HPLC analyses were performed at 254 nm. The ESI-MS spectra were recorded by direct injection at a 5 μL min–1 flow rate in an Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with a HESI source. The working conditions were as follows: positive polarity, spray voltage of 3.4 kV, capillary temperature of 290 °C, S-lens RF level 50. The sheath and the auxiliary gases were set at 24 and 5 (arbitrary units), respectively. For acquisition and analysis, Xcalibur 4.2 software (Thermo) was used. For spectra acquisition, a nominal resolution (at m/z 200) of 140,000 was used. Yields refer to isolated and purified products derived from non-optimized procedures. Compound 2-chloro-5-methoxybenzoic acid was synthesized as previously reported. (32)

4.1.1. General Procedure for the Synthesis of Compounds 19–22, 46

Commercially available 3-bromophenol 18 or 4-bromophenol 43 (250 mg, 1 equiv) and 2-chloro-3-trifluoromethylpyridine 14, 2-chloro-4-trifluoromethylpyridine 15, 2-chloro-5-trifluoromethylpyridine 16, or 2-chloro-6-trifluoromethylpyridine 17 (1 equiv) were mixed in anhydrous DMF (3.7 mL) and treated with anhydrous potassium carbonate (2 equiv). The mixture was stirred at 110 °C overnight, then cooled at room temperature, and partitioned between water and ethyl acetate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. Silica gel column chromatography (1–5% EtOAc in n-hexane or petroleum ether) afforded the title compounds.
4.1.1.1. 2-(3-Bromophenoxy)-3-(trifluoromethyl)pyridine (19)
Light yellow oil. 82% yield from 14 and 18. 1H NMR (CDCl3) δ (ppm): 7.08–7.17 (m, 2H), 7.29 (t, 1H, J = 8.1 Hz), 7.34–7.42 (m, 2H), 7.97–8.04 (m, 1H), 8.28–8.34 (m, 1H).
4.1.1.2. 2-(3-Bromophenoxy)-4-(trifluoromethyl)pyridine (20)
Light yellow oil. 99% yield from 15 and 18. 1H-NMR (CDCl3) δ (ppm): 7.11 (ddd, 1H, J = 8.1, 2.2, 1.0 Hz), 7.17–7.20 (m, 1H), 7.21–7.25 (m, 1H), 7.30 (t, 1H, J = 8.1 Hz), 7.34 (t, 1H, J = 2.0 Hz), 7.39 (ddd, 1H, J = 8.0, 1.8, 1.0 Hz), 8.33 (d, 1H, J = 5.2 Hz).
4.1.1.3. 2-(3-Bromophenoxy)-5-(trifluoromethyl)pyridine (21)
Colorless oil. 77% yield from 16 and 18. 1H-NMR (CDCl3) δ (ppm): 7.04 (d, 1H, J = 8.7 Hz), 7.11 (ddd, 1H, J = 8.2, 2.3, 1.0 Hz), 7.30 (t, 1H, J = 8.1 Hz), 7.34 (t, 1H, J = 2.1 Hz), 7.40 (ddd, 1H, J = 8.0, 1.8, 1.0 Hz), 7.93 (dd, 1H, J = 8.6, 2.2 Hz), 8.42–8.47 (m, 1H).
4.1.1.4. 2-(3-Bromophenoxy)-6-(trifluoromethyl)pyridine (22)
Light yellow oil. 82% yield from 17 and 18. 1H-NMR (CDCl3) δ (ppm): 7.07 (d, 1H, J = 8.5 Hz), 7.11–7.17 (m, 1H), 7.26–7.31 (m, 1H), 7.34–7.39 (m, 2H), 7.41 (d, 1H, J = 7.4 Hz), 7.86 (t, 1H, J = 8.0 Hz).
4.1.1.5. 2-(4-Bromophenoxy)-5-(trifluoromethyl)pyridine (46)
Colorless liquid. 84% yield from 16 and 43. 1H-NMR (CDCl3) δ (ppm): 7.01–7.08 (m, 3H), 7.54 (AA’XX’, 2H, JAX = 8.8 Hz, JAA’/XX’ = 2.6 Hz), 7.92 (dd, 1H, J = 8.7, 2.5 Hz), 8.40–8.45 (m, 1H).

4.1.2. General Procedure for the Synthesis of Compounds 44 and 45

A vial was loaded with K3PO4 (2 equiv) and commercially available 3-bromophenol 18 (600 mg, 2 equiv). Then, in an inert atmosphere, copper(I) iodide (0.1 equiv) in anhydrous DMSO (1.4 mL) and commercially available 2-chloropyridine 41 or bromobenzene 42 (1 equiv) were added. The vial was sealed, and the reaction mixture was stirred at 130 °C. After the reaction mixture was heated for 24 h, it was cooled to room temperature and the workup consisted in the filtration of the reaction mixture through a Celite pad, washing it repeatedly with EtOAc. The filtrate was concentrated under vacuum to give a crude residue, which was then purified by flash column chromatography (silica gel, 2–5% EtOAc in n-hexane or petroleum ether), to give the desired compounds.
4.1.2.1. 2-(3-Bromophenoxy)pyridine (44)
Colorless oil. 32% yield from 41 and 18. 1H NMR (CDCl3) δ (ppm): 6.94 (dt, 1H, J = 8.3, 0.8 Hz), 7.05 (ddd, 1H, J = 7.2, 5.0, 0.9 Hz), 7.09 (ddd, 1H, J = 8.1, 2.3, 1.1 Hz), 7.27 (t, 1H, J = 8.0 Hz), 7.29–7.36 (m, 2H), 7.73 (ddd, 1H, J = 8.8, 6.8, 2.0 Hz), 8.22 (ddd, 1H, J = 5.0, 2.0, 0.7 Hz).
4.1.2.2. 1-Bromo-3-phenoxybenzene (45)
Colorless oil. 11% yield from 42 and 18. 1H NMR (CDCl3) δ (ppm): 6.94 (ddd, 1H, J = 7.7, 2.3, 1.5 Hz), 6.99–7.05 (m, 2H), 7.12–7.24 (m, 4H), 7.33–7.40 (m, 2H).

4.1.3. General Procedure for the Synthesis of Compounds 23–26, 47–49

tert-Butyl-4-methylenepiperidine-1-carboxylate (1.25 equiv) in anhydrous toluene (2.2 mL) was treated with 9-BBN (0.5 M in THF, 1.25 equiv) and heated at 115 °C for 1 h. The reaction mixture was cooled and treated with NaOH (3.2 M aqueous solution, 3 equiv) followed by Pd(PPh3)4 (0.03 equiv). Finally, intermediates 19–22 and 44–46 (370 mg, 1 equiv) in anhydrous toluene (0.9 mL) and tetrabutylammonium iodide (0.5 equiv) were added. The reaction mixture was placed under argon and heated at 115 °C. After 18 h, the mixture was cooled and partitioned between EtOAc and saturated aqueous NaHCO3. The organic layer was separated and dried over sodium sulfate, filtered, and concentrated to obtain a crude, which was purified by silica gel column chromatography (5–20% EtOAc in n-hexane or petroleum ether) to give the desired products.
4.1.3.1. tert-Butyl-4-(3-((3-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidine-1-carboxylate (23)
Yellow oil. 46% yield from 19. 1H-NMR (CDCl3) δ (ppm): 1.44 (s, 9H), 1.49–1.91 (bm, 5H), 2.55 (d, 2H, J = 6.8 Hz), 2.64 (td, 2H, J = 13.0, 2.5 Hz), 4.02–4.11 (m, 2H), 6.95 (t, 1H, J = 1.8 Hz), 6.98–7-04 (m, 2H), 7.08 (ddd, 1H, J = 7.5, 5.0, 0.7 Hz), 7.33 (t, 1H, J = 7.9 Hz), 7.96–8.01 (m, 1H), 8.26–8.31 (m, 1H).
4.1.3.2. tert-Butyl-4-(3-((4-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidine-1-carboxylate (24)
Orange oil. 99% yield from 20. 1H-NMR (CDCl3) δ (ppm): 1.45 (s, 9H), 1.59–1.74 (m, 3H), 1.75–1.92 (m, 1H), 2.56 (d, 2H, J = 6.8 Hz), 2.64 (td, 2H, J = 12.9, 2.2 Hz), 4.02–4.12 (m, 2H), 4.31–4.43 (bm, 1H), 6.92 (t, 1H, J = 1.9 Hz), 6.99 (ddd, 1H, J = 8.1, 2.3, 0.9 Hz), 7.01–7.05 (m, 1H), 7.10–7.14 (m, 1H), 7.17–7.22 (m, 1H), 7.34 (t, 1H, J = 7.8 Hz), 8.33 (d, 1H, J = 5.2 Hz).
4.1.3.3. tert-Butyl-4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidine-1-carboxylate (25)
Colorless oil. 99% yield from 21. 1H-NMR (CDCl3) δ (ppm): 1.05–1.23 (m, 3H), 1.45 (s, 9H), 1.60–1.74 (m, 3H), 2.56 (d, 2H, J = 6.8 Hz), 2.59–2.70 (bm, 2H), 4.00–4.15 (bm, 1H), 6.91–6.94 (m, 1H), 6.97–7.01 (m, 2H), 7.01–7.06 (m, 1H), 7.34 (t, 1H, J = 7.9 Hz), 7.90 (dd, 1H, J = 8.6, 2.1 Hz), 8.42–8.47 (m, 1H).
4.1.3.4. tert-Butyl-4-(3-((6-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidine-1-carboxylate (26)
Colorless oil. 97% yield from 22. 1H-NMR (CDCl3) δ (ppm): 1.45 (s, 9H), 1.50–1.73 (bm, 3H), 1.78–1.91 (bm, 2H), 2.54 (d, 2H, J = 6.8 Hz), 2.64 (td, 2H, J = 12.6, 2.6 Hz), 4.03–4.12 (m, 2H), 6.95–7.06 (m, 4H), 7.31 (t, 1H, J = 7.8 Hz), 7.37 (d, 1H, J = 7.4 Hz), 7.82 (t, 1H, J = 7.9 Hz).
4.1.3.5. tert-Butyl-4-(3-(pyridin-2-yloxy)benzyl)piperidine-1-carboxylate (47)
Colorless oil. 30% yield from 44. 1H-NMR (CDCl3) δ (ppm): 1.46 (s, 9H), 1.48–1.77 (m, 5H), 2.54 (d, 2H, J = 6.9 Hz), 2.58–2.70 (m, 2H), 3.95–4.18 (bm, 2H), 6.85–6.93 (m, 3H), 6.95–7.02 (m, 2H), 7.30 (t, 1H, J = 7.8 Hz), 7.65–7.72 (m, 1H), 8.21 (ddd, 1H, J = 5.0, 2.1, 0.8 Hz).
4.1.3.6. tert-Butyl 4-(3-phenoxybenzyl)piperidine-1-carboxylate (48)
Light yellow oil. 78% yield from 45. 1H-NMR (CDCl3) δ (ppm): 1.45 (s, 9H), 1.51–1.71 (m, 4H), 1.83–1.91 (m, 1H), 2.48–2.56 (m, 2H), 2.64 (td, 2H, J = 12.9, 2.2), 4.01–4.12 (m, 2H), 6.79–6.85 (m, 2H), 6.86–6.90 (m, 1H), 6.97–7.03 (m, 2H), 7.07–7.15 (m, 1H), 7.23 (t, 1H, J = 7.8 Hz), 7.27–7.36 (m, 2H).
4.1.3.7. tert-Butyl-4-(4-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidine-1-carboxylate (49)
Colorless oil. 70% yield from 46. 1H-NMR (CDCl3) δ (ppm): 1.10–1.22 (bm, 1H), 1.45 (s, 9H), 1.59–1.71 (bm, 3H), 1.80–1.92 (bm, 1H), 2.56 (d, 2H, J = 6.9 Hz), 2.60–2.70 (m, 2H), 4.04–4.13 (m, 2H), 6.99 (d, 1H, J = 6.3 Hz), 7.06 (d, 2H, J = 8.4 Hz), 7.19 (d, 2H, J = 8.4 Hz), 7.89 (dd, 1H, J = 8.7, 2.4 Hz), 8.41–8.47 (m, 1H).

4.1.4. General Procedure for the Synthesis of Compounds 27–30, 50–52

N-Boc-piperidine intermediates 23–26 and 47–49 (490 mg, 1 equiv) were dissolved in methanol (1.7 mL) and dichloromethane (1.7 mL), treated dropwise with HCl (4.0 M in dioxane, 6 equiv), and stirred at room temperature for 1 h. Toluene (1.8 mL) was added, and the reaction mixture was concentrated under nitrogen flux. A second evaporation from toluene (1.8 mL) followed by high vacuum afforded the title compounds, which were used in the next step without further purification.
4.1.4.1. 2-(3-(Piperidin-4-ylmethyl)phenoxy)-3-(trifluoromethyl)pyridine Hydrochloride (27)
White solid. 99% yield from 23. 1H-NMR (D2O) δ (ppm): 1.32–2.02 (m, 5H), 2.64 (d, 2H, J = 5.6 Hz), 2.83–3.01 (m, 2H), 3.31–3.48 (m, 2H), 6.96–7.12 (m, 2H), 7.12–7.24 (m, 1H), 7.24–7.36 (m, 1H), 7.37–7.50 (m, 1H), 8.14–8.28 (m, 2H).
4.1.4.2. 2-(3-(Piperidin-4-ylmethyl)phenoxy)-4-(trifluoromethyl)pyridine Hydrochloride (28)
White solid. 99% yield from 24. 1H-NMR (D2O) δ (ppm): 1.30–2.03 (m, 4H), 2.66 (d, 2H, J = 6.8 Hz), 2.87–3.02 (m, 2H), 3.37–3.46 (m, 2H), 4.29–4.47 (bm, 1H), 7.03–7.11 (m, 2H), 7.20 (d, 1H, J = 7.5 Hz), 7.30–7.35 (m, 1H), 7.41–7.50 (m, 2H), 8.30 (d, 1H, J = 5.2 Hz).
4.1.4.3. 2-(3-(Piperidin-4-ylmethyl)phenoxy)-5-(trifluoromethyl)pyridine Hydrochloride (29)
Light yellow solid. 99% yield from 25. 1H-NMR (D2O) δ (ppm): 1.31–1.95 (m, 4H), 2.63 (d, 2H, J = 6.7 Hz), 2.85–2.98 (m, 2H), 3.34–3.44 (m, 2H), 4.33–4.42 (bm, 1H), 7.01–7.08 (m, 2H), 7.12 (d, 1H, J = 8.8 Hz), 7.18 (d, 1H, J = 7.8 Hz), 7.42 (t, 1H, J = 7.8 Hz), 8.12 (dd, 1H, J = 8.5, 2.3 Hz), 8.37–8.43 (m, 1H).
4.1.4.4. 2-(3-(Piperidin-4-ylmethyl)phenoxy)-6-(trifluoromethyl)pyridine Hydrochloride (30)
White solid. 99% yield from 26. 1H-NMR (D2O) δ (ppm): 1.30–1.95 (bm, 5H), 2.62 (d, 2H, J = 6.4 Hz), 2.83–2.98 (bm, 2H), 3.32–3.45 (bm, 2H), 7.00–7.09 (m, 2H), 7.11–7.20 (m, 2H), 7.41 (t, 1H, J = 8.2 Hz), 7.59 (d, 1H, J = 7.4 Hz), 8.01 (t, 1H, J = 7.6 Hz).
4.1.4.5. 2-(3-(Piperidin-4-ylmethyl)phenoxy)pyridine Hydrochloride (50)
Yellow solid. 90% yield from 47. 1H-NMR (D2O) δ (ppm): 1.35–1.53 (m, 3H), 1.79–2.00 (m, 2H), 2.68 (d, 2H, J = 7.0 Hz), 2.93 (td, 2H, J = 12.9, 2.5 Hz), 3.35–3.45 (m, 2H), 7.14–7.20 (m, 3H), 7.28–7.32 (m, 1H), 7.47–7.55 (m, 2H), 8.31 (ddd, 1H, J = 8.8, 7.0, 1.9 Hz), 8.34–8.38 (m, 1H).
4.1.4.6. 4-(3-Phenoxybenzyl)Piperidine Hydrochloride (51)
Yellow oil. 99% yield from 48. 1H-NMR (D2O) δ (ppm): 1.80–1.97 (m, 4H), 2.25–2.40 (bm, 1H), 2.56–2.68 (m, 2H), 2.87–3.01 (m, 2H), 3.34–3.48 (m, 2H), 6.89–7.00 (m, 2H), 7.03–7.13 (m, 2H), 7.15–7.33 (m, 3H), 7.34–7.50 (m, 2H).
4.1.4.7. 2-(4-(Piperidin-4-Ylmethyl)Phenoxy)-5-(Trifluoromethyl)Pyridine Hydrochloride (52)
White solid. 91% yield from 49. 1H-NMR (D2O) δ (ppm): 1.36–1.52 (m, 2H), 1.84–1.98 (m, 3H), 2.65 (d, 2H, J = 6.7 Hz), 2.87–3.00 (m, 2H), 3.35–3.46 (m, 2H), 7.08–7.16 (m, 3H), 7.32 (d, 2H, J = 8.5 Hz), 8.12 (dd, 1H, J = 8.8, 2.6 Hz), 8.37–8.42 (m, 1H).

4.1.5. General Procedure for the Synthesis of Compounds 31–40, 53–55

HATU (1.05 equiv) was added to a solution of the appropriate commercially available or in-house synthesized benzoic acid (3-methoxybenzoic acid for 31, 37–39, 53–55, 2-fluoro-5-methoxybenzoic acid for 32 and 40, 4-fluoro-3-methoxybenzoic acid for 33, 2-chloro-5-methoxybenzoic acid for 34, 4-chloro-3-methoxybenzoic acid for 35, and 4-bromo-5-methoxybenzoic acid for 36; 1 equiv) in dry DMF (5.7 mL), and then DIPEA (4 equiv) was added dropwise. The resulting mixture was stirred at room temperature for 30 min, and then piperidine hydrochlorides 27–30 and 50–52 (460 mg, 1 equiv) were added and left under stirring at room temperature until consumption of starting material (TLC). After this time, the residue was diluted with water and extracted with EtOAc. The organic layer was repeatedly washed with brine and dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified with a flash column chromatography (silica gel, mixtures from 8:2 to 6:4 of n-hexane or petroleum ether/ethyl acetate), and pure fractions containing the desired compounds were evaporated to dryness affording the amides.
4.1.5.1. (3-Methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (31)
Yellow oil. 66% yield from 29 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.50–1.69 (m, 3H), 1.74–1.88 (bm, 2H), 2.53–2.64 (m, 2H), 2.65–2.80 (bm, 1H), 2.84–3.02 (bm, 1H), 3.70–3.79 (bm, 1H), 3.81 (s, 3H), 4.64–4.77 (bm, 1H), 6.90–6.95 (m, 4H), 6.97–7.02 (m, 2H), 7.02–7.06 (m, 1H), 7.24–7.32 (m, 1H), 7.35 (t, 1H, J = 7.8 Hz), 7.89 (dd, 1H, J = 8.7, 2.5 Hz), 8.41–8.45 (m, 1H).
4.1.5.2. (2-Fluoro-5-methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (32)
Yellow oil. 41% yield from 29 and 2-fluoro-5-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.58–1.68 (bm, 3H), 1.75–1.86 (bm, 2H), 2.52–2.65 (m, 2H), 2.67–2.78 (m, 1H), 2.80–3.12 (bm, 1H), 3.53–3.64 (m, 1H), 3.78 (s, 3H), 4.70–4.79 (m, 1H), 6.80–7.08 (m, 7H), 7.34 (t, 1H, J = 7.8 Hz), 7.90 (dd, 1H, J = 8.6, 2.6 Hz), 8.41–8.46 (m, 1H).
4.1.5.3. (4-Fluoro-3-methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (33)
Yellow solid. 53% yield from 29 and 4-fluoro-3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.10–1.40 (m, 2H), 1.60–1.90 (m, 3H), 2.60 (d, 2H, J = 7.2 Hz), 2.61–3.06 (bm, 2H), 3.70–3.84 (bm, 1H), 3.90 (s, 3H), 4.60–4.75 (bm, 1H), 6.90 (ddd, 1H, J = 8.2, 4.3, 2.0 Hz), 6.92–6.95 (m, 1H), 6.98–7.10 (m, 5H), 7.35 (t, 1H, J = 7.8 Hz), 7.90 (dd, 1H, J = 8.7, 2.5 Hz), 8.40–8.47 (m, 1H).
4.1.5.4. (2-Chloro-5-methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (34)
Light yellow oil. 75% yield from 29 and 2-chloro-5-methoxybenzoic acid. 1H-NMR (CDCl3; asterisk denotes isomer peaks) δ (ppm): 1.20–1.48 (bm, 3H), 1.72–1.88 (bm, 2H), 2.51–2.64 (m, 2H), 2.65–2.79 (m, 1H), 2.82–2.94 (m, 1H), 2.98–3.10* (m, 1H), 3.37–3.48 (m,1H), 3.77* (s, H), 3.80 (s, 3H), 4.68–4.82 (m, 1H), 6.74 (d, 1H, J = 2.9 Hz), 6.80–6.87 (m, 2H), 6.90–6.95 (m, 1H), 6.96–7.07 (m, 2H), 7.23–7.30 (m, 1H), 7.34 (t, 1H, J = 7.9 Hz), 7.89 (dd, 1H, J = 8.9, 2.5 Hz), 8.40–8.46 (m, 1H).
4.1.5.5. (4-Chloro-3-methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (35)
Yellow oil. 48% yield from 29 and 4-chloro-3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.08–1.33 (bm, 2H), 1.60–1.90 (bm, 3H), 2.61 (d, 2H, J = 7.4 Hz), 2.66–2.83 (bm, 1H), 2.84–3.05 (bm, 1H), 3.67–3.83 (bm, 1H), 3.91 (s, 3H), 4.59–4.77 (bm, 1H), 6.88 (dd, 1H J = 8.0, 1.8 Hz), 6.93 (t, 1H, J = 1.9 Hz), 6.97–7.07 (m, 4H), 7.32–7.39 (m, 2H), 7.90 (dd, 1H, J = 8.4, 2.6 Hz), 8.41–8.46 (m, 1H).
4.1.5.6. (4-Bromo-3-methoxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (36)
Yellow solid. 54% yield from 29 and 4-bromo-5-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.10–1.40 (bm, 2H), 1.60–1.80 (bm, 3H), 2.60 (d, 2H, J = 6.5 Hz), 2.65–2.82 (bm, 1H), 2.85–3.04 (bm, 1H), 3.65–3.80 (bm, 1H), 3.91 (s, 3H), 4.60–4.73 (bm, 1H), 6.81 (dd, 1H, J = 8.0, 1.8 Hz), 6.90–6.96 (m, 2H), 6.98–7.07 (m, 3H), 7.35 (t, 1H, J = 7.9 Hz), 7.54 (d, 1H, J = 8.0 Hz), 7.90 (dd, 1H, J = 8.5, 2.4 Hz), 8.41–8.46 (m, 1H).
4.1.5.7. (3-Methoxyphenyl)(4-(3-((3-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (37)
Light yellow oil. 48% yield from 27 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.08–1.37 (bm, 2H), 1.66–1.91 (bm, 3H), 2.59 (d, 2H, J = 5.8 Hz), 2.65–3.03 (bm, 2H), 3.62–3.83 (bm, 1H), 3.81 (s, 3H), 4.59–4.78 (bm, 1H), 6.89–6.97 (m, 4H), 6.98–7.05 (m, 2H), 7.08 (dd, 1H, J = 7.6, 5.2 Hz), 7.26–7.31 (m, 1H), 7.33 (t, 1H, J = 7.9 Hz), 7.96–8.01 (m, 1H), 8.25–8.31 (m, 1H).
4.1.5.8. (3-Methoxyphenyl)(4-(3-((4-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (38)
Yellow oil. 44% yield from 28 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.11–1.35 (bm, 1H), 1.65–1.92 (bm, 4H), 2.60 (d, 2H, J = 6.6 Hz), 2.68–3.06 (bm, 2H), 3.62–3.98 (bm, 1H), 3.82 (s, 3H), 4.50–4.88 (bm, 1H), 6.90–6.97 (m, 4H), 7.00 (dd, 1H, J = 8.2, 2.0 Hz), 7.02–7.06 (m, 1H), 7.13 (s, 1H), 7.20 (d, 1H, J = 5.4 Hz), 7.26–7.32 (m, 1H), 7.34 (t, 1H, J = 7.8 Hz), 8.32 (d, 1H, J = 5.2 Hz).
4.1.5.9. (3-Methoxyphenyl)(4-(3-((6-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (39)
Yellow oil. 61% yield from 30 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.50–1.88 (bm, 6H), 2.50–3.00 (bm, 4H), 3.62–3.84 (bm, 1H), 3.82 (s, 3H), 6.89–7.08 (m, 7H), 7.27–7.41 (m, 3H), 7.83 (t, 1H, J = 7.8 Hz).
4.1.5.10. (2-Fluoro-5-methoxyphenyl)(4-(3-((4-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (40)
Yellow oil, 72% yield from 28 and 2-fluoro-5-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.21–1.36 (bm, 1H), 1.56–1.68 (bm, 2H), 1.74–1.86 (bm, 2H), 2.52–2.66 (m, 2H), 2.66–2.78 (m, 1H), 2.82–3.12 (bm, 1H), 3.51–3.61 (m, 1H), 3.78 (s, 3H), 4.68–4.89 (m, 1H), 6.81–6.90 (m, 2H), 6.91–6.94 (m, 1H), 6.95–7.01 (m, 2H), 7.01–7.05 (m, 1H), 7.11–7.15 (m, 1H), 7.18–7.21 (m, 1H), 7.34 (t, 1H, J = 7.9 Hz), 8.32 (d, 1H, J = 5.2 Hz). 13C NMR (CDCl3) δ (ppm): 31.84, 32.63, 38.28, 42.31, 42.87, 47.45, 55.98, 108.09 (q, J = 4.1 Hz), 113.14 (d, J = 3.8 Hz), 114.12 (q, J = 3.2 Hz), 116.58 (d, J = 23.6 Hz), 116.81 (d, J = 8.0 Hz), 119.09, 122.01, 122.61 (q, J = 273.1 Hz), 124.97 (d, J = 19.9 Hz), 126.26, 129.79, 141.85 (q, J = 34.2 Hz), 142.22, 149.16, 152.47 (d, J = 240.0 Hz), 153.48, 156.07 (d, J = 2.0 Hz), 164.34. 164.98. HPLC analysis: retention time = 14.186 min; peak area, 95% (254 nm). HRMS: m/z for C26H25F4N2O3 [M + H]+ calculated: 489.17958, found: 489.17963.
4.1.5.11. (3-Methoxyphenyl)(4-(3-(pyridin-2-yloxy)benzyl)piperidin-1-yl)methanone (53)
Yellow oil. 53% yield from 50 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.55–1.87 (bm, 5H), 2.50–3.00 (bm, 4H), 3.65–3.80 (bm, 1H), 3.81 (s, 3H), 4.61–4.78 (bm, 1H), 6.85–7.04 (m, 8H), 7.26–7.35 (m, 2H), 7.70 (ddd, 1H, J = 8.8, 6.7, 1.6 Hz), 8.21 (ddd, 1H, J = 5.0, 2.0, 0.7 Hz).
4.1.5.12. (3-Methoxyphenyl)(4-(3-phenoxybenzyl)piperidin-1-yl)methanone (54)
Light yellow oil. 67% yield from 51 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.10–1.41 (bm, 1H), 1.50–1.87 (bm, 4H), 2.54 (d, 2H, J = 7.2 Hz), 2.61–3.00 (bm, 2H), 3.66–3.87 (bm, 1H), 3.81 (s, 3H), 4.51–4.81 (bm, 1H), 6.80–6.90 (m, 3H), 6.90–6.95 (m, 3H), 6.97–7.02 (m, 2H), 7.07–7.15 (m, 1H), 7.21 (t, 1H, J = 7.4 Hz), 7.25–7.36 (m, 3H).
4.1.5.13. (3-Methoxyphenyl)(4-(4-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (55)
Light yellow oil. 72% yield from 52 and 3-methoxybenzoic acid. 1H-NMR (CDCl3) δ (ppm): 1.14–1.37 (bm, 3H), 1.72–1.89 (bm, 2H), 2.59 (d, 2H, J = 7.2 Hz), 2.66–3.00 (bm, 2H), 3.69–3.87 (bm, 1H), 3.82 (s, 3H), 4.59–4.82 (bm, 1H), 6.90–6.97 (m, 3H), 7.00 (d, 1H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.5 Hz), 7.19 (d, 2H, J = 8.5 Hz), 7.30 (dd, 1H, J = 9.1, 7.4 Hz), 7.89 (dd, 1H, J = 8.6, 2.3 Hz), 8.41–8.46 (m, 1H).

4.1.6. General Procedure for the Synthesis of Compounds 7–9, 10a–e, 11a–c, 12, 13

A solution of O-methylated amides 31–40 and 53–55 (345 mg, 0.738 mmol) in anhydrous CH2Cl2 (8.6 mL) was cooled to −10 °C and treated dropwise with a 1.0 M solution of BBr3 in CH2Cl2 (2.3 mL) under argon. The mixture was left under stirring at the same temperature for 5 min and then at 0 °C for 1 h and finally at room temperature until the starting material was consumed (TLC). The mixture was then diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, dried, and concentrated. The crude product was purified by flash chromatography over silica gel. Elution with n-hexane/EtOAc (4:6 to 6:4) or CHCl3/MeOH (95:5 to 99:1) mixtures afforded the desired compounds.
4.1.6.1. (3-Hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (7)
White solid, 59% yield from 31. 1H-NMR (DMSO-d6) δ (ppm): 1.00–1.20 (bm, 2H), 1.46–1.70 (bm, 2H), 1.73–1.87 (bm, 1H), 2.56 (d, 2H, J = 6.8 Hz), 2.60–2.80 (bm, 1H), 2.86–3.00 (bm, 1H), 3.50–3.66 (bm, 1H), 4.30–4.50 (bm, 1H), 6.68–6.75 (m, 2H), 6.80 (ddd, 1H, J = 8.2, 2.5, 0.9 Hz), 6.99–7.05 (m, 2H), 7.09 (d, 1H, J = 7.8 Hz), 7.17–7.23 (m, 2H), 7.35 (dd, 1H, J = 8.6, 7.7 Hz), 8.22 (dd, 1H, J = 8.9, 2.4 Hz), 8.54–8.60 (m, 1H), 9.65 (exchangeable s, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.27, 31.99, 37.29, 41.44, 41.67, 47.06, 111.65, 113.35, 116.14, 116.96, 118.97, 120.28 (q, J = 32.6 Hz), 121.94, 123.90 (q, J = 271.4 Hz), 126.03, 129.48, 129.53, 137.56 (q, J = 3.1 Hz), 137.71, 142.28, 145.34 (q, J = 4.4 Hz), 152.84, 157.22, 165.60, 168.74. HPLC analysis: retention time = 12.879 min; peak area, 98% (254 nm). HRMS: m/z for C25H24F3N2O3 [M + H]+ calculated: 457.17335, found: 457.17307.
4.1.6.2. (3-Hydroxyphenyl)(4-(3-(pyridin-2-yloxy)benzyl)piperidin-1-yl)methanone (8)
White solid, 50% yield from 53. 1H-NMR (DMSO-d6) δ (ppm): 1.00–1.20 (bm, 2H), 1.44–1.87 (bm, 3H), 2.54 (d, 2H, J = 7.1 Hz), 2.60–2.80 (bm, 1H), 2.81–3.02 (bm, 1H), 3.45–3.66 (bm, 1H), 4.30–4.48 (bm, 1H), 6.67–6.75 (m, 2H), 6.80 (ddd, 1H, J = 8.1, 2.5, 0.9 Hz), 6.90–6.95 (m, 2H), 6.96–7.05 (m, 2H), 7.12 (ddd, 1H, J = 7.2, 5.0, 0.9 Hz), 7.20 (t, 1H, J = 7.9 Hz), 7.31 (tt, 1H, J = 8.8, 7.6 Hz), 7.84 (ddd, 1H, J = 8.8, 6.8, 1.6 Hz), 8.15 (ddd, 1H, J = 5.0, 2.0, 0.8 Hz), 9.65 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.24, 31.96, 37.29, 41.47, 41.70, 47.02, 111.44, 113.32, 116.11, 116.94, 118.47, 118.94, 121.50, 125.10, 129.29, 129.45, 137.68, 140.07, 141.97, 147.44, 153.82, 157.19, 163.01, 168.71.
HPLC analysis: retention time = 11.703 min; peak area, 95% (254 nm). HRMS: m/z for C24H25N2O3 [M + H]+ calculated: 389.18597, found: 389.18588.
4.1.6.3. (3-Hydroxyphenyl)(4-(3-phenoxybenzyl)piperidin-1-yl)methanone (9)
White solid, 62% yield from 54. 1H-NMR (DMSO-d6) δ (ppm): 1.00–1.20 (bm, 2H), 1.43–1.84 (bm, 3H), 2.50–2.57 (m, 2H), 2.60–2.80 (bm, 1H), 2.80–3.00 (bm, 1H), 3.46–3.64 (bm, 1H), 4.30–4.50 (bm, 1H), 6.67–7.76 (m, 2H), 6.78–6.86 (m, 3H), 6.94–7.01 (m, 2H), 7.12 (tt, 1H, J = 7.4, 1.1 Hz), 7.15–7.23 (m, 2H), 7.24–7.31 (m, 1H), 7.35–7.41 (m, 2H), 9.64 (exchangeable s, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.20, 31.43, 37.33, 41.50, 41.81, 47.13, 113.39, 116.20, 117.02, 118.49 (2C), 119.33, 123.32, 124.26, 128.19, 129.53, 129.73, 130.02 (2C), 137.74, 142.41, 156.52, 156.79, 157.26, 168.82. HPLC analysis: retention time = 12.997 min; peak area, 95% (254 nm). HRMS: m/z for C25H26NO3 [M + H]+ calculated: 388.19072, found: 388.19031.
4.1.6.4. (2-Fluoro-5-hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (10a)
White solid, 52% yield from 32. 1H-NMR (DMSO-d6) δ (ppm): 1.02–1.16 (bm, 2H), 1.49–1.60 (bm, 1H), 1.62–1.73 (bm, 1H), 1.73–1.88 (bm, 1H), 2.55 (d, 2H, J = 6.9 Hz), 2.65–2.77 (m, 1H), 2.90–3.04 (bm, 1H), 3.35–3.49 (m, 1H), 4.40–4.49 (m, 1H), 6.57–6.67 (bm, 1H), 6.75–6.83 (m, 1H), 6.99–7.12 (m, 4H), 7.20 (d, 1H, J = 8.7 Hz), 7.35 (dd, 1H, J = 8.7, 7.6 Hz), 8.22 (dd, 1H, J = 9.0, 2.4 Hz), 8.54–8.58 (m, 1H), 9.64 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.23, 32.01, 37.17, 41.17, 41.59, 46.55, 111.67, 113.91, 116.34 (d, J = 23.2 Hz), 117.07 (d, J = 7.5 Hz), 118.99, 120.29 (q, J = 32.4 Hz), 121.97, 123.91 (q, J = 271.4 Hz), 124.90 (d, J = 20.4 Hz), 126.05, 129.54, 137.57 (q, J = 3.3 Hz), 142.24, 145.34 (q, J = 4.3 Hz) 150.67 (d, J = 235.1 Hz), 152.86, 153.71 (d, J = 1.9 Hz), 163.64, 165.62. HPLC analysis: retention time = 13.049 min; peak area, 95% (254 nm). HRMS: m/z for C25H23F4N2O3 [M + H]+ calculated: 475.16393, found: 475.16394.
4.1.6.5. (4-Fluoro-3-hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (10b)
White solid, 56% yield from 33. 1H-NMR (DMSO-d6) δ (ppm): 1.07–1.19 (m, 2H), 1.49–1.72 (bm, 2H), 1.74–1.86 (bm, 1H), 2.56 (d, 2H, J = 6.9 Hz), 2.62–2.82 (bm, 1H), 2.82–3.01 (bm, 1H), 3.50–3.68 (bm, 1H), 4.30–4.45 (bm, 1H), 6.76 (ddd, 1H, J = 8.3, 4.3, 2.1 Hz), 6.91 (dd, 1H, J = 8.5, 2.0 Hz), 7.00–7.04 (m, 2H), 7.09 (d, 1H, J = 7.7 Hz), 7.13–7.22 (m, 2H), 7.36 (dd, 1H, J = 8.7, 7.6 Hz), 8.22 (dd, 1H, J = 8.8, 2.6 Hz), 8.54–8.58 (m, 1H), 10.16 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.35, 37.26, 41.65, 47.11, 111.65, 116.00 (d, J = 18.9 Hz), 116.35 (d, J = 3.4 Hz), 117.86 (d, J = 6.9 Hz), 118.97, 120.27 (q, J = 32.5 Hz), 121.93, 123.89 (q, J = 271.4 Hz), 126.01, 129.52, 132.91 (d, J = 3.7 Hz), 137.56 (q, J = 3.1 Hz), 142.26, 144.80 (d, J = 12.5 Hz), 145.34 (q, J = 4.4 Hz), 151.36 (d, J = 243.6 Hz), 152.84, 165.59, 167.99. HPLC analysis: retention time = 13.029 min; peak area, 98% (254 nm). HRMS: m/z for C25H23F4N2O3 [M + H]+ calculated: 475.16393, found: 475.16397.
4.1.6.6. (2-Chloro-5-hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (10c)
White solid, 57% yield from 34. 1H-NMR (DMSO-d6, asterisk denotes isomer peaks) δ (ppm): 1.00–1.20 (bm, 1H), 1.47–1.58 (bm, 1H), 1.63–1.72 (bm, 1H), 1.73–1.87 (bm, 1H), 2.54 (d, 2H, J = 2.4 Hz), 2.56* (d, 2H, J = 2.8 Hz), 2.64–2.77 (bm, 1H), 2.87–3.02 (m, 1H), 3.19–3.30 (m, 2H), 4.36–4.52 (bm, 1H), 6.59* (d, 1H, J = 2.8 Hz), 6.66 (d, 1H, J = 2.9 Hz), 6.75–6.82 (m, 1H), 6.98–7.04 (m, 2H), 7.08 (d, 1H, J = 7.7 Hz), 7.20 (d, 1H, J = 9.0 Hz), 7.26 (dd, 1H, J = 8.7, 2.6 Hz), 7.31–7.39 (m, 1H), 8.22 (dd, 1H, J = 8.7, 2.5 Hz), 8.53–8.59 (m, 1H), 9.97 (exchangeable bs, 1H). 13C NMR (DMSO-d6, asterisk denotes isomer peaks) δ (ppm): 31.11*, 31.18, 31.77, 32.01*, 37.12, 37.18*, 40.90, 41.61*, 45.95*, 46.57, 111.66, 114.00, 117.12*, 117.24, 118.26, 119.00, 120.29 (J = 32.4 Hz), 121.96, 123.91 (q, J = 271.5 Hz), 126.04, 129.55, 130.23*, 130.29, 136.87*, 137.03, 137.56, 142.22, 145.36 (q, J = 4.1 Hz), 152.85, 156.51, 156.55*, 165.11*, 165.21, 165.62. HPLC analysis: retention time = 13.300 min; peak area, 95% (254 nm). HRMS: m/z for C25H23ClF3N2O3 [M + H]+ calculated: 491.13438, found: 491.13416.
4.1.6.7. (4-Chloro-3-hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (10d)
White solid, 54% yield from 35. 1H-NMR (DMSO-d6) δ (ppm): 1.01–1.20 (bm, 2H), 1.44–1.88 (bm, 3H), 2.56 (d, 2H, J = 6.3 Hz), 2.60–2.79 (bm, 1H), 2.81–3.04 (bm, 1H), 3.46–3.68 (bm, 1H), 4.30–4.46 (bm, 1H), 6.73–6.79 (m, 1H), 6.91 (d, 1H, J = 2.0 Hz), 6.98–7.05 (m, 2H), 7.09 (d, 1H, J = 7.6 Hz), 7.20 (d, 1H, J = 8.8 Hz), 7.32–7.40 (m, 2H), 8.22 (dd, 1H, J = 8.9, 2.4 Hz), 8.54–8.60 (m, 1H), 10.50 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.30, 31.99, 37.26, 41.68, 47.15, 111.68, 114.86, 118.19, 119.01, 120.31 (q, J = 32.5 Hz) ,120.65, 121.96, 123.92 (q, J = 271.6 Hz), 126.04, 129.56, 129.86, 136.18, 137.59 (q, J = 3.2 Hz), 142.28, 145.36 (q, J = 4.3 Hz), 152.87, 153.00, 165.62, 167.83. HPLC analysis: retention time = 13.419 min; peak area, 98% (254 nm). HRMS: m/z for C25H23ClF3N2O3 [M + H]+ calculated: 491.13438, found: 491.13437.
4.1.6.8. (4-Bromo-3-hydroxyphenyl)(4-(3-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (10e)
White solid, 46% yield from 36. 1H-NMR (DMSO-d6) δ (ppm): 1.03–1.18 (bm, 2H), 1.47–1.89 (bm, 3H), 2.56 (d, 2H, J = 6.6 Hz), 2.61–2.76 (bm, 1H), 2.85–3.05 (bm, 1H), 3.50–3.64 (bm, 1H), 4.34–4.45 (bm, 1H), 6.70 (dd, 1H, J = 8.1, 1.9 Hz), 6.89 (d, 1H, J = 1.9 Hz), 6.89–7.05 (m, 2H), 7.09 (d, 1H, J = 7.4 Hz), 7.20 (d, 1H, J = 8.7 Hz), 7.36 (dd, 1H, J = 8.6, 7.7 Hz), 7.51 (d, 1H, J = 8.0 Hz), 8.22 (dd, 1H, J = 9.2, 2.6 Hz), 8.54–8.59 (m, 1H), 10.56 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.22, 32.04, 37.29, 41.71, 47.20, 110.32, 111.71, 114.53, 118.59, 119.05, 120.33 (q, J = 32.6 Hz), 121.99, 123.95 (q, J = 271.4 Hz), 126.07, 129.59, 132.91, 136.88, 137.62 (q, J = 3.0 Hz), 142.31, 145.38 (q, J = 4.4 Hz), 152.89, 154.07, 165.64, 165.65, 167.87. HPLC analysis: retention time = 13.548 min; peak area, 99% (254 nm). HRMS: m/z for C25H23BrF3N2O3 [M + H]+ calculated: 535.08387, found: 535.08398.
4.1.6.9. (3-Hydroxyphenyl)(4-(3-((3-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (11a)
White solid, 52% yield from 37. 1H-NMR (DMSO-d6) δ (ppm): 1.02–1.20 (bm, 2H), 1.48–1.71 (bm, 2H), 1.72–1.88 (bm, 1H), 2.56 (d, 2H, J = 7.0 Hz), 2.62–2.75 (bm, 1H), 2.81–3.01 (bm, 1H), 3.48–3.67 (bm, 1H), 4.31–4.51 (bm, 1H), 6.67–6.71 (m, 1H), 6.73 (d, 1H, J = 7.5 Hz), 6.80 (dd, 1H, J = 8.3, 2.3 Hz), 6.95–7.01 (m, 2H), 7.09 (d, 1H, J = 7.6 Hz), 7.20 (t, 1H, J = 7.8 Hz), 7.29–7.37 (m, 2H), 8.22–8.38 (m, 1H), 8.34–8.40 (m, 1H), 9.65 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.32, 32.02, 37.36, 41.46, 41.69, 47.12, 112.59 (q, J = 32.9 Hz), 113.38, 116.17, 117.00, 118.80, 118.99, 121.91, 122.98 (q, J = 217.8 Hz), 125.95, 129.38, 129.51, 137.73, 137.82 (q, J = 4.8 Hz), 142.21, 151.58, 152.85, 157.24, 159.52, 168.77. HPLC analysis: retention time = 12.692 min; peak area, 97% (254 nm). HRMS: m/z for C25H24F3N2O3 [M + H]+ calculated: 457.17335, found: 457.17334.
4.1.6.10. (3-Hydroxyphenyl)(4-(3-((4-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (11b)
White solid, 61% yield from 38. 1H-NMR (DMSO-d6) δ (ppm): 1.01–1.21 (bm, 2H), 1.48–1.72 (bm, 2H), 1.73–1.88 (bm, 1H), 2.56 (d, 2H, J = 6.9 Hz), 2.60–2.80 (bm, 1H), 2.82–3-01 (bm, 1H), 3.46–3.69 (bm, 1H), 4.29–4.49 (bm, 1H), 6.68–6.71 (m, 1H), 6.72 (dt, 1H, J = 7.4, 1.2 Hz), 6.80 (ddd, 1H, J = 8.2, 2.5, 1.0 Hz), 6.98–7.03 (m, 2H), 7.08 (d, 1H, J = 7.6 Hz), 7.20 (t, 1H, J = 7.8 Hz), 7.35 (tt, 1H, J = 8.7, 7.6 Hz), 7.38–7.41 (m, 1H), 7.46–7.50 (m, 1H), 8.40 (d, 1H, J = 5.2 Hz), 9.63 (exchangeable s, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.27, 32.01, 37.21, 41.47, 41.69, 47.06, 107.68 (q, J = 3.9 Hz), 113.39, 114.27 (q, J = 3.2 Hz), 116.17, 116.97, 118.80, 121.81, 122.56 (q, J = 273.4 Hz), 125.84, 129.46, 129.50, 137.71, 140.28 (q, J = 33.5 Hz), 142.20, 149.51, 153.11, 157.26, 163.72, 168.78. HPLC analysis: retention time = 12.915 min; peak area, 98% (254 nm). HRMS: m/z for C25H24F3N2O3 [M + H]+ calculated: 457.17335, found: 457.17343.
4.1.6.11. (3-Hydroxyphenyl)(4-(3-((6-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (11c)
White solid, 60% yield from 39. 1H-NMR (DMSO-d6) δ (ppm): 1.01–1.20 (bm, 2H), 1.46–1.87 (bm, 3H), 2.55 (d, 2H, J = 7.3 Hz), 2.60–2.78 (bm, 1H), 2.80–3.00 (bm, 1H), 3.50–3.64 (bm, 1H), 4.32–4.50 (bm, 1H), 6.67–6.76 (m, 2H), 6.80 (dd, 1H, J = 8.1, 1.7 Hz), 6.98–7.05 (m, 2H), 7.08 (d, 1H, J = 7.6 Hz), 7.20 (t, 1H, J = 7.8 Hz), 7.30 (d, 1H, J = 8.4 Hz), 7.36 (t, 1H, J = 7.9 Hz), 7.62 (d, 1H, J = 7.4 Hz), 8.11 (t, 1H, J = 7.9 Hz), 9.64 (exchangeable s, 1H). 13C-NMR (DMSO-d6) δ (ppm): 31.29, 31.96, 37.48, 41.49, 41.75, 47.07, 113.39, 115.62, 115.65, 116.16, 116.97, 118.29, 120.74 (q, J = 273.9 Hz), 121.75, 125.86, 129.46, 129.62, 137.72, 142.16, 142.24, 144.23 (q, J = 34.3 Hz), 152.88, 157.26, 162.99, 168.77. HPLC analysis: retention time = 12.843 min; peak area, 95% (254 nm). HRMS: m/z for C25H24F3N2O3 [M + H]+ calculated: 457.17335, found: 457.17355.
4.1.6.12. (3-Hydroxyphenyl)(4-(4-((5-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (12)
White solid, 29% yield from 55. 1H-NMR (DMSO-d6) δ (ppm): 1.05–1.21 (bm, 2H), 1.47–1.61 (bm, 1H), 1.62–1.74 (bm, 1H), 1.75–1.88 (bm, 1H), 2.56 (d, 2H, J = 6.9 Hz), 2.63–2.79 (bm, 1H), 2.86–3.04 (bm, 1H), 3.49–3.69 (bm, 1H), 4.36–4.51 (bm, 1H), 6.68–6.72 (m, 1H), 6.74 (d, 1H, J = 7.6 Hz), 6.78–6.84 (m, 1H), 7.11 (d, 2H, J = 8.5 Hz), 7.17–7.22 (m, 2H), 7.25 (d, 2H, J = 8.5 Hz), 8.21 (dd, 1H, J = 8.9, 2.6 Hz), 8.52–8.59 (m, 1H), 9.67 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.38, 32.10, 37.46, 41.38, 47.12, 111.64, 113.38, 116.17, 117.01, 120.25 (q, J = 32.6 Hz), 121.25 (2C), 123.93 (q, J = 271.5 Hz), 129.52, 130.30 (2C), 137.15, 137.53 (q, J = 3.1 Hz), 137.75, 145.29 (q, J = 8.7 Hz), 151.04, 157.25, 165.68, 168.78. HPLC analysis: retention time = 12.933 min; peak area, 99% (254 nm). HRMS: m/z for C25H24F3N2O3 [M + H]+ calculated: 457.17335, found: 457.17331.
4.1.6.13. (2-Fluoro-5-hydroxyphenyl)(4-(3-((4-(trifluoromethyl)pyridin-2-yl)oxy)benzyl)piperidin-1-yl)methanone (13)
Light-yellow solid, 66% yield from 40. 1H-NMR (DMSO-d6) δ (ppm): 0.99–1.18 (m, 2H), 1.49–1.59 (m, 1H), 1.62–1.72 (m, 1H), 1.73–1.86 (m, 1H), 2.55 (d, 2H, J = 7.0 Hz), 2.70 (td, 1H, J = 12.6, 2.4 Hz), 2.91–3.03 (bm, 1H), 3.34–3.44 (m, 1H), 4.39–4.49 (m, 1H), 6.58–6.66 (m, 1H), 6.75–6.82 (m, 1H), 6.98–7.04 (m, 2H), 7.05–7.11 (m, 2H), 7.35 (dd, 1H, J = 8.6, 7.7 Hz), 7.38–7.42 (m, 1H), 7.48 (dd, 1H, J = 5.2, 0.9 Hz), 8.40 (d, 1H, J = 5.2 Hz), 9.65 (exchangeable bs, 1H). 13C NMR (DMSO-d6) δ (ppm): 31.23, 32.03, 37.20, 41.17, 41.61, 46.56, 107.72 (q, J = 3.9 Hz), 113.95, 114.31 (q, J = 3.3 Hz), 116.37 (d, J = 23.2 Hz), 117.09 (d, J = 7.7 Hz), 118.85, 121.85, 122.59 (q, J = 273.4 Hz), 124.91 (d, J = 20.4 Hz), 125.87, 129.53, 140.28 (q, J = 33.5 Hz), 142.18, 149.54, 150.70 (d, J = 233.2 Hz), 153.12, 153.73 (d, J = 1.9 Hz), 163.65, 163.74. HPLC analysis: retention time = 13.100 min; peak area, 98% (254 nm). HRMS: m/z for C25H23F4N2O3 [M + H]+ calculated: 475.16393, found: 475.16434.

4.2. MAGL Inhibition Assay

Human recombinant MAGL and 4-nitrophenylacetate (4-NPA) substrates were purchased from Cayman Chemical. IC50 values were generated in 96-well microtiter plates. The MAGL reaction was carried out at room temperature, at a final volume of 200 μL in 10 mM Tris buffer, pH 7.2, containing 1 mM EDTA, and 0.1 mg/mL bovine serum albumin (BSA). A total of 150 μL of 4-NPA 133.3 μM was added to 10 μL of DMSO containing the appropriate amount of compound. The reaction was started by adding 40 μL of MAGL (11 ng/well) so that the assay was linear over 30 min. The final concentration of the compounds analyzed ranged for from 320 to 0.02 nM. After 30 min from the start of the reaction, the absorbance values were measured using Victor X3 Microplates Reader (PerkinElmer) at 405 nm. Two reactions were also performed: one reaction containing no compounds and the second containing neither compound nor MAGL. IC50 values were derived from experimental data using the sigmoidal dose–response fitting of GraphPad Prism software. Final values were obtained from duplicates of three independent experiments. To remove possible false-positive results, a blank analysis was performed for each compound concentration, and the final absorbance results were obtained by subtracting the absorbance produced by the presence of all the components except MAGL under the same conditions. In the enzyme kinetics experiments, compound 13 was tested in the presence of scalar concentrations of 4-NPA. It was added in scalar amounts (concentration range = 10–1.25 nM) to a reaction mixture containing scalar concentrations of 4-NPA (15–1400 μM). Finally, the MAGL solution was added (11 ng/well). MAGL activity was measured by recording the increase in absorbance of 4-nitrophenol using Victor X3 Microplates Reader (PerkinElmer). The experimental data were analyzed by nonlinear regression analysis with GraphPad Prism software, using second order polynomial regression analysis and by applying the mixed model inhibition fit.

4.3. DTT Interference Assay

The inhibition assay was the same as described above, with the exception that prior to the addition of 40 μL of MAGL (11 ng/well), the compound–substrate mixture was incubated for 15 min in the presence of DTT at a 10 μM concentration.

4.4. MAGL Preincubation Assay

The MAGL reaction was conducted under the same conditions reported above. A total of 150 μL of MAGL (11 ng/well) was added to 10 μL of DMSO containing the appropriate amount of compound. After 0, 30, and 60 min of incubation time, the reaction was started by adding 40 μL of 4-NPA 500 μM. The enzyme activity was then measured after 30 min from the start of the reaction according to the procedure described above. Final values were obtained from triplicates of two independent experiments.

4.5. MAGL Dilution Assay

MAGL enzyme (880 ng in 75 μL of Tris buffer, pH 7.2) was incubated for 60 min at room temperature with 5 μL of compound 13 (concentration of 320 nM in the mixture) dissolved in DMSO. The MAGL–inhibitor mixture was then diluted 40-fold with the buffer. After 15 min of incubation, the reaction was started on a 160 μL aliquot by the addition of 40 μL of 4-NPA 500 μM and the enzyme activity was measured according to the procedure described above. Final values were obtained from triplicates of two independent experiments.

4.6. FAAH Inhibition Assay

Human recombinant FAAH and AMC arachidonoylamide substrates were purchased from Cayman Chemical. The FAAH reaction was carried out at room temperature (final volume of 200 μL in 125 mM Tris buffer, pH 9.0, containing 1 mM EDTA and 0.1 mg/mL BSA). A total of 150 μL of AMC arachidonoylamide (13.3 μM) (final concentration = 10 μM) was added to 10 μL of DMSO containing the appropriate amount of compound. The reaction was started adding 40 μL of FAAH (0.9 μg/well) so that the assay was linear over 30 min. The final concentration of the compounds analyzed ranged for from 200 to 0.0128 μM. After the reaction had proceeded for 30 min, fluorescence values were measured using a Victor X3 PerkinElmer instrument at an excitation wavelength of 340 nm and an emission of 460 nm. As for the MAGL assay, two reactions were also performed: one reaction containing no compound and the second one containing neither the inhibitor nor enzyme. IC50 values were derived from experimental data using the sigmoidal dose–response fitting of GraphPad Prism software. To remove possible false-positive results, a blank analysis was carried out for each compound concentration, and the final fluorescence results were obtained by subtracting the fluorescence produced by the presence of all the components except FAAH under the same conditions.

4.7. CB1 and CB2 Binding Assay

Binding assays to cannabinoid receptor 1 and 2 (CB1 and CB2) were performed as previously described. (8) Briefly, clean membranes expressing hCB1 or hCB2 were resuspended in binding buffer (50 mM Tris–HCl, 2.5 mM EDTA, 5 mM MgCl2, 0.5% fatty acid-free BSA, pH 7.4) and incubated with vehicle or compounds and 0.5 nM [3H]CP55,940 for 90 min at 30 °C. Nonspecific binding was determined in the presence of 10 μM WIN55,512. After incubation, membranes were filtered through a pre-soaked 96-well microplate bonded with GF/B filters under vacuum and washed 12 times with 150 μL of ice-cold binding buffer. The radioactivity was measured, and the results are expressed as [3H]CP55,940 binding. Compound 13 was tested, at a screening concentration of 10 μM, in two independent experiments, each performed in triplicate.

4.8. Competitive Activity-Based Protein Profiling (ABPP)

A TAMRA-Fluorophosphonate serine hydrolase probe (TAMRA-FP, ActivX, Thermo Scientific) was used to fluorescently label serine hydrolases of mouse brain membrane preparations, while the binding of the TAMRA-FP was competed with different serine hydrolase inhibitors. Snap-frozen mouse half brains were homogenized each in 1 mL of extraction buffer (EB: 50 mM Tris–HCl, 3 mM MgCl2, 1 mM EGTA, pH 7.4) using a Mini-Bead Beater (BioSpec Products). The homogenate was centrifuged at 800g and 4 °C for 10 min. The supernatant was collected and kept on ice, while the pellet was resuspended in 1 mL of EB. The centrifugation with collection of the supernatant and resuspension was repeated four times. The collected supernatants were centrifuged at 16000g for 20 min at 4 °C. Afterward, the supernatants were discarded, and the pellets were resuspended with a syringe and needle (25G, 0.5 × 16 mm, NIPRO) and combined in 700 μL of 50 mM Tris–HCl (pH 7.4). The protein amount of the membrane preparation was accessed with a Pierce BCA protein assay (Thermo Scientific) according to the manufacturer’s instructions, and the membrane preparations were stored at −80 °C until use. For the ABPP, the mouse brain membrane preparations were diluted to 4 mg/mL in PBS and 19.5 μL was pre-incubated for 25 min at 25 °C with 0.5 μL of DMSO (vehicle control) or one of the following inhibitors (final concentration in brackets): 13 (10 μM, MAGL inhibitor), JZL184 (10 μM, MAGL inhibitor, Cayman Chemical Company), URB597 (4 μM, FAAH inhibitor, Cayman Chemical Company), WWL70 (10 μM, ABHD6 inhibitor, Cayman Chemical Company), THL (30 μM, ABHD6 and 12 inhibitor, Orlistat, Cayman Chemical Company) or MAFP (5 μM, serine hydrolase inhibitor, Abcam Biochemicals). TAMRA-FP (125 nM final concentration) was added to the samples and incubated for 25 min at 25 °C. The reaction was stopped by adding 6.5 μL of 4× Laemmli buffer with an incubation of 3 min at 25 °C followed by 10 min at 90 °C. The samples were cooled down, centrifuged for 1 min at 10000g, and separated by electrophoresis in a 12% SDS-polyacrylamide gel (120 V, 180 min). The fluorescent signal in the gel was recorded with a Typhoon FLA 9500 (GE Healthcare Bio-Sciences AB) in TAMRA settings. The comparability of the loaded protein amount was afterward confirmed by a Coomassie staining of the gel. The presented results were confirmed in two additional independent repetitions of the ABPP experiment.

4.9. Docking Calculations

The crystal structure of the hMAGL protein (5ZUN PDB code (30)) was taken from the Protein Data Bank. (50) After adding hydrogen atoms, the protein was minimized using Amber20 software (51) and the ff14SB force field at 300 K. The complex was placed in a rectangular parallelepiped waterbox; the TIP3P explicit solvent model for water was used, and the complex was solvated with a 10 Å water cap. Sodium ions were added as counterions to neutralize the system. Two steps of minimization were then carried out. In the first stage, we kept the protein fixed with a position restraint of 500 kcal/mol·Å2 and we solely minimized the positions of the water molecules. In the second stage, we minimized the entire system through 5000 steps of steepest descent followed by a conjugate gradient (CG) until a convergence of 0.05 kcal/Å mol. The energy minimized receptor was then used for the docking studies relative to compound 11b, which were performed with AUTODOCK 4.0 software. (52) The ligand was built with Maestro (53) and then subjected to energy minimization performed with Macromodel (54) until a convergence value of 0.05 kcal/Å mol, by employing the CG algorithm, MMFFs force field, and a distance-dependent dielectric constant of 1.0. AUTODOCK tools were used to automatically identify the torsion angles in the ligand, add the solvent model, and assign partial atomic charges to the protein and ligands (Kollmann and Gasteiger charges, respectively). The docking site used for calculations was defined in such a way as to contain all residues within a 10 Å shell from the reference ligand in the X-ray crystal structure. The energetic maps were calculated using a grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant. The ligand was subjected to 200 runs of AUTODOCK search using the Lamarckian Genetic Algorithm, following a robust protocol, (29,55) whose reliability was also tested with a self-docking study that produced an RMSD of 1.45 Å between the predicted and experimental conformations of the co-crystallized ligand in the reference X-ray structure. For each docking run, 10,000,000 steps of energy evaluations were performed, the number of individuals in the initial population was set to 500, and a maximum of 10,000,000 generations was simulated. An RMS cutoff of 2.0 Å was used for pose clustering. All other settings were left as their defaults. Clusters with a population lower than 10 conformers were not considered. For the docking calculations performed on compounds 7–9, 10a-e, 11a, 11c, 12, 13, and 40, the same protocols of ligand construction, preparation, and docking were used, with the only exception that the average structure of the hMAGL-11b complex obtained after molecular dynamics simulations (see Section 4.3) was used as the receptor for docking studies and the best docked conformation belonging to the best cluster of solutions obtained (top-scored pose) was considered for each ligand.

4.10. MD Simulations

MD simulations were performed using Amber20 (51) and were carried out using the ff14SB force field. General Amber force field (GAFF) parameters were used for the ligand, whose partial charges were assigned using the Antechamber suite of Amber20, based on the AM1-BCC method. Each analyzed MAGL–ligand complex produced by docking was placed at the center of a rectangular parallelepiped box and solvated with a 15 Å water cap, generated using the TIP3P explicit solvent model. Sodium ions were then added to neutralize the obtained system, which was then energy-minimized using the same two-step protocol employed for the initial minimization of the receptor. The minimized complexes were used as input structures for the MD simulations, which were run using Particle Mesh Ewald (PME) electrostatics, a cutoff of 10 Å for the non-bonded interactions, and periodic boundary conditions. The SHAKE algorithm was used to constrain all bonds involving hydrogen atoms, and a time step of 2.0 fs was thus used for the simulation. Initially, a MD heating stage of 0.5 ns, in which the temperature of the system was raised from 0 to 300 K, was performed using constant-volume periodic boundary conditions. An initial equilibration stage of constant-pressure periodic boundary MD was run for 50 ns, keeping the temperature of the system at the constant value of 300 K with the Langevin thermostat. A second constant-pressure step of 500 ns was then performed at 300 K for optimal relaxation of the ligand–protein binding conformation and complex equilibration. In all these steps all α carbons of the protein were subjected to a harmonic potential of 10 kcal/mol Å2. Finally, a production step of 500 ns was performed, maintaining the same temperature and pressure conditions but removing any harmonic restraint, thus leaving the system totally free. In total, each analyzed complex was thus subjected to 1.05 μs of MD simulation. The final structures of the different MAGL–ligand complexes corresponded to the average of the last 500 ns of MD simulation minimized by the CG method until a convergence of 0.05 kcal/mol Å2. The average structures were obtained using the Cpptraj program (56) implemented in Amber20, which was also used for RMSD and H-bond analyses.

4.11. Binding Energy Evaluations

The evaluation of the binding free energy associated with all hMAGL–ligand complexes analyzed through MD simulations was carried out using Amber20 as previously described. (57,58) The trajectories relative to the last 500 ns of each simulation were extracted and used for the calculation, for a total of 500 snapshots (at time intervals of 1 ns). Van der Waals electrostatic and internal interactions were calculated with the SANDER module of Amber20, and the MOLSURF program was employed to estimate the nonpolar energies, while polar energies were calculated using the Poisson–Boltzmann methods with the MM-PBSA module of Amber20. A dielectric constant of 80 was used to represent the water phase in all calculations. For the gas phase, 10 different values of dielectric constant were used, ranging from 1 to 10. A total of 10 different binding free energy evaluations were thus performed for each of the analyzed ligand–protein complexes.

4.12. Evaluation of MAGL mRNA Expression in Human Normal and Tumor Tissues and Correlation with Survival in Pancreatic Cancer

The mRNA expression of MAGL was evaluated using the web-based genomics analysis and visualization platform GEPIA, analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects. (45) Moreover, we performed a correlation of MAGL mRNA expression with overall survival using TCGA-PAAD data of pancreatic cancer specimens (Tumor pancreatic adenocarcinoma TCGA dataset 178-rsem-tcgars) in R2 (R2: Genomics analysis and Visualization Platform, http://r2.amc.nl).

4.13. Cell Culture

The pancreatic cancer cell line SUIT-2 (JCRB1094, Tokyo, Japan) and the primary cell cultures PDAC2 and PDAC3 were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% newborn calf serum, penicillin (50 IU/mL), and streptomycin (50 μg/mL) from Gibco (Gaithersburg, MD). In addition, the ductal immortalized normal hTERT-HPNE cells obtained from ATCC (Manassas, VA, USA) were cultured in DMEM medium with 5% FBS and 10 ng/mL human recombinant EGF. The cells were grown at 37 °C, 5% CO2 and were frequently tested for mycoplasma contamination with the MycoAlert Mycoplasma Detection Kit (Westburg, Leusden, The Netherlands).

4.14. Evaluation of MAGL mRNA Expression in Pancreatic Cancer Cells

RNA-sequencing analyses for PDAC2 and PDAC3 were performed, as described by Firuzi et al. (59) Raw data were pre-processed for quality filtering and adapter trimming using the FASTX Toolkit (version 0.7) and subsequently mapped to the Human genome (GRCh38) using the STAR alignment tool (version 2.5.3a). We obtained ∼90% of reads mapped to the Human Genome per sample. Gene counts in fragments per kilobase of transcript per million mapped reads (FPKM) normalization were computed using the CuffLinks algorithm, and plots were generated with R version 3.5.0. SUIT-2 mRNA expression data was obtained from the Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle).

4.15. Drugs and Chemicals

Gemcitabine was a generous gift from Eli-Lilly (Indianapolis, IN) and was dissolved in sterile water, while MAGL inhibitors were solubilized in DMSO and diluted in culture medium before use. All other chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands).

4.16. Growth Inhibition Studies

The inhibitory effects on cell growth were evaluated by sulforhodamine B (SRB) assay. Cells were seeded in 96 well plates at a density of 5000 per well. After 24 h, once the cell monolayer was formed, cells were treated for 72 h with MAGL inhibitors (0.1–50 μM) or gemcitabine (1–1250 nM). Cells were then incubated for 72 h, fixed with trichloroacetic acid at 4 °C, washed with deionized water, and then dried at RT. After the fixation, the plate was stained with SRB, washed with acetic acid solution, and left to dry again. SRB was resuspended in a Tris base solution and its absorption was measured at 490 and 540 nm, as described previously. (60) Finally, the half-maximal response concentration (IC50) was calculated with GraphPad Prism version 9 (GraphPad PRISM, Intuitive Software for Science, San Diego, CA).

4.17. Analysis of Cell Migration

Pancreatic cancer cells were seeded in a 96 well plate at a density of 25,000 cells/well to form a confluent monolayer after 24 h. Subsequently, the monolayer was wounded by a 96-well pin tool scratcher. Detached cells were washed away with phosphate-buffered saline (PBS). Medium only or medium containing a concentration of 5 times (5×) the IC50 of each drug was added to the wells: 36 μM for 13, 14 μM for JZL-184, and 17 μM for ABX-1431. Bright-field images were taken with the software Universal Grab 6.3 digital on a Leica DMI300B microscope (Leica Microsystems, Eindhoven, The Netherlands) at different time points, to be analyzed with Scratch Assay 6.2 software (Digital Cell imaging Labs, Keerbergen, Belgium) as described previously. (60)

4.18. Apoptosis Assays

First, cells were seeded in a 96 well plate at a density of 5000 per well. After 24 h, cells were treated with drugs at the concentration of IC50 for 72 h. At the end of treatment, cells were fixed with paraformaldehyde, washed, and stained with annexin V/FITC (Apoptest, VPS Diagnostic, Hoeven, the Netherlands) in binding buffer BBA (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). After a wash with BBA, the fluorescence signal was measured by a plate reader (BioTek Instruments Inc., Winooski, VT) with excitation and emission at 485 and 535 nm, respectively. The values were normalized on cells number stained by crystal violet solution (for a solution of 100 mL: 750 mg of violet crystal powder, 250 mg of NaCl, 4.7 mL of 37% formaldehyde, 50 mL of ethanol and 45.3 mL of bidistilled water). The dye was solubilized in PBS containing 1% SDS and measured photometrically at 595 nm absorbance. Then, to assess whether caspase-3, an enzyme involved in the effector phase of apoptosis, is a downstream target of MAGL inhibitors, its enzymatic activity was measured by a specific spectrofluorimetric activity assay (Human Active Caspase-3 Immunoassay Quantikine ELISA, Catalog Number KM300, R&D Systems, Inc., Minneapolis, MN). Briefly, cells were plated in 6-well plates (5 × 105 cells/ml) and exposed to the drugs for 24 h at 5× IC50 or for 72 h at their IC50. At the end of drug incubation, cell extracts were diluted and mixed to the reagents according to the manufacturers’ protocol. Absorbance was measured at 450 nm, subtracting readings at 540 nm. Relative caspase activity was calculated using a standard curve with human recombinant caspase-3.

4.19. Evaluation of Pharmacological Interaction with Gemcitabine

The pharmacological interaction between compound 13 and gemcitabine was evaluated by the median drug effect analysis method, as described previously. (61) Compound 13 was added at the inhibitory concentration of 50%, while gemcitabine was added in a drug range between 0 and 1250 nM. The combination index (CI) was calculated to compare cell growth inhibition of the combination and each drug alone. Data analysis was carried out using CalcuSyn software (Biosoft, Oxford, UK). A CI of below 0.8 indicates a synergetic cytotoxic effect. A CI between 0.8 and 1.2 indicates an additive effect and above 1.2 indicates an antagonistic effect of the combination therapy.

4.20. PCR Assays to Evaluate Key Determinants in Migration, Apoptosis Induction, and Gemcitabine Activity

Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to evaluate the gene expression of MMP9, BCL-2, BCL-x, and hENT1, using β-actin, and GAPDH as housekeeping genes. The cells were seeded at 3 × 103 to 5 × 103 in a 6-well flat bottom plate with 2 mL medium per well and incubated with drugs at 5× IC50 for 24 h. Total RNA was extracted using the TRIzol Reagent (15596–026, ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocol. One microgram of RNA was reverse transcribed using first-strand cDNA synthesis (First Strand cDNA Synthesis Kit; ThermoFisher #K1612) on a Bio-Rad machine C100 Thermal Cycler. Real-time qPCR quantification was performed using specific TaqMan detection probes and primers (TaqMan Universal PCR Master Mix #4304437; Thermo Fisher Scientific, USA) with the ABIPRISM-7500 instrument (Applied Biosystems, Foster City, CA), as described previously. (60) Data obtained were analyzed according to the 2–ΔΔCt method.

4.21. Statistics

All experiments were performed in triplicate and repeated at least twice. Data were expressed as mean values ± standard deviation (SD) or standard error of the mean (SEM) and analyzed by Student’s t test or ANOVA performed by GraphPad Prism 9 software. The level of significance was p < 0.05.

4.22. In Vitro ADME Assays

4.22.1. Chemicals

All solvents and reagents were from Sigma-Aldrich Srl (Milan, Italy). Dodecane was purchased from Fluka (Milan, Italy). Pooled male donors 20 mg/mL HLMs were from BD Gentest-Biosciences (San Jose, California). Milli-Q quality water (Millipore, Milford, MA, USA) was used. Hydrophobic filter plates (MultiScreen-IP, clear plates, 0.45 mm diameter pore size), 96-well microplates, and 96-well UV-transparent microplates were obtained from Millipore (Bedford, MA, USA).

4.22.2. UV/LC–MS Methods

LC analyses for water solubility were performed by an LC–MS/MS system consisting of a Varian apparatus (Varian Inc) including a vacuum solvent degassing unit, two pumps (212-LC), a Triple Quadrupole MSD (Mod. 320-LC) mass spectrometer with ES interface, and Varian MS Workstation System Control Vers. 6.9 software. Chromatographic separation was obtained using a Kinetex C18 column (150 × 4.6 mm) with a 5 μm particle size and gradient elution with a binary solution; (eluent A: ACN, eluent B: water, both eluents were acidified with formic acid 0.1% v/v). The analysis started with 5% of A (from t = 0 to t = 1 min), then A was increased to 95% (from t = 1 to t = 10 min), then kept at 95% (from t = 10 to t = 19 min), and finally returned to 5% of eluent A in 1.0 min. The flow rate was 0.6 mL/min, and injection volumes were 10 μL. The instrument operated in positive mode, and parameters were detector 1450 V, drying gas pressure 35.0 psi, desolvation temperature 300.0 °C, nebulizing gas 45.0 psi, needle 5550 V and shield 350 V. Nitrogen was used as a nebulizer gas and drying gas. Collision-induced dissociation was performed using argon as the collision gas at a pressure of 1.8 mTorr in the collision cell. The transitions as well as the capillary voltage and the collision energy used are appropriated for each tested compound. Quantification of the single compound was made by comparison with appropriate calibration curves realized with standard solutions in methanol. LC analyses of PAMPA, metabolic stability, and stability tests (in MeOH, PBS, human plasma) were performed by UV/LC–MS with an Agilent 1100 LC/MSD VL system ((G1946C) Agilent Technologies, Palo Alto, CA) using a Phenomenex Kinetex C18–100 Å (150 × 4.6 mm, 5 μm particle size) at room temperature. Analyses were carried out with the same chromatographic conditions reported above.

4.22.3. Water Solubility

Solid 5c and 13 (1 mg) were added to 1 mL of distilled water. Each sample was mixed at room temperature in a shaker water bath for 24 h. (62) The resulting suspension was filtered through a 0.45 μm nylon filter (Acrodisc), and the solubilized compound was quantified in triplicate using the LC–MS/MS method reported above, by comparison with the appropriate calibration curve that was obtained from samples of the compound dissolved in methanol at different concentrations.

4.22.4. Parallel Artificial Membrane Permeability Assay (PAMPA)

Each “donor solution” was prepared from a solution of the appropriate compound (DMSO, 1 mM) diluted with phosphate buffer (pH 7.4, 0.025 M) up to a final concentration of 0.5 mM. The donor wells were filled with 150 μL of “donor solution”. The filters were coated with 10 μL of 1% (w/v) dodecane solution of phosphatidylcholine, and the lower wells were filled with 300 μL of “acceptor solution” (50% v/v DMSO and phosphate buffer). The sandwich plate was assembled and incubated for 5 h at room temperature with gentle shaking. After the incubation time, plates were separated and the amount of compound in both the donor and acceptor wells was measured by UV/LC–MS. For each compound, the determination was performed in three independent experiments. Permeability (Papp) was calculated according to the following equation obtained from the literature equation (63,64) with some modification in order to obtain permeability values in cm/s:
where VA is the volume in the acceptor well (cm3), VD is the volume in the donor well (cm3), A is the “effective area” of the membrane (cm2), t is the incubation time (s), and r is the ratio between drug concentration in the acceptor and equilibrium concentration of the drug in the total volume (VD + VA). Drug concentration was estimated by using the peak area integration. Membrane retentions (%MR) were calculated according to the following equation:
where r is the ratio between drug concentration in the acceptor and equilibrium concentration, and D, A, and Eq represent drug concentration in the donor, acceptor, and equilibrium solutions, respectively.

4.22.5. Metabolic Stability in HLMs (Human Liver Microsomes)

DMSO solutions of 5c and 13 were incubated at 37 °C for 1 h in presence of human liver microsomes (0.2 mg/mL, 5 μL), an NADPH regenerating system (NADPH 0.2 mM, NADPH+ 1 mM, d-glucose-6-phosphate 4 mM, 4 unit/mL glucose-6-phosphate dehydrogenase and MgCl2 48 mM), and phosphate buffer (pH 7.4, 25 mM, up to a final volume of 500 μL). The reaction was cooled down in ice and quenched by adding acetonitrile (1.0 mL). After centrifugation (4000 rpm for 10 min), the supernatant was taken, dried under nitrogen flow, and suspended in 100 μL of methanol and the parent drug and metabolites were subsequently determined by UV/LC–MS. The percentage of nonmetabolized compounds was calculated by comparison with reference solutions. For each compound, the determination was performed in three independent experiments.

4.22.6. Stability Test

4.22.6.1. In Polar Solvents
Each compound was dissolved at RT in MeOH or PBS (25 mM, pH 7.4) up to a final concentration of 500 μM. Aliquot samples (20 μL) were taken at fixed time points (0.0, 4.0, 8.0, and 24.0 h) and were analyzed by UV/LC–MS. For each compound, the determination was performed in three independent experiments.
4.22.6.2. In Human Plasma
The incubation mixture (total volume of 2.0 mL) was constituted by the following components: pooled human plasma (1.5 mL, 55.7 mg protein/mL), (65) HEPES buffer (1.4 mL, 25 mM, 140 mM NaCl pH 7.4), and 100 μL of each compound in DMSO (3.0 mM). The solution was mixed in a test tube that was incubated at 37 °C. At set time points (0.0, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, and 24.0 h), samples of 100 μL were taken, mixed with 400 μL of cold acetonitrile, and centrifuged at 5000 rpm for 15 min. (66) The supernatant was removed and analyzed by UV/LC–MS. For each compound, the determination was performed in three independent experiments.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01806.

  • RMSD analysis of ligand disposition in the MAGL-11b complexes during the MD; MM-PBSA results for the eight different MAGL-11b complexes; correlation of ligand activity and binding energy obtained using different εint values; activities and best correlated binding energy values predicted for the analyzed ligands; IC50 values of compound 13 toward CB1, CB2, and FAAH; MM-PBSA (εint = 4) results for MAGL-11b and MAGL-13 complexes; minimized average structures of MAGL in complex with 7 and 12; RP-HPLC traces of the final compounds; 1H and 13C-NMR spectra of the final compounds; ESI-HRMS spectra of the final compounds; analysis of the mechanism of MAGL inhibition of JZL-184; inhibition of the activity of MAGL and competitive binding of compound 13; minimized average structures of MAGL in complex with 11b superimposed with 5b; minimized average structures of MAGL in complex with 13 (PDF)

  • Molecular formula strings and associated biochemical data for the reported compounds (CSV)

  • PDB ID of the crystal structure of MAGL (PDB)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Giulia Bononi - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    • Miriana Di Stefano - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy;  Department of Life Sciences, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy;  Orcidhttps://orcid.org/0000-0001-6727-5816
    • Giulio Poli - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    • Gabriella Ortore - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    • Philip Meier - Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland
    • Francesca Masetto - Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, DeBoelelaan 1117, 1081HV Amsterdam, The Netherlands
    • Isabella Caligiuri - Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy
    • Flavio Rizzolio - Pathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081 Aviano, Italy;  Department of Molecular Sciences and Nanosystems, Ca’ Foscari University, 30123 Venezia, Italy
    • Marco Macchia - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
    • Andrea Chicca - Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland;  Orcidhttps://orcid.org/0000-0001-9593-636X
    • Amir Avan - Metabolic Syndrome Research Center, Mashhad University of Medical Science, Mashhad 91886-17871, Iran
    • Elisa Giovannetti - Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, DeBoelelaan 1117, 1081HV Amsterdam, The Netherlands;  Cancer Pharmacology Lab, Fondazione Pisana per la Scienza, via Giovannini 13, 56017 San Giuliano Terme, Pisa, Italy
    • Chiara Vagaggini - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
    • Annalaura Brai - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy;  Orcidhttps://orcid.org/0000-0001-6395-9348
    • Elena Dreassi - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy
    • Massimo Valoti - Department of Life Sciences, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy;  Orcidhttps://orcid.org/0000-0002-7240-3576
    • Filippo Minutolo - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy;  Center for Instrument Sharing of the University of Pisa (CISUP), Lungarno Pacinotti 43, 56126 Pisa, Italy
    • Jürg Gertsch - Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012 Bern, Switzerland
    • Tiziano Tuccinardi - Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy;  Center for Instrument Sharing of the University of Pisa (CISUP), Lungarno Pacinotti 43, 56126 Pisa, Italy;  Orcidhttps://orcid.org/0000-0002-6205-4069
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We are grateful to the University of Pisa (Progetti di Ricerca di Ateneo, prog. PRA-2020-58), MIUR (PRIN 2017, project 2017SA5837) and the Italian Ministry of Health – Ricerca Finalizzata 2016 - NET-2016-02363765 for funding. This work was also supported by the CCA Foundation 2018 grant, KWF Dutch Cancer Society grant (KWF project#19571), and AIRC/IG-grant-2020 (E.G.). We thank Lisa Baldoni and Francesco Scialpi for their support to the synthesis of some compounds. We acknowledge Center for Instrument Sharing of the University of Pisa (CISUP) for the acquisition and elaboration of the high-resolution mass spectra.

ABBREVIATIONS

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2-AG

2-arachidonoylglycerol

4-NPA

4-nitrophenylacetate

9-BBN

9-borabicyclo[3.3.1]nonane

AA

arachidonic acid

ABHD6

α/β hydrolase-6

ABHD12

α/β hydrolase-12

ABPP

activity-based protein profiling

AEA

anandamide

BSA

bovine serum albumin

CB

cannabinoid receptor

CG

conjugate gradient

DIPEA

N,N-diisopropylethylamine

DMF

N,N-dimethylformamide

DTT

1,4-dithio-dl-threitol

eCBs

endocannabinoids

FAAH

fatty acid amide hydrolase

FPKM

fragments per kilobase million

GAFF

general Amber force field

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

hENT1

human equilibrative nucleoside transporter 1

MAGL

monoacylglycerol lipase

MD

molecular dynamics

MM-PBSA

molecular mechanics Poisson–Boltzmann surface area

MMP9

matrix metalloproteinase 9

PAAD

pancreatic adenocarcinoma

PDAC

pancreatic ductal adenocarcinoma

PME

Particle Mesh Ewald

RMSD

root-mean square deviation

SRB

sulforhodamine B

TBAI

tetrabutylammonium iodide

References

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This article references 66 other publications.

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    Rajesh, M.; Pan, H.; Mukhopadhyay, P.; Batkai, S.; Osei-Hyiaman, D.; Hasko, G.; Liaudet, L.; Gao, B.; Pacher, P. Pivotal Advance: Cannabinoid-2 Receptor Agonist HU-308 Protects against Hepatic Ischemia/Reperfusion Injury by Attenuating Oxidative Stress, Inflammatory Response, and Apoptosis. J. Leukocyte Biol. 2007, 82, 1382– 1389,  DOI: 10.1189/jlb.0307180
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    Pivotal advance: cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress, inflammatory response, and apoptosis
    Rajesh, Mohanraj; Pan, Hao; Mukhopadhyay, Partha; Batkai, Sandor; Osei-Hyiaman, Douglas; Hasko, Gyorgy; Liaudet, Lucas; Gao, Bin; Pacher, Pal
    Journal of Leukocyte Biology (2007), 82 (6), 1382-1389CODEN: JLBIE7; ISSN:0741-5400. (Federation of American Societies for Experimental Biology)
    In this study, we have investigated the role of the cannabinoid CB2 (CB2) receptor in an in vivo mouse model of hepatic ischemia/reperfusion (I/R) injury. In addn., we have assessed the role of the CB2 receptor in TNF-α-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and in the adhesion of human neutrophils to HLSECs in vitro. The potent CB2 receptor agonist HU-308, given prior to the induction of I/R, significantly attenuated the extent of liver damage (measured by blood serum alanine aminotransferase and lactate dehydrogenase) and decreased blood serum and tissue TNF-α, MIP-1α, and MIP-2 levels, tissue lipid peroxidn., neutrophil infiltration, DNA fragmentation, and caspase 3 activity. The protective effect of HU-308 against liver damage was also preserved when given right after the ischemic episode. HU-308 also attenuated the TNF-α-induced ICAM-1 and VCAM-1 expression in HLSECs, which expressed CB2 receptors, and the adhesion of human neutrophils to HLSECs in vitro. These findings suggest that selective CB2 receptor agonists may represent a novel, protective strategy against I/R injury by attenuating oxidative stress, inflammatory response, and apoptosis.
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  2. 2
    Calignano, A.; La Rana, G.; Giuffrida, A.; Piomelli, D. Control of Pain Initiation by Endogenous Cannabinoids. Nature 1998, 394, 277– 281,  DOI: 10.1038/28393
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    2
    Control of pain initiation by endogenous cannabinoids
    Calignano, Antonio; La Rana, Giovanna; Giuffrida, Andrea; Piomelli, Daniele
    Nature (London) (1998), 394 (6690), 277-281CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
    The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here the authors show that anandamide attenuates the pain behavior produced by chem. damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compds. at synergistically reducing pain responses 100-fold more potently than does each compd. alone. Gas-chromatog./mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behavior produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.
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  3. 3
    Sánchez, A. J.; García-Merino, A. Neuroprotective Agents: Cannabinoids. Clin. Immunol. 2012, 142, 57– 67,  DOI: 10.1016/j.clim.2011.02.010
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    Neuroprotective agents: Cannabinoids
    Sanchez, A. J.; Garcia-Merino, A.
    Clinical Immunology (Amsterdam, Netherlands) (2012), 142 (1), 57-67CODEN: CLIIFY; ISSN:1521-6616. (Elsevier B.V.)
    A review. Chronic inflammation and neurodegeneration are the main pathol. traits of multiple sclerosis that coexist in all stages of the disease course, with complex and still nonclarified relationships. Currently licensed medications have efficacy to control aspects related to inflammation, but have been unable to modify pure progression. Exptl. work has provided robust evidence of the immunomodulatory and neuroprotective properties that cannabinoids exert in animal models of multiple sclerosis. Through activation of the CB2 receptor, cannabinoids modulate peripheral blood lymphocytes, interfere with migration across the blood-brain barrier and control microglial/macrophage activation. CB1 receptors present in neural cells have a fundamental role in direct neuroprotection against several insults, mainly excitotoxicity. In multiple sclerosis, several reports have documented the disturbance of the endocannabinoid system. Considering the actions demonstrated exptl., cannabinoids might be promising agents to target the main aspects of the human disease.
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  4. 4
    Pertwee, R. G.; Ross, R. A. Cannabinoid Receptors and Their Ligands. Prostaglandins, Leukotrienes Essent. Fatty Acids 2002, 66, 101– 121,  DOI: 10.1054/plef.2001.0341
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    4
    Cannabinoid receptors and their ligands
    Pertwee, R. G.; Ross, R. A.
    Prostaglandins, Leukotrienes and Essential Fatty Acids (2002), 66 (2&3), 101-121CODEN: PLEAEU; ISSN:0952-3278. (Churchill Livingstone)
    A review. There are at least two types of cannabinoid receptors, CB1 and CB2, both coupled to G proteins. CB1 receptors exist primarily on central and peripheral neurons, one of their functions being to modulate neurotransmitter release. CB2 receptors are present mainly on immune cells. Their roles are proving more difficult to establish but seem to include the modulation of cytokine release. Endogenous agonists for cannabinoid receptors (endocannabinoids) have also been discovered, the most important being arachidonoyl ethanolamide (anandamide), 2-arachidonoyl glycerol and 2-arachidonyl glyceryl ether. Other endocannabinoids and cannabinoid receptor types may also exist. Although anandamide can act through CB1 and CB2 receptors, it is also a vanilloid receptor agonist and some of its metabolites may possess yet other important modes of action. The discovery of the system of cannabinoid receptors and endocannabinoids that constitutes the "endocannabinoid system" has prompted the development of CB1- and CB2-selective agonists and antagonists/inverse agonists. CB1/CB2 agonists are already used clin., as anti-emetics or to stimulate appetite. Potential therapeutic uses of cannabinoid receptor agonists include the management of multiple sclerosis/spinal cord injury, pain, inflammatory disorders, glaucoma, bronchial asthma, vasodilation that accompanies advanced cirrhosis, and cancer. Following their release onto cannabinoid receptors, endocannabinoids are removed from the extracellular space by membrane transport and then degraded by intracellular enzymic hydrolysis. Inhibitors of both these processes have been developed. Such inhibitors have therapeutic potential as animal data suggest that released endocannabinoids mediate redns. both in inflammatory pain and in the spasticity and tremor of multiple sclerosis. So too have CB1 receptor antagonists, for example for the suppression of appetite and the management of cognitive dysfunction or schizophrenia.
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  5. 5
    Piomelli, D. The Molecular Logic of Endocannabinoid Signalling. Nat. Rev. Neurosci. 2003, 4, 873– 884,  DOI: 10.1038/nrn1247
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    5
    The molecular logic of endocannabinoid signalling
    Piomelli, Daniele
    Nature Reviews Neuroscience (2003), 4 (11), 873-884CODEN: NRNAAN; ISSN:1471-003X. (Nature Publishing Group)
    A review. The endocannabinoids are a family of lipid messengers that engage the cell surface receptors that are targeted by Δ9-tetrahydrocannabinol, the active principle in marijuana (Cannabis). They are made on demand through cleavage of membrane precursors and are involved in various short-range signaling processes. In the brain, they combine with CB1 cannabinoid receptors on axon terminals to regulate ion channel activity and neurotransmitter release. Their ability to modulate synaptic efficacy has a wide range of functional consequences and provides unique therapeutic possibilities.
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  6. 6
    Ahn, K.; McKinney, M. K.; Cravatt, B. F. Enzymatic Pathways That Regulate Endocannabinoid Signaling in the Nervous System. Chem. Rev. 2008, 108, 1687– 1707,  DOI: 10.1021/cr0782067
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    6
    Enzymatic pathways that regulate endocannabinoid signaling in the nervous system
    Ahn, Kay; McKinney, Michele K.; Cravatt, Benjamin F.
    Chemical Reviews (Washington, DC, United States) (2008), 108 (5), 1687-1707CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Recent advances in the understanding of the compn. and regulation of endocannabinoid metabolic pathways, esp. with respect to the nervous system, are reviewed. The current state of the understanding of the 4 major pathways for endocannabinoid metab. are discussed. These include (1) anandamide degrdn.; (2) anandamide biosynthesis; (3) 2-arachidonoylglycerol (2-AG) degrdn.; and (4) 2-AG biosynthesis. Some of these pathways, such as anandamide degrdn., are relatively well characterized, at least in terms of the participating enzymes, esp. fatty acid amide hydrolase (FAAH), and the advent of specific research tools to probe their function in vivo. For the other pathways, candidate enzymes have been identified, but the specific roles that these proteins play in regulating endocannabinoid metab. in vivo remains to be elucidated.
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  7. 7
    Blankman, J. L.; Simon, G. M.; Cravatt, B. F. A Comprehensive Profile of Brain Enzymes That Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol. Chem. Biol. 2007, 14, 1347– 1356,  DOI: 10.1016/j.chembiol.2007.11.006
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    7
    A Comprehensive Profile of Brain Enzymes that Hydrolyze the Endocannabinoid 2-Arachidonoylglycerol
    Blankman, Jacqueline L.; Simon, Gabriel M.; Cravatt, Benjamin F.
    Chemistry & Biology (Cambridge, MA, United States) (2007), 14 (12), 1347-1356CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Endogenous ligands for cannabinoid receptors ("endocannabinoids") include the lipid transmitters anandamide and 2-arachidonoylglycerol (2-AG). Endocannabinoids modulate a diverse set of physiol. processes and are tightly regulated by enzymic biosynthesis and degrdn. Termination of anandamide signaling by fatty acid amide hydrolase (FAAH) is well characterized, but less is known about the inactivation of 2-AG, which can be hydrolyzed by multiple enzymes in vitro, including FAAH and monoacylglycerol lipase (MAGL). Here, we have taken a functional proteomic approach to comprehensively map 2-AG hydrolases in the mouse brain. Our data reveal that ∼85% of brain 2-AG hydrolase activity can be ascribed to MAGL, and that the remaining 15% is mostly catalyzed by two uncharacterized enzymes, ABHD6 and ABHD12. Interestingly, MAGL, ABHD6, and ABHD12 display distinct subcellular distributions, suggesting that they may control different pools of 2-AG in the nervous system.
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  8. 8
    Deng, H.; Li, W. Monoacylglycerol Lipase Inhibitors: Modulators for Lipid Metabolism in Cancer Malignancy, Neurological and Metabolic Disorders. Acta Pharm. Sin. B 2020, 10, 582– 602,  DOI: 10.1016/j.apsb.2019.10.006
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    8
    Monoacylglycerol lipase inhibitors: modulators for lipid metabolism in cancer malignancy, neurological and metabolic disorders
    Deng, Hui; Li, Weimin
    Acta Pharmaceutica Sinica B (2020), 10 (4), 582-602CODEN: APSBCW; ISSN:2211-3835. (Elsevier B.V.)
    A review. Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays a crucial role catalyzing the hydrolysis of monoglycerides into glycerol and fatty acids. It links the endocannabinoid and eicosanoid systems together by degrdn. of the abundant endocannabinoid 2-arachidaoylglycerol into arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. MAGL inhibitors have been considered as important agents in many therapeutic fields, including anti-nociceptive, anxiolytic, anti-inflammatory, and even anti-cancer. Currently, ABX-1431, a first-in-class inhibitor of MAGL, is entering clin. phase 2 studies for neurol. disorders and other diseases. This review summarizes the diverse (patho)physiol. roles of MAGL and will provide an overview on the development of MAGL inhibitors. Although a large no. of MAGL inhibitors have been reported, novel inhibitors are still required, particularly reversible ones.
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  9. 9
    Long, J. Z.; Li, W.; Booker, L.; Burston, J. J.; Kinsey, S. G.; Schlosburg, J. E.; Pavón, F. J.; Serrano, A. M.; Selley, D. E.; Parsons, L. H.; Lichtman, A. H.; Cravatt, B. F. Selective Blockade of 2-Arachidonoylglycerol Hydrolysis Produces Cannabinoid Behavioral Effects. Nat. Chem. Biol. 2009, 5, 37– 44,  DOI: 10.1038/nchembio.129
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    9
    Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects
    Long, Jonathan Z.; Li, Weiwei; Booker, Lamont; Burston, James J.; Kinsey, Steven G.; Schlosburg, Joel E.; Pavon, Franciso J.; Serrano, Antonia M.; Selley, Dana E.; Parsons, Loren H.; Lichtman, Aron H.; Cravatt, Benjamin F.
    Nature Chemical Biology (2009), 5 (1), 37-44CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymic hydrolysis, a process that for anandamide is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects obsd. with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metab. Here, we report a potent and selective inhibitor of MAGL called JZL184 that, upon administration to mice, raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically assocd. with the pharmacol. of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.
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  10. 10
    Nomura, D. K.; Morrison, B. E.; Blankman, J. L.; Long, J. Z.; Kinsey, S. G.; Marcondes, M. C. G.; Ward, A. M.; Hahn, Y. K.; Lichtman, A. H.; Conti, B.; Cravatt, B. F. Endocannabinoid Hydrolysis Generates Brain Prostaglandins That Promote Neuroinflammation. Science 2011, 334, 809– 813,  DOI: 10.1126/science.1209200
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    10
    Endocannabinoid Hydrolysis Generates Brain Prostaglandins That Promote Neuroinflammation
    Nomura, Daniel K.; Morrison, Bradley E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Kinsey, Steven G.; Marcondes, Maria Cecilia G.; Ward, Anna M.; Hahn, Yun Kyung; Lichtman, Aron H.; Conti, Bruno; Cravatt, Benjamin F.
    Science (Washington, DC, United States) (2011), 334 (6057), 809-813CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Phospholipase A2(PLA2) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in brain, where monoacyl-glycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic PLA2. These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.
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  11. 11
    Pisanti, S.; Picardi, P.; D’Alessandro, A.; Laezza, C.; Bifulco, M. The Endocannabinoid Signaling System in Cancer. Trends Pharmacol. Sci. 2013, 34, 273– 282,  DOI: 10.1016/j.tips.2013.03.003
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    11
    The endocannabinoid signaling system in cancer
    Pisanti, Simona; Picardi, Paola; D'Alessandro, Alba; Laezza, Chiara; Bifulco, Maurizio
    Trends in Pharmacological Sciences (2013), 34 (5), 273-282CODEN: TPHSDY; ISSN:0165-6147. (Elsevier Ltd.)
    A review. Changes in lipid metab. are intimately related to cancer. Several classes of bioactive lipids play roles in the regulation of signaling pathways involved in neoplastic transformation and tumor growth and progression. The endocannabinoid system, comprising lipid-derived endocannabinoids, their G-protein-coupled receptors (GPCRs), and the enzymes for their metab., is emerging as a promising therapeutic target in cancer. This report highlights the main signaling pathways for the antitumor effects of the endocannabinoid system in cancer and its basic role in cancer pathogenesis, and discusses the alternative view of cannabinoid receptors as tumor promoters. We focus on new players in the antitumor action of the endocannabinoid system and on emerging crosstalk among cannabinoid receptors and other membrane or nuclear receptors involved in cancer. We also discuss the enzyme MAGL, a key player in endocannabinoid metab. that was recently recognized as a marker of tumor lipogenic phenotype.
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  12. 12
    Strangman, N. M.; Patrick, S. L.; Hohmann, A. G.; Tsou, K.; Walker, J. M. Evidence for a Role of Endogenous Cannabinoids in the Modulation of Acute and Tonic Pain Sensitivity. Brain Res. 1998, 813, 323– 328,  DOI: 10.1016/S0006-8993(98)01031-2
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    12
    Evidence for a role of endogenous cannabinoids in the modulation of acute and tonic pain sensitivity
    Strangman, Nicole M.; Patrick, Saundra L.; Hohmann, Andrea G.; Tsou, Kang; Walker, J. Michael
    Brain Research (1998), 813 (2), 323-328CODEN: BRREAP; ISSN:0006-8993. (Elsevier Science B.V.)
    The competitive CB1 receptor antagonist SR 141716A was used to test the hypothesis that endogenous cannabinoids modulate tonic pain sensitivity. Pretreatment with the antagonist significantly enhanced the response to a chem. nociceptive stimulus in the formalin test. Posttreatment with the antagonist 5 min following the induction of tonic pain produced hyperalgesia during the tonic phase only. These findings suggest that endogenous cannabinoids serve naturally to modulate the maintenance of pain following repeated noxious stimulation.
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  13. 13
    Guindon, J.; Desroches, J.; Beaulieu, P. The Antinociceptive Effects of Intraplantar Injections of 2-Arachidonoyl Glycerol Are Mediated by Cannabinoid CB 2 Receptors. Br. J. Pharmacol. 2007, 150, 693– 701,  DOI: 10.1038/sj.bjp.0706990
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    13
    The antinociceptive effects of intraplantar injections of 2-arachidonoyl glycerol are mediated by cannabinoid CB2 receptors
    Guindon, J.; Desroches, J.; Beaulieu, P.
    British Journal of Pharmacology (2007), 150 (6), 693-701CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
    Background and purpose:2-arachidonoyl glycerol (2-AG) is an endogenous cannabinoid with central antinociceptive properties. Its degrdn. is catalyzed by monoacylglycerol lipase (MGL) whose activity is inhibited by URB602, a new synthetic compd. The peripheral antinociceptive effects of 2-AG and URB602 in an inflammatory model of pain are not yet detd. We have evaluated these effects with and without the cannabinoid CB1 (AM251) and CB2 (AM630) receptor antagonists.Exptl. approach:Inflammation was induced in rat hind paws by intraplantar injection of formalin. Nociception was assessed behaviorally over the next 60 min, in 19 exptl. groups: (1) control; (2-6) 2-AG (0.01-100 μg); (7) AM251 (80 μg); (8) AM251+2-AG (10 μg); (9) AM630 (25 μg); (10) AM630+2-AG (10 μg); (11-16) URB602 (0.1-500 μg); (17) 2-AG+URB602 (ED50); (18) AM251+URB602 (ED50); (19) AM630+URB602 (ED50). Drugs were injected s.c. in the dorsal surface of the hind paw (50 μl), 15 min before formalin injection into the same paw. Key results:2-AG and URB602 produced dose-dependent antinociceptive effects for the late phases of the formalin test with ED50 of 0.65±0.455 μg and 68±14.3 μg, resp. Their combination at ED50 doses produced an additive antinociceptive effect. These effects were inhibited by AM630 but not by AM251 for 2-AG and by the two cannabinoid antagonists for URB602. Conclusions and implications:Locally injected 2-AG and URB602 decreased pain behavior in a dose-dependent manner in an inflammatory model of pain. The antinociceptive effect of 2-AG was mediated by the CB2 receptor.
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  14. 14
    Ignatowska-Jankowska, B. M.; Ghosh, S.; Crowe, M. S.; Kinsey, S. G.; Niphakis, M. J.; Abdullah, R. A.; Tao, Q.; O’ Neal, S. T.; Walentiny, D. M.; Wiley, J. L.; Cravatt, B. F.; Lichtman, A. H. In Vivo Characterization of the Highly Selective Monoacylglycerol Lipase Inhibitor KML29: Antinociceptive Activity without Cannabimimetic Side Effects. Br. J. Pharmacol. 2014, 171, 1392– 1407,  DOI: 10.1111/bph.12298
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    14
    In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects
    Ignatowska-Jankowska, B. M.; Ghosh, S.; Crowe, M. S.; Kinsey, S. G.; Niphakis, M. J.; Abdullah, R. A.; Tao, Q.; O'Neal, S. T.; Walentiny, D. M.; Wiley, J. L.; Cravatt, B. F.; Lichtman, A. H.
    British Journal of Pharmacology (2014), 171 (6), 1392-1407CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
    Background and Purpose: Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compd., KML29 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate). Exptl. Approach : In the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric hemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temp., catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm. Key Results: KML29 attenuated carrageenan-induced paw edema and completely reversed carrageenan-induced mech. allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB1) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB1 and CB2 receptors played differential roles in these pharmacol. effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Δ9-tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (-/-) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects. Conclusions and Implications : These results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behavior without occurrence of cannabimimetic side effects.
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  15. 15
    Kinsey, S. G.; Long, J. Z.; Cravatt, B. F.; Lichtman, A. H. Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibitors Produce Anti-Allodynic Effects in Mice Through Distinct Cannabinoid Receptor Mechanisms. J. Pain 2010, 11, 1420– 1428,  DOI: 10.1016/j.jpain.2010.04.001
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    15
    Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibitors Produce Anti-Allodynic Effects in Mice Through Distinct Cannabinoid Receptor Mechanisms
    Kinsey, Steven G.; Long, Jonathan Z.; Cravatt, Benjamin F.; Lichtman, Aron H.
    Journal of Pain (2010), 11 (12), 1420-1428CODEN: JPOAB5; ISSN:1526-5900. (Elsevier)
    The endocannabinoids anandamide and 2-arachidonoylglycerol are predominantly regulated by the resp. catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Inhibition of these enzymes elevates endocannabinoid levels and attenuates neuropathic pain. In the present study, CB1 and CB2 receptor-deficient mice were subjected to chronic constriction injury (CCI) of the sciatic nerve to examine the relative contribution of each receptor for the anti-allodynic effects of the FAAH inhibitor, PF-3845, and the MAGL inhibitor, JZL184. CCI caused marked hypersensitivity to mech. and cold stimuli, which was not altered by deletion of either the CB1 or CB2 receptor, but was attenuated by gabapentin, as well as by each enzyme inhibitor. Whereas PF-3845 lacked anti-allodynic efficacy in both knockout lines, JZL184 did not produce anti-allodynic effects in CB1 (-/-) mice, but retained its anti-allodynic effects in CB2 (-/-) mice. These data indicate that FAAH and MAGL inhibitors reduce nerve injury-related hyperalgesic states through distinct cannabinoid receptor mechanisms of action. In conclusion, although endogenous cannabinoids do not appear to play a tonic role in long-term expression of neuropathic pain states, both FAAH and MAGL represent potential therapeutic targets for the development of pharmacol. agents to treat chronic pain resulting from nerve injury. Perspective: This article presents data addressing the cannabinoid receptor mechanisms underlying the anti-allodynic actions of endocannabinoid catabolic enzyme inhibitors in the mouse sciatic nerve ligation model. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors reduced allodynia through distinct cannabinoid receptor mechanisms. These enzymes offer potential targets to treat neuropathic pain.
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  16. 16
    Jaiswal, S.; Akhilesh; Uniyal, A.; Tiwari, V.; Raja Ayyannan, S. Synthesis and Evaluation of Dual Fatty Acid Amide Hydrolase-Monoacylglycerol Lipase Inhibition and Antinociceptive Activities of 4-Methylsulfonylaniline-Derived Semicarbazones. Bioorg. Med. Chem. 2022, 60, 116698,  DOI: 10.1016/j.bmc.2022.116698
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    16
    Synthesis and evaluation of dual fatty acid amide hydrolase-monoacylglycerol lipase inhibition and antinociceptive activities of 4-methylsulfonylaniline-derived semicarbazones
    Jaiswal, Shivani; Akhilesh; Uniyal, Ankit; Tiwari, Vinod; Raja Ayyannan, Senthil
    Bioorganic & Medicinal Chemistry (2022), 60 (), 116698CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    We designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones I [R1 = H, Me, Ph; R2 = H, 2-OH, 4-F, etc.] and II [R1 = H, allyl, propargyl; R2 = H, Cl] and evaluated for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition properties. Most of the compds. showed potency toward both enzymes with leading FAAH selectivity. Compd. (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC50 = 11 nM) and MAGL (IC50 = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Mol. docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π-π stacking via Ph ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by mol. simulation studies. PAMPA assay revealed that the lead compd. was suitable for blood-brain penetration. The lead compd. showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo expts. unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compd. exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compd. can be a potential template for the development of agents for neuropathic pain.
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  17. 17
    Nomura, D. K.; Long, J. Z.; Niessen, S.; Hoover, H. S.; Ng, S. W.; Cravatt, B. F. Monoacylglycerol Lipase Regulates a Fatty Acid Network That Promotes Cancer Pathogenesis. Cell 2010, 140, 49– 61,  DOI: 10.1016/j.cell.2009.11.027
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    17
    Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis
    Nomura, Daniel K.; Long, Jonathan Z.; Niessen, Sherry; Hoover, Heather S.; Ng, Shu-Wing; Cravatt, Benjamin F.
    Cell (Cambridge, MA, United States) (2010), 140 (1), 49-61CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Tumor cells display progressive changes in metab. that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.
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  18. 18
    Schlosburg, J. E.; Blankman, J. L.; Long, J. Z.; Nomura, D. K.; Pan, B.; Kinsey, S. G.; Nguyen, P. T.; Ramesh, D.; Booker, L.; Burston, J. J.; Thomas, E. A.; Selley, D. E.; Sim-Selley, L. J.; Liu, Q. S.; Lichtman, A. H.; Cravatt, B. F. Chronic Monoacylglycerol Lipase Blockade Causes Functional Antagonism of the Endocannabinoid System. Nat. Neurosci. 2010, 13, 1113– 1119,  DOI: 10.1038/nn.2616
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    18
    Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system
    Schlosburg, Joel E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Nomura, Daniel K.; Pan, Bin; Kinsey, Steven G.; Nguyen, Peter T.; Ramesh, Divya; Booker, Lamont; Burston, James J.; Thomas, Elizabeth A.; Selley, Dana E.; Sim-Selley, Laura J.; Liu, Qing-song; Lichtman, Aron H.; Cravatt, Benjamin F.
    Nature Neuroscience (2010), 13 (9), 1113-1119CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
    Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacol. tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused phys. dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and assocd. with agonism and functional antagonism of the brain cannabinoid system, resp.
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  19. 19
    Chanda, P. K.; Gao, Y.; Mark, L.; Btesh, J.; Strassle, B. W.; Lu, P.; Piesla, M. J.; Zhang, M.-Y.; Bingham, B.; Uveges, A.; Kowal, D.; Garbe, D.; Kouranova, E. V.; Ring, R. H.; Bates, B.; Pangalos, M. N.; Kennedy, J. D.; Whiteside, G. T.; Samad, T. A. Monoacylglycerol Lipase Activity Is a Critical Modulator of the Tone and Integrity of the Endocannabinoid System. Mol. Pharmacol. 2010, 78, 996– 1003,  DOI: 10.1124/mol.110.068304
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    19
    Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system
    Chanda, Pranab K.; Gao, Ying; Mark, Lilly; Btesh, Joan; Strassle, Brian W.; Lu, Peimin; Piesla, Michael J.; Zhang, Mei-Yi; Bingham, Brendan; Uveges, Albert; Kowal, Dianne; Garbe, David; Kouranova, Evguenia V.; Ring, Robert H.; Bates, Brian; Pangalos, Menelas N.; Kennedy, Jeffrey D.; Whiteside, Garth T.; Samad, Tarek A.
    Molecular Pharmacology (2010), 78 (6), 996-1003CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
    Endocannabinoids are lipid mols. that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiol. processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymic biosynthesis and degrdn. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant redn. of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.
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  20. 20
    Taschler, U.; Radner, F. P. W.; Heier, C.; Schreiber, R.; Schweiger, M.; Schoiswohl, G.; Preiss-Landl, K.; Jaeger, D.; Reiter, B.; Koefeler, H. C.; Wojciechowski, J.; Theussl, C.; Penninger, J. M.; Lass, A.; Haemmerle, G.; Zechner, R.; Zimmermann, R. Monoglyceride Lipase Deficiency in Mice Impairs Lipolysis and Attenuates Diet-Induced Insulin Resistance. J. Biol. Chem. 2011, 286, 17467– 17477,  DOI: 10.1074/jbc.M110.215434
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    20
    Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance
    Taschler, Ulrike; Radner, Franz P. W.; Heier, Christoph; Schreiber, Renate; Schweiger, Martina; Schoiswohl, Gabriele; Preiss-Landl, Karina; Jaeger, Doris; Reiter, Birgit; Koefeler, Harald C.; Wojciechowski, Jacek; Theussl, Christian; Penninger, Josef M.; Lass, Achim; Haemmerle, Guenter; Zechner, Rudolf; Zimmermann, Robert
    Journal of Biological Chemistry (2011), 286 (20), 17467-17477CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Monoglyceride lipase (MGL) influences energy metab. by at least two mechanisms. First, it hydrolyzes monoacylglycerols (MG) into fatty acids and glycerol. These products can be used for energy prodn. or synthetic reactions. Second, MGL degrades 2-arachidonoyl glycerol (2-AG), the most abundant endogenous ligand of cannabinoid receptors (CBR). Activation of CBR affects energy homeostasis by central orexigenic stimuli, by promoting lipid storage, and by reducing energy expenditure. To characterize the metabolic role of MGL in vivo, we generated an MGL-deficient mouse model (MGL-ko). These mice exhibit a redn. in MG hydrolase activity and a concomitant increase in MG levels in adipose tissue, brain, and liver. In adipose tissue, the lack of MGL activity is partially compensated by hormone-sensitive lipase. Nonetheless, fasted MGL-ko mice exhibit reduced plasma glycerol and triacylglycerol, as well as liver triacylglycerol levels indicative for impaired lipolysis. Despite a strong elevation of 2-AG levels, MGL-ko mice exhibit normal food intake, fat mass, and energy expenditure. Yet mice lacking MGL show a pharmacol. tolerance to the CBR agonist CP 55,940 suggesting that the elevated 2-AG levels are functionally antagonized by desensitization of CBR. Interestingly, however, MGL-ko mice receiving a high fat diet exhibit significantly improved glucose tolerance and insulin sensitivity in comparison with wild-type controls despite equal wt. gain. In conclusion, our observations implicate that MGL deficiency impairs lipolysis and attenuates diet-induced insulin resistance. Defective degrdn. of 2-AG does not provoke cannabinoid-like effects on feeding behavior, lipid storage, and energy expenditure, which may be explained by desensitization of CBR.
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  21. 21
    Ghosh, S.; Wise, L. E.; Chen, Y.; Gujjar, R.; Mahadevan, A.; Cravatt, B. F.; Lichtman, A. H. The Monoacylglycerol Lipase Inhibitor JZL184 Suppresses Inflammatory Pain in the Mouse Carrageenan Model. Life Sci. 2013, 92, 498– 505,  DOI: 10.1016/j.lfs.2012.06.020
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    21
    The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model
    Ghosh, Sudeshna; Wise, Laura E.; Chen, Yugang; Gujjar, Ramesh; Mahadevan, Anu; Cravatt, Benjamin F.; Lichtman, Aron H.
    Life Sciences (2013), 92 (8-9), 498-505CODEN: LIFSAK; ISSN:0024-3205. (Elsevier B.V.)
    The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test.The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the resp. CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40 mg/kg) was administered for six days to assess tolerance.JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mech. allodynia. Complementary genetic and pharmacol. approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy.These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.
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  22. 22
    Schlosburg, J. E.; Kinsey, S. G.; Ignatowska-Jankowska, B.; Ramesh, D.; Abdullah, R. A.; Tao, Q.; Booker, L.; Long, J. Z.; Selley, D. E.; Cravatt, B. F.; Lichtman, A. H. Prolonged Monoacylglycerol Lipase Blockade Causes Equivalent Cannabinoid Receptor Type 1 Receptor–Mediated Adaptations in Fatty Acid Amide Hydrolase Wild-Type and Knockout Mice. J. Pharmacol. Exp. Ther. 2014, 350, 196– 204,  DOI: 10.1124/jpet.114.212753
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    22
    Prolonged monoacylglycerol lipase blockade causes equivalent cannabinoid receptor type 1 receptor-mediated adaptations in fatty acid amide hydrolase wild-type and knockout mice
    Schlosburg, Joel E.; Kinsey, Steven G.; Ignatowska-Jankowska, Bogna; Ramesh, Divya; Abdullah, Rehab A.; Tao, Qing; Booker, Lamont; Long, Jonathan Z.; Selley, Dana E.; Cravatt, Benjamin F.; Lichtman, Aron H.
    Journal of Pharmacology and Experimental Therapeutics (2014), 350 (2), 196-204, 9 pp.CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
    Complementary genetic and pharmacol. approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the resp. endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiol. roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and mol. signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacol. effects of Δ9-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding, and dependence as indicated by rimonabant-pptd. withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.
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  23. 23
    Muccioli, G. G.; Labar, G.; Lambert, D. M. CAY10499, a Novel Monoglyceride Lipase Inhibitor Evidenced by an Expeditious MGL Assay. ChemBioChem 2008, 9, 2704– 2710,  DOI: 10.1002/cbic.200800428
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    23
    CAY10499, a novel monoglyceride lipase inhibitor evidenced by an expeditious MGL assay
    Muccioli, Giulio G.; Labar, Geoffray; Lambert, Didier M.
    ChemBioChem (2008), 9 (16), 2704-2710CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Monoglyceride lipase (MGL) plays a major role in the metab. of the lipid transmitter 2-arachidonoylglycerol (2-AG). This endocannabinoid is known to mediate a large no. of physiol. processes, and its regulation is thought to be of great therapeutic potential. However, the no. of available monoglyceride lipase inhibitors is limited, mostly due to the lack of rapid and accurate pharmacol. assays for the enzyme. We have developed a 96-well-format assay for MGL using a nonradiolabeled substrate, 4-nitrophenylacetate. The IC50 values that were obtained for known inhibitors of MGL using 4-nitrophenylacetate were similar to those reported by using the radiolabeled form of an endogenous substrate, 2-oleoylglycerol. In a first small-scale screening, we identified CAY10499 as a novel monoglyceride lipase inhibitor. Thus, we report the characterization of this submicromolar inhibitor, which acts on MGL through an unprecedented mechanism for inhibitors of this enzyme.
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  24. 24
    Cisar, J. S.; Weber, O. D.; Clapper, J. R.; Blankman, J. L.; Henry, C. L.; Simon, G. M.; Alexander, J. P.; Jones, T. K.; Ezekowitz, R. A. B.; O’Neill, G. P.; Grice, C. A. Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders. J. Med. Chem. 2018, 61, 9062– 9084,  DOI: 10.1021/acs.jmedchem.8b00951
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    24
    Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders
    Cisar, Justin S.; Weber, Olivia D.; Clapper, Jason R.; Blankman, Jacqueline L.; Henry, Cassandra L.; Simon, Gabriel M.; Alexander, Jessica P.; Jones, Todd K.; Ezekowitz, R. Alan B.; O'Neill, Gary P.; Grice, Cheryl A.
    Journal of Medicinal Chemistry (2018), 61 (20), 9062-9084CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacol. inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding redns. in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling expts. verify the exquisite selectivity of 28 for MGLL vs. other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain 2-AG concns., and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clin. trials.
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  25. 25
    King, A. R.; Dotsey, E. Y.; Lodola, A.; Jung, K. M.; Ghomian, A.; Qiu, Y.; Fu, J.; Mor, M.; Piomelli, D. Discovery of Potent and Reversible Monoacylglycerol Lipase Inhibitors. Chem. Biol. 2009, 16, 1045– 1052,  DOI: 10.1016/j.chembiol.2009.09.012
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    25
    Discovery of Potent and Reversible Monoacylglycerol Lipase Inhibitors
    King, Alvin R.; Dotsey, Emmanuel Y.; Lodola, Alessio; Jung, Kwang Mook; Ghomian, Azar; Qiu, Yan; Fu, Jin; Mor, Marco; Piomelli, Daniele
    Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (10), 1045-1052CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Summary: Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biol. deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chem. scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concn., IC50 = 93 nM and 315 nM, resp.) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys201 and Cys208) flanking such pocket. This previously unrecognized regulatory region might offer a mol. target for potent and reversible inhibitors of MGL.
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  26. 26
    Schalk-Hihi, C.; Schubert, C.; Alexander, R.; Bayoumy, S.; Clemente, J. C.; Deckman, I.; DesJarlais, R. L.; Dzordzorme, K. C.; Flores, C. M.; Grasberger, B.; Kranz, J. K.; Lewandowski, F.; Liu, L.; Ma, H.; Maguire, D.; Macielag, M. J.; McDonnell, M. E.; Haarlander, T. M.; Miller, R.; Milligan, C.; Reynolds, C.; Kuo, L. C. Crystal Structure of a Soluble Form of Human Monoglyceride Lipase in Complex with an Inhibitor at 1.35 Å Resolution. Protein Sci. 2011, 20, 670– 683,  DOI: 10.1002/pro.596
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    26
    Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution
    Schalk-Hihi, Celine; Schubert, Carsten; Alexander, Richard; Bayoumy, Shariff; Clemente, Jose C.; Deckman, Ingrid; Des Jarlais, Renee L.; Dzordzorme, Keli C.; Flores, Christopher M.; Grasberger, Bruce; Kranz, James K.; Lewandowski, Frank; Liu, Li; Ma, Hongchang; Maguire, Diane; Macielag, Mark J.; McDonnell, Mark E.; Haarlander, Tara Mezzasalma; Miller, Robyn; Milligan, Cindy; Reynolds, Charles; Kuo, Lawrence C.
    Protein Science (2011), 20 (4), 670-683CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    A high-resoln. structure of a ligand-bound, sol. form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Anal. of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissocn. from the membrane upon completion of the cycle. In addn., the study outlines a successful approach to transform membrane assocd. proteins, which tend to aggregate upon purifn., into a monomeric and sol. form.
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  27. 27
    Hernández-Torres, G.; Cipriano, M.; Hedán, E.; Björklund, E.; Canales, A.; Zian, D.; Feliú, A.; Mecha, M.; Guaza, C.; Fowler, C. J.; Ortega-Gutiárrez, S.; López-Rodríguez, M. L. A Reversible and Selective Inhibitor of Monoacylglycerol Lipase Ameliorates Multiple Sclerosis. Angew. Chem., Int. Ed. 2014, 53, 13765– 13770,  DOI: 10.1002/anie.201407807
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    27
    A Reversible and Selective Inhibitor of Monoacylglycerol Lipase Ameliorates Multiple Sclerosis
    Hernandez-Torres, Gloria; Cipriano, Mariateresa; Heden, Erika; Bjoerklund, Emmelie; Canales, Angeles; Zian, Debora; Feliu, Ana; Mecha, Miriam; Guaza, Carmen; Fowler, Christopher J.; Ortega-Gutierrez, Silvia; Lopez-Rodriguez, Maria L.
    Angewandte Chemie, International Edition (2014), 53 (50), 13765-13770CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacol. tolerance. Hence, addnl. classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC50=0.18 μM) which is active in vivo and ameliorates the clin. progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB1-mediated side effects. These results support the interest in MAGL as a target for the treatment of MS.
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  28. 28
    Aghazadeh Tabrizi, M.; Baraldi, P. G.; Baraldi, S.; Ruggiero, E.; De Stefano, L.; Rizzolio, F.; Di Cesare Mannelli, L.; Ghelardini, C.; Chicca, A.; Lapillo, M.; Gertsch, J.; Manera, C.; Macchia, M.; Martinelli, A.; Granchi, C.; Minutolo, F.; Tuccinardi, T. Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors. J. Med. Chem. 2018, 61, 1340– 1354,  DOI: 10.1021/acs.jmedchem.7b01845
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    28
    Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors
    Aghazadeh-Tabrizi, Mojgan; Baraldi, Pier Giovanni; Baraldi, Stefania; Ruggiero, Emanuela; De Stefano, Lucia; Rizzolio, Flavio; Di Cesare Mannelli, Lorenzo; Ghelardini, Carla; Chicca, Andrea; Lapillo, Margherita; Gertsch, Jurg; Manera, Clementina; Macchia, Marco; Martinelli, Adriano; Granchi, Carlotta; Minutolo, Filippo; Tuccinardi, Tiziano
    Journal of Medicinal Chemistry (2018), 61 (3), 1340-1354CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the degrdn. of the endocannabinoid neurotransmitter 2-arachidonoylglycerol, which is implicated in many physiol. processes. Beyond the possible utilization of MAGL inhibitors as anti-inflammatory, antinociceptive, and anticancer agents, their application has encountered obstacles due to the unwanted effects caused by the irreversible inhibition of this enzyme. The possible application of reversible MAGL inhibitors has only recently been explored, mainly due to the deficiency of known compds. possessing efficient reversible inhibitory activities. The authors report a new series of reversible MAGL inhibitors. Among them, compd. 26 ((4-benzylpiperidin-1-yl)(5-(4-hydroxyphenyl)-1-(3-methylbenzyl)-1H-pyrazol-3-yl)methanone) showed to be a potent MAGL inhibitor (IC50 = 0.51 μM, Ki = 412 nM) with a good selectivity vs. fatty acid amide hydrolase (FAAH), α/β-hydrolase domain-contg. 6 (ABHD6), and 12 (ABHD12). Interestingly, this compd. also possesses antiproliferative activities against two different cancer cell lines and relieves the neuropathic hypersensitivity induced in vivo by oxaliplatin.
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  29. 29
    Bononi, G.; Granchi, C.; Lapillo, M.; Giannotti, M.; Nieri, D.; Fortunato, S.; El Boustani, M.; Caligiuri, I.; Poli, G.; Carlson, K. E.; Kim, S. H.; Macchia, M.; Martinelli, A.; Rizzolio, F.; Chicca, A.; Katzenellenbogen, J. A.; Minutolo, F.; Tuccinardi, T. Discovery of Long-Chain Salicylketoxime Derivatives as Monoacylglycerol Lipase (MAGL) Inhibitors. Eur. J. Med. Chem. 2018, 157, 817– 836,  DOI: 10.1016/j.ejmech.2018.08.038
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    29
    Discovery of long-chain salicylketoxime derivatives as monoacylglycerol lipase (MAGL) inhibitors
    Bononi, Giulia; Granchi, Carlotta; Lapillo, Margherita; Giannotti, Massimiliano; Nieri, Daniela; Fortunato, Serena; Boustani, Maguie El; Caligiuri, Isabella; Poli, Giulio; Carlson, Kathryn E.; Kim, Sung Hoon; Macchia, Marco; Martinelli, Adriano; Rizzolio, Flavio; Chicca, Andrea; Katzenellenbogen, John A.; Minutolo, Filippo; Tuccinardi, Tiziano
    European Journal of Medicinal Chemistry (2018), 157 (), 817-836CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    Monoacylglycerol lipase (MAGL) is the enzyme hydrolyzing the endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid and glycerol. Therefore, MAGL is implicated in many physiol. processes involving the regulation of the endocannabinoid system and eicosanoid network. MAGL inhibition represents a potential therapeutic target for many diseases, including cancer. Nowadays, most MAGL inhibitors inhibit this enzyme by an irreversible mechanism of action, potentially leading to unwanted side effects from chronic treatment. Herein, we report the discovery of long-chain salicylketoxime derivs. as potent and reversible MAGL inhibitors. The compds. herein described are characterized by a good target selectivity for MAGL and by antiproliferative activities against a series of cancer cell lines. Finally, modeling studies suggest a reasonable hypothetical binding mode for this class of compds.
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  30. 30
    Aida, J.; Fushimi, M.; Kusumoto, T.; Sugiyama, H.; Arimura, N.; Ikeda, S.; Sasaki, M.; Sogabe, S.; Aoyama, K.; Koike, T. Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-Ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors. J. Med. Chem. 2018, 61, 9205– 9217,  DOI: 10.1021/acs.jmedchem.8b00824
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    30
    Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors
    Aida, Jumpei; Fushimi, Makoto; Kusumoto, Tomokazu; Sugiyama, Hideyuki; Arimura, Naoto; Ikeda, Shuhei; Sasaki, Masako; Sogabe, Satoshi; Aoyama, Kazunobu; Koike, Tatsuki
    Journal of Medicinal Chemistry (2018), 61 (20), 9205-9217CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biol. active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, authors report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, they identified a potent and reversible MAGL inhibitor, compd., 1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one (R)-3t. Oral administration of compd. (R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.
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  31. 31
    Granchi, C.; Rizzolio, F.; Palazzolo, S.; Carmignani, S.; MacChia, M.; Saccomanni, G.; Manera, C.; Martinelli, A.; Minutolo, F.; Tuccinardi, T. Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors. J. Med. Chem. 2016, 59, 10299– 10314,  DOI: 10.1021/acs.jmedchem.6b01459
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    31
    Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors
    Granchi, Carlotta; Rizzolio, Flavio; Palazzolo, Stefano; Carmignani, Sara; Macchia, Marco; Saccomanni, Giuseppe; Manera, Clementina; Martinelli, Adriano; Minutolo, Filippo; Tuccinardi, Tiziano
    Journal of Medicinal Chemistry (2016), 59 (22), 10299-10314CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Monoacylglycerol lipase (MAGL) inhibitors are considered potential therapeutic agents for a variety of pathol. conditions, including several types of cancer. Many MAGL inhibitors are reported in literature; however, most of them showed an irreversible mechanism of action, which caused important side effects. The use of reversible MAGL inhibitors has been only partially investigated so far, mainly because of the lack of compds. with good MAGL reversible inhibition properties. In this study, starting from the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) lead compd. that showed a reversible mechanism of MAGL inhibition (Ki = 8.6 μM), the authors started its structural optimization and the authors developed a new potent and selective MAGL inhibitor ((4-(4-Chlorobenzoyl)piperidin-1-yl)(3-hydroxyphenyl)methanone (17b), Ki = 0.65 μM). Furthermore, modeling studies suggested that the binding interactions of this compd. replace a structural water mol. reproducing its H-bonds in the MAGL binding site, thus identifying a new key anchoring point for the development of new MAGL inhibitors.
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  32. 32
    Granchi, C.; Lapillo, M.; Glasmacher, S.; Bononi, G.; Licari, C.; Poli, G.; el Boustani, M.; Caligiuri, I.; Rizzolio, F.; Gertsch, J.; Macchia, M.; Minutolo, F.; Tuccinardi, T.; Chicca, A. Optimization of a Benzoylpiperidine Class Identifies a Highly Potent and Selective Reversible Monoacylglycerol Lipase (MAGL) Inhibitor. J. Med. Chem. 2019, 62, 1932– 1958,  DOI: 10.1021/acs.jmedchem.8b01483
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    32
    Optimization of a Benzoylpiperidine Class Identifies a Highly Potent and Selective Reversible Monoacylglycerol Lipase (MAGL) Inhibitor
    Granchi, Carlotta; Lapillo, Margherita; Glasmacher, Sandra; Bononi, Giulia; Licari, Cristina; Poli, Giulio; El Boustani, Maguie; Caligiuri, Isabella; Rizzolio, Flavio; Gertsch, Jurg; Macchia, Marco; Minutolo, Filippo; Tuccinardi, Tiziano; Chicca, Andrea
    Journal of Medicinal Chemistry (2019), 62 (4), 1932-1958CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Monoacylglycerol lipase (MAGL) is the enzyme degrading the endocannabinoid 2-arachidonoylglycerol, and it is involved in several physiol. and pathol. processes. The therapeutic potential of MAGL is linked to several diseases, including cancer. The development of MAGL inhibitors has been greatly limited by the side effects assocd. with the prolonged MAGL inactivation. Importantly, it could be preferable to use reversible MAGL inhibitors in vivo, but nowadays only few reversible compds. have been developed. In the present study, structural optimization of a previously developed class of MAGL inhibitors led to the identification of compd. 23, which proved to be a very potent reversible MAGL inhibitor (IC50 = 80 nM), selective for MAGL over the other main components of the endocannabinoid system, endowed of a promising antiproliferative activity in a series of cancer cell lines and able to block MAGL both in cell-based as well as in vivo assays.
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  33. 33
    Tuccinardi, T.; Granchi, C.; Rizzolio, F.; Caligiuri, I.; Battistello, V.; Toffoli, G.; Minutolo, F.; Macchia, M.; Martinelli, A. Identification and Characterization of a New Reversible MAGL Inhibitor. Bioorg. Med. Chem. 2014, 22, 3285– 3291,  DOI: 10.1016/j.bmc.2014.04.057
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    33
    Identification and characterization of a new reversible MAGL inhibitor
    Tuccinardi, Tiziano; Granchi, Carlotta; Rizzolio, Flavio; Caligiuri, Isabella; Battistello, Vittoria; Toffoli, Giuseppe; Minutolo, Filippo; Macchia, Marco; Martinelli, Adriano
    Bioorganic & Medicinal Chemistry (2014), 22 (13), 3285-3291CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    Monoacylglycerol lipase is a serine hydrolase that play a major role in the degrdn. of 2-arachidonoylglycerol, an endocannabinoid neurotransmitter implicated in several physiol. processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors detd. an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compds. possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening anal. for the identification of reversible MAGL inhibitors. Among the screened compds., the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (Ki = 8.6 μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging addnl. screenings of other com. databases.
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  34. 34
    Granchi, C.; Bononi, G.; Ferrisi, R.; Gori, E.; Mantini, G.; Glasmacher, S.; Poli, G.; Palazzolo, S.; Caligiuri, I.; Rizzolio, F.; Canzonieri, V.; Perin, T.; Gertsch, J.; Sodi, A.; Giovannetti, E.; Macchia, M.; Minutolo, F.; Tuccinardi, T.; Chicca, A. Design, Synthesis and Biological Evaluation of Second-Generation Benzoylpiperidine Derivatives as Reversible Monoacylglycerol Lipase (MAGL) Inhibitors. Eur. J. Med. Chem. 2021, 209, 112857,  DOI: 10.1016/j.ejmech.2020.112857
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    34
    Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors
    Granchi, Carlotta; Bononi, Giulia; Ferrisi, Rebecca; Gori, Eleonora; Mantini, Giulia; Glasmacher, Sandra; Poli, Giulio; Palazzolo, Stefano; Caligiuri, Isabella; Rizzolio, Flavio; Canzonieri, Vincenzo; Perin, Tiziana; Gertsch, Jurg; Sodi, Andrea; Giovannetti, Elisa; Macchia, Marco; Minutolo, Filippo; Tuccinardi, Tiziano; Chicca, Andrea
    European Journal of Medicinal Chemistry (2021), 209 (), 112857CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    An interesting enzyme of the endocannabinoid system is monoacylglycerol lipase (MAGL). This enzyme, which metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG), has attracted great interest due to its involvement in several physiol. and pathol. processes, such as cancer progression. Exptl. evidences highlighted some drawbacks assocd. with the use of irreversible MAGL inhibitors in vivo, therefore the research field concerning reversible inhibitors is rapidly growing. In the present manuscript, the class of benzoylpiperidine-based MAGL inhibitors was further expanded and optimized. Enzymic assays identified some compds. in the low nanomolar range and steered mol. dynamics simulations predicted the dissocn. itinerary of one of the best compds. from the enzyme, confirming the obsd. structure-activity relationship. Biol. evaluation, including assays in intact U937 cells and competitive activity-based protein profiling expts. in mouse brain membranes, confirmed the selectivity of the selected compds. for MAGL vs. other components of the endocannabinoid system. Future studies on the potential use of these compds. in the clin. setting are also supported by the inhibition of cell growth obsd. both in cancer organoids derived from high grade serous ovarian cancer patients and in pancreatic ductal adenocarcinoma primary cells, which showed genetic and histol. features very similar to the primary tumors.
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  35. 35
    Bononi, G.; Tonarini, G.; Poli, G.; Barravecchia, I.; Caligiuri, I.; Macchia, M.; Rizzolio, F.; Demontis, G. C.; Minutolo, F.; Granchi, C.; Tuccinardi, T. Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Diphenylsulfide-Benzoylpiperidine Scaffold. Eur. J. Med. Chem. 2021, 223, 113679,  DOI: 10.1016/j.ejmech.2021.113679
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    35
    Monoacylglycerol lipase (MAGL) inhibitors based on a diphenylsulfide-benzoylpiperidine scaffold
    Bononi, Giulia; Tonarini, Giacomo; Poli, Giulio; Barravecchia, Ivana; Caligiuri, Isabella; Macchia, Marco; Rizzolio, Flavio; Demontis, Gian Carlo; Minutolo, Filippo; Granchi, Carlotta; Tuccinardi, Tiziano
    European Journal of Medicinal Chemistry (2021), 223 (), 113679CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    Monoacylglycerol lipase (MAGL) is an enzyme belonging to the endocannabinoid system that mainly metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG). Numerous studies have shown the involvement of this enzyme in various pathol. conditions such as pain, cancer progression, Parkinson's and Alzheimer's disease, thus encouraging the development of new MAGL modulators. In this context, we developed new diphenylsulfide-benzoylpiperidine derivs. characterized by a high enzymic MAGL inhibition activity in the low nanomolar range, a reversible mechanism of action and selectivity. The three most active compds. I, wherein R = CF3, Cl, OCF3, induced an appreciable inhibition of cell viability in a panel of nine cancer cell lines, with IC50 values ranging between 0.32 and 10μM, thus highlighting their potential as novel anticancer agents.
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  36. 36
    Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.; Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith, S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Van Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Discovery and Characterization of a Highly Selective FAAH Inhibitor That Reduces Inflammatory Pain. Chem. Biol. 2009, 16, 411– 420,  DOI: 10.1016/j.chembiol.2009.02.013
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    Discovery and Characterization of a Highly Selective FAAH Inhibitor that Reduces Inflammatory Pain
    Ahn, Kay; Johnson, Douglas S.; Mileni, Mauro; Beidler, David; Long, Jonathan Z.; McKinney, Michele K.; Weerapana, Eranthie; Sadagopan, Nalini; Liimatta, Marya; Smith, Sarah E.; Lazerwith, Scott; Stiff, Cory; Kamtekar, Satwik; Bhattacharya, Keshab; Zhang, Yanhua; Swaney, Stephen; Van Becelaere, Keri; Stevens, Raymond C.; Cravatt, Benjamin F.
    Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (4), 411-420CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Summary: Endocannabinoids are lipid signaling mols. that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as detd. by activity-based protein profiling; raises brain anandamide levels for up to 24 h; and produces significant cannabinoid receptor-dependent redns. in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacol. tool for in vivo characterization of the endocannabinoid system.
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    Niphakis, M. J.; Cravatt, B. F. Enzyme Inhibitor Discovery by Activity-Based Protein Profiling. Annu. Rev. Biochem. 2014, 341,  DOI: 10.1146/annurev-biochem-060713-035708
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    Enzyme inhibitor discovery by activity-based protein profiling
    Niphakis, Micah J.; Cravatt, Benjamin F.
    Annual Review of Biochemistry (2014), 83 (), 341-377CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    Eukaryotic and prokaryotic organisms possess huge nos. of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biol. systems. Here, we discuss how the chem. proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochem. and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiol. and disease. These studies demonstrate the value of selective chem. probes as drivers of biol. inquiry.
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    Blankman, J. L.; Cravatt, B. F. Chemical Probes of Endocannabinoid Metabolism. Pharmacol. Rev. 2013, 65, 849– 871,  DOI: 10.1124/pr.112.006387
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    Chemical probes of endocannabinoid metabolism
    Blankman, Jacqueline L.; Cravatt, Benjamin F.
    Pharmacological Reviews (2013), 65 (2), 849-871, 23 pp.CODEN: PAREAQ; ISSN:1521-0081. (American Society for Pharmacology and Experimental Therapeutics)
    A review. The endocannabinoid signaling system regulates diverse physiol. processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which activate the two major cannabinoid receptors, CB1 and CB2. Anandamide and 2-AG signaling pathways in the nervous system are terminated by enzymic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resp. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacol. application to investigate the function of anandamide and 2-AG signaling pathways in preclinicalmodels of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and 2-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.
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  39. 39
    Piomelli, D.; Tarzia, G.; Duranti, A.; Tontini, A.; Mor, M.; Compton, T. R.; Dasse, O.; Monaghan, E. P.; Parrott, J. A.; Putman, D. Pharmacological Profile of the Selective FAAH Inhibitor KDS-4103 (URB597). CNS Drug Rev. 2006, 12, 21– 38,  DOI: 10.1111/j.1527-3458.2006.00021.x
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    39
    Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597)
    Piomelli, Daniele; Tarzia, Giorgio; Duranti, Andrea; Tontini, Andrea; Mor, Marco; Compton, Timothy R.; Dasse, Olivier; Monaghan, Edward P.; Parrott, Jeff A.; Putman, David
    CNS Drug Reviews (2006), 12 (1), 21-38CODEN: CDREFB; ISSN:1080-563X. (Blackwell Publishing, Inc.)
    A review. In the present article, we review the pharmacol. properties of KDS-4103 (URB597), a highly potent and selective inhibitor of the enzyme fatty-acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In vitro, KDS-4103 inhibits FAAH activity with median inhibitory concns. (IC50) of 5 nM in rat brain membranes and 3 nM in human liver microsomes. In vivo, KDS-4103 inhibits rat brain FAAH activity after i.p. administration with a median ID (ID50) of 0.15 mg/kg. The compd. does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice KDS-4103 elicits significant, anxiolytic-like, anti-depressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Δ9-tetrahydrocannabinol (Δ9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB1 receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were obsd. in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.
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  40. 40
    Li, W.; Blankman, J. L.; Cravatt, B. F. A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases. J. Am. Chem. Soc. 2007, 129, 9594– 9595,  DOI: 10.1021/ja073650c
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    40
    A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases
    Li, Weiwei; Blankman, Jacqueline L.; Cravatt, Benjamin F.
    Journal of the American Chemical Society (2007), 129 (31), 9594-9595CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Hydrolytic enzymes constitute one of the largest and most diverse protein classes in nature and play key roles in nearly all physiol. and pathol. processes. The mammalian serine hydrolase superfamily contains a remarkable no. of uncharacterized members, with at least 40-50% of these enzymes lacking exptl. verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacol. tools to selectively perturb their activity. We describe herein a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine hydrolases and its application to the brain-enriched enzyme α/β-hydrolase domain 6. We anticipate that the methods described herein will facilitate the development of selective chem. probes to annotate the metabolic and (patho)physiol. functions of many of the uncharacterized serine hydrolases that currently populate eukaryotic and prokaryotic proteomes.
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    Hoover, H. S.; Blankman, J. L.; Niessen, S.; Cravatt, B. F. Selectivity of Inhibitors of Endocannabinoid Biosynthesis Evaluated by Activity-Based Protein Profiling. Bioorg. Med. Chem. Lett. 2008, 18, 5838– 5841,  DOI: 10.1016/j.bmcl.2008.06.091
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    Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling
    Hoover, Heather S.; Blankman, Jacqueline L.; Niessen, Sherry; Cravatt, Benjamin F.
    Bioorganic & Medicinal Chemistry Letters (2008), 18 (22), 5838-5841CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
    The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacol. studies using inhibitors of the 2-AG biosynthetic enzymes diacylglycerol lipase α and β (DAGL-α/β). Here, we show by competitive activity-based protein profiling that the DAGL-α/β inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was obsd. for THL and RHC80267, suggesting that pharmacol. effects obsd. with both agents may be viewed as good initial evidence for DAGL-dependent events.
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    Deutsch, D. G.; Omeir, R.; Arreaza, G.; Salehani, D.; Prestwich, G. D.; Huang, Z.; Howlett, A. Methyl Arachidonyl Fluorophosphonate: A Potent Irreversible Inhibitor of Anandamide Amidase. Biochem. Pharmacol. 1997, 53, 255– 260,  DOI: 10.1016/S0006-2952(96)00830-1
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    42
    Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase
    Deutsch, Dale G.; Omeir, Romelda; Arreaza, Gladys; Salehani, David; Prestwich, Glenn D.; Huang, Zheng; Howlett, Allyn
    Biochemical Pharmacology (1997), 53 (3), 255-260CODEN: BCPCA6; ISSN:0006-2952. (Elsevier)
    Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described. In the current study, Me arachidonyl fluorophosphate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approx. 1000-fold more potent than Arach-CF3, MAFP demonstrated selectively towards anandamide amidase for which it was approx. 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, resp. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addn., can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonyl ethanolamide amidase.
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  43. 43
    Baggelaar, M. P.; Janssen, F. J.; van Esbroeck, A. C. M.; den Dulk, H.; Allarà, M.; Hoogendoorn, S.; McGuire, R.; Florea, B. I.; Meeuwenoord, N.; van den Elst, H.; van der Marel, G. A.; Brouwer, J.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M. Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain. Angew. Chem., Int. Ed. 2013, 52, 12081– 12085,  DOI: 10.1002/anie.201306295
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    Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain
    Baggelaar, Marc P.; Janssen, Freek J.; van Esbroeck, Annelot C. M.; den Dulk, Hans; Allara, Marco; Hoogendoorn, Sascha; McGuire, Ross; Florea, Bogdan I.; Meeuwenoord, Nico; van den Elst, Hans; van der Marel, Gijsbert A.; Brouwer, Jaap; Di Marzo, Vincenzo; Overkleeft, Herman S.; van der Stelt, Mario
    Angewandte Chemie, International Edition (2013), 52 (46), 12081-12085CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A strategy that combines a knowledge-based in silico design approach and the development of novel activity-based probes for the detection of endogenous diacylglycerol lipase-α (DAGL-α) is presented. This approach resulted in the rapid identification of new DAGL-α inhibitors with high selectivity in the brain proteome.
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    Poli, G.; Granchi, C.; Rizzolio, F.; Tuccinardi, T. Application of MM-PBSA Methods in Virtual Screening. Molecules 2020, 25, 1971,  DOI: 10.3390/molecules25081971
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    Application of MM-PBSA methods in virtual screening
    Poli, Giulio; Granchi, Carlotta; Rizzolio, Flavio; Tuccinardi, Tiziano
    Molecules (2020), 25 (8), 1971CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    A review. Computer-aided drug design techniques are today largely applied in medicinal chem. In particular, receptor-based virtual screening (VS) studies, in which mol. docking represents the gold std. in silico approach, constitute a powerful strategy for identifying novel hit compds. active against the desired target receptor. Nevertheless, the need for improving the ability of docking in discriminating true active ligands from inactive compds., thus boosting VS hit rates, is still pressing. In this context, the use of binding free energy evaluation approaches can represent a profitable tool for rescoring ligand-protein complexes predicted by docking based on more reliable estns. of ligand-protein binding affinities than those obtained with simple scoring functions. In the present review, we focused our attention on the Mol. Mechanics-Poisson Boltzman Surface Area (MM-PBSA) method for the calcn. of binding free energies and its application in VS studies. We provided examples of successful applications of this method in VS campaigns and evaluation studies in which the reliability of this approach has been assessed, thus providing useful guidelines for employing this approach in VS.
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    Tang, Z.; Li, C.; Kang, B.; Gao, G.; Li, C.; Zhang, Z. GEPIA: A Web Server for Cancer and Normal Gene Expression Profiling and Interactive Analyses. Nucleic Acids Res. 2017, 45, W98– W102,  DOI: 10.1093/nar/gkx247
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    GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses
    Tang, Zefang; Li, Chenwei; Kang, Boxi; Gao, Ge; Li, Cheng; Zhang, Zemin
    Nucleic Acids Research (2017), 45 (W1), W98-W102CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Tremendous amt. of RNA sequencing data have been produced by large consortium projects such as TCGA and GTEx, creating new opportunities for data mining and deeper understanding of gene functions. While certain existing web servers are valuable and widely used, many expression anal. functions needed by exptl. biologists are still not adequately addressed by these tools. We introduce GEPIA (Gene Expression Profiling Interactive Anal.), a web-based tool to deliver fast and customizable functionalities based on TCGA and GTEx data. GEPIA provides key interactive and customizable functions including differential expression anal., profiling plotting, correlation anal., patient survival anal., similar gene detection and dimensionality redn. anal. The comprehensive expression analyses with simple clicking through GEPIA greatly facilitate data mining in wide research areas, scientific discussion and the therapeutic discovery process. GEPIA fills in the gap between cancer genomics big data and the delivery of integrated information to end users, thus helping unleash the value of the current data resources. GEPIA is available at http://gepia.cancer-pku.cn/.
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  46. 46
    Avan, A.; Caretti, V.; Funel, N.; Galvani, E.; Maftouh, M.; Honeywell, R. J.; Lagerweij, T.; Van Tellingen, O.; Campani, D.; Fuchs, D.; Verheul, H. M.; Schuurhuis, G.-J.; Boggi, U.; Peters, G. J.; Würdinger, T.; Giovannetti, E. Crizotinib Inhibits Metabolic Inactivation of Gemcitabine in C-Met–Driven Pancreatic Carcinoma. Cancer Res. 2013, 73, 6745– 6756,  DOI: 10.1158/0008-5472.CAN-13-0837
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    Crizotinib Inhibits Metabolic Inactivation of Gemcitabine in c-Met-driven Pancreatic Carcinoma
    Avan, Amir; Caretti, Viola; Funel, Niccola; Galvani, Elena; Maftouh, Mina; Honeywell, Richard J.; Lagerweij, Tonny; Van Tellingen, Olaf; Campani, Daniela; Fuchs, Dieter; Verheul, Henk M.; Schuurhuis, Gerrit-Jan; Boggi, Ugo; Peters, Godefridus J.; Wuerdinger, Thomas; Giovannetti, Elisa
    Cancer Research (2013), 73 (22), 6745-6756CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclin. tests fail in these patients, emphasizing the need of improved preclin. models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathol. and immunohistochem. analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochem. evidence revealed an increased copy no. and overexpression of c-Met in one of the models; we examd. the preclin. efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concns. of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathol., and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clin. evaluation for PDAC treatment.
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    Yin, J.; Kim, S. S.; Choi, E.; Oh, Y. T.; Lin, W.; Kim, T.-H.; Sa, J. K.; Hong, J. H.; Park, S. H.; Kwon, H. J.; Jin, X.; You, Y.; Kim, J. H.; Kim, H.; Son, J.; Lee, J.; Nam, D.-H.; Choi, K. S.; Shi, B.; Gwak, H.-S.; Yoo, H.; Iavarone, A.; Kim, J. H.; Park, J. B. ARS2/MAGL Signaling in Glioblastoma Stem Cells Promotes Self-Renewal and M2-like Polarization of Tumor-Associated Macrophages. Nat. Commun. 2020, 11, 2978,  DOI: 10.1038/s41467-020-16789-2
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    ARS2/MAGL signaling in glioblastoma stem cells promotes self-renewal and M2-like polarization of tumor-associated macrophages
    Yin, Jinlong; Kim, Sung Soo; Choi, Eunji; Oh, Young Taek; Lin, Weiwei; Kim, Tae-Hoon; Sa, Jason K.; Hong, Jun Hee; Park, Se Hwan; Kwon, Hyung Joon; Jin, Xiong; You, Yeonhee; Kim, Ji Hye; Kim, Hyunggee; Son, Jaekyoung; Lee, Jeongwu; Nam, Do-Hyun; Choi, Kui Son; Shi, Bingyang; Gwak, Ho-Shin; Yoo, Heon; Iavarone, Antonio; Kim, Jong Heon; Park, Jong Bae
    Nature Communications (2020), 11 (1), 2978CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: The interplay between glioblastoma stem cells (GSCs) and tumor-assocd. macrophages (TAMs) promotes progression of glioblastoma multiforme (GBM). However, the detailed mol. mechanisms underlying the relationship between these two cell types remain unclear. Here, we demonstrate that ARS2 (arsenite-resistance protein 2), a zinc finger protein that is essential for early mammalian development, plays crit. roles in GSC maintenance and M2-like TAM polarization. ARS2 directly activates its novel transcriptional target MGLL, encoding monoacylglycerol lipase (MAGL), to regulate the self-renewal and tumorigenicity of GSCs through prodn. of prostaglandin E2 (PGE2), which stimulates β-catenin activation of GSC and M2-like TAM polarization. We identify M2-like signature downregulated by which MAGL-specific inhibitor, JZL184, increased survival rate significantly in the mouse xenograft model by blocking PGE2 prodn. Taken together, our results suggest that blocking the interplay between GSCs and TAMs by targeting ARS2/MAGL signaling offers a potentially novel therapeutic option for GBM patients.
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    Zhang, J.; Liu, Z.; Lian, Z.; Liao, R.; Chen, Y.; Qin, Y.; Wang, J.; Jiang, Q.; Wang, X.; Gong, J. Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma. Sci. Rep. 2016, 6, 35784,  DOI: 10.1038/srep35784
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    48
    Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma
    Zhang, Junyong; Liu, Zuojin; Lian, Zhengrong; Liao, Rui; Chen, Yi; Qin, Yi; Wang, Jinlong; Jiang, Qing; Wang, Xiaobo; Gong, Jianping
    Scientific Reports (2016), 6 (), 35784CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Monoacylglycerol lipase (MAGL) is a key enzyme in lipid metab. that is demonstrated to be involved in tumor progression through both energy supply of fatty acid (FA) oxidn. and enhancing cancer cell malignance. The aim of this study was to investigate whether MAGL could be a potential therapeutic target and prognostic indicator for hepatocellular carcinoma (HCC). To evaluate the relationship between MAGL levels and clin. characteristics, a tissue microarray (TMA) of 353 human HCC samples was performed. MAGL levels in HCC samples were closely linked to the degree of malignancy and patient prognosis. RNA interference, specific pharmacol. inhibitor JZL-184 and gene knock-in of MAGL were utilized to investigate the effects of MAGL on HCC cell proliferation, apoptosis, and invasion. MAGL played important roles in both proliferation and invasion of HCC cells through mechanisms that involved prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA). JZL-184 administration significantly inhibited tumor growth in mice. Furthermore, we confirmed that promoter methylation of large tumor suppressor kinase 1 (LATS1) resulted in dysfunction of the Hippo signal pathway, which induced overexpression of MAGL in HCC. These results indicate that MAGL could be a potentially novel therapeutic target and prognostic indicator for HCC.
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    Randazzo, O.; Papini, F.; Mantini, G.; Gregori, A.; Parrino, B.; Liu, D. S. K.; Cascioferro, S.; Carbone, D.; Peters, G. J.; Frampton, A. E.; Garajova, I.; Giovannetti, E. “Open Sesame?:” Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing Its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer. Cancers 2020, 12, 3206,  DOI: 10.3390/cancers12113206
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    "Open sesame?": biomarker status of the human equilibrative nucleoside transporter-1 and molecular mechanisms influencing its expression and activity in the uptake and cytotoxicity of gemcitabine in pancreatic cancer
    Randazzo, Ornella; Papini, Filippo; Mantini, Giulia; Gregori, Alessandro; Parrino, Barbara; Liu, Daniel S. K.; Cascioferro, Stella; Carbone, Daniela; Peters, Godefridus J.; Frampton, Adam E.; Garajova, Ingrid; Giovannetti, Elisa
    Cancers (2020), 12 (11), 3206CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
    Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analog, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal anal. of hENT-1 status evaluated its predictive role by both immunohistochem. (with five different antibodies), and quant.-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels obsd. with this antibody were assocd. with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this anal. and lists mol. factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.
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    Berman, H. M.; Battistuz, T.; Bhat, T. N.; Bluhm, W. F.; Bourne, P. E.; Burkhardt, K.; Feng, Z.; Gilliland, G. L.; Iype, L.; Jain, S.; Fagan, P.; Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, B.; Thanki, N.; Weissig, H.; Westbrook, J. D.; Zardecki, C. The Protein Data Bank. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002, 58, 899– 907,  DOI: 10.1107/S0907444902003451
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    The Protein Data Bank
    Berman, Helen M.; Battistuz, Tammy; Bhat, T. N.; Bluhm, Wolfgang F.; Bourne, Philip E.; Burkhardt, Kyle; Feng, Zukang; Gilliland, Gary L.; Iype, Lisa; Jain, Shri; Fagan, Phoebe; Marvin, Jessica; Padilla, David; Ravichandran, Veerasamy; Schneider, Bohdan; Thanki, Narmada; Weissig, Helge; Westbrook, John D.; Zardecki, Christine
    Acta Crystallographica, Section D: Biological Crystallography (2002), D58 (6, No. 1), 899-907CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Munksgaard)
    The Protein Data Bank [PDB; Berman, Westbrook et al. (2000), Nucleic Acids Res. 28, 235-242; http://www.pdb.org/] is the single worldwide archive of primary structural data of biol. macromols. Many secondary sources of information are derived from PDB data. It is the starting point for studies in structural bioinformatics. This article describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information and plans for the future development of the resource. The reader should come away with an understanding of the scope of the PDB and what is provided by the resource.
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    Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber Biomolecular Simulation Programs. J. Comput. Chem. 2005, 26, 1668– 1688,  DOI: 10.1002/jcc.20290
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    The amber biomolecular simulation programs
    Case, David A.; Cheatham, Thomas E., III; Darden, Tom; Gohlke, Holger; Luo, Ray; Merz, Kenneth M., Jr.; Onufriev, Alexey; Simmerling, Carlos; Wang, Bing; Woods, Robert J.
    Journal of Computational Chemistry (2005), 26 (16), 1668-1688CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    The authors describe the development, current features, and some directions for future development of the Amber package of computer programs. This package evolved from a program that was constructed in the late 1970s to do Assisted Model Building with Energy Refinement, and now contains a group of programs embodying a no. of powerful tools of modern computational chem., focused on mol. dynamics and free energy calcns. of proteins, nucleic acids, and carbohydrates.
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    Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J. Comput. Chem. 2009, 30, 2785– 2791,  DOI: 10.1002/jcc.21256
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    AutoDock and AutoDockTools: Automated docking with selective receptor flexibility
    Morris, Garrett M.; Huey, Ruth; Lindstrom, William; Sanner, Michel F.; Belew, Richard K.; Goodsell, David S.; Olson, Arthur J.
    Journal of Computational Chemistry (2009), 30 (16), 2785-2791CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking expt. with 188 diverse ligand-protein complexes and a cross-docking expt. using flexible sidechains in 87 HIV protease complexes. We also report its utility in anal. of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009.
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    Poli, G.; Gelain, A.; Porta, F.; Asai, A.; Martinelli, A.; Tuccinardi, T. Identification of a New STAT3 Dimerization Inhibitor through a Pharmacophore-Based Virtual Screening Approach. J. Enzyme Inhib. Med. Chem. 2016, 31, 1011– 1017,  DOI: 10.3109/14756366.2015.1079184
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    Identification of a new STAT3 dimerization inhibitor through a pharmacophore-based virtual screening approach
    Poli, Giulio; Gelain, Arianna; Porta, Federica; Asai, Akira; Martinelli, Adriano; Tuccinardi, Tiziano
    Journal of Enzyme Inhibition and Medicinal Chemistry (2016), 31 (6), 1011-1017CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
    Signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell growth regulation and survival. An aberrant STAT3 activation and/or expression is implied in various solid and blood tumors as well as in other pathologies like rheumatoid arthritis and pulmonary fibrosis, thus making the search for STAT3 inhibitors a growing field of study. With the aim of identifying new inhibitors of STAT3 dimerization, we screened a database including more than 1 320 000 com. available compds. using a receptor-based pharmacophore model comprising the key protein-protein interactions identified in the STAT3 dimer and refining the search through docking and mol. dynamic simulations studies. STAT3 binding assays revealed a significant STAT3 inhibitory activity and selectivity vs. Grb2 for one of the four top-scored compds., thus verifying the reliability of the virtual screening workflow. Moreover, such compd. could already be considered as a lead for the development of new and more potent STAT3 dimerization inhibitors.
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    Roe, D. R.; Cheatham, T. E., Jr. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013, 9, 3084– 3095,  DOI: 10.1021/ct400341p
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    PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data
    Roe, Daniel R.; Cheatham, Thomas E.
    Journal of Chemical Theory and Computation (2013), 9 (7), 3084-3095CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the anal. and processing of time series of three-dimensional at. positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange mol. dynamics), image with periodic boundary conditions, create av. structures, strip subsets of the system, and perform calcns. such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
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    Poli, G.; Lapillo, M.; Jha, V.; Mouawad, N.; Caligiuri, I.; Macchia, M.; Minutolo, F.; Rizzolio, F.; Tuccinardi, T.; Granchi, C. Computationally Driven Discovery of Phenyl(Piperazin-1-Yl)Methanone Derivatives as Reversible Monoacylglycerol Lipase (MAGL) Inhibitors. J. Enzyme Inhib. Med. Chem. 2019, 34, 589– 596,  DOI: 10.1080/14756366.2019.1571271
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    Computationally driven discovery of phenyl(piperazin-1-yl)methanone derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors
    Poli, Giulio; Lapillo, Margherita; Jha, Vibhu; Mouawad, Nayla; Caligiuri, Isabella; Macchia, Marco; Minutolo, Filippo; Rizzolio, Flavio; Tuccinardi, Tiziano; Granchi, Carlotta
    Journal of Enzyme Inhibition and Medicinal Chemistry (2019), 34 (1), 589-596CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
    Monoacylglycerol lipase (MAGL) is an attractive therapeutic target for many pathologies, including neurodegenerative diseases, cancer as well as chronic pain and inflammatory pathologies. The identification of reversible MAGL inhibitors, devoid of the side effects assocd. to prolonged MAGL inactivation, is a hot topic in medicinal chem. In this study, a novel phenyl(piperazin-1-yl)methanone inhibitor of MAGL was identified through a virtual screening protocol based on a fingerprint-driven consensus docking (CD) approach. Mol. modeling and preliminary structure-based hit optimization studies allowed the discovery of deriv., which showed an efficient reversible MAGL inhibition (IC50 = 6.1 μM) and a promising antiproliferative activity on breast and ovarian cancer cell lines (IC50 of 31-72 μM), thus representing a lead for the development of new and more potent reversible MAGL inhibitors. Moreover, the obtained results confirmed the reliability of the fingerprint-driven CD approach herein developed.
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    Poli, G.; Lapillo, M.; Granchi, C.; Caciolla, J.; Mouawad, N.; Caligiuri, I.; Rizzolio, F.; Langer, T.; Minutolo, F.; Tuccinardi, T. Binding Investigation and Preliminary Optimisation of the 3-Amino-1,2,4-Triazin-5(2H)-One Core for the Development of New Fyn Inhibitors. J. Enzyme Inhib. Med. Chem. 2018, 33, 956– 961,  DOI: 10.1080/14756366.2018.1469017
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    Poli, Giulio; Lapillo, Margherita; Granchi, Carlotta; Caciolla, Jessica; Mouawad, Nayla; Caligiuri, Isabella; Rizzolio, Flavio; Langer, Thierry; Minutolo, Filippo; Tuccinardi, Tiziano
    Journal of Enzyme Inhibition and Medicinal Chemistry (2018), 33 (1), 956-961CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
    Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (), a hit compd. that showed a micromolar inhibition of Fyn (IC50 = 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimization. This anal. led us to confirm the hypothesised binding mode of and to identify a new deriv. that is about 6-fold more active than (compd. , IC50 = 0.76 μM).
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    Firuzi, O.; Che, P. P.; El Hassouni, B.; Buijs, M.; Coppola, S.; Löhr, M.; Funel, N.; Heuchel, R.; Carnevale, I.; Schmidt, T.; Mantini, G.; Avan, A.; Saso, L.; Peters, G. J.; Giovannetti, E. Role of C-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells. Cancers 2019, 11, 638,  DOI: 10.3390/cancers11050638
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    Cancers (2019), 11 (5), 638CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
    Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochem. ELISA (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examd. by luciferase assay, while spheroids' architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for "stage specific embryonic antigen-4" (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, contg. increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids contg. both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Expts. with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.
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    Massihnia, D.; Avan, A.; Funel, N.; Maftouh, M.; van Krieken, A.; Granchi, C.; Raktoe, R.; Boggi, U.; Aicher, B.; Minutolo, F.; Russo, A.; Leon, L. G.; Peters, G. J.; Giovannetti, E. Phospho-Akt Overexpression Is Prognostic and Can Be Used to Tailor the Synergistic Interaction of Akt Inhibitors with Gemcitabine in Pancreatic Cancer. J. Hematol. Oncol. 2017, 10, 9,  DOI: 10.1186/s13045-016-0371-1
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    Massihnia, Daniela; Avan, Amir; Funel, Niccola; Maftouh, Mina; van Krieken, Anne; Granchi, Carlotta; Raktoe, Rajiv; Boggi, Ugo; Aicher, Babette; Minutolo, Filippo; Russo, Antonio; Leon, Leticia G.; Peters, Godefridus J.; Giovannetti, Elisa
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    Background: There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), assocd. with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated mol. mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine. Methods: Phospho-Akt expression was evaluated by immunohistochem. in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quant.-RT-PCR and immunocytochem. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacol. interaction with gemcitabine was detd. with combination index (CI) method. Results: Immunohistochem. of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-freesurvival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was addnl. reduced by the combination with gemcitabine. This combination significantly increased apoptosis, assocd. with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells. Conclusions: These data support the anal. of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.
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    El Hassouni, B.; Mantini, G.; Li Petri, G.; Capula, M.; Boyd, L.; Weinstein, H. N. W.; Vallás-Marti, A.; Kouwenhoven, M. C. M.; Giovannetti, E.; Westerman, B. A.; Peters, G. J.; EORTC PAMM Group To Combine or Not Combine: Drug Interactions and Tools for Their Analysis. Reflections from the EORTC-PAMM Course on Preclinical and Early-Phase Clinical Pharmacology. Anticancer Res. 2019, 39, 3303– 3309,  DOI: 10.21873/anticanres.13472
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    A review. Combination therapies are used in the clinic to achieve cure, better efficacy and to circumvent resistant disease in patients. Initial assessment of the effect of such combinations, usually of two agents, is frequently performed using in vitro assays. In this , we give a short summary of the types of analyses that were presented during the Preclin. and Early-phase Clin. Pharmacol. Course of the Pharmacol. and Mol. Mechanisms Group, European Organization for Research and Treatment on Cancer, that can be used to det. the efficacy of drug combinations. The effect of a combination treatment can be calcd. using math. equations based on either the Loewe additivity or Bliss independence model, or a combination of both, such as Chou and Talalay's median-drug effect model. Interactions can be additive, synergistic (more than additive), or antagonistic (less than additive). Software packages CalcuSyn (also available as CompuSyn) and Combenefit are designed to calc. the extent of the combined effects. Interestingly, the application of machine-learning methods in the prediction of combination treatments, which can include pharmacogenomic, genetic, metabolomic and proteomic profiles, might contribute to further refinement of combination regimens. However, more research is needed to apply appropriate rules of machine learning methods to ensure correct predictive models.
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    High throughput UV method for the estimation of thermodynamic solubility and the determination of the solubility in biorelevant media
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    The growing interest for high quality soly. data in the early stages of drug discovery suggested a detailed optimization of exptl. conditions for a 96-well HTS UV method in order to obtain soly. values close to thermodn. soly. measured by shake-flask method. Results have shown that soly. data obtained by the HTS approach were highly dependent on shaking intensity and incubation times due to the formation of supersatd. solns. resulting from the diln. of DMSO stock solns. in aq. buffer. Thus, careful exptl. set-up was developed to improve the quality and the reproducibility of the HTS method. Moreover, the early qual. prediction of bioavailability and absorption of orally administered drugs require more and more biorelevant soly. values in drug discovery programs. Thus, the optimized HTS method was also adapted to measure soly. directly in FaSSIF and FeSSIF media. The versatile HTS UV approach presented in this paper provides a unique and reliable way to det. soly. in various exptl. conditions.
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    This study reports on a novel, high-throughput assay, designed to predict passive, transcellular permeability in early drug discovery. The assay is carried out in 96-well microtiterplates and measures the ability of compds. to diffuse from a donor to an acceptor compartment which are sepd. by a 9-10 μm hexadecane liq. layer. A set of 32 well-characterized, chem. diverse drugs was used to validate the method. The permeability values derived from the flux factors between donor and acceptor compartments show a good correlation with gastrointestinal absorption in humans. For comparison, correlations based on exptl. or calcd. octanol/water distribution coeffs. (log Do/w,6.8) were significantly lower. In addn., this simple and robust assay allows detn. of pH permeability profiles, crit. information to predict gastrointestinal absorption of ionizable drugs and difficult to obtain from cell culture expts. Correction for the unstirred water layer effect allows to differentiate between effective and intrinsic membrane permeability and opens up the dynamic range of the method. In addn., alkane/water partition coeffs. can be derived from intrinsic membrane permeabilities, making this assay the first high-throughput method able to measure alkane/water log P in the microtiterplate format.
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    Synthesis and biological characterization of protease-activated prodrugs of doxazolidine
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    Journal of Medicinal Chemistry (2012), 55 (14), 6595-6607CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clin. impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common assocn. with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (av. IC50 of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.
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Cited By

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  1. Alessandro Gregori, Cecilia Bergonzini, Mjriam Capula, Giulia Mantini, Fatemeh Khojasteh-Leylakoohi, Annalisa Comandatore, Ghazaleh Khalili-Tanha, Alireza Khooei, Luca Morelli, Amir Avan, Erik H. Danen, Thomas Schmidt, Elisa Giovannetti. Prognostic Significance of Integrin Subunit Alpha 2 (ITGA2) and Role of Mechanical Cues in Resistance to Gemcitabine in Pancreatic Ductal Adenocarcinoma (PDAC). Cancers 2023, 15 (3) , 628. https://doi.org/10.3390/cancers15030628
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  • Abstract

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    Figure 1

    Figure 1. Structures of some representative synthetic reversible MAGL inhibitors.

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    Figure 2

    Figure 2. Design of the new benzylpiperidine derivative 7. The moiety deriving from FAAH inhibitor 6 is highlighted in blue, and the moiety deriving from our MAGL inhibitor 5a is highlighted in red.

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    Figure 3

    Figure 3. Newly synthesized benzylpiperidine derivatives 8, 9, 10a–e, 11a–c, 12, and 13. The modified moieties compared to parent compound 7 are highlighted in blue, in red, or with a dashed square.

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    Scheme 1

    Scheme 1. Synthesis of Compounds 7, 10a–e, 11a–c, and 13a
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    aReagents and conditions: (a) anhydrous K2CO3, anhydrous DMF, 110 °C, overnight [77–99%]; (b) i. tert-butyl 4-methylenepiperidine-1-carboxylate, 9-BBN 0.5 M solution in THF, anhydrous toluene, 115 °C, 1 h; ii. aq. 3.2 M NaOH, Pd(PPh3)4, TBAI, anhydrous toluene, 115 °C, 18 h [46–99%]; (c) HCl 4.0 M solution in dioxane, anhydrous MeOH, anhydrous CH2Cl2, RT, 1 h [99%]; (d) properly substituted benzoic acid, HATU, DIPEA, anhydrous DMF, RT, 3–12 h [41–75%]; (e) BBr3 1 M solution in CH2Cl2, anhydrous CH2Cl2, −10 to 0 °C, then RT, 1–3 h [46–66%].

    Scheme 2

    Scheme 2. Synthesis of Compounds 8, 9, and 12a
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    aReagents and conditions: (a) for compounds 41 and 42: K3PO4, CuI, anhydrous DMSO, 130 °C, 24 h [11–32%]; for compound 16: anhydrous K2CO3, anhydrous DMF, 110 °C, overnight [84%]; (b) i. tert-butyl 4-methylenepiperidine-1-carboxylate, 9-BBN 0.5 M solution in THF, anhydrous toluene, 115 °C, 1 h; ii. aq. 3.2 M NaOH, Pd(PPh3)4, TBAI, anhydrous toluene, 115 °C, 18 h [30–78%]; (c) HCl 4.0 M solution in dioxane, anhydrous MeOH, anhydrous CH2Cl2, RT, 1 h [90–99%]; (d) 3-methoxybenzoic acid, HATU, DIPEA, anhydrous DMF, RT, 3–12 h [53–72%]; (e) BBr3 1 M solution in CH2Cl2, anhydrous CH2Cl2, −10 to 0 °C, then RT, 1–3 h [29–62%].

    Figure 4

    Figure 4. Analysis of the mechanism of MAGL inhibition of compound 13. (A) Effect of DTT on MAGL inhibition activity. (B) IC50 (nM) values at different preincubation times with MAGL (0, 30, and 60 min). (C) Dilution assay: the first two columns indicate the inhibition percentage of the compound at concentrations of 320 and 8 nM. The third column indicates the inhibition percentage of the compound after dilution (final concentration = 8 nM).

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    Figure 5

    Figure 5. ABPP with fluorescent labeling of serine hydrolases in mouse brain membrane homogenates using a TAMRA-FP serine hydrolase probe and different inhibitors as controls. The mouse brain membranes (4 mg/mL) were pre-incubated for 25 min with either DMSO, 13 (10 μM, MAGL inhibitor), JZL-184 (10 μM, MAGL inhibitor), (9) URB597 (4 μM, FAAH inhibitor), (39) WWL70 (10 μM, ABHD6 inhibitor), (40) THL (30 μM, ABHD6 and ABHD12 inhibitor), (41) or MAFP (5 μM, unselective serine hydrolase inhibitor). (42) After additional incubation with TAMRA-FP (125 nM) for 25 min, the samples were separated in SDS-PAGE. A representative image of the TAMRA-FP signal after SDS-PAGE is shown. The presented results could be observed in three independent experiments.

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    Figure 6

    Figure 6. Minimized average structure of hMAGL in complex with compound 11b in the predicted binding pose. The protein residues surrounding the ligand are shown. Ligand–protein hydrogen bonds are highlighted with black lines. The inner surface of the protein binding site is shown in gray (PDB code 5ZUN).

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    Figure 7

    Figure 7. Correlation between the compound’s activities expressed as pIC50 values and the binding energies estimated using the best MM-PBSA protocol (εint = 4) expressed in kcal/mol.

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    Figure 8

    Figure 8. MAGL gene expression levels. (A) MAGL mRNA is more expressed in cancer tissues than in normal tissues (http://gepia.cancer-pku.cn/detail.php?gene=MAGL). Pancreatic cancer tissues are among the tumor tissues with the highest expression levels of MAGL. (B) MAGL mRNA expression is a prognostic factor in pancreatic cancer. The expression cutoff between patients with high versus low expression of MAGL (5132, RNA expression units) was obtained by the “R2: Genomics Analysis and Visualization Platform”. (C) Two primary pancreatic cancer cell cultures (PDAC2 and PDAC3) originating from patients undergoing surgery for pancreatic cancer showed significantly different expression levels of MAGL mRNA.

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    Figure 9

    Figure 9. Antiproliferative and pro-apoptotic effects of MAGL inhibitor 13. (A) IC50 of compound 13 in different pancreatic cancer models and in the immortalized ductal cells HPNE. (B) Representative curves of PDAC3 cells growth inhibitory effects of 13, JZL-184 and ABX-1431, as control. (C) Induction of apoptosis and (D) levels of active caspase-3 in PDAC3 cells treated with 13, gemcitabine, JZL-184, and ABX-1431 for 72 h, compared to control/untreated cells (value = 1, as illustrated by the dashed line). Measurements were performed in triplicate, and data are presented as means ± SEM. *p < 0.05 versus control; #p < 0.05 versus gemcitabine.

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    Figure 10

    Figure 10. Antimigratory effects of MAGL inhibitors. Statistical evaluation of the results of the wound-healing/migration assay on the PDAC3 cells 20 h after scratch induction and treatment. The percentages of scratch closure for control, 13-, JZL-184-, or ABX-1431-treated cells were compared with one-way analysis of variance (ANOVA)/t test. *p < 0.05 versus control.

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    Figure 11

    Figure 11. Combination assay and modulation of gene expression. (A) CI values of gemcitabine (GEM) combined with compound 13 at IC50 and IC25. The upper line represents an antagonistic CI > 1.2, the lower bar represents a synergistic CI < 0.8. (B) Combined results of different PCR experiments, evaluating the effect of GEM, 13, and JZL-184 on potential determinants of apoptosis induction, migration, and synergistic interaction with gemcitabine compared to control/untreated cells (value = 1, as illustrated by the dashed line). Measurements were performed in triplicate, and data are presented as means ± SEM.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntlynsb0%253D&md5=009abe68416cf28a18cfeb381aed47ac
    13. 13
      Guindon, J.; Desroches, J.; Beaulieu, P. The Antinociceptive Effects of Intraplantar Injections of 2-Arachidonoyl Glycerol Are Mediated by Cannabinoid CB 2 Receptors. Br. J. Pharmacol. 2007, 150, 693– 701,  DOI: 10.1038/sj.bjp.0706990
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      13
      The antinociceptive effects of intraplantar injections of 2-arachidonoyl glycerol are mediated by cannabinoid CB2 receptors
      Guindon, J.; Desroches, J.; Beaulieu, P.
      British Journal of Pharmacology (2007), 150 (6), 693-701CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
      Background and purpose:2-arachidonoyl glycerol (2-AG) is an endogenous cannabinoid with central antinociceptive properties. Its degrdn. is catalyzed by monoacylglycerol lipase (MGL) whose activity is inhibited by URB602, a new synthetic compd. The peripheral antinociceptive effects of 2-AG and URB602 in an inflammatory model of pain are not yet detd. We have evaluated these effects with and without the cannabinoid CB1 (AM251) and CB2 (AM630) receptor antagonists.Exptl. approach:Inflammation was induced in rat hind paws by intraplantar injection of formalin. Nociception was assessed behaviorally over the next 60 min, in 19 exptl. groups: (1) control; (2-6) 2-AG (0.01-100 μg); (7) AM251 (80 μg); (8) AM251+2-AG (10 μg); (9) AM630 (25 μg); (10) AM630+2-AG (10 μg); (11-16) URB602 (0.1-500 μg); (17) 2-AG+URB602 (ED50); (18) AM251+URB602 (ED50); (19) AM630+URB602 (ED50). Drugs were injected s.c. in the dorsal surface of the hind paw (50 μl), 15 min before formalin injection into the same paw. Key results:2-AG and URB602 produced dose-dependent antinociceptive effects for the late phases of the formalin test with ED50 of 0.65±0.455 μg and 68±14.3 μg, resp. Their combination at ED50 doses produced an additive antinociceptive effect. These effects were inhibited by AM630 but not by AM251 for 2-AG and by the two cannabinoid antagonists for URB602. Conclusions and implications:Locally injected 2-AG and URB602 decreased pain behavior in a dose-dependent manner in an inflammatory model of pain. The antinociceptive effect of 2-AG was mediated by the CB2 receptor.
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    14. 14
      Ignatowska-Jankowska, B. M.; Ghosh, S.; Crowe, M. S.; Kinsey, S. G.; Niphakis, M. J.; Abdullah, R. A.; Tao, Q.; O’ Neal, S. T.; Walentiny, D. M.; Wiley, J. L.; Cravatt, B. F.; Lichtman, A. H. In Vivo Characterization of the Highly Selective Monoacylglycerol Lipase Inhibitor KML29: Antinociceptive Activity without Cannabimimetic Side Effects. Br. J. Pharmacol. 2014, 171, 1392– 1407,  DOI: 10.1111/bph.12298
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      14
      In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects
      Ignatowska-Jankowska, B. M.; Ghosh, S.; Crowe, M. S.; Kinsey, S. G.; Niphakis, M. J.; Abdullah, R. A.; Tao, Q.; O'Neal, S. T.; Walentiny, D. M.; Wiley, J. L.; Cravatt, B. F.; Lichtman, A. H.
      British Journal of Pharmacology (2014), 171 (6), 1392-1407CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
      Background and Purpose: Since monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compd., KML29 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate). Exptl. Approach : In the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric hemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temp., catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm. Key Results: KML29 attenuated carrageenan-induced paw edema and completely reversed carrageenan-induced mech. allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB1) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB1 and CB2 receptors played differential roles in these pharmacol. effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Δ9-tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (-/-) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects. Conclusions and Implications : These results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behavior without occurrence of cannabimimetic side effects.
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    15. 15
      Kinsey, S. G.; Long, J. Z.; Cravatt, B. F.; Lichtman, A. H. Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibitors Produce Anti-Allodynic Effects in Mice Through Distinct Cannabinoid Receptor Mechanisms. J. Pain 2010, 11, 1420– 1428,  DOI: 10.1016/j.jpain.2010.04.001
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      15
      Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibitors Produce Anti-Allodynic Effects in Mice Through Distinct Cannabinoid Receptor Mechanisms
      Kinsey, Steven G.; Long, Jonathan Z.; Cravatt, Benjamin F.; Lichtman, Aron H.
      Journal of Pain (2010), 11 (12), 1420-1428CODEN: JPOAB5; ISSN:1526-5900. (Elsevier)
      The endocannabinoids anandamide and 2-arachidonoylglycerol are predominantly regulated by the resp. catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Inhibition of these enzymes elevates endocannabinoid levels and attenuates neuropathic pain. In the present study, CB1 and CB2 receptor-deficient mice were subjected to chronic constriction injury (CCI) of the sciatic nerve to examine the relative contribution of each receptor for the anti-allodynic effects of the FAAH inhibitor, PF-3845, and the MAGL inhibitor, JZL184. CCI caused marked hypersensitivity to mech. and cold stimuli, which was not altered by deletion of either the CB1 or CB2 receptor, but was attenuated by gabapentin, as well as by each enzyme inhibitor. Whereas PF-3845 lacked anti-allodynic efficacy in both knockout lines, JZL184 did not produce anti-allodynic effects in CB1 (-/-) mice, but retained its anti-allodynic effects in CB2 (-/-) mice. These data indicate that FAAH and MAGL inhibitors reduce nerve injury-related hyperalgesic states through distinct cannabinoid receptor mechanisms of action. In conclusion, although endogenous cannabinoids do not appear to play a tonic role in long-term expression of neuropathic pain states, both FAAH and MAGL represent potential therapeutic targets for the development of pharmacol. agents to treat chronic pain resulting from nerve injury. Perspective: This article presents data addressing the cannabinoid receptor mechanisms underlying the anti-allodynic actions of endocannabinoid catabolic enzyme inhibitors in the mouse sciatic nerve ligation model. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors reduced allodynia through distinct cannabinoid receptor mechanisms. These enzymes offer potential targets to treat neuropathic pain.
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    16. 16
      Jaiswal, S.; Akhilesh; Uniyal, A.; Tiwari, V.; Raja Ayyannan, S. Synthesis and Evaluation of Dual Fatty Acid Amide Hydrolase-Monoacylglycerol Lipase Inhibition and Antinociceptive Activities of 4-Methylsulfonylaniline-Derived Semicarbazones. Bioorg. Med. Chem. 2022, 60, 116698,  DOI: 10.1016/j.bmc.2022.116698
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      16
      Synthesis and evaluation of dual fatty acid amide hydrolase-monoacylglycerol lipase inhibition and antinociceptive activities of 4-methylsulfonylaniline-derived semicarbazones
      Jaiswal, Shivani; Akhilesh; Uniyal, Ankit; Tiwari, Vinod; Raja Ayyannan, Senthil
      Bioorganic & Medicinal Chemistry (2022), 60 (), 116698CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      We designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones I [R1 = H, Me, Ph; R2 = H, 2-OH, 4-F, etc.] and II [R1 = H, allyl, propargyl; R2 = H, Cl] and evaluated for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibition properties. Most of the compds. showed potency toward both enzymes with leading FAAH selectivity. Compd. (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC50 = 11 nM) and MAGL (IC50 = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Mol. docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π-π stacking via Ph ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by mol. simulation studies. PAMPA assay revealed that the lead compd. was suitable for blood-brain penetration. The lead compd. showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo expts. unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compd. exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compd. can be a potential template for the development of agents for neuropathic pain.
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    17. 17
      Nomura, D. K.; Long, J. Z.; Niessen, S.; Hoover, H. S.; Ng, S. W.; Cravatt, B. F. Monoacylglycerol Lipase Regulates a Fatty Acid Network That Promotes Cancer Pathogenesis. Cell 2010, 140, 49– 61,  DOI: 10.1016/j.cell.2009.11.027
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      17
      Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis
      Nomura, Daniel K.; Long, Jonathan Z.; Niessen, Sherry; Hoover, Heather S.; Ng, Shu-Wing; Cravatt, Benjamin F.
      Cell (Cambridge, MA, United States) (2010), 140 (1), 49-61CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Tumor cells display progressive changes in metab. that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.
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    18. 18
      Schlosburg, J. E.; Blankman, J. L.; Long, J. Z.; Nomura, D. K.; Pan, B.; Kinsey, S. G.; Nguyen, P. T.; Ramesh, D.; Booker, L.; Burston, J. J.; Thomas, E. A.; Selley, D. E.; Sim-Selley, L. J.; Liu, Q. S.; Lichtman, A. H.; Cravatt, B. F. Chronic Monoacylglycerol Lipase Blockade Causes Functional Antagonism of the Endocannabinoid System. Nat. Neurosci. 2010, 13, 1113– 1119,  DOI: 10.1038/nn.2616
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      18
      Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system
      Schlosburg, Joel E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Nomura, Daniel K.; Pan, Bin; Kinsey, Steven G.; Nguyen, Peter T.; Ramesh, Divya; Booker, Lamont; Burston, James J.; Thomas, Elizabeth A.; Selley, Dana E.; Sim-Selley, Laura J.; Liu, Qing-song; Lichtman, Aron H.; Cravatt, Benjamin F.
      Nature Neuroscience (2010), 13 (9), 1113-1119CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
      Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacol. tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused phys. dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and assocd. with agonism and functional antagonism of the brain cannabinoid system, resp.
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    19. 19
      Chanda, P. K.; Gao, Y.; Mark, L.; Btesh, J.; Strassle, B. W.; Lu, P.; Piesla, M. J.; Zhang, M.-Y.; Bingham, B.; Uveges, A.; Kowal, D.; Garbe, D.; Kouranova, E. V.; Ring, R. H.; Bates, B.; Pangalos, M. N.; Kennedy, J. D.; Whiteside, G. T.; Samad, T. A. Monoacylglycerol Lipase Activity Is a Critical Modulator of the Tone and Integrity of the Endocannabinoid System. Mol. Pharmacol. 2010, 78, 996– 1003,  DOI: 10.1124/mol.110.068304
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      19
      Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system
      Chanda, Pranab K.; Gao, Ying; Mark, Lilly; Btesh, Joan; Strassle, Brian W.; Lu, Peimin; Piesla, Michael J.; Zhang, Mei-Yi; Bingham, Brendan; Uveges, Albert; Kowal, Dianne; Garbe, David; Kouranova, Evguenia V.; Ring, Robert H.; Bates, Brian; Pangalos, Menelas N.; Kennedy, Jeffrey D.; Whiteside, Garth T.; Samad, Tarek A.
      Molecular Pharmacology (2010), 78 (6), 996-1003CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)
      Endocannabinoids are lipid mols. that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiol. processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymic biosynthesis and degrdn. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant redn. of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.
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    20. 20
      Taschler, U.; Radner, F. P. W.; Heier, C.; Schreiber, R.; Schweiger, M.; Schoiswohl, G.; Preiss-Landl, K.; Jaeger, D.; Reiter, B.; Koefeler, H. C.; Wojciechowski, J.; Theussl, C.; Penninger, J. M.; Lass, A.; Haemmerle, G.; Zechner, R.; Zimmermann, R. Monoglyceride Lipase Deficiency in Mice Impairs Lipolysis and Attenuates Diet-Induced Insulin Resistance. J. Biol. Chem. 2011, 286, 17467– 17477,  DOI: 10.1074/jbc.M110.215434
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      20
      Monoglyceride lipase deficiency in mice impairs lipolysis and attenuates diet-induced insulin resistance
      Taschler, Ulrike; Radner, Franz P. W.; Heier, Christoph; Schreiber, Renate; Schweiger, Martina; Schoiswohl, Gabriele; Preiss-Landl, Karina; Jaeger, Doris; Reiter, Birgit; Koefeler, Harald C.; Wojciechowski, Jacek; Theussl, Christian; Penninger, Josef M.; Lass, Achim; Haemmerle, Guenter; Zechner, Rudolf; Zimmermann, Robert
      Journal of Biological Chemistry (2011), 286 (20), 17467-17477CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Monoglyceride lipase (MGL) influences energy metab. by at least two mechanisms. First, it hydrolyzes monoacylglycerols (MG) into fatty acids and glycerol. These products can be used for energy prodn. or synthetic reactions. Second, MGL degrades 2-arachidonoyl glycerol (2-AG), the most abundant endogenous ligand of cannabinoid receptors (CBR). Activation of CBR affects energy homeostasis by central orexigenic stimuli, by promoting lipid storage, and by reducing energy expenditure. To characterize the metabolic role of MGL in vivo, we generated an MGL-deficient mouse model (MGL-ko). These mice exhibit a redn. in MG hydrolase activity and a concomitant increase in MG levels in adipose tissue, brain, and liver. In adipose tissue, the lack of MGL activity is partially compensated by hormone-sensitive lipase. Nonetheless, fasted MGL-ko mice exhibit reduced plasma glycerol and triacylglycerol, as well as liver triacylglycerol levels indicative for impaired lipolysis. Despite a strong elevation of 2-AG levels, MGL-ko mice exhibit normal food intake, fat mass, and energy expenditure. Yet mice lacking MGL show a pharmacol. tolerance to the CBR agonist CP 55,940 suggesting that the elevated 2-AG levels are functionally antagonized by desensitization of CBR. Interestingly, however, MGL-ko mice receiving a high fat diet exhibit significantly improved glucose tolerance and insulin sensitivity in comparison with wild-type controls despite equal wt. gain. In conclusion, our observations implicate that MGL deficiency impairs lipolysis and attenuates diet-induced insulin resistance. Defective degrdn. of 2-AG does not provoke cannabinoid-like effects on feeding behavior, lipid storage, and energy expenditure, which may be explained by desensitization of CBR.
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    21. 21
      Ghosh, S.; Wise, L. E.; Chen, Y.; Gujjar, R.; Mahadevan, A.; Cravatt, B. F.; Lichtman, A. H. The Monoacylglycerol Lipase Inhibitor JZL184 Suppresses Inflammatory Pain in the Mouse Carrageenan Model. Life Sci. 2013, 92, 498– 505,  DOI: 10.1016/j.lfs.2012.06.020
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      21
      The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model
      Ghosh, Sudeshna; Wise, Laura E.; Chen, Yugang; Gujjar, Ramesh; Mahadevan, Anu; Cravatt, Benjamin F.; Lichtman, Aron H.
      Life Sciences (2013), 92 (8-9), 498-505CODEN: LIFSAK; ISSN:0024-3205. (Elsevier B.V.)
      The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test.The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the resp. CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40 mg/kg) was administered for six days to assess tolerance.JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mech. allodynia. Complementary genetic and pharmacol. approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy.These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.
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    22. 22
      Schlosburg, J. E.; Kinsey, S. G.; Ignatowska-Jankowska, B.; Ramesh, D.; Abdullah, R. A.; Tao, Q.; Booker, L.; Long, J. Z.; Selley, D. E.; Cravatt, B. F.; Lichtman, A. H. Prolonged Monoacylglycerol Lipase Blockade Causes Equivalent Cannabinoid Receptor Type 1 Receptor–Mediated Adaptations in Fatty Acid Amide Hydrolase Wild-Type and Knockout Mice. J. Pharmacol. Exp. Ther. 2014, 350, 196– 204,  DOI: 10.1124/jpet.114.212753
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      22
      Prolonged monoacylglycerol lipase blockade causes equivalent cannabinoid receptor type 1 receptor-mediated adaptations in fatty acid amide hydrolase wild-type and knockout mice
      Schlosburg, Joel E.; Kinsey, Steven G.; Ignatowska-Jankowska, Bogna; Ramesh, Divya; Abdullah, Rehab A.; Tao, Qing; Booker, Lamont; Long, Jonathan Z.; Selley, Dana E.; Cravatt, Benjamin F.; Lichtman, Aron H.
      Journal of Pharmacology and Experimental Therapeutics (2014), 350 (2), 196-204, 9 pp.CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)
      Complementary genetic and pharmacol. approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the resp. endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiol. roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and mol. signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacol. effects of Δ9-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding, and dependence as indicated by rimonabant-pptd. withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.
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    23. 23
      Muccioli, G. G.; Labar, G.; Lambert, D. M. CAY10499, a Novel Monoglyceride Lipase Inhibitor Evidenced by an Expeditious MGL Assay. ChemBioChem 2008, 9, 2704– 2710,  DOI: 10.1002/cbic.200800428
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      23
      CAY10499, a novel monoglyceride lipase inhibitor evidenced by an expeditious MGL assay
      Muccioli, Giulio G.; Labar, Geoffray; Lambert, Didier M.
      ChemBioChem (2008), 9 (16), 2704-2710CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Monoglyceride lipase (MGL) plays a major role in the metab. of the lipid transmitter 2-arachidonoylglycerol (2-AG). This endocannabinoid is known to mediate a large no. of physiol. processes, and its regulation is thought to be of great therapeutic potential. However, the no. of available monoglyceride lipase inhibitors is limited, mostly due to the lack of rapid and accurate pharmacol. assays for the enzyme. We have developed a 96-well-format assay for MGL using a nonradiolabeled substrate, 4-nitrophenylacetate. The IC50 values that were obtained for known inhibitors of MGL using 4-nitrophenylacetate were similar to those reported by using the radiolabeled form of an endogenous substrate, 2-oleoylglycerol. In a first small-scale screening, we identified CAY10499 as a novel monoglyceride lipase inhibitor. Thus, we report the characterization of this submicromolar inhibitor, which acts on MGL through an unprecedented mechanism for inhibitors of this enzyme.
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    24. 24
      Cisar, J. S.; Weber, O. D.; Clapper, J. R.; Blankman, J. L.; Henry, C. L.; Simon, G. M.; Alexander, J. P.; Jones, T. K.; Ezekowitz, R. A. B.; O’Neill, G. P.; Grice, C. A. Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders. J. Med. Chem. 2018, 61, 9062– 9084,  DOI: 10.1021/acs.jmedchem.8b00951
      [ACS Full Text ACS Full Text], [CAS], Google Scholar
      24
      Identification of ABX-1431, a Selective Inhibitor of Monoacylglycerol Lipase and Clinical Candidate for Treatment of Neurological Disorders
      Cisar, Justin S.; Weber, Olivia D.; Clapper, Jason R.; Blankman, Jacqueline L.; Henry, Cassandra L.; Simon, Gabriel M.; Alexander, Jessica P.; Jones, Todd K.; Ezekowitz, R. Alan B.; O'Neill, Gary P.; Grice, Cheryl A.
      Journal of Medicinal Chemistry (2018), 61 (20), 9062-9084CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacol. inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding redns. in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling expts. verify the exquisite selectivity of 28 for MGLL vs. other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain 2-AG concns., and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clin. trials.
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    25. 25
      King, A. R.; Dotsey, E. Y.; Lodola, A.; Jung, K. M.; Ghomian, A.; Qiu, Y.; Fu, J.; Mor, M.; Piomelli, D. Discovery of Potent and Reversible Monoacylglycerol Lipase Inhibitors. Chem. Biol. 2009, 16, 1045– 1052,  DOI: 10.1016/j.chembiol.2009.09.012
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      25
      Discovery of Potent and Reversible Monoacylglycerol Lipase Inhibitors
      King, Alvin R.; Dotsey, Emmanuel Y.; Lodola, Alessio; Jung, Kwang Mook; Ghomian, Azar; Qiu, Yan; Fu, Jin; Mor, Marco; Piomelli, Daniele
      Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (10), 1045-1052CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Summary: Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biol. deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chem. scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concn., IC50 = 93 nM and 315 nM, resp.) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys201 and Cys208) flanking such pocket. This previously unrecognized regulatory region might offer a mol. target for potent and reversible inhibitors of MGL.
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    26. 26
      Schalk-Hihi, C.; Schubert, C.; Alexander, R.; Bayoumy, S.; Clemente, J. C.; Deckman, I.; DesJarlais, R. L.; Dzordzorme, K. C.; Flores, C. M.; Grasberger, B.; Kranz, J. K.; Lewandowski, F.; Liu, L.; Ma, H.; Maguire, D.; Macielag, M. J.; McDonnell, M. E.; Haarlander, T. M.; Miller, R.; Milligan, C.; Reynolds, C.; Kuo, L. C. Crystal Structure of a Soluble Form of Human Monoglyceride Lipase in Complex with an Inhibitor at 1.35 Å Resolution. Protein Sci. 2011, 20, 670– 683,  DOI: 10.1002/pro.596
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      26
      Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution
      Schalk-Hihi, Celine; Schubert, Carsten; Alexander, Richard; Bayoumy, Shariff; Clemente, Jose C.; Deckman, Ingrid; Des Jarlais, Renee L.; Dzordzorme, Keli C.; Flores, Christopher M.; Grasberger, Bruce; Kranz, James K.; Lewandowski, Frank; Liu, Li; Ma, Hongchang; Maguire, Diane; Macielag, Mark J.; McDonnell, Mark E.; Haarlander, Tara Mezzasalma; Miller, Robyn; Milligan, Cindy; Reynolds, Charles; Kuo, Lawrence C.
      Protein Science (2011), 20 (4), 670-683CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      A high-resoln. structure of a ligand-bound, sol. form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Anal. of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissocn. from the membrane upon completion of the cycle. In addn., the study outlines a successful approach to transform membrane assocd. proteins, which tend to aggregate upon purifn., into a monomeric and sol. form.
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    27. 27
      Hernández-Torres, G.; Cipriano, M.; Hedán, E.; Björklund, E.; Canales, A.; Zian, D.; Feliú, A.; Mecha, M.; Guaza, C.; Fowler, C. J.; Ortega-Gutiárrez, S.; López-Rodríguez, M. L. A Reversible and Selective Inhibitor of Monoacylglycerol Lipase Ameliorates Multiple Sclerosis. Angew. Chem., Int. Ed. 2014, 53, 13765– 13770,  DOI: 10.1002/anie.201407807
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      27
      A Reversible and Selective Inhibitor of Monoacylglycerol Lipase Ameliorates Multiple Sclerosis
      Hernandez-Torres, Gloria; Cipriano, Mariateresa; Heden, Erika; Bjoerklund, Emmelie; Canales, Angeles; Zian, Debora; Feliu, Ana; Mecha, Miriam; Guaza, Carmen; Fowler, Christopher J.; Ortega-Gutierrez, Silvia; Lopez-Rodriguez, Maria L.
      Angewandte Chemie, International Edition (2014), 53 (50), 13765-13770CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacol. tolerance. Hence, addnl. classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC50=0.18 μM) which is active in vivo and ameliorates the clin. progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB1-mediated side effects. These results support the interest in MAGL as a target for the treatment of MS.
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    28. 28
      Aghazadeh Tabrizi, M.; Baraldi, P. G.; Baraldi, S.; Ruggiero, E.; De Stefano, L.; Rizzolio, F.; Di Cesare Mannelli, L.; Ghelardini, C.; Chicca, A.; Lapillo, M.; Gertsch, J.; Manera, C.; Macchia, M.; Martinelli, A.; Granchi, C.; Minutolo, F.; Tuccinardi, T. Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors. J. Med. Chem. 2018, 61, 1340– 1354,  DOI: 10.1021/acs.jmedchem.7b01845
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      28
      Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors
      Aghazadeh-Tabrizi, Mojgan; Baraldi, Pier Giovanni; Baraldi, Stefania; Ruggiero, Emanuela; De Stefano, Lucia; Rizzolio, Flavio; Di Cesare Mannelli, Lorenzo; Ghelardini, Carla; Chicca, Andrea; Lapillo, Margherita; Gertsch, Jurg; Manera, Clementina; Macchia, Marco; Martinelli, Adriano; Granchi, Carlotta; Minutolo, Filippo; Tuccinardi, Tiziano
      Journal of Medicinal Chemistry (2018), 61 (3), 1340-1354CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the degrdn. of the endocannabinoid neurotransmitter 2-arachidonoylglycerol, which is implicated in many physiol. processes. Beyond the possible utilization of MAGL inhibitors as anti-inflammatory, antinociceptive, and anticancer agents, their application has encountered obstacles due to the unwanted effects caused by the irreversible inhibition of this enzyme. The possible application of reversible MAGL inhibitors has only recently been explored, mainly due to the deficiency of known compds. possessing efficient reversible inhibitory activities. The authors report a new series of reversible MAGL inhibitors. Among them, compd. 26 ((4-benzylpiperidin-1-yl)(5-(4-hydroxyphenyl)-1-(3-methylbenzyl)-1H-pyrazol-3-yl)methanone) showed to be a potent MAGL inhibitor (IC50 = 0.51 μM, Ki = 412 nM) with a good selectivity vs. fatty acid amide hydrolase (FAAH), α/β-hydrolase domain-contg. 6 (ABHD6), and 12 (ABHD12). Interestingly, this compd. also possesses antiproliferative activities against two different cancer cell lines and relieves the neuropathic hypersensitivity induced in vivo by oxaliplatin.
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    29. 29
      Bononi, G.; Granchi, C.; Lapillo, M.; Giannotti, M.; Nieri, D.; Fortunato, S.; El Boustani, M.; Caligiuri, I.; Poli, G.; Carlson, K. E.; Kim, S. H.; Macchia, M.; Martinelli, A.; Rizzolio, F.; Chicca, A.; Katzenellenbogen, J. A.; Minutolo, F.; Tuccinardi, T. Discovery of Long-Chain Salicylketoxime Derivatives as Monoacylglycerol Lipase (MAGL) Inhibitors. Eur. J. Med. Chem. 2018, 157, 817– 836,  DOI: 10.1016/j.ejmech.2018.08.038
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      29
      Discovery of long-chain salicylketoxime derivatives as monoacylglycerol lipase (MAGL) inhibitors
      Bononi, Giulia; Granchi, Carlotta; Lapillo, Margherita; Giannotti, Massimiliano; Nieri, Daniela; Fortunato, Serena; Boustani, Maguie El; Caligiuri, Isabella; Poli, Giulio; Carlson, Kathryn E.; Kim, Sung Hoon; Macchia, Marco; Martinelli, Adriano; Rizzolio, Flavio; Chicca, Andrea; Katzenellenbogen, John A.; Minutolo, Filippo; Tuccinardi, Tiziano
      European Journal of Medicinal Chemistry (2018), 157 (), 817-836CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      Monoacylglycerol lipase (MAGL) is the enzyme hydrolyzing the endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid and glycerol. Therefore, MAGL is implicated in many physiol. processes involving the regulation of the endocannabinoid system and eicosanoid network. MAGL inhibition represents a potential therapeutic target for many diseases, including cancer. Nowadays, most MAGL inhibitors inhibit this enzyme by an irreversible mechanism of action, potentially leading to unwanted side effects from chronic treatment. Herein, we report the discovery of long-chain salicylketoxime derivs. as potent and reversible MAGL inhibitors. The compds. herein described are characterized by a good target selectivity for MAGL and by antiproliferative activities against a series of cancer cell lines. Finally, modeling studies suggest a reasonable hypothetical binding mode for this class of compds.
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    30. 30
      Aida, J.; Fushimi, M.; Kusumoto, T.; Sugiyama, H.; Arimura, N.; Ikeda, S.; Sasaki, M.; Sogabe, S.; Aoyama, K.; Koike, T. Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-Ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors. J. Med. Chem. 2018, 61, 9205– 9217,  DOI: 10.1021/acs.jmedchem.8b00824
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      30
      Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors
      Aida, Jumpei; Fushimi, Makoto; Kusumoto, Tomokazu; Sugiyama, Hideyuki; Arimura, Naoto; Ikeda, Shuhei; Sasaki, Masako; Sogabe, Satoshi; Aoyama, Kazunobu; Koike, Tatsuki
      Journal of Medicinal Chemistry (2018), 61 (20), 9205-9217CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biol. active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, authors report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, they identified a potent and reversible MAGL inhibitor, compd., 1-[3-Fluoro-5-(2-methylpyridin-3-yl)phenyl]-4-[4-(pyrimidin-2-yl)piperazin-1-yl]pyrrolidin-2-one (R)-3t. Oral administration of compd. (R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.
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    31. 31
      Granchi, C.; Rizzolio, F.; Palazzolo, S.; Carmignani, S.; MacChia, M.; Saccomanni, G.; Manera, C.; Martinelli, A.; Minutolo, F.; Tuccinardi, T. Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors. J. Med. Chem. 2016, 59, 10299– 10314,  DOI: 10.1021/acs.jmedchem.6b01459
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      31
      Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors
      Granchi, Carlotta; Rizzolio, Flavio; Palazzolo, Stefano; Carmignani, Sara; Macchia, Marco; Saccomanni, Giuseppe; Manera, Clementina; Martinelli, Adriano; Minutolo, Filippo; Tuccinardi, Tiziano
      Journal of Medicinal Chemistry (2016), 59 (22), 10299-10314CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Monoacylglycerol lipase (MAGL) inhibitors are considered potential therapeutic agents for a variety of pathol. conditions, including several types of cancer. Many MAGL inhibitors are reported in literature; however, most of them showed an irreversible mechanism of action, which caused important side effects. The use of reversible MAGL inhibitors has been only partially investigated so far, mainly because of the lack of compds. with good MAGL reversible inhibition properties. In this study, starting from the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) lead compd. that showed a reversible mechanism of MAGL inhibition (Ki = 8.6 μM), the authors started its structural optimization and the authors developed a new potent and selective MAGL inhibitor ((4-(4-Chlorobenzoyl)piperidin-1-yl)(3-hydroxyphenyl)methanone (17b), Ki = 0.65 μM). Furthermore, modeling studies suggested that the binding interactions of this compd. replace a structural water mol. reproducing its H-bonds in the MAGL binding site, thus identifying a new key anchoring point for the development of new MAGL inhibitors.
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    32. 32
      Granchi, C.; Lapillo, M.; Glasmacher, S.; Bononi, G.; Licari, C.; Poli, G.; el Boustani, M.; Caligiuri, I.; Rizzolio, F.; Gertsch, J.; Macchia, M.; Minutolo, F.; Tuccinardi, T.; Chicca, A. Optimization of a Benzoylpiperidine Class Identifies a Highly Potent and Selective Reversible Monoacylglycerol Lipase (MAGL) Inhibitor. J. Med. Chem. 2019, 62, 1932– 1958,  DOI: 10.1021/acs.jmedchem.8b01483
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      32
      Optimization of a Benzoylpiperidine Class Identifies a Highly Potent and Selective Reversible Monoacylglycerol Lipase (MAGL) Inhibitor
      Granchi, Carlotta; Lapillo, Margherita; Glasmacher, Sandra; Bononi, Giulia; Licari, Cristina; Poli, Giulio; El Boustani, Maguie; Caligiuri, Isabella; Rizzolio, Flavio; Gertsch, Jurg; Macchia, Marco; Minutolo, Filippo; Tuccinardi, Tiziano; Chicca, Andrea
      Journal of Medicinal Chemistry (2019), 62 (4), 1932-1958CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Monoacylglycerol lipase (MAGL) is the enzyme degrading the endocannabinoid 2-arachidonoylglycerol, and it is involved in several physiol. and pathol. processes. The therapeutic potential of MAGL is linked to several diseases, including cancer. The development of MAGL inhibitors has been greatly limited by the side effects assocd. with the prolonged MAGL inactivation. Importantly, it could be preferable to use reversible MAGL inhibitors in vivo, but nowadays only few reversible compds. have been developed. In the present study, structural optimization of a previously developed class of MAGL inhibitors led to the identification of compd. 23, which proved to be a very potent reversible MAGL inhibitor (IC50 = 80 nM), selective for MAGL over the other main components of the endocannabinoid system, endowed of a promising antiproliferative activity in a series of cancer cell lines and able to block MAGL both in cell-based as well as in vivo assays.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVWisr0%253D&md5=04dedd9a69d80382563ff19ea0124788
    33. 33
      Tuccinardi, T.; Granchi, C.; Rizzolio, F.; Caligiuri, I.; Battistello, V.; Toffoli, G.; Minutolo, F.; Macchia, M.; Martinelli, A. Identification and Characterization of a New Reversible MAGL Inhibitor. Bioorg. Med. Chem. 2014, 22, 3285– 3291,  DOI: 10.1016/j.bmc.2014.04.057
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      33
      Identification and characterization of a new reversible MAGL inhibitor
      Tuccinardi, Tiziano; Granchi, Carlotta; Rizzolio, Flavio; Caligiuri, Isabella; Battistello, Vittoria; Toffoli, Giuseppe; Minutolo, Filippo; Macchia, Marco; Martinelli, Adriano
      Bioorganic & Medicinal Chemistry (2014), 22 (13), 3285-3291CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      Monoacylglycerol lipase is a serine hydrolase that play a major role in the degrdn. of 2-arachidonoylglycerol, an endocannabinoid neurotransmitter implicated in several physiol. processes. Recent studies have shown the possible role of MAGL inhibitors as anti-inflammatory, anti-nociceptive and anti-cancer agents. The use of irreversible MAGL inhibitors detd. an unwanted chronic MAGL inactivation, which acquires a functional antagonism function of the endocannabinoid system. However, the application of reversible MAGL inhibitors has not yet been explored, mainly due to the scarcity of known compds. possessing efficient reversible inhibitory activities. In this study we reported the first virtual screening anal. for the identification of reversible MAGL inhibitors. Among the screened compds., the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) is a promising reversible MAGL inhibitor lead (Ki = 8.6 μM), which may be used for the future development of a new class of MAGL inhibitors. Furthermore, the results demonstrate the validity of the methodologies that we followed, encouraging addnl. screenings of other com. databases.
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    34. 34
      Granchi, C.; Bononi, G.; Ferrisi, R.; Gori, E.; Mantini, G.; Glasmacher, S.; Poli, G.; Palazzolo, S.; Caligiuri, I.; Rizzolio, F.; Canzonieri, V.; Perin, T.; Gertsch, J.; Sodi, A.; Giovannetti, E.; Macchia, M.; Minutolo, F.; Tuccinardi, T.; Chicca, A. Design, Synthesis and Biological Evaluation of Second-Generation Benzoylpiperidine Derivatives as Reversible Monoacylglycerol Lipase (MAGL) Inhibitors. Eur. J. Med. Chem. 2021, 209, 112857,  DOI: 10.1016/j.ejmech.2020.112857
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      34
      Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors
      Granchi, Carlotta; Bononi, Giulia; Ferrisi, Rebecca; Gori, Eleonora; Mantini, Giulia; Glasmacher, Sandra; Poli, Giulio; Palazzolo, Stefano; Caligiuri, Isabella; Rizzolio, Flavio; Canzonieri, Vincenzo; Perin, Tiziana; Gertsch, Jurg; Sodi, Andrea; Giovannetti, Elisa; Macchia, Marco; Minutolo, Filippo; Tuccinardi, Tiziano; Chicca, Andrea
      European Journal of Medicinal Chemistry (2021), 209 (), 112857CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      An interesting enzyme of the endocannabinoid system is monoacylglycerol lipase (MAGL). This enzyme, which metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG), has attracted great interest due to its involvement in several physiol. and pathol. processes, such as cancer progression. Exptl. evidences highlighted some drawbacks assocd. with the use of irreversible MAGL inhibitors in vivo, therefore the research field concerning reversible inhibitors is rapidly growing. In the present manuscript, the class of benzoylpiperidine-based MAGL inhibitors was further expanded and optimized. Enzymic assays identified some compds. in the low nanomolar range and steered mol. dynamics simulations predicted the dissocn. itinerary of one of the best compds. from the enzyme, confirming the obsd. structure-activity relationship. Biol. evaluation, including assays in intact U937 cells and competitive activity-based protein profiling expts. in mouse brain membranes, confirmed the selectivity of the selected compds. for MAGL vs. other components of the endocannabinoid system. Future studies on the potential use of these compds. in the clin. setting are also supported by the inhibition of cell growth obsd. both in cancer organoids derived from high grade serous ovarian cancer patients and in pancreatic ductal adenocarcinoma primary cells, which showed genetic and histol. features very similar to the primary tumors.
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    35. 35
      Bononi, G.; Tonarini, G.; Poli, G.; Barravecchia, I.; Caligiuri, I.; Macchia, M.; Rizzolio, F.; Demontis, G. C.; Minutolo, F.; Granchi, C.; Tuccinardi, T. Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Diphenylsulfide-Benzoylpiperidine Scaffold. Eur. J. Med. Chem. 2021, 223, 113679,  DOI: 10.1016/j.ejmech.2021.113679
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      35
      Monoacylglycerol lipase (MAGL) inhibitors based on a diphenylsulfide-benzoylpiperidine scaffold
      Bononi, Giulia; Tonarini, Giacomo; Poli, Giulio; Barravecchia, Ivana; Caligiuri, Isabella; Macchia, Marco; Rizzolio, Flavio; Demontis, Gian Carlo; Minutolo, Filippo; Granchi, Carlotta; Tuccinardi, Tiziano
      European Journal of Medicinal Chemistry (2021), 223 (), 113679CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      Monoacylglycerol lipase (MAGL) is an enzyme belonging to the endocannabinoid system that mainly metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG). Numerous studies have shown the involvement of this enzyme in various pathol. conditions such as pain, cancer progression, Parkinson's and Alzheimer's disease, thus encouraging the development of new MAGL modulators. In this context, we developed new diphenylsulfide-benzoylpiperidine derivs. characterized by a high enzymic MAGL inhibition activity in the low nanomolar range, a reversible mechanism of action and selectivity. The three most active compds. I, wherein R = CF3, Cl, OCF3, induced an appreciable inhibition of cell viability in a panel of nine cancer cell lines, with IC50 values ranging between 0.32 and 10μM, thus highlighting their potential as novel anticancer agents.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1amtrrO&md5=09db0118b344eee9e4f883494a35888c
    36. 36
      Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.; Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith, S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Van Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Discovery and Characterization of a Highly Selective FAAH Inhibitor That Reduces Inflammatory Pain. Chem. Biol. 2009, 16, 411– 420,  DOI: 10.1016/j.chembiol.2009.02.013
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      36
      Discovery and Characterization of a Highly Selective FAAH Inhibitor that Reduces Inflammatory Pain
      Ahn, Kay; Johnson, Douglas S.; Mileni, Mauro; Beidler, David; Long, Jonathan Z.; McKinney, Michele K.; Weerapana, Eranthie; Sadagopan, Nalini; Liimatta, Marya; Smith, Sarah E.; Lazerwith, Scott; Stiff, Cory; Kamtekar, Satwik; Bhattacharya, Keshab; Zhang, Yanhua; Swaney, Stephen; Van Becelaere, Keri; Stevens, Raymond C.; Cravatt, Benjamin F.
      Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (4), 411-420CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Summary: Endocannabinoids are lipid signaling mols. that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as detd. by activity-based protein profiling; raises brain anandamide levels for up to 24 h; and produces significant cannabinoid receptor-dependent redns. in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacol. tool for in vivo characterization of the endocannabinoid system.
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    37. 37
      Niphakis, M. J.; Cravatt, B. F. Enzyme Inhibitor Discovery by Activity-Based Protein Profiling. Annu. Rev. Biochem. 2014, 341,  DOI: 10.1146/annurev-biochem-060713-035708
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      Enzyme inhibitor discovery by activity-based protein profiling
      Niphakis, Micah J.; Cravatt, Benjamin F.
      Annual Review of Biochemistry (2014), 83 (), 341-377CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      Eukaryotic and prokaryotic organisms possess huge nos. of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biol. systems. Here, we discuss how the chem. proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochem. and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiol. and disease. These studies demonstrate the value of selective chem. probes as drivers of biol. inquiry.
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    38. 38
      Blankman, J. L.; Cravatt, B. F. Chemical Probes of Endocannabinoid Metabolism. Pharmacol. Rev. 2013, 65, 849– 871,  DOI: 10.1124/pr.112.006387
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      Chemical probes of endocannabinoid metabolism
      Blankman, Jacqueline L.; Cravatt, Benjamin F.
      Pharmacological Reviews (2013), 65 (2), 849-871, 23 pp.CODEN: PAREAQ; ISSN:1521-0081. (American Society for Pharmacology and Experimental Therapeutics)
      A review. The endocannabinoid signaling system regulates diverse physiol. processes and has attracted considerable attention as a potential pharmaceutical target for treating diseases, such as pain, anxiety/depression, and metabolic disorders. The principal ligands of the endocannabinoid system are the lipid transmitters N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which activate the two major cannabinoid receptors, CB1 and CB2. Anandamide and 2-AG signaling pathways in the nervous system are terminated by enzymic hydrolysis mediated primarily by the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), resp. In this review, we will discuss the development of FAAH and MAGL inhibitors and their pharmacol. application to investigate the function of anandamide and 2-AG signaling pathways in preclinicalmodels of neurobehavioral processes, such as pain, anxiety, and addiction. We will place emphasis on how these studies are beginning to discern the different roles played by anandamide and 2-AG in the nervous system and the resulting implications for advancing endocannabinoid hydrolase inhibitors as next-generation therapeutics.
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    39. 39
      Piomelli, D.; Tarzia, G.; Duranti, A.; Tontini, A.; Mor, M.; Compton, T. R.; Dasse, O.; Monaghan, E. P.; Parrott, J. A.; Putman, D. Pharmacological Profile of the Selective FAAH Inhibitor KDS-4103 (URB597). CNS Drug Rev. 2006, 12, 21– 38,  DOI: 10.1111/j.1527-3458.2006.00021.x
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      39
      Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597)
      Piomelli, Daniele; Tarzia, Giorgio; Duranti, Andrea; Tontini, Andrea; Mor, Marco; Compton, Timothy R.; Dasse, Olivier; Monaghan, Edward P.; Parrott, Jeff A.; Putman, David
      CNS Drug Reviews (2006), 12 (1), 21-38CODEN: CDREFB; ISSN:1080-563X. (Blackwell Publishing, Inc.)
      A review. In the present article, we review the pharmacol. properties of KDS-4103 (URB597), a highly potent and selective inhibitor of the enzyme fatty-acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In vitro, KDS-4103 inhibits FAAH activity with median inhibitory concns. (IC50) of 5 nM in rat brain membranes and 3 nM in human liver microsomes. In vivo, KDS-4103 inhibits rat brain FAAH activity after i.p. administration with a median ID (ID50) of 0.15 mg/kg. The compd. does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice KDS-4103 elicits significant, anxiolytic-like, anti-depressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Δ9-tetrahydrocannabinol (Δ9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB1 receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were obsd. in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.
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    40. 40
      Li, W.; Blankman, J. L.; Cravatt, B. F. A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases. J. Am. Chem. Soc. 2007, 129, 9594– 9595,  DOI: 10.1021/ja073650c
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      40
      A Functional Proteomic Strategy to Discover Inhibitors for Uncharacterized Hydrolases
      Li, Weiwei; Blankman, Jacqueline L.; Cravatt, Benjamin F.
      Journal of the American Chemical Society (2007), 129 (31), 9594-9595CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Hydrolytic enzymes constitute one of the largest and most diverse protein classes in nature and play key roles in nearly all physiol. and pathol. processes. The mammalian serine hydrolase superfamily contains a remarkable no. of uncharacterized members, with at least 40-50% of these enzymes lacking exptl. verified endogenous substrates and products. Assignment of metabolic and cellular functions to these enzymes requires the development of pharmacol. tools to selectively perturb their activity. We describe herein a functional proteomic strategy to systematically develop potent and selective inhibitors for uncharacterized serine hydrolases and its application to the brain-enriched enzyme α/β-hydrolase domain 6. We anticipate that the methods described herein will facilitate the development of selective chem. probes to annotate the metabolic and (patho)physiol. functions of many of the uncharacterized serine hydrolases that currently populate eukaryotic and prokaryotic proteomes.
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    41. 41
      Hoover, H. S.; Blankman, J. L.; Niessen, S.; Cravatt, B. F. Selectivity of Inhibitors of Endocannabinoid Biosynthesis Evaluated by Activity-Based Protein Profiling. Bioorg. Med. Chem. Lett. 2008, 18, 5838– 5841,  DOI: 10.1016/j.bmcl.2008.06.091
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      Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling
      Hoover, Heather S.; Blankman, Jacqueline L.; Niessen, Sherry; Cravatt, Benjamin F.
      Bioorganic & Medicinal Chemistry Letters (2008), 18 (22), 5838-5841CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
      The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacol. studies using inhibitors of the 2-AG biosynthetic enzymes diacylglycerol lipase α and β (DAGL-α/β). Here, we show by competitive activity-based protein profiling that the DAGL-α/β inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was obsd. for THL and RHC80267, suggesting that pharmacol. effects obsd. with both agents may be viewed as good initial evidence for DAGL-dependent events.
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    42. 42
      Deutsch, D. G.; Omeir, R.; Arreaza, G.; Salehani, D.; Prestwich, G. D.; Huang, Z.; Howlett, A. Methyl Arachidonyl Fluorophosphonate: A Potent Irreversible Inhibitor of Anandamide Amidase. Biochem. Pharmacol. 1997, 53, 255– 260,  DOI: 10.1016/S0006-2952(96)00830-1
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      42
      Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase
      Deutsch, Dale G.; Omeir, Romelda; Arreaza, Gladys; Salehani, David; Prestwich, Glenn D.; Huang, Zheng; Howlett, Allyn
      Biochemical Pharmacology (1997), 53 (3), 255-260CODEN: BCPCA6; ISSN:0006-2952. (Elsevier)
      Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described. In the current study, Me arachidonyl fluorophosphate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approx. 1000-fold more potent than Arach-CF3, MAFP demonstrated selectively towards anandamide amidase for which it was approx. 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, resp. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addn., can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonyl ethanolamide amidase.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXhs1Ggt78%253D&md5=8b67ceff9bb0ba13826e4574e5f4ef38
    43. 43
      Baggelaar, M. P.; Janssen, F. J.; van Esbroeck, A. C. M.; den Dulk, H.; Allarà, M.; Hoogendoorn, S.; McGuire, R.; Florea, B. I.; Meeuwenoord, N.; van den Elst, H.; van der Marel, G. A.; Brouwer, J.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M. Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain. Angew. Chem., Int. Ed. 2013, 52, 12081– 12085,  DOI: 10.1002/anie.201306295
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      Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain
      Baggelaar, Marc P.; Janssen, Freek J.; van Esbroeck, Annelot C. M.; den Dulk, Hans; Allara, Marco; Hoogendoorn, Sascha; McGuire, Ross; Florea, Bogdan I.; Meeuwenoord, Nico; van den Elst, Hans; van der Marel, Gijsbert A.; Brouwer, Jaap; Di Marzo, Vincenzo; Overkleeft, Herman S.; van der Stelt, Mario
      Angewandte Chemie, International Edition (2013), 52 (46), 12081-12085CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A strategy that combines a knowledge-based in silico design approach and the development of novel activity-based probes for the detection of endogenous diacylglycerol lipase-α (DAGL-α) is presented. This approach resulted in the rapid identification of new DAGL-α inhibitors with high selectivity in the brain proteome.
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    44. 44
      Poli, G.; Granchi, C.; Rizzolio, F.; Tuccinardi, T. Application of MM-PBSA Methods in Virtual Screening. Molecules 2020, 25, 1971,  DOI: 10.3390/molecules25081971
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      Application of MM-PBSA methods in virtual screening
      Poli, Giulio; Granchi, Carlotta; Rizzolio, Flavio; Tuccinardi, Tiziano
      Molecules (2020), 25 (8), 1971CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      A review. Computer-aided drug design techniques are today largely applied in medicinal chem. In particular, receptor-based virtual screening (VS) studies, in which mol. docking represents the gold std. in silico approach, constitute a powerful strategy for identifying novel hit compds. active against the desired target receptor. Nevertheless, the need for improving the ability of docking in discriminating true active ligands from inactive compds., thus boosting VS hit rates, is still pressing. In this context, the use of binding free energy evaluation approaches can represent a profitable tool for rescoring ligand-protein complexes predicted by docking based on more reliable estns. of ligand-protein binding affinities than those obtained with simple scoring functions. In the present review, we focused our attention on the Mol. Mechanics-Poisson Boltzman Surface Area (MM-PBSA) method for the calcn. of binding free energies and its application in VS studies. We provided examples of successful applications of this method in VS campaigns and evaluation studies in which the reliability of this approach has been assessed, thus providing useful guidelines for employing this approach in VS.
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    45. 45
      Tang, Z.; Li, C.; Kang, B.; Gao, G.; Li, C.; Zhang, Z. GEPIA: A Web Server for Cancer and Normal Gene Expression Profiling and Interactive Analyses. Nucleic Acids Res. 2017, 45, W98– W102,  DOI: 10.1093/nar/gkx247
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      GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses
      Tang, Zefang; Li, Chenwei; Kang, Boxi; Gao, Ge; Li, Cheng; Zhang, Zemin
      Nucleic Acids Research (2017), 45 (W1), W98-W102CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Tremendous amt. of RNA sequencing data have been produced by large consortium projects such as TCGA and GTEx, creating new opportunities for data mining and deeper understanding of gene functions. While certain existing web servers are valuable and widely used, many expression anal. functions needed by exptl. biologists are still not adequately addressed by these tools. We introduce GEPIA (Gene Expression Profiling Interactive Anal.), a web-based tool to deliver fast and customizable functionalities based on TCGA and GTEx data. GEPIA provides key interactive and customizable functions including differential expression anal., profiling plotting, correlation anal., patient survival anal., similar gene detection and dimensionality redn. anal. The comprehensive expression analyses with simple clicking through GEPIA greatly facilitate data mining in wide research areas, scientific discussion and the therapeutic discovery process. GEPIA fills in the gap between cancer genomics big data and the delivery of integrated information to end users, thus helping unleash the value of the current data resources. GEPIA is available at http://gepia.cancer-pku.cn/.
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    46. 46
      Avan, A.; Caretti, V.; Funel, N.; Galvani, E.; Maftouh, M.; Honeywell, R. J.; Lagerweij, T.; Van Tellingen, O.; Campani, D.; Fuchs, D.; Verheul, H. M.; Schuurhuis, G.-J.; Boggi, U.; Peters, G. J.; Würdinger, T.; Giovannetti, E. Crizotinib Inhibits Metabolic Inactivation of Gemcitabine in C-Met–Driven Pancreatic Carcinoma. Cancer Res. 2013, 73, 6745– 6756,  DOI: 10.1158/0008-5472.CAN-13-0837
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      Crizotinib Inhibits Metabolic Inactivation of Gemcitabine in c-Met-driven Pancreatic Carcinoma
      Avan, Amir; Caretti, Viola; Funel, Niccola; Galvani, Elena; Maftouh, Mina; Honeywell, Richard J.; Lagerweij, Tonny; Van Tellingen, Olaf; Campani, Daniela; Fuchs, Dieter; Verheul, Henk M.; Schuurhuis, Gerrit-Jan; Boggi, Ugo; Peters, Godefridus J.; Wuerdinger, Thomas; Giovannetti, Elisa
      Cancer Research (2013), 73 (22), 6745-6756CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclin. tests fail in these patients, emphasizing the need of improved preclin. models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathol. and immunohistochem. analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochem. evidence revealed an increased copy no. and overexpression of c-Met in one of the models; we examd. the preclin. efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concns. of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathol., and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clin. evaluation for PDAC treatment.
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    47. 47
      Yin, J.; Kim, S. S.; Choi, E.; Oh, Y. T.; Lin, W.; Kim, T.-H.; Sa, J. K.; Hong, J. H.; Park, S. H.; Kwon, H. J.; Jin, X.; You, Y.; Kim, J. H.; Kim, H.; Son, J.; Lee, J.; Nam, D.-H.; Choi, K. S.; Shi, B.; Gwak, H.-S.; Yoo, H.; Iavarone, A.; Kim, J. H.; Park, J. B. ARS2/MAGL Signaling in Glioblastoma Stem Cells Promotes Self-Renewal and M2-like Polarization of Tumor-Associated Macrophages. Nat. Commun. 2020, 11, 2978,  DOI: 10.1038/s41467-020-16789-2
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      ARS2/MAGL signaling in glioblastoma stem cells promotes self-renewal and M2-like polarization of tumor-associated macrophages
      Yin, Jinlong; Kim, Sung Soo; Choi, Eunji; Oh, Young Taek; Lin, Weiwei; Kim, Tae-Hoon; Sa, Jason K.; Hong, Jun Hee; Park, Se Hwan; Kwon, Hyung Joon; Jin, Xiong; You, Yeonhee; Kim, Ji Hye; Kim, Hyunggee; Son, Jaekyoung; Lee, Jeongwu; Nam, Do-Hyun; Choi, Kui Son; Shi, Bingyang; Gwak, Ho-Shin; Yoo, Heon; Iavarone, Antonio; Kim, Jong Heon; Park, Jong Bae
      Nature Communications (2020), 11 (1), 2978CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: The interplay between glioblastoma stem cells (GSCs) and tumor-assocd. macrophages (TAMs) promotes progression of glioblastoma multiforme (GBM). However, the detailed mol. mechanisms underlying the relationship between these two cell types remain unclear. Here, we demonstrate that ARS2 (arsenite-resistance protein 2), a zinc finger protein that is essential for early mammalian development, plays crit. roles in GSC maintenance and M2-like TAM polarization. ARS2 directly activates its novel transcriptional target MGLL, encoding monoacylglycerol lipase (MAGL), to regulate the self-renewal and tumorigenicity of GSCs through prodn. of prostaglandin E2 (PGE2), which stimulates β-catenin activation of GSC and M2-like TAM polarization. We identify M2-like signature downregulated by which MAGL-specific inhibitor, JZL184, increased survival rate significantly in the mouse xenograft model by blocking PGE2 prodn. Taken together, our results suggest that blocking the interplay between GSCs and TAMs by targeting ARS2/MAGL signaling offers a potentially novel therapeutic option for GBM patients.
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    48. 48
      Zhang, J.; Liu, Z.; Lian, Z.; Liao, R.; Chen, Y.; Qin, Y.; Wang, J.; Jiang, Q.; Wang, X.; Gong, J. Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma. Sci. Rep. 2016, 6, 35784,  DOI: 10.1038/srep35784
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      48
      Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma
      Zhang, Junyong; Liu, Zuojin; Lian, Zhengrong; Liao, Rui; Chen, Yi; Qin, Yi; Wang, Jinlong; Jiang, Qing; Wang, Xiaobo; Gong, Jianping
      Scientific Reports (2016), 6 (), 35784CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Monoacylglycerol lipase (MAGL) is a key enzyme in lipid metab. that is demonstrated to be involved in tumor progression through both energy supply of fatty acid (FA) oxidn. and enhancing cancer cell malignance. The aim of this study was to investigate whether MAGL could be a potential therapeutic target and prognostic indicator for hepatocellular carcinoma (HCC). To evaluate the relationship between MAGL levels and clin. characteristics, a tissue microarray (TMA) of 353 human HCC samples was performed. MAGL levels in HCC samples were closely linked to the degree of malignancy and patient prognosis. RNA interference, specific pharmacol. inhibitor JZL-184 and gene knock-in of MAGL were utilized to investigate the effects of MAGL on HCC cell proliferation, apoptosis, and invasion. MAGL played important roles in both proliferation and invasion of HCC cells through mechanisms that involved prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA). JZL-184 administration significantly inhibited tumor growth in mice. Furthermore, we confirmed that promoter methylation of large tumor suppressor kinase 1 (LATS1) resulted in dysfunction of the Hippo signal pathway, which induced overexpression of MAGL in HCC. These results indicate that MAGL could be a potentially novel therapeutic target and prognostic indicator for HCC.
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    49. 49
      Randazzo, O.; Papini, F.; Mantini, G.; Gregori, A.; Parrino, B.; Liu, D. S. K.; Cascioferro, S.; Carbone, D.; Peters, G. J.; Frampton, A. E.; Garajova, I.; Giovannetti, E. “Open Sesame?:” Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing Its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer. Cancers 2020, 12, 3206,  DOI: 10.3390/cancers12113206
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      "Open sesame?": biomarker status of the human equilibrative nucleoside transporter-1 and molecular mechanisms influencing its expression and activity in the uptake and cytotoxicity of gemcitabine in pancreatic cancer
      Randazzo, Ornella; Papini, Filippo; Mantini, Giulia; Gregori, Alessandro; Parrino, Barbara; Liu, Daniel S. K.; Cascioferro, Stella; Carbone, Daniela; Peters, Godefridus J.; Frampton, Adam E.; Garajova, Ingrid; Giovannetti, Elisa
      Cancers (2020), 12 (11), 3206CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
      Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analog, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal anal. of hENT-1 status evaluated its predictive role by both immunohistochem. (with five different antibodies), and quant.-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels obsd. with this antibody were assocd. with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this anal. and lists mol. factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.
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      Berman, H. M.; Battistuz, T.; Bhat, T. N.; Bluhm, W. F.; Bourne, P. E.; Burkhardt, K.; Feng, Z.; Gilliland, G. L.; Iype, L.; Jain, S.; Fagan, P.; Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, B.; Thanki, N.; Weissig, H.; Westbrook, J. D.; Zardecki, C. The Protein Data Bank. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002, 58, 899– 907,  DOI: 10.1107/S0907444902003451
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      The Protein Data Bank [PDB; Berman, Westbrook et al. (2000), Nucleic Acids Res. 28, 235-242; http://www.pdb.org/] is the single worldwide archive of primary structural data of biol. macromols. Many secondary sources of information are derived from PDB data. It is the starting point for studies in structural bioinformatics. This article describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information and plans for the future development of the resource. The reader should come away with an understanding of the scope of the PDB and what is provided by the resource.
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      Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber Biomolecular Simulation Programs. J. Comput. Chem. 2005, 26, 1668– 1688,  DOI: 10.1002/jcc.20290
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      The authors describe the development, current features, and some directions for future development of the Amber package of computer programs. This package evolved from a program that was constructed in the late 1970s to do Assisted Model Building with Energy Refinement, and now contains a group of programs embodying a no. of powerful tools of modern computational chem., focused on mol. dynamics and free energy calcns. of proteins, nucleic acids, and carbohydrates.
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      Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J. Comput. Chem. 2009, 30, 2785– 2791,  DOI: 10.1002/jcc.21256
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      Morris, Garrett M.; Huey, Ruth; Lindstrom, William; Sanner, Michel F.; Belew, Richard K.; Goodsell, David S.; Olson, Arthur J.
      Journal of Computational Chemistry (2009), 30 (16), 2785-2791CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking expt. with 188 diverse ligand-protein complexes and a cross-docking expt. using flexible sidechains in 87 HIV protease complexes. We also report its utility in anal. of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009.
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      Maestro. Schrödinger, LLC, New York, NY 2016.
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      Macromodel. Schrödinger, LLC, New York, NY 2009.
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      Poli, G.; Gelain, A.; Porta, F.; Asai, A.; Martinelli, A.; Tuccinardi, T. Identification of a New STAT3 Dimerization Inhibitor through a Pharmacophore-Based Virtual Screening Approach. J. Enzyme Inhib. Med. Chem. 2016, 31, 1011– 1017,  DOI: 10.3109/14756366.2015.1079184
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      Identification of a new STAT3 dimerization inhibitor through a pharmacophore-based virtual screening approach
      Poli, Giulio; Gelain, Arianna; Porta, Federica; Asai, Akira; Martinelli, Adriano; Tuccinardi, Tiziano
      Journal of Enzyme Inhibition and Medicinal Chemistry (2016), 31 (6), 1011-1017CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
      Signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell growth regulation and survival. An aberrant STAT3 activation and/or expression is implied in various solid and blood tumors as well as in other pathologies like rheumatoid arthritis and pulmonary fibrosis, thus making the search for STAT3 inhibitors a growing field of study. With the aim of identifying new inhibitors of STAT3 dimerization, we screened a database including more than 1 320 000 com. available compds. using a receptor-based pharmacophore model comprising the key protein-protein interactions identified in the STAT3 dimer and refining the search through docking and mol. dynamic simulations studies. STAT3 binding assays revealed a significant STAT3 inhibitory activity and selectivity vs. Grb2 for one of the four top-scored compds., thus verifying the reliability of the virtual screening workflow. Moreover, such compd. could already be considered as a lead for the development of new and more potent STAT3 dimerization inhibitors.
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      Roe, D. R.; Cheatham, T. E., Jr. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013, 9, 3084– 3095,  DOI: 10.1021/ct400341p
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      PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data
      Roe, Daniel R.; Cheatham, Thomas E.
      Journal of Chemical Theory and Computation (2013), 9 (7), 3084-3095CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the anal. and processing of time series of three-dimensional at. positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange mol. dynamics), image with periodic boundary conditions, create av. structures, strip subsets of the system, and perform calcns. such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
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      Poli, G.; Lapillo, M.; Jha, V.; Mouawad, N.; Caligiuri, I.; Macchia, M.; Minutolo, F.; Rizzolio, F.; Tuccinardi, T.; Granchi, C. Computationally Driven Discovery of Phenyl(Piperazin-1-Yl)Methanone Derivatives as Reversible Monoacylglycerol Lipase (MAGL) Inhibitors. J. Enzyme Inhib. Med. Chem. 2019, 34, 589– 596,  DOI: 10.1080/14756366.2019.1571271
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      Computationally driven discovery of phenyl(piperazin-1-yl)methanone derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors
      Poli, Giulio; Lapillo, Margherita; Jha, Vibhu; Mouawad, Nayla; Caligiuri, Isabella; Macchia, Marco; Minutolo, Filippo; Rizzolio, Flavio; Tuccinardi, Tiziano; Granchi, Carlotta
      Journal of Enzyme Inhibition and Medicinal Chemistry (2019), 34 (1), 589-596CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
      Monoacylglycerol lipase (MAGL) is an attractive therapeutic target for many pathologies, including neurodegenerative diseases, cancer as well as chronic pain and inflammatory pathologies. The identification of reversible MAGL inhibitors, devoid of the side effects assocd. to prolonged MAGL inactivation, is a hot topic in medicinal chem. In this study, a novel phenyl(piperazin-1-yl)methanone inhibitor of MAGL was identified through a virtual screening protocol based on a fingerprint-driven consensus docking (CD) approach. Mol. modeling and preliminary structure-based hit optimization studies allowed the discovery of deriv., which showed an efficient reversible MAGL inhibition (IC50 = 6.1 μM) and a promising antiproliferative activity on breast and ovarian cancer cell lines (IC50 of 31-72 μM), thus representing a lead for the development of new and more potent reversible MAGL inhibitors. Moreover, the obtained results confirmed the reliability of the fingerprint-driven CD approach herein developed.
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    58. 58
      Poli, G.; Lapillo, M.; Granchi, C.; Caciolla, J.; Mouawad, N.; Caligiuri, I.; Rizzolio, F.; Langer, T.; Minutolo, F.; Tuccinardi, T. Binding Investigation and Preliminary Optimisation of the 3-Amino-1,2,4-Triazin-5(2H)-One Core for the Development of New Fyn Inhibitors. J. Enzyme Inhib. Med. Chem. 2018, 33, 956– 961,  DOI: 10.1080/14756366.2018.1469017
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      Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors
      Poli, Giulio; Lapillo, Margherita; Granchi, Carlotta; Caciolla, Jessica; Mouawad, Nayla; Caligiuri, Isabella; Rizzolio, Flavio; Langer, Thierry; Minutolo, Filippo; Tuccinardi, Tiziano
      Journal of Enzyme Inhibition and Medicinal Chemistry (2018), 33 (1), 956-961CODEN: JEIMAZ; ISSN:1475-6366. (Taylor & Francis Ltd.)
      Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (), a hit compd. that showed a micromolar inhibition of Fyn (IC50 = 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimization. This anal. led us to confirm the hypothesised binding mode of and to identify a new deriv. that is about 6-fold more active than (compd. , IC50 = 0.76 μM).
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    59. 59
      Firuzi, O.; Che, P. P.; El Hassouni, B.; Buijs, M.; Coppola, S.; Löhr, M.; Funel, N.; Heuchel, R.; Carnevale, I.; Schmidt, T.; Mantini, G.; Avan, A.; Saso, L.; Peters, G. J.; Giovannetti, E. Role of C-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells. Cancers 2019, 11, 638,  DOI: 10.3390/cancers11050638
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      Role of c-MET inhibitors in overcoming drug resistance in spheroid models of primary human pancreatic cancer and stellate cells
      Firuzi, Omidreza; Che, Pei Pei; El Hassouni, Btissame; Buijs, Mark; Coppola, Stefano; Loehr, Matthias; Funel, Niccola; Heuchel, Rainer; Carnevale, Ilaria; Schmidt, Thomas; Mantini, Giulia; Avan, Amir; Saso, Luciano; Peters, Godefridus J.; Giovannetti, Elisa
      Cancers (2019), 11 (5), 638CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
      Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochem. ELISA (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examd. by luciferase assay, while spheroids' architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for "stage specific embryonic antigen-4" (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, contg. increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids contg. both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Expts. with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.
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    60. 60
      Massihnia, D.; Avan, A.; Funel, N.; Maftouh, M.; van Krieken, A.; Granchi, C.; Raktoe, R.; Boggi, U.; Aicher, B.; Minutolo, F.; Russo, A.; Leon, L. G.; Peters, G. J.; Giovannetti, E. Phospho-Akt Overexpression Is Prognostic and Can Be Used to Tailor the Synergistic Interaction of Akt Inhibitors with Gemcitabine in Pancreatic Cancer. J. Hematol. Oncol. 2017, 10, 9,  DOI: 10.1186/s13045-016-0371-1
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      Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer
      Massihnia, Daniela; Avan, Amir; Funel, Niccola; Maftouh, Mina; van Krieken, Anne; Granchi, Carlotta; Raktoe, Rajiv; Boggi, Ugo; Aicher, Babette; Minutolo, Filippo; Russo, Antonio; Leon, Leticia G.; Peters, Godefridus J.; Giovannetti, Elisa
      Journal of Hematology & Oncology (2017), 10 (), 9/1-9/17CODEN: JHOOAO; ISSN:1756-8722. (BioMed Central Ltd.)
      Background: There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), assocd. with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated mol. mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine. Methods: Phospho-Akt expression was evaluated by immunohistochem. in tissue microarrays (TMAs) with specimens tissue from radically-resected patients (n = 100). Data were analyzed by Fisher and log-rank test. In vitro studies were performed in 14 PDAC cells, including seven primary cultures, characterized for their Akt1 mRNA and phospho-Akt/Akt levels by quant.-RT-PCR and immunocytochem. Growth inhibitory effects of Akt inhibitors and gemcitabine were evaluated by SRB assay, whereas modulation of Akt and phospho-Akt was investigated by Western blotting and ELISA. Cell cycle perturbation, apoptosis-induction, and anti-migratory behaviors were studied by flow cytometry, AnnexinV, membrane potential, and migration assay, while pharmacol. interaction with gemcitabine was detd. with combination index (CI) method. Results: Immunohistochem. of TMAs revealed a correlation between phospho-Akt expression and worse outcome, particularly in patients with the highest phospho-Akt levels, who had significantly shorter overall and progression-freesurvival. Similar expression levels were detected in LPC028 primary cells, while LPC006 were characterized by low phospho-Akt. Remarkably, Akt inhibitors reduced cancer cell growth in monolayers and spheroids and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028, while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a reduced expression of ribonucleotide reductase, potentially facilitating gemcitabine cytotoxicity. Inhibition of Akt decreased cell migration and invasion, which was addnl. reduced by the combination with gemcitabine. This combination significantly increased apoptosis, assocd. with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. However, targeting the key glucose transporter Glut1 resulted in similar apoptosis induction in LPC006 cells. Conclusions: These data support the anal. of phospho-Akt expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies.
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    61. 61
      El Hassouni, B.; Mantini, G.; Li Petri, G.; Capula, M.; Boyd, L.; Weinstein, H. N. W.; Vallás-Marti, A.; Kouwenhoven, M. C. M.; Giovannetti, E.; Westerman, B. A.; Peters, G. J.; EORTC PAMM Group To Combine or Not Combine: Drug Interactions and Tools for Their Analysis. Reflections from the EORTC-PAMM Course on Preclinical and Early-Phase Clinical Pharmacology. Anticancer Res. 2019, 39, 3303– 3309,  DOI: 10.21873/anticanres.13472
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      To combine or not combine: drug interactions and tools for their analysis. Reflections from the EORTC-PAMM course on preclinical and early-phase clinical pharmacology
      El Hassouni, Btissame; Mantini, Giulia; Petri, Giovanna Li; Capula, Mjriam; Boyd, Lenka; Weinstein, Hannah N. W.; Valles-marti, Andrea; Kouwenhoven, Mathilde C. M.; Giovannetti, Elisa; Westerman, Bart A.; Peters, Godefridus J.
      Anticancer Research (2019), 39 (7), 3303-3309CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)
      A review. Combination therapies are used in the clinic to achieve cure, better efficacy and to circumvent resistant disease in patients. Initial assessment of the effect of such combinations, usually of two agents, is frequently performed using in vitro assays. In this , we give a short summary of the types of analyses that were presented during the Preclin. and Early-phase Clin. Pharmacol. Course of the Pharmacol. and Mol. Mechanisms Group, European Organization for Research and Treatment on Cancer, that can be used to det. the efficacy of drug combinations. The effect of a combination treatment can be calcd. using math. equations based on either the Loewe additivity or Bliss independence model, or a combination of both, such as Chou and Talalay's median-drug effect model. Interactions can be additive, synergistic (more than additive), or antagonistic (less than additive). Software packages CalcuSyn (also available as CompuSyn) and Combenefit are designed to calc. the extent of the combined effects. Interestingly, the application of machine-learning methods in the prediction of combination treatments, which can include pharmacogenomic, genetic, metabolomic and proteomic profiles, might contribute to further refinement of combination regimens. However, more research is needed to apply appropriate rules of machine learning methods to ensure correct predictive models.
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      Bard, B.; Martel, S.; Carrupt, P.-A. High Throughput UV Method for the Estimation of Thermodynamic Solubility and the Determination of the Solubility in Biorelevant Media. Eur. J. Pharm. Sci. 2008, 33, 230– 240,  DOI: 10.1016/j.ejps.2007.12.002
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      High throughput UV method for the estimation of thermodynamic solubility and the determination of the solubility in biorelevant media
      Bard, Bruno; Martel, Sophie; Carrupt, Pierre-Alain
      European Journal of Pharmaceutical Sciences (2008), 33 (3), 230-240CODEN: EPSCED; ISSN:0928-0987. (Elsevier B.V.)
      The growing interest for high quality soly. data in the early stages of drug discovery suggested a detailed optimization of exptl. conditions for a 96-well HTS UV method in order to obtain soly. values close to thermodn. soly. measured by shake-flask method. Results have shown that soly. data obtained by the HTS approach were highly dependent on shaking intensity and incubation times due to the formation of supersatd. solns. resulting from the diln. of DMSO stock solns. in aq. buffer. Thus, careful exptl. set-up was developed to improve the quality and the reproducibility of the HTS method. Moreover, the early qual. prediction of bioavailability and absorption of orally administered drugs require more and more biorelevant soly. values in drug discovery programs. Thus, the optimized HTS method was also adapted to measure soly. directly in FaSSIF and FeSSIF media. The versatile HTS UV approach presented in this paper provides a unique and reliable way to det. soly. in various exptl. conditions.
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      Wohnsland, F.; Faller, B. High-Throughput Permeability PH Profile and High-Throughput Alkane/Water Log P with Artificial Membranes. J. Med. Chem. 2001, 44, 923– 930,  DOI: 10.1021/jm001020e
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      High-Throughput Permeability pH Profile and High-Throughput Alkane/Water log P with Artificial Membranes
      Wohnsland, Frank; Faller, Bernard
      Journal of Medicinal Chemistry (2001), 44 (6), 923-930CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      This study reports on a novel, high-throughput assay, designed to predict passive, transcellular permeability in early drug discovery. The assay is carried out in 96-well microtiterplates and measures the ability of compds. to diffuse from a donor to an acceptor compartment which are sepd. by a 9-10 μm hexadecane liq. layer. A set of 32 well-characterized, chem. diverse drugs was used to validate the method. The permeability values derived from the flux factors between donor and acceptor compartments show a good correlation with gastrointestinal absorption in humans. For comparison, correlations based on exptl. or calcd. octanol/water distribution coeffs. (log Do/w,6.8) were significantly lower. In addn., this simple and robust assay allows detn. of pH permeability profiles, crit. information to predict gastrointestinal absorption of ionizable drugs and difficult to obtain from cell culture expts. Correction for the unstirred water layer effect allows to differentiate between effective and intrinsic membrane permeability and opens up the dynamic range of the method. In addn., alkane/water partition coeffs. can be derived from intrinsic membrane permeabilities, making this assay the first high-throughput method able to measure alkane/water log P in the microtiterplate format.
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      Sugano, K.; Hamada, H.; Machida, M.; Ushio, H. High Throughput Prediction of Oral Absorption: Improvement of the Composition of the Lipid Solution Used in Parallel Artificial Membrane Permeation Assay. J. Biomol. Screen. 2001, 6, 189– 196,  DOI: 10.1177/108705710100600309
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      High throughput prediction of oral absorption: Improvement of the composition of the lipid solution used in parallel artificial membrane permeation assay
      Sugano, Kiyohiko; Hamada, Hirokazu; Machida, Minoru; Ushio, Hidetoshi
      Journal of Biomolecular Screening (2001), 6 (3), 189-196CODEN: JBISF3; ISSN:1087-0571. (Mary Ann Liebert, Inc.)
      The purpose of the present study was to improve the compn. of the lipid soln. used in parallel artificial membrane permeation assay for the precise prediction of oral absorption. We modified the compn. of lipid soln., which was used to make a lipid membrane on the filter support. First, we changed the chain length of org. solvent (PC/alkyldienes [C7-C10]). A neg. charge was then added to the membrane to mimic the intestinal membrane (PC/stearic acid/1,7-octadiene and PC/PE/PS/PI/cholesterol/1,7-octadiene). Finally, we examd. the predictability of the PC/PE/PS/PI/CHO/1,7-octadiene membrane using structurally diverse compds. Permeability coeffs. of tested compds. were increased as the chain length of alkyldiene became shorter. The addn. of a neg. charge to the membrane increased the permeability of the basic compds. However, the neg. charged membrane with stearic acid showed different permeability profiles from PC/PE/PS/PI/CHO. The predictability of the PC/PE/PS/PI/CHO/1,7-octadiene membrane was adequate (r = 0.858, n = 31) for use during the early stages of the drug discovery/development process.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltFeiu78%253D&md5=519957734416eb6d7e76d6913f0b9587
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      Bradford, M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248– 254,  DOI: 10.1006/abio.1976.9999
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      A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
      Bradford, Marion M.
      Analytical Biochemistry (1976), 72 (1-2), 248-54CODEN: ANBCA2; ISSN:0003-2697.
      A protein detn. method that involves the binding of coomassie Brilliant Blue G 250 to protein is described. The binding of the dye to protein causes a shift in the absorption max. of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm that is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in ∼ 2 min with good color stability for 1 hr. There is little or no interference from cations such as Na+ or K+ nor from carbohydrates such as sucrose. A small amt. of color is developed in the presence of strongly alk. buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amts. of detergents such as Na dodecyl sulfate, Triton X 100, and commercial glassware detergents. Interference by small amts. of detergent may be eliminated by the use of proper control.
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      Barthel, B. L.; Rudnicki, D. L.; Kirby, T. P.; Colvin, S. M.; Burkhart, D. J.; Koch, T. H. Synthesis and Biological Characterization of Protease-Activated Prodrugs of Doxazolidine. J. Med. Chem. 2012, 55, 6595– 6607,  DOI: 10.1021/jm300714p
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      Synthesis and biological characterization of protease-activated prodrugs of doxazolidine
      Barthel, Benjamin L.; Rudnicki, Daniel L.; Kirby, Thomas Price; Colvin, Sean M.; Burkhart, David J.; Koch, Tad H.
      Journal of Medicinal Chemistry (2012), 55 (14), 6595-6607CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clin. impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common assocn. with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (av. IC50 of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.
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  • Supporting Information

    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01806.

    • RMSD analysis of ligand disposition in the MAGL-11b complexes during the MD; MM-PBSA results for the eight different MAGL-11b complexes; correlation of ligand activity and binding energy obtained using different εint values; activities and best correlated binding energy values predicted for the analyzed ligands; IC50 values of compound 13 toward CB1, CB2, and FAAH; MM-PBSA (εint = 4) results for MAGL-11b and MAGL-13 complexes; minimized average structures of MAGL in complex with 7 and 12; RP-HPLC traces of the final compounds; 1H and 13C-NMR spectra of the final compounds; ESI-HRMS spectra of the final compounds; analysis of the mechanism of MAGL inhibition of JZL-184; inhibition of the activity of MAGL and competitive binding of compound 13; minimized average structures of MAGL in complex with 11b superimposed with 5b; minimized average structures of MAGL in complex with 13 (PDF)

    • Molecular formula strings and associated biochemical data for the reported compounds (CSV)

    • PDB ID of the crystal structure of MAGL (PDB)


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