Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as

Subscriber access provided by READING UNIV

Article

Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors Mojgan Aghazadeh Tabrizi, Pier Giovanni Baraldi, Stefania Baraldi, Emanuela Ruggiero, Lucia De Stefano, Flavio Rizzolio, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Andrea Chicca, Margherita Lapillo, Jürg Gertsch, Clementina Manera, Marco Macchia, Adriano Martinelli, Carlotta Granchi, Filippo Minutolo, and Tiziano Tuccinardi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01845 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors Mojgan Aghazadeh Tabrizi,§ Pier Giovanni Baraldi,§ Stefania Baraldi,§ Emanuela Ruggiero,§ Lucia De Stefano,⊥,ϕ Flavio Rizzolio,‡,ϕ Lorenzo Di Cesare Mannelli,ǁ Carla Ghelardini,ǁ Andrea Chicca,# Margherita Lapillo,†,# Jürg Gertsch,# Clementina Manera,† Marco Macchia,† Adriano Martinelli,† Carlotta Granchi,† Filippo Minutolo,† and Tiziano Tuccinardi†,ψ,* §

Department of Chemical and Pharmaceutical Sciences, University of Ferrara, 44121 Ferrara, Italy

⊥

Graduate School in Chemistry, University of Trieste, 34127 Trieste, Italy

†

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy

ϕ

Division of Experimental and Clinical Pharmacology, Department of Molecular Biology and Translational Research, National Cancer Institute and Center for Molecular Biomedicine, 33081 Aviano (PN), Italy

‡

Department of Molecular Science and Nanosystems, Ca' Foscari Università di Venezia, 30172 Venezia-Mestre, Italy

ǁ

Department of Neuroscience, Psychology, Drug Research and Child Health, Section of Pharmacology and Toxicology, University of Firenze, 50139 Firenze, Italy #

Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH3012 Bern, Switzerland ψ

Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, 19122 Philadelphia, PA, USA

1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 49

ABSTRACT Monoacylglycerol lipase (MAGL) is a serine hydrolase that plays an important role in the degradation of the endocannabinoid neurotransmitter 2-arachidonoylglycerol, which is implicated in many physiological processes. Beyond the possible utilization of MAGL inhibitors as antiinflammatory, anti-nociceptive and anti-cancer 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 compounds possessing efficient reversible inhibitory activities. In this work, we report a new series of reversible MAGL inhibitors. Among them, compound 26 showed to be a potent MAGL inhibitor (IC50 = 0.51 µM, Ki = 412 nM) with a good selectivity versus fatty acid amide hydrolase (FAAH), α/β-hydrolase domain-containing 6 (ABHD6) and 12 (ABHD12). Interestingly, this compound also possesses antiproliferative activities against two different cancer cell lines and relieves the neuropathic hypersensitivity induced in vivo by oxaliplatin.

INTRODUCTION The endocannabinoid system is characterized by a set of neuromodulatory lipids and their receptors, which are involved in a multiplicity of physiological and pathological conditions. Two different types of G protein-coupled receptors (GPCRs) have been discovered and named as central cannabinoid (CB1) and peripheral cannabinoid (CB2) receptors.1 Endocannabinoid signaling regulates various phases of mammalian physiological processes, including pain perception, feeding, emotional state, learning and memory.2-4 Endocannabinoids are hypothesized to act as retrograde messengers, where stimulation of the postsynaptic neuron activates the biosynthesis of endocannabinoids, which are released and transported to activate CB1 receptors expressed mainly in the CNS on the presynaptic terminal.5 Endocannabinoids are biosynthesized on demand from 2 ACS Paragon Plus Environment

Page 3 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


phospholipid precursors at the plasma membrane levels and then released in the extracellular milieu.6 After activating the receptors, endocannabinoids are transported into the cytoplasm via facilitated diffusion and degraded by specific enzymes. Several evidence in vitro and in vivo support the existence of a putative endocannabinoid membrane transporter which mediates the bidirectional movement of endocannabinoids through the plasma membrane7, 8 in cooperation with other proteins such as hydrolytic enzymes and intracellular carrier proteins.9,

10

The complex

enzymatic cascades that regulate endocannabinoid production and inactivation include different hydrolytic enzymes, such as the fatty acid amide hydrolase (FAAH), N-acylethanolamine hydrolyzing acid amidase (NAAA) and monoacylglycerol lipase (MAGL).3,

11

The selective

diacylglycerol lipases (DAGL) α and β are thought to catalyze the formation of 2arachidonoylglycerol (2-AG), and MAGL degrades this compound into arachidonate and glycerol. Anandamide (AEA) is a partial CB1 agonist and a weak partial CB2 agonist, whereas 2-AG acts as a full CB2 agonist.12 The 2-AG levels in the brain are about three orders of magnitude higher than AEA levels, although the relevance of this difference on their signaling actions is still uncertain, mainly taking into consideration that their basal extracellular levels, as measured by in vivo microdialysis, are within 2- to 5-fold.13,

14

In addition to extracellular CB receptors,

endocannabinoids interact with other receptors like the vanilloid receptor 1 (VR1), the transient potential vanilloid type 1 channels (TRPV1) and other specific GPCRs such as opioid or GPR55 and GPR35 receptors.15 The discovery that the endocannabinoids work together with different receptor sites has contributed to the identification of this signaling system as a flexible tool to control diverse functions in the cells and tissues of the organism. Numerous studies suggest the potential use of selective inhibitors implicated in enzymatic degradation of endocannabinoids that could provide selective therapeutic chances.16 To reduce the problems connected with CB1 agonists, an amplification of the actions of AEA and 2-AG by inhibiting their enzymatic degradation has emerged as a potential strategy to develop the endocannabinoid system for medicinal purposes. Pharmacological inhibition of FAAH and MAGL was found to decrease pain, inflammation, 3 ACS Paragon Plus Environment


Page 4 of 49

anxiety, and depression in rodent models without the undesired side effects in motility and behavior observed with direct CB1 agonists.3 First generation MAGL inhibitors include URB602 (compound 1), N-Arachidonoyl maleimide (NAM, compound 2), and OMDM169 (compound 3).17 Carbamate compound 1 (Figure 1) was reported as a MAGL inhibitor with relatively low potency (IC50 = 28 µM)18 and it was shown to be equally potent against FAAH in vitro.19,

20

Furthermore, its

administration was shown to attenuate nociception in rodent models of acute, inflammatory, and neuropathic pain.21, 22 Compound 2 (Figure 1) is an irreversible MAGL inhibitor (IC50 = 140 nM) that was found to decrease 2-AG hydrolase activity of rat cerebellar membranes.23 NAM is not selective due to its chemically reactive maleimide functional group that will likely react with many cysteine-containing proteins in vivo, thus limiting its use in physiologic studies. Another carbamate-based derivative 3 (OMDM169, Figure 1) is a MAGL competitive inhibitor (IC50 = 0.89 µM)24 that increased the 2-AG levels at the site of formalin-induced paw inflammation but it also inhibited pancreatic lipase and DAGL-α, because of its structural similarity with tetrahydrolipstatin. Selective pharmacological tools able to interrupt the in vivo MAGL activity have only become accessible within the last few years. By now, they have been used to demonstrate the role of this enzyme in 2-AG signaling termination and the potential translational use of targeting MAGL in the treatment of nervous system disorders such as pain, anxiety, drug addiction, nausea, and neuroinflammation.25-27 Furthermore, MAGL is upregulated in aggressive cancer cells and primary tumors and its inhibition in aggressive breast, ovarian and melanoma cancer cells impairs cell migration, invasiveness and tumorigenicity.28 Confirmation of MAGL as the primary brain 2-AG hydrolase was achieved by the generation of a selective and in vivo active MAGL inhibitor, JZL184 (4, Figure 1). This piperidine carbamate is a potent and selective MAGL inhibitor (IC50 = 8 nM) that when administered to mice, elevated brain 2-AG levels leading to several cannabinoid-related behavioral effects.29 Almost all the reported compounds are characterized by an irreversible MAGL inhibition mechanism and as reported by Scholsburg et al., the irreversible inhibition of MAGL produces cross-tolerance to CB1 agonists in mice after repeated administrations.30 Furthermore, 4 ACS Paragon Plus Environment

Page 5 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chronic MAGL blockade causes physical dependence, impairs endocannabinoid-dependent synaptic plasticity and desensitizes brain CB1 receptors.30 Considering these drawbacks associated with an irreversible MAGL inhibition, the development of reversible inhibitors could represent a promising alternative strategy; however, their use has been investigated only partially so far, mainly because of the lack of compounds with good MAGL reversible inhibition properties. In 2012 Cisneros et al. reported a series of oxirane derivatives characterized by a dual MAGL/FAAH reversible inhibition activity;31 whereas in 2014 Hernandez-Torres et al. reported the reversible MAGL inhibitor benzo[d][1,3]dioxol-5-ylmethyl 6-([1,1′-biphenyl]-4-yl)-hexanoate 5 (compound c21, Figure 1). Interestingly, this compound was also tested in vivo and its therapeutic effects were not accompanied by catalepsy or other motor impairments that have been observed instead after the administration of irreversible MAGL inhibitors.32 In 2015 Patel et al. developed a series of Loratadine analogues that showed potent and reversible inhibition of MAGL, accompanied also by selectivity towards human FAAH. Furthermore, one of these derivatives proved to possess antagonistic activity towards the Histamine H1 receptor and showed selectivity against cannabinoid receptors, α/β-hydrolase domain-containing 6 (ABHD6) and α/β-hydrolase domain-containing 12 (ABHD12).33 Finally, very recently Granchi et al. reported a benzoylpiperidine derivative characterized by a good MAGL inhibitory activity (IC50 = 0.84 µM) and a reversible mechanism of inhibition.34



Page 6 of 49

O NH

O

O

O

O

O

O

N O

N H

10

O

1, URB602

3, OMDM169

2, NAM O

O

O

O OH

O O

O 2N

N

O

5 O

O

O 5, c21

4, JZL-184

Figure 1. Representative MAGL inhibitors. In the present study, we report the development of a diphenylpyrazole series as a new class of MAGL reversible inhibitors. RESULTS AND DISCUSSION From the analysis of the main available data concerning MAGL reversible inhibition,34-36 some pharmacophoric elements seem to be important for inhibitory activity: a) a carbonyl group able to interact into the oxyanion hole of the enzyme, b) an aromatic portion comprising also an heterocyclic nucleus, involved in van der Waals interactions in a closed hydrophobic region of the binding site (red region of Figure S1) and c) a second aromatic portion which interacts in the open large hydrophobic cavity of MAGL (green region of Figure S1). Following this binding analysis, we hypothesized that the 1,5-diphenylpyrazole scaffold, properly substituted with a carbonyl group and different fragments, could represent the starting point for the identification of new reversible MAGL inhibitors. Following these indications, we synthesized three different diphenylpyrazole derivatives (6-8, Table 1) characterized by different substituents. The compounds were thus tested for their MAGL inhibition activity together with the CAY10499 derivative,37 which was used as a 6 ACS Paragon Plus Environment

Page 7 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reference compound. As shown in Table 1, the (5-(4-hydroxyphenyl)-1-phenyl-1H-pyrazol-3-yl)(4(pyrimidin-2-yl)piperazin-1-yl)methanone (8) showed an appreciable activity with an IC50 value of 6.8 µM, whereas the other two compounds were inactive.

Table 1. MAGL inhibitory activities of diphenylpyrazole derivatives. compd

Structure

IC50 (µM)

6

>> 200

7

>> 200

8

6.8 ± 0.4

CAY10499

0.14 ± 0.02

As shown in Figure 2, the docking results for this compound highlight the presence of two H-bonds between the carbonyl oxygen of the compound and the nitrogen backbone of A51 and M123 in the oxyanion hole. The piperazinylpyrimidine fragment is inserted into the closed hydrophobic region of the binding site and shows lipophilic interactions with I179, L184, V270 and a π-π interaction



Page 8 of 49

with Y194. The N-phenyl ring shows lipophilic interactions with L148, A151, L213 and L241, whereas the 5-phenol ring shows lipophilic interactions with L205 and a weak H-bond with S176.

Figure 2. Docking of compound 8 into MAGL.

Using this compound as a starting hit, a first round of structural investigation has been carried out by maintaining the central pyrazole core fixed and varying: 1) the substituent present on the nitrogen atom (phenyl or methyl group) of the pyrazole ring (R2, Table 2); 2) the portion linked to the carbonyl group in position 3 of the pyrazole ring (R3, Table 2) and 3) the position of the hydroxyl group of the phenolic ring in position 5 (R1, Table 2). As shown in Table 2, the substitution of the 1-phenyl ring with a methyl group determined an important decrease of activity (compound 9) compared to 8, as well as the shift of the p-hydroxy group to the meta position (compound 10). The replacement of the pyrimidine with a phenyl ring determined a slight increase of activity (11, IC50 = 4.7 µM), but when a benzyl group was inserted in place of the phenyl the activity was about two-fold lower (14, IC50 = 12 µM). When pyrimidine was replaced with a methyl group (12) or the entire piperazinylpyrimidine fragment with a morpholine ring (13) a ten-fold decrease of activity was observed. Conversely, the best result was achieved by the replacement of the piperazinylpyrimidine with the 4-benzylpiperidine, which led to a 3.5 fold increase of the activity (15, IC50 = 2.0 µM). Finally, the replacement of the 4-benzylpiperidine with larger 8 ACS Paragon Plus Environment

Page 9 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dimension groups such as the 1-benzylpiperidin-4-amine of compound 16 and the 2-(4-(pyrimidin2-yl)piperazin-1-yl)ethanamine of compound 17 led to a completely loss of activity.

Table 2. MAGL inhibitory activities of 8 derivatives. O R3 N N R2

R1

compd

R1

R2

R3

8

4-OH

9

4-OH

10

3-OH

48 ± 2

11

4-OH

4.7 ± 0.1

12

4-OH

71 ± 3

13

4-OH

61 ± 15

14

4-OH

12 ± 1

15

4-OH

2.0 ± 0.1

16

4-OH

>> 200

IC50 (µM) 6.8 ± 0.4

-CH3

132 ± 20



17

Page 10 of 49

>> 200

4-OH

As shown in Figure 3, with respect to the piperazinylpirimidine of derivative 8, the 4benzylpiperidine of 15 is inserted deeper inside the closed hydrophobic region of the binding site and shows stronger lipophilic interactions with L184, V270 and the π-π interaction with Y194. This different disposition determines also a slight different orientation of the 5-(4-hydroxyphenyl)-1phenyl-1H-pyrazole fragment with the formation of an H-bond between the hydroxyl group and the oxygen backbone of P178.

Figure 3. Docking of compound 15 (green) into MAGL. Compound 8 (cyan) is displayed as a reference compound.

In order to verify the role of the p-hydroxyl in the binding process, this group was removed or replaced with a chlorine atom. As shown in Table 3, in both cases (compounds 19 and 18), the compounds showed a decrease of MAGL inhibitory activity (IC50 value of 13 and 11 µM, respectively), supporting the possible attractive interaction of the p-hydroxy group with the enzyme, as suggested by modeling studies. The comparison between the disposition of compounds 8 and 15 10 ACS Paragon Plus Environment

Page 11 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


suggests that the phenyl ring of 15 bound to the nitrogen atom in position 1 of the pyrazole ring appears to be shifted towards the central region of the open binding cavity and could have weaker lipophilic interactions with L148, A151 and L241, when compared to the phenyl ring of analogue 8. On these bases, the phenyl ring of 15 was substituted with different groups, such as the p-methoxy (20), p-chloro (21) and p-methyl (22) and with different aromatic portions such as the benzyl (23), phenylethyl (24) and phenylpropyl (25) fragments. The enzymatic assays revealed that the introduction of the benzyl group such as in compound 23 determined the highest increase of inhibitory activity (IC50 = 0.81 µM, Table 3). Then, in order to maximize the lipophilic interactions of this fragment, methyl groups in para and meta position of the benzyl moiety were introduced (26, 27 and 28). Finally, a naphtylmethyl group (29) was inserted in the place of the benzyl group. As shown in Table 3, the meta-methyl substitution of the benzyl ring determined a further slight improvement of the MAGL inhibitory activity, with a resulting IC50 of 0.51 µM (compound 26).

Table 3. MAGL inhibitory activities of 4-benzylpiperidines (compound 15 derivatives). O N

R1

N N

R

2

compd

R1

R2

18

Cl

11 ± 3

19

H

13 ± 1

20

OH

3.2 ± 0.1

21

OH

1.4 ± 0.2

IC50 (µM)



Page 12 of 49

22

OH

1.5 ± 0.1

23

OH

0.81 ± 0.02

24

OH

4.3 ± 0.4

25

OH

1.6 ± 0.1

26

OH

0.51 ± 0.03

27

OH

1.1 ± 0.1

28

OH

1.4 ± 0.1

29

OH

1.6 ± 0.3

As shown in Figure 4, the binding disposition of compound 26 is very similar to that observed for 15, maintaining the lipophilic interactions of the 4-benzylpiperidine with L184, V270 and the π-π interaction with Y194, the interaction of the carbonyl group in the oxyanion hole and the H-bond between the p-hydroxyl group and the oxygen backbone of P178. Differently from 15, the mmethylbenzyl fragment points towards the surface of the binding site and shows stronger lipophilic interactions with in particular A151 and L241.


Page 13 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Docking of compound 26 (orange) into MAGL. Compound 8 (cyan) is displayed as a reference compound.

With the aim of evaluating the reversible or irreversible mechanism of inhibition, the effects of dilution and preincubation on the inhibitory ability of compound 26 were evaluated. In the dilution experiments, in case of an irreversible inhibition, the potency should not decrease after dilution. Differently, in case of a reversible inhibition, the potency level should be substantially reduced after dilution.38 In our experiment, the inhibition produced by preincubation with a 20 µM concentration of 26 was measured after a 40X dilution and compared to the potency observed by a 20 µM and a 0.5 µM of compound 26. As shown in Figure 5A, 26 showed a reversible inhibition mechanism, as the inhibition produced by 0.5 µM of the compound was similar to that of 40X dilution, and was different to that produced by the compound at a concentration of 20 µM. As a second test, the activity of 26 was assayed at different preincubation times of the compound with MAGL. In this assay, the compound is incubated with the enzyme 30 and 60 minutes before the addition of the substrate and the observed IC50 is then compared with that obtained without a preincubation of the compound with the enzyme. An irreversible inhibitor will show a higher potency with a higher incubation time whereas a reversible inhibitor will show a constant inhibition potency independent from the incubation time. As shown in Figure 5B, this assay confirmed the reversible property of 26, as it did not show any significant increase in inhibition potency with different incubation times. 13 ACS Paragon Plus Environment


Page 14 of 49

Figure 5. Compound 26-MAGL inhibition analysis. A) Dilution assay: the first two columns indicate the inhibition percentage of compound 26 at a concentration of 20 µM and 0.5 µM. The third column indicates the inhibition percentage of compound 26 after dilution (final concentration = 0.5 µM). B) IC50 (µM) values of 26 at different preincubation times with MAGL (0 min, 30 min and 60 min).

Once confirmed the reversible mechanism of compound 26, its inhibition mode was then determined by evaluating Michaelis-Menten kinetics with various inhibitor concentrations. The 14 ACS Paragon Plus Environment

Page 15 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dataset was plotted as substrate concentration versus MAGL activity and evaluated by using the mixed-model inhibition fit of GraphPad Prism 5.0, thus including competitive, uncompetitive, and noncompetitive inhibition terms. This analysis produces the Vmax, Km, Ki and the α value, a parameter consisting of a positive number (α is always greater than zero) that can be used as an indicator of the mechanism of inhibition. In fact, when α is smaller than one, then the mixed model becomes nearly identical to an uncompetitive model; when it is equal to one, then the mixed-model is identical to a noncompetitive inhibition; finally, when α has a very large value, then the mixedmodel becomes identical to a competitive inhibition. As shown in Figure 6, the Michaelis-Mententype curve resulted in Km values of 0.21 ± 0.07 mM, Vmax value of 42 ± 1 µmol•(min-1mg-1), a Ki value for 26 of 412 ± 19 nM and an α value greater than 100000, thus supporting a competitive behavior for the selected compound.

Figure 6. Inhibition of the activity of MAGL and competitive nature of compound 26.

Selectivity analysis. With the aim of evaluating the selectivity of compound 26 versus the other major endocannabinoid degrading enzymes, its activities against FAAH, ABHD6 and ABHD12 were tested. As shown in Table 4, compound 26 did not show any significant inhibition of FAAH and ABHD12 at the concentration of 10 µM (7% and 32% of inhibition for FAAH and ABHD12, 15 ACS Paragon Plus Environment


Page 16 of 49

respectively), while ABHD6 was actually inhibited with an IC50 value of 7.1 ± 2.3 µM. Thus compound 26 behaves as MAGL inhibitor showing good selectivity over FAAH, ABHD6 and ABHD12 (see the Experimental section for details). Finally, since the diarylpyrazoles are a wellknown class of cannabinoid receptor ligands,39 the possible affinities of compound 26 for the cannabinoid receptors CB1 and CB2 were also evaluated. As reported in Table 4 this compound did not show a significant binding to any of the two cannabinoid receptor subtypes.

Table 4. Biological activity of compound 26 on the major components of the endocannabinoid system. Effects on FAAH, ABHD6, ABHD12, CB1 and CB2 receptors are expressed as IC50 value (mean ± SD, µM). In bracket the % enzymatic inhibition (FAAH and ABHD12) or receptor binding (CB1 and CB2 receptors) at 10 µM is reported. compd

FAAH

ABHD6

ABHD12

CB1 binding

CB2 binding

26

> 10 (7%)

7.1 ± 2.3

> 10 (32%)

> 10 (22%)

> 10 (35%)

Antiproliferative assays. Compound 26 was further tested in in vitro experiments to evaluate its antiproliferative potency against cancer cells, together with compound CAY10499, which was used as the reference compound. As suggested by literature data, MAGL is an optimal candidate target for ovarian cancer.28 Western blot analysis has recently highlighted that MAGL is overexpressed in OVCAR3 and CAOV3 compared to OVSAHO and COV318 cell lines.34 These cell lines were selected since they represent more closely high-grade serous ovarian cancer (HG-SOC),40 a fatal tumor. At the molecular level, all cell lines are mutated in the TP53 gene, BRCA2 gene (OVSAHO) or the cell cycle pathway (OVSAHO, COV318, OVCAR3), which are common events in HG-SOC. Therefore, the antiproliferative activity of the two compounds was tested against these four cell lines, together with the noncancerous human fibroblast lung cells (MRC5). As shown in Table 5, compound 26 caused a good inhibition of cell viability, with IC50 values of 12 and 11 µM in the 16 ACS Paragon Plus Environment

Page 17 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


OVCAR3 and CAOV3 cell lines, respectively, whereas it proved to be about 7-fold less potent against ovarian cancer cells that do not overexpress MAGL, such as OVSAHO and COV318 cells. Furthermore, 26 proved to be inactive also against noncancerous human fibroblast lung cells (MRC5, IC50 > 100 µM). The covalent reference inhibitor CAY10499 showed inhibition of cell viability in all the four ovarian cancer cell lines, with IC50 values ranging from 23 to 77 µM without any significant discrimination between the two MAGL-overexpressing cell lines OVCAR3 and CAOV3 and the other two lines, OVSAHO and COV318.

Table 5. Cell growth inhibitory activities (IC50) of compounds 24 and CAY10499. IC50 values (µM) OVSAHO

OVCAR3

COV318

CAOV3

MRC5

26

90 ± 6

12 ± 2

78 ± 6

11 ± 1

> 100

CAY10499

23 ± 2

43 ± 6

59 ± 4

77 ± 7

> 100

Antinociceptive effects of 26 in animal model of neuropathic pain. The effect of 26 was evaluated in a mouse model of nociceptive behavior caused by the chemotherapeutic agent, oxaliplatin (cold plate test). The pain threshold measurements of oxaliplatin-treated animals are shown in Figure 7. The cold plate test allows to evaluate the response to a thermal non noxious stimulus defined as allodynia-like. On day 15, oxaliplatin decreased the licking latency to 10.0 ± 0.5 s in comparison to control mice (17.6 ± 0.4 s). A single treatment p.o. with 26 relieved pain dosedependently. The lower dose of 10 mg/kg induced a significant effect 30 min after the administration. At 30 mg/kg the compound reduced oxaliplatin-induced neuropathic pain 30 and 45 min after treatment and in particular, after 30 min it increased pain threshold to a value similar to that shown by control animals (vehicle + vehicle).



Page 18 of 49

Figure 7. Effect of 26 on oxaliplatin-induced neuropathic pain. Oxaliplatin (2.4 mg/kg) was dissolved in 5% glucose solution and administered i.p. on days 1-2, 5-9, 12-14. On day 15, 26 (10 and 30 mg/kg) was suspended in carboxymethylcellulose (CMC) and p.o. administered. Painrelated behavior (i.e. lifting and licking of the hind paw) was observed by the cold plate test and the time (s) of the first sign was recorded. **P

Discovery of 1,5-Diphenylpyrazole-3-Carboxamide Derivatives as

Recommend Documents